Spermatogenesis

Biology and Clinical Implications

http://taylorandfrancis.com
 Spermatogenesis
Biology and Clinical Implications

Edited by
C. Yan Cheng, PhD
Senior Scientist, Center for Biomedical Research
The Population Council, New York City, New York, USA
CRC Press
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Library of Congress Cataloging‑in‑Publication Data

Names: Cheng, C. Yan, editor.

Title: Spermatogenesis : biology and clinical implications / edited by C. Yan Cheng.
Other titles: Spermatogenesis (Cheng)
Description: Boca Raton, FL : CRC Press, [2019] | Includes bibliographical references and index.
Identifiers: LCCN 2018003483| ISBN 9781498764117 (pack- hardback and ebook : alk. paper) |
ISBN 9780429488634 (ebook)
Subjects: | MESH: Spermatogenesis | Testis--physiology | Fertility--physiology
Classification: LCC QP255 | NLM WJ 834 | DDC 612.6/1--dc23
LC record available at https://lccn.loc.gov/2018003483

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 Dedicated to:
My wife Po-lee and our children
Yan-ho and Chin-ho
http://taylorandfrancis.com
Contents

Preface ix
Contributors xi

1 Golgi apparatus regulation of differentiation: A case study for male germ cells of the rat testis 1
Louis Hermo, Regiana L. Oliveira, Charles E. Smith, Catherine E. Au, and John J. M. Bergeron
2 Androgen regulation of spermatogenesis 40
William H. Walker
3 Testicular immunoregulation: The role of Tyro3, Axl, and Mer receptor tyrosine kinases and pattern recognition receptors 52
Fei Wang, Qian Jiang, and Daishu Han
4 Inflammation and Spermatogenesis 63
Maria Susana Theas, Patricia Verónica Jacobo, Cecilia Valeria Pérez, Vanesa Anabella Guazzone, and Livia Lustig
5 Junctional adhesion molecule (JAM) family: Recent findings and their role and regulation in spermatogenesis 73
Kun Huang and Wing-Yee Lui
6 Sertoli cell immune regulation within the testis 81
Gurvinder Kaur, Kandis Wright, Robin Hannah Greer, Karl Mueller, Allan Haynes, and Jannette M. Dufour
7 The mitotic phase of spermatogenesis: Recent advances and perspectives 94
Kin Lam Fok and Hsiao Chang Chan
8 Genetics of mammalian meiosis 106
Fang Yang and P. Jeremy Wang
9 Roles of membrane and nuclear estrogen receptors in spermatogenesis 116
Paul S. Cooke, Manjunatha K. Nanjappa, Sergei G. Tevosian, and Rex A. Hess
10 Regulation of fertility and infertility in humans 123
Nahid Punjani, Ryan Flannigan, and Peter N. Schlegel
11 Male infertility: Evaluation and treatment 139
Ryan Flannigan and Marc Goldstein
12 Effects of chemical pollutants on spermatogenesis and implications in male infertility 155
Chris KC Wong
13 Advances in our understanding of human spermatogenesis 168
Qing Wen, Elizabeth I. Tang, Tito Jesus, Bruno Silvestrini, and C. Yan Cheng
14 A look into the testis as a reservoir for HIV and ZIKV—A reproductive biologist’s perspective 183
Elizabeth I. Tang, Christopher L. Robinson, Chi Nok Chong, Shuibing Chen, and C. Yan Cheng
15 Cytoskeletons (F-actin) and spermatogenesis 191
Liza O’Donnell and Peter G. Stanton
16 Roles of mTOR signaling in spermatogenesis 201
Lan Ye and Ke Zheng
17 Does planar cell polarity matter during spermatogenesis? 211
Linxi Li, Haiqi Chen, Qingquan Lian, Ren-Shan Ge, and C. Yan Cheng
18 Computational characterization and integrative analysis of proteins involved in spermatogenesis 220
Pranitha Jenardhanan, Manivel Panneerselvam, and Premendu P. Mathur
19 Effects of chemical exposures on testis cell-cell interactions and endocrine function 234
Rachel C. Knight, Jennifer R. Panizzi, and Benson T. Akingbemi
20 Environmental toxicants on Leydig cell function 245
Leping Ye, Xiaoheng Li, Xiaomin Chen, Qingquan Lian, and Ren-Shan Ge

Index 268

vii
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Preface
Over the last two decades, there have been major advances
in the study of spermatogenesis. However, a number of
these advances come from unexpected groups of inves-
tigators in other fields. These include investigators who
specialize in studying stem cell biology, clinical urolo-
gists and endocrinologists, toxicologists, as well as chem-
ists, biologists, and clinicians. The rationale behind such
as unexpected surge of interest in spermatogenesis is as
follows.
First, there is an intensive interest in the field to under-
stand the biology of spermatogonial stem cells (SSCs) in
the testis, since investigators wish to understand if SSCs in
rodents (Asingle spermatogonia) or humans (Adark spermato-
gonia)1–6 can be used to develop functional spermatids
in vitro,7–9 which would serve as a useful model to study
meiosis. These studies thus shed light on understanding
the molecular mechanism(s) that regulate meiosis, which
remains largely unknown in the testis besides the morpho-
logical events. There is also interest in the field to establish
an in vitro testis model to study testis biology.7,8 In addition,
researchers in the field are trying to use SSCs to develop
other mammalian cells and/or tissues, such as insulin-
producing pancreatic cells10 for tissue transplants. For
instance, a recent report has provided information neces-
sary to develop functional haploid spermatids from human
germline stem cells using a three-dimentional-induced
system.11 This information thus will be exceedingly help-
ful for clinicians to help men who cannot father their own
children due to nonobstructive azoospermia (NOA). In
short, many cell biologists are drawn to the field of sper-
matogenesis to make important discoveries and advances.
Second, there is an increasing number of clinician-based
scientists who are attracted to the field of spermatogenesis
because they need to provide better care to their patients.
For instance, many patients with NOA who cannot produce
functional spermatids were later found to possess normal
SSCs in their testes. The recent report on the production of
functional haploid spermatids from SSCs in men11 will pro-
vide hope to NOA patients since many of these men would
like to use their own functional haploid spermatids for in
vitro fertilization to father their own children using eggs
from their spouses. Furthermore, this technique has been
shown in farm animals, as reported in a recent study in
which functional haploid spermatids were produced using
an in vitro system from SSCs isolated from goat testes.12
Third, some toxicologists and clinicians are also
attracted to the field of studying spermatogenesis because
studies have shown that the testis is a highly sensitive
endocrine organ to environmental toxicants,13–15 so that
more studies are warranted in order to provide treatments
to affected individuals.
Lastly, there is interest in the field of developing non-
hormonal contraceptives for men so that men can play a
more active role in family planning among couples in both
developed and developing countries. This has sparked
interest, drawing chemists, biologists, clinicians, and
pharmacologists into the field to better understand sper-
matogenesis, so that better new molecules can be synthe-
sized and tested prior to clinical studies.16–18
In this book, I attempt to provide a balanced over-
view of the topic of spermatogenesis from the perspec-
tive of a reproductive biologist, while also providing
some thought-provoking concepts on the latest develop-
ments in the field, which will benefit both clinical and
basic research scientists in the field. I have organized
this book so that the opening chapters are devoted to the
basic aspects of hormonal and nonhormonal regulation
of spermatogenesis, immunological regulation of sper-
matogenesis and other recently identified novel pathways,
and meiosis and role of receptors. These are then followed
by chapters devoted to the biology of human fertility and
infertility, with a chapter on the role of the testis that
serves as a reservoir for HIV and Zika virus. Thereafter,
we have chapters on the role of the cytoskeleton, polarity,
and mTOR signaling, and new developments in the field
using computational approaches to study spermatogen-
esis. We conclude with chapters on the role of environ-
mental toxicants that impede spermatogenesis. In short,
all efforts were made to have some of the best investiga-
tors in their fields to update our latest developments in
corresponding fields. As such, this book will serve as an
important reference work for investigators who wish to
encompass different aspects of the rapidly developing
fields pertinent to the study of spermatogenesis. This is
the goal of this work.
I am grateful and indebted to all the colleagues who
worked with me selflessly for the past year. I am particu-
larly indebted to Robert Peden and Kyle Meyer at the pub-
lisher’s office, who has provided the needed support and
patience during the preparation of this work. I am aslo
grateful to Amy Rodriguez who has worked selflessly to
assist us in copy-editing all the chapters and preparing all
the proofs for the publication of this book.
C. Yan Cheng
New York City, New York
REFERENCES
1. Ehmcke J, Schlatt S. A revised model for spermato-
gonial expansion in man: Lessons from non-human
primates. Reproduction. 2006;132:673–680.
ix
x x
2. Ehmcke J, Wistuba J, Schlatt S. Spermatogonial stem
cells: Questions, models and perspectives. Human
Reprod Update. 2006;12:275–282.
3. de Rooij DG. The spermatogonial stem cell niche.
Microsc Res Tech. 2009;72:580–585.
4. de Rooij DG, Russell LD. All you wanted to know
about spermatogonia but were afraid to ask. J Androl.
2000;21:776–798.
5. Oatley JM, Brinster RL. The germline stem cell niche
unit in mammalian testes. Physiol Rev. 2012;​92:577–595.
6. Phillips BT, Gassei K, Orwig KE. Spermatogonial
stem cell regulation and spermatogenesis. Philos
Trans R Soc Lond B Biol Sci. 2010;365:1663–1678.
7. Sato T, Katagiri K, Gohbara A et al. In vitro produc-
tion of functional sperm in cultured neonatal mouse
testes. Nature. 2011;471:504–507.
8. Sato T, Katagiri K, Yoknishi T et al. In vitro produc-
tion of fertile sperm from murine spermatogonial
stem cell lines. Nat Commun. 2011;2:472 (DOI:10.1038​
/ncomms1478).
9. Zhou Q, Wang M, Yuan Y et al. Complete meio-
sis from embryonic stem cell-derived germ cells in
vitro. Cell Stem Cell. 2016;18:330–340.
10. Skurikhin EG, Pakhomova AV, Ermakova NN et
al. Role of tissue-specific stem and progenitor cells
in the regeneration of the pancreas and testicu-
lar tissue in diabetic disorders. Bull Exp Biol Med.
2016;162:146–152.
11. Sun M, Yuan Q, Niu M et al. Efficient generation of
functional haploid spermatids from human germline
stem cells by three-dimensional-induced system. Cell
Death Differ. 2018;25:747–764.
12. Deng SL, Zhang Y, Yu K et al. Melatonin up-regulates
the expression of the GATA-4 transcription factor
and increases testosterone secretion from Leydig
cells through RORα signaling in an in vitro goal sper-
matogonial stem cell differentation culture system.
Oncotarget. 2017;8:110592–110605.
13. Sedha S, Kumar S, Shukla S. Role of oxidative stress
in male reproductive dysfunctions with reference to
phthalate compounds. Urol J. 2015;12:2304–2316.
14. Gao Y, Mruk DD, Cheng CY. Sertoli cells are the
target of environmental toxicants in the testis—A
mechanistic and therapeutic insight. Expert Opin Ther
Targets 2015;19:1073–1090.
15. Moffit JS, Her LS, Mineo AM, Knight BL, Phillips
JA, Thibodeau MS. Assessment of inhibin B as a
biomarker of testicular injury following administra-
tion of carbendazim, cetrorelix, or 1,2-dibromo-3-­
chloropropane in Wistar han rats. Birth Defects Res B
Dev Reprod Toxicol. 2013;98:17–28.
16. Roth MY, Amory JK. Beyond the condom: Frontiers
in male contraception. Semin Reprod Med. 2016;34:​
183–190.
17. Gu X, Gupta V, Yang Y et al. Structure–activity
studies of N-butyl-1-deoxynojirimycin (NB-DNJ)
analogues: Discovery of potent and selective amino-
cyclopentitol inhibitors of GBA1 and GBA2. Chem
Med Chem. 2017;12:1977–1984.
18. Schonbrunn E, Betzi S, Alam R et al. Development
of highly potent and selective diaminothiazole
inhibitors of cyclin-dependent kinases. J Med Chem.
2013;56:3768–3782.
Contributors

Benson T. Akingbemi C. Yan Cheng

Department of Anatomy, Physiology, and Pharmacology The Mary M. Wohlford Laboratory for Male Contraceptive
Auburn University Research
Auburn, Alabama Center for Biomedical Research
Population Council
Catherine E. Au New York City, New York
Department of Medicine
McGill University Hospital Research Institute Chi Nok Chong
Montreal, Canada Department of Surgery
Weill Cornell Medical College
John J. M. Bergeron New York City, New York
Department of Medicine
McGill University Hospital Research Institute Paul S. Cooke
Montreal, Canada Department of Physiological Sciences
University of Florida
Hsiao Chang Chan Gainesville, Florida
Epithelial Cell Biology Research Center
School of Biomedical Sciences, Faculty of Medicine Jannette M. Dufour
The Chinese University of Hong Kong Cell Biology and Biochemistry
Hong Kong, China Texas Tech University Health Sciences Center
and Lubbock, Texas
School of Biomedical Sciences Core Laboratory
Shenzhen Research Institute Ryan Flannigan
The Chinese University of Hong Kong Department of Urology
Shenzhen, China Weill Cornell Medicine
and New York City, New York
Sichuan University–The Chinese University of Hong Kong
Joint Laboratory for Reproductive Medicine Kin Lam Fok
West China Second University Hospital Epithelial Cell Biology Research Center
Chengdu, China School of Biomedical Sciences, Faculty of Medicine
The Chinese University of Hong Kong
Haiqi Chen Hong Kong, China
The Mary M. Wohlford Laboratory for Male Contraceptive and
Research School of Biomedical Sciences Core Laboratory
Center for Biomedical Research Shenzhen Research Institute
Population Council The Chinese University of Hong Kong
New York City, New York Shenzhen, China

Shuibing Chen Robin Hannah Greer

Department of Surgery Department of Cell Biology and Biochemistry
Weill Cornell Medical College Texas Tech University Health Sciences Center
New York City, New York Lubbock, Texas

Xiaomin Chen Ren-Shan Ge

The Second Affiliated Hospital and Yuying Children’s Hospital The Second Affiliated Hospital and Yuying Children’s Hospital
Wenzhou Medical University and
Wenzhou, Zhejiang Institute of Reproductive Biomedicine
Wenzhou Medical University
Zhejiang, China

xi
xii Contributors

Marc Goldstein Tito Jesus

Department of Urology The Mary M. Wohlford Laboratory for Male Contraceptive
Weill Cornell Medicine Research
New York City, New York Center for Biomedical Research
Population Council
Vanesa Anabella Guazzone New York City, New York
Department of Cellular Biology
University of Buenos Aires Qian Jiang
and Institute of Basic Medical Sciences
Cátedra II de Histología Chinese Academy of Medical Sciences
and and
Instituto de Investigaciones Biomédícas (INBIOMED) School of Basic Medicine
Buenos Aires, Argentina Peking Union Medical College
Beijing, China
Daishu Han
Institute of Basic Medical Sciences Gurvinder Kaur
Chinese Academy of Medical Sciences Department of Cell Biology and Biochemistry
and Texas Tech University Health Sciences Center
School of Basic Medicine Lubbock, Texas
Peking Union Medical College
Beijing, China Rachel C. Knight
Department of Anatomy, Physiology, and Pharmacology
Allan Haynes Auburn University
Department of Urology Auburn, Alabama
Texas Tech University Health Sciences Center
Lubbock, Texas Linxi Li
The Mary M. Wohlford Laboratory for Male Contraceptive
Louis Hermo Research
Department of Anatomy and Cell Biology Center for Biomedical Research
McGill University Population Council
Montreal, Canada New York City, New York
and
Rex A. Hess Institute of Reproductive Biomedicine
Department of Comparative Biosciences Wenzhou Medical University
University of Illinois at Urbana-Champaign Zhejiang, China
Urbana, Illinois
Xiaoheng Li
Kun Huang The Second Affiliated Hospital and Yuying Children’s Hospital
School of Biological Sciences Wenzhou Medical University
The University of Hong Kong Zhejiang, China
Hong Kong, China
Qingquan Lian
Patricia Verónica Jacobo The Second Affiliated Hospital and Yuying Children’s Hospital
Universidad de Buenos Aires. Facultad de Medicina Wenzhou Medical University
Departamento de Biología Celular Zhejiang, China
Cátedra II de Histología
and Wing-Yee Lui
CONICET-Universidad de Buenos Aires School of Biological Sciences
Instituto de Investigaciones Biomédícas (INBIOMED) The University of Hong Kong
Buenos Aires, Argentina Hong Kong, China

Pranitha Jenardhanan
Centre for Bioinformatics
Pondicherry University
Pondicherry, India
 Contributors xiii

Livia Lustig Christopher L. Robinson

Department of Cellular Biology Department of Surgery
University of Buenos Aires Weill Cornell Medical College
and New York City, New York
Cátedra II de Histología
and Peter N. Schlegel
Instituto de Investigaciones Biomédícas (INBIOMED) Department of Urology
Buenos Aires, Argentina Weill Cornell Medicine
New York City, New York
Premendu P. Mathur
KIIT University Bruno Silvestrini
Odisha, India S.B.M. Srl Pharmaceuticals
Rome, Italy
Karl Mueller
Department of Cell Biology and Biochemistry Charles E. Smith
Texas Tech University Health Sciences Center McGill University
Lubbock, Texas Department of Anatomy and Cell Biology
Montreal, Canada
Manjunatha K. Nanjappa
Department of Physiological Sciences Peter G. Stanton
University of Florida Hudson Institute of Medical Research
Gainesville, Florida and
Department of Molecular and Translational Science
Liza O’Donnell Monash University
Hudson Institute of Medical Research Clayton, Australia
and
Department of Molecular and Translational Science Elizabeth I. Tang
Monash University The Mary M. Wohlford Laboratory for Male Contraceptive
Clayton, Australia Research
Center for Biomedical Research
Regiana L. Oliveira Population Council
Department of Anatomy and Cell Biology New York City, New York
McGill University
Montreal, Canada Sergei G. Tevosian
Department of Physiological Sciences
Manivel Panneerselvam University of Florida
Centre for Bioinformatics Gainesville, Florida
Pondicherry University
Pondicherry, India Maria Susana Theas
Department of Cellular Biology
Jennifer R. Panizzi University of Buenos Aires
Department of Anatomy, Physiology, and Pharmacology and
Auburn University Cátedra II de Histología
Auburn, Alabama and
Instituto de Investigaciones Biomédícas (INBIOMED)
Cecilia Valeria Pérez Buenos Aires, Argentina
CONICET – University of Buenos Aires
and William H. Walker
Instituto de Investigaciones Biomédícas (INBIOMED) Department of Obstetrics, Gynecology and Reproductive
Buenos Aires, Argentina Sciences
University of Pittsburgh School of Medicine and Magee-
Nahid Punjani Womens Research Institute
Department of Surgery Pittsburgh, Pennsylvania
Division of Urology
University of Western Ontario
London, Canada
xiv Contributors

Fei Wang Kandis Wright

Institute of Basic Medical Sciences Department of Cell Biology and Biochemistry
Chinese Academy of Medical Sciences Texas Tech University Health Sciences Center
School of Basic Medicine Lubbock, Texas
Peking Union Medical College
Beijing, China Fang Yang
Department of Biomedical Sciences
P. Jeremy Wang University of Pennsylvania
Department of Biomedical Sciences Philadelphia, Pennsylvania
University of Pennsylvania
Philadelphia, Pennsylvania Lan Ye
State Key Laboratory of Reproductive Medicine
Qing Wen Nanjing Medical University
The Mary M. Wohlford Laboratory for Male Contraceptive Nanjing, China
Research
Center for Biomedical Research Leping Ye
Population Council The Second Affiliated Hospital and Yuying Children’s Hospital
New York City, New York Wenzhou Medical University
Zhejiang, China
Chris KC Wong
Croucher Institute for Environmental Sciences Ke Zheng
Department of Biology State Key Laboratory of Reproductive Medicine
Hong Kong Baptist University Nanjing Medical University
Hong Kong, China Nanjing, China
Golgi apparatus regulation
of differentiation
A case study for male germ cells of the rat testis
LOUIS HERMO, REGIANA L. OLIVEIRA, CHARLES E. SMITH, CATHERINE E. AU,
and JOHN J. M. BERGERON
INTRODUCTION
The Golgi apparatus is found in each of the estimated
more than 200 different cell types that make up the human
body.1 Golgi apparatus structure visualized by standard
thin sectioning techniques at the electron microscope
(EM) level, which reached its apogee in the latter part of
the last century, can identify solely and unambiguously
any of the differentiated cell types in a given species.
Remarkably, its structure is strictly conserved with the
number and size of cisternae in Golgi stacks and overall
size of the Golgi apparatus under strict control for each of
the many differentiated cell types of a given organ of the
human body. This conjecture has been illustrated here for
the Golgi apparatus using the well-described differentia-
tion of male germ cells in the adult rat testis.
SPERMATOGENESIS: A DISCOVERY BASED
ON THE GOLGI APPARATUS
In the mid-1950s, detailed light microscope (LM) observa-
tions elucidated the complexity of the differentiation path-
way of male germ cells in seminiferous tubules of the testis,
a process referred to as spermatogenesis. The periodic acid
Schiff (PAS) technique2 was the breakthrough that enabled
both the steps of germ cell differentiation to be uncovered
as well as the discovery of stem cells (Figure 1.1).
The PAS stain is specific to vicinyl hydroxyls and when
applied to the testis reveals staining of the Golgi appara-
tus and Golgi derived acrosome of germ cells.3 The reac-
tion due to carbohydrate modifications on glycoproteins
is modulated by Golgi localized glycosyltransferases. This
method provides a precise mapping of how germ cells
associate with each  other in cross sections of seminifer-
ous tubules of the testis, as first described by Leblond and
Clermont3 (Figure 1.2).
In the seminiferous epithelium, germ cells at different
steps of differentiation associate with each other to form
specific cellular associations referred to as stages of the
cycle of the seminiferous epithelium. The PAS method was
the first test in defining the significance of the Golgi appa-
ratus in understanding the differentiation process of any
organ undergoing renewal. Even more important was the
first identification of stem cells identified as a constantly
replenished population undergoing cell division to gener-
ate other stem cells as well as those destined for further
differentiation. The stem cell renewal theory that resulted
1
from this seminal paper is celebrated today as the found-
ing paradigm in modern stem cell biology.4
DEFINING SPERMATOGENESIS
Spermatogenesis is defined as a temporal event whereby
germ cells in the seminiferous epithelium of the testis
undergo a differentiation process into maturing sperma-
tozoa.3,5–10 It occurs over a span of several weeks and is
characterized by a period of mitotic, meiotic, and post-
meiotic events.3,6 During the mitotic or proliferation
phase, spermatogonia undergo several mitotic divisions
to gradually increase their numbers and eventually form
spermatocytes. The latter undergoing two meiotic divi-
sions form haploid spermatids, which metamorphose into
spermatozoa involving a series of dramatic structural and
functional modifications defined as the 19 steps of sper-
miogenesis, a subdivision of spermatogenesis.10
Cross-sectional profiles of the numerous seminiferous
tubules comprising the testis reveal several concentric lay-
ers of germ cells (Figures 1.2 and 1.3).11 These layers consist
of spermatogonia and early spermatocytes localizing to
the base of the epithelium, more advanced spermatocytes
and early spermatids in the mid region, and late sperma-
tids prior to release closest to the lumen. Through the PAS
stain, the different germ cells undergoing differentiation
associate with each other to form specific cellular associa-
tions or stages of their differentiating process. In the rat, 14
different stages were identified and represented by roman
numerals I–XIV. Moreover, in any given cross-sectional
tubule profile constituting a specific stage, the different
germ cell layers were all at the same step of development,
defining what was termed as a generation of germ cells.
In any given tubular cross-sectional profile, four or five
distinct generations of germ cells could be seen, depend-
ing on the particular stage of the seminiferous epithelium
at that precise area of the tubule. Stages I–VIII show five
generations of germ cells, while stages IX–XIV present
four3,6,10 (Figures 1.2, 1.3).
The different generations of germ cells were shown to
be constant for each of the 14 stages of the adult rat tes-
tis.3 Moreover, each generation defining a distinct stage
(e.g., stage I) in the living organism had to be advancing
in differentiation to give rise to the next stage (e.g., stage
II), until all 14 stages appeared in that given area of the
tubule. Considering that spermatozoa have to be released
1
2  Golgi apparatus regulation of differentiation

Glycoconjugate

HO2SHN HIO4 RCH (OH) O2SHN

C NH2 + 2R-CHO C NH

HO2SHN Aldehyde RCH (OH) O2SHN

SO3H
Schiff’s reagent Quinoid chromophore (red ~ purplish blue)

Figure 1.1  Chemical ingredients for the Schiff reaction. (Reproduced with permission from Wako Chemicals USA.)

Stage I Stage II Stage VIII Stage IX

19
RB
15
16
1
8
2 9
P P

S L
A PI
Stage III In In Stage X A
Stage IV Stage XI

10
3 4 11
P
16 17
P
L

In A A L
A Stage XII A Stage XIII
InM
Stage VI Di
Stage V

6
Di
518
Di
17
P P
Z
A
B B BM
Stage VII Stage XIV
18
14
7
IM
IIM
P Di
S II
P
A PI
A

Figure 1.2  Schematic drawings of portions of seminiferous tubules, each representing one of the 14 well-defined stages of the
cycle of the seminiferous epithelium as revealed from PAS-hematoxylin stained preparations. Stages are represented by roman
numerals. Spermatogonia: type A, intermediate (In) and B; InM and BM, mitosis of In and B spermatogonia; preleptotene (Pl), lep-
totene (L), zygotene (Z), pachytene (P), diplotene/diakinesis (Di) spermatocytes and secondary spermatocytes (II). IM and IIM, first
and second meiotic divisions. Arabic numbers represent the 19 steps of differentiation of spermatids undergoing spermiogenesis.
RB, residual body. (Reproduced with permission from American Physiological Society. Clermont, Y. Physiol Rev. 1972;52(1):198–236.)
 Golgi apparatus and acrosome formation  3

58
15 16 16 17 17 18 19 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14

P P P P P P P P P P P P Di II

In In In M B BM PI PI L L L Z Z P
A4M
A1 A1 A1 A1 A1 A1 A1 A1M A2 A2 A2M A3 A3M
I II III IV V VI VII VIII IX X XI XII XIII XIV
40 17 10 12 29 27 58 30 7 7 7 31 18 15

Figure 1.3  Schematic diagram of the different cellular associations or stages of the cycle of the seminiferous epithelium in adult
rats. Drawings as revealed from PAS-hematoxylin stained preparations. The stages are indicated by roman numerals I–XIV. The verti-
cal columns illustrate the different cell types present in each stage. The cells illustrated are spermatogonia: types A1–4, intermediate
(In), and type B; spermatocytes: preleptotene, leptotene, zygotene, pachytene, and diplotene spermatocytes; and spermatids at
steps 1–19 of spermiogenesis. M next to spermatogonia indicates that these cells undergo mitosis, while II at stage XIV indicates
secondary spermatocytes undergoing meiotic division. (Reproduced with permission from Wiley. Dym, M and Clermont, Y. Am J
Anat. 1970;128:265–282.)

periodically from the seminiferous epithelium, the idea of
a cycle of the seminiferous epithelium was proposed. The
sequential occurrence of all 14 stages that occur over time
in a given area of the tubule was defined as a cycle of the
seminiferous epithelium3 (Figure 1.3).11
Using a variety of techniques, the duration of each stage
of the cycle of the seminiferous epithelium was calculated
and shown to be constant but varying in time for differ-
ent stages. Some stages lasted for a short period of time
(i.e.,  stages IX and X [9 hours]), while others lasted lon-
ger (i.e., stage VII [58 hours]) (Figure 1.3). The duration
of each stage was calculated as well as the total duration
of the cycle; in rats it lasts about 13 days and that of the
complete process of spermatogenesis about 65 days.6,12
The entire process of spermatogenesis varies for different
species and even within similar species. A comprehensive
analysis of the stages of the cycle of the rat and several
other species is provided in a complete book on the testis.13
DEFINING SPERMIOGENESIS
Spermiogenesis is a timely event defining the last phase
of germ cell differentiation. It begins after the second
meiotic division of spermatocytes with the formation of
haploid spermatids and follows them through their meta-
morphosis into spermatozoa. It is subdivided into distinct
steps with 19 being identified in rats; the entire process
extends over 22.7 days (Figure 1.3). During spermiogenesis,
many genes/proteins are expressed, some for the first time,
with implications in the diverse dynamic events occurring
at this time point of germ cell development. These events
include elaboration of the acrosome and flagellum, nuclear
condensation, and modification of specific organelles, such
as the Golgi apparatus and endoplasmic reticulum (ER),
leading to the formation of spermatozoa destined to be
released from the seminiferous epithelium with the cyto-
plasmic droplet attached but the residual body detached
and subject to disposal by the Sertoli cells.9,10,14,15
GOLGI APPARATUS AND ACROSOME FORMATION
The most conspicuous event that defines spermatids is the
formation of the acrosome, a membrane-bound lysosomal
structure made by the Golgi apparatus. Steps of spermatid
differentiation as defined by Golgi identification and acro-
some formation were discovered through the PAS reac-
tion, which alone enabled a distinction in the rat among
the transformation of early round spermatids (steps 1–8)
to late elongating spermatids (steps 9–19).3,10 The PAS
staining of the Golgi apparatus and the acrosome is found
in the original illustration from Yves Clermont,16 shown
here in Figure 1.4.
4  Golgi apparatus regulation of differentiation

2 3
6

8
10

13 12

15
19b

17 19a RB

Figure 1.4  Schematic representation of the role of the Golgi apparatus in acrosome formation during spermiogenesis as visual-
ized by the PAS method. At step 2, the Golgi apparatus, seen as a small magenta-colored bubble, has formed two small dense proac-
rosomic granules placed next to the nucleus, which at step 3 have fused together to form a single larger dense granule. At step 6 the
acrosome has grown in size and covers a sizeable portion of the nucleus. At step 8 the nucleus occupies an eccentric position next to
one pole of the nucleus, while the Golgi apparatus becomes spherical in shape and floats freely in the cytoplasm. From steps 10–19
the nucleus becomes gradually compacted, elongated, and sickle-shaped. The spherical Golgi apparatus is still evident as a reddish
bubble in the vast cytoplasm of these cells. At step 19a, the cytoplasm has displaced itself along the tail to form the residual body
seen hanging as a sac onto the sperm at the junction of its head and tail. At step 19b, the residual body has completely liberated
itself from the sperm, while a small dilated focal area of cytoplasm at the neck region of the tail constitutes the Hermes body (cyto-
plasmic droplet). It reveals the presence of a small reddish remnant of the Golgi apparatus, as does the residual body (RB) depicted
in step 19a. (Reproduced with permission from Wiley: Hess, RA et al., Andrology, 2015; 3(6):1015–1021.)

As visualized in a LM, small proacrosomic granules
derived from the hemispherical Golgi apparatus appear
adjacent to the spherical nucleus from steps 1–3 (Figure
1.4). These granules fuse with one another and with con-
tinued production create a larger acrosome (step 4). The
latter gradually applies itself to the nuclear surface, and
with time and progressive enlargement, it spans more and
more of the nuclear surface (steps 5, 6) until its formation
is completed, at which time it covers almost half of the
nuclear circumference (step 7).
After completion, the nucleus and overlying acrosome
of step 7 spermatids shifts from its central position in the
cytoplasm to become eccentric and apply itself directly to
one pole of the plasma membrane. This event constitutes
the formation of step 8 spermatids and coincides with
the change in shape of the Golgi apparatus from crescen-
tic and elongated and closely apposed to the acrosome to
spherical and removed from it. The spherical Golgi there-
after migrates and floats freely in the expanded cytoplas-
mic lobe of differentiating elongating spermatids.
 Defining Golgi proteins localized to spermatogonia: TMED7/p27, TMED4/p25, and TM9SF3  5
During the ensuing decades, the paradigm of the
“cycle”  and the description of germ cells recognizable
by standard light microscopy formed the foundation
for the accretion of knowledge on the subcellular struc-
tures and molecular changes accompanying germ cell
differentiation.10,14,17–23
GOLGI APPARATUS STRUCTURE AND FUNCTION
DISTINGUISHES SPERMATOGONIA FROM
SPERMATOCYTES AND SPERMATIDS
The structure of the Golgi apparatus of spermatogonia and
spermatocytes has been described by EM analysis10,24 but
the majority of earlier studies were done on spermatids
during acrosome formation (i.e., steps 1–7 of spermiogen-
esis10,14,24–29). During this time, the hemispherical Golgi
is closely associated with one pole of the nucleus as it is
actively involved in laying down the membrane bound
acrosome onto its surface. Thereafter, beginning at step 8,
the Golgi apparatus becomes spherical and migrates freely
into the cytoplasmic lobe.9,29 This event coincides with
cessation of transcription in the nucleus30 and with the
replacement in the nucleus of histones by protamines.31
Nevertheless, in elongating spermatids the Golgi appara-
tus continues to be involved in glycoprotein synthesis.32,33
During steps 16–18 spermatids, the Golgi apparatus frag-
ments and becomes vacuolated,29 with individual flattened
cisternae dispersing into the cytoplasm.34 At the last step
of spermiogenesis, two cytoplasmic distensions of the step
19 spermatid become prominent; that is, the cytoplasmic
droplet, recently renamed the Hermes body35 and residual
bodies. The dispersed Golgi cisternae congregate into the
Hermes body (Figure 1.5).34
Historically, the very first EM visualization of Golgi
apparatus for any cell type was done for spermatids36 as
well as very early three-dimensional (3D) images of Golgi
apparatus structure.28 The early spermatid Golgi appa-
ratus is a continuous structure with alternating com-
pact stacked and noncompact tubular interconnecting
regions24,28,37 with an overall appearance in mammalian
cells to that of a ribbon. The term Golgi ribbon was first
introduced by Rambourg and Clermont38 and its usage has
been adopted universally by the scientific community to
define the conserved properties of Golgi apparatus struc-
ture in all mammalian cell types.39–44
The structure of the early spermatid Golgi has been
described in several studies by Clermont et al.37 and visu-
alized diagrammatically (Figure 1.6).9 The stacked regions
of the Golgi ribbon consist of a highly anastomotic Cis
Golgi network (CGN), followed by nine closely apposed
flattened cisternae. Morphologically distinct thickened
cisternae appear on the trans face of the Golgi ribbon and
form trans Golgi networks (TGNs). The latter are close
to the developing acrosome apposed to the nucleus. The
TGNs often reveal a peeling off configuration from the
stack with dilated edges showing a bristle coat. The area
in between the trans face of the Golgi apparatus and the
developing acrosome contains vesicular and tubular pro-
files decorated with fuzz or bristle coats, some of which
fuse with the acrosome.29,45,46 Two types of β-COP, coat-
omer protein complexers, are found associated with the
Golgi apparatus and in the acrosomal membrane of sper-
matids.46 EM cytochemical markers indicate that nico-
tinamide adenine dinucleotide phosphatase (NADPase)
localizes to middle cisternae, thiamine pyrophospha-
tase (TPPase) to the transmost cisternae, and cytidine
5-monophosphatase (CMPase) to the TGNs (Figure
1.6).24,48,49 Because the acrosome also shows a cytochemi-
cal reaction for CMPase, TPPase, and other phosphatases
such as orotidine 50-monophosphatase, it is suggested
that these respective Golgi cisternal elements contribute
directly to acrosome formation (Figure 1.6).14,24
MOLECULAR DISSECTION OF GOLGI APPARATUS
DURING SPERMATOGENESIS
Following an unbiased strategy known as the cell map,50
a profile of Golgi protein expression was determined in
germ cells.51 First, a reproducible method to isolate Golgi
apparatus selectively from male germ cells of whole adult
rat testis homogenates was developed.51 Then by quantita-
tive proteomics, a characterization of over 1000 different
proteins in this subcellular fraction was elucidated, with
several representing novel Golgi proteins as well as known
Golgi proteins.
Several of the proteins identified were characterized
for the first time requiring the generation of antibodies to
a subset to assure that they represented bona fide Golgi
proteins.51 In total, with monospecific antibodies to 20
different proteins obtained from different sources51 and
concluded to be Golgi localized by LM immunocytochem-
ical localization, a map of their expression profile during
spermatogenesis was realized.
The insight gained from the detailed analysis of this data
will be summarized by an illustration of spermatogene-
sis truncated to 30 of the 64 distinct cell types identified
(Figure 1.7). Here germ cells are represented undergoing
mitosis, meiosis, acrosome formation (steps 1–7), posta-
crosome Golgi migration (steps 8–18), and ending at the
terminal step of spermiogenesis, step 19 spermatids when
the Hermes body is formed (Figure 1.7).
DEFINING GOLGI PROTEINS LOCALIZED TO
SPERMATOGONIA: TMED7/P27, TMED4/P25,
AND TM9SF3
The periodic renewal of type A spermatogonia was recog-
nized early in the last century,52 but the complex process
of spermatogonial renewal was only beginning to be fully
understood 50 years later. In the early 1950s, it was the
inspirational work of Clermont and Leblond4 that first
defined stem cells and paved the way for all subsequent
significant contributions on their process of renewal that
over the course of the next 60 years has yielded an exten-
sive amount of information.10,18,53–62
While there are many descriptions of markers for undif-
ferentiated and differentiated spermatogonia,10 those that
demarcate the Golgi apparatus of these cells are clearly lack-
ing. Three of the 20 proteins characterized by Au et al.,51
6  Golgi apparatus regulation of differentiation

An

CD

S
CD CD
Step 19 CD

RB

Step 18

IS
Sertoli Caput CD
G CL ED
cell
RT

Tubule

Step 10 Corpus

N
Testis
AS
G
Step 7
Cauda

Figure 1.5  Diagrammatic representation of the shape, position, and fate of the Golgi apparatus of early and late spermatids dur-
ing spermiogenesis and formation and fate of the cytoplasmic droplet (CD) of sperm as they pass through the epididymal duct. From
steps 1–7, the Golgi (G) forms the acrosomic system (AS) applied to the nucleus (N). From steps 8–16, the Golgi becomes spherical
and occupies the cytoplasmic lobe (CL). In steps 17 and 18, the Golgi apparatus undergoes fragmentation whereupon it is seen as
many randomly dispersed individual Golgi saccular elements (S) floating freely in the CL. At step 19 the Golgi cisternae congregate
into the CD while other organelles enter the residual body (RB), which is phagocytosed by Sertoli cells. The CD is positioned at the
neck region of the sperm flagellum as it forms in the testis at step 19 and remains there as sperm pass through the rete testis (RT),
efferent ducts (ED), and initial segment (IS). In the caput epididymidis, the CD displaces itself along the tail to the annulus (An). In
the corpus it detaches from the sperm leaving most sperm free of CDs in the cauda. (Reproduced with permission from Oko, R et al.,
J Cell Biol. 1993;123(4):809–821.)

although widely distributed throughout spermatogenesis,
clearly labeled the Golgi apparatus of spermatogonia. These
were two members of the transmembrane emp24 domain
(TMEDp24) family of Golgi (GOLD domain) integral
membrane proteins, TMED4/p25 and TMED7/p27, along
with one member of the TM9SF (transmembrane 9 super
family) family of proteins, TM9SF3 (Figure 1.8a).
The TMEDp24 family of Golgi (GOLD domain) mem-
brane proteins regulate trafficking, cargo selection, and
lipid makeup of Golgi-derived carriers.63–66 They are regu-
lated during differentiation and development in many cell
types.67–69 Hence TMED4/p25 and TMED7/p27 proteins
may regulate germ cell differentiation with their expres-
sion in the Golgi apparatus of germ cells commencing in
 MG160 and GBF1  7

NCZ

VA ER

W VA

CIS-element S
S
TGN
NADPase + W
E
M R
TPPase + ER
CMPase +

E
R
ER

Acrosome

Head cap

Nucleus

Figure 1.6  Schematic representation of the Golgi apparatus of a step 7 spermatid. Represented are stacks of Golgi cisternae
(S) forming the compact zone and dilated tubules extending from the cisternal edges serving to join adjacent stacks that form the
noncompact zone (NCZ). The Golgi stack consists of an anastomotic tubular network on the cis face called the CIS-element, mid cis-
ternae stained for NADPase, a trans cisterna stained for TPPase, and two CMPase+ cisternae called the trans Golgi networks (TGN’s).
The latter reveal bristle-coated buds at their edges and a peeling-off configuration. Endoplasmic reticulum (ER) is seen overlying the
cis element, applied to trans cisterna and the TGN, as well as in the NCZ and core or medulla (M) of the Golgi apparatus. Pan-shaped
spaces of the cis cisternae form wells (W). Vesicular aggregates (VA) also appear on the cis face of the Golgi apparatus. (Reproduced
with permission from Clermont, Y et al. Cell biology of mammalian spermiogenesis. In Desjardins C and Ewing L, eds. Cell and
Molecular Biology of the Testis. New York: Oxford University Press, 1993.)

spermatogonia but also extending into late spermatids
(Figure 1.8a and b).
Three members of the TM9SF family of proteins of
unknown function were noted in the isolated germ cell
Golgi fraction, with TM9SF3 localized.51 In yeast, a
related protein to TM9SF3, emp70 (endomembrane pro-
tein 70), has been considered as endocytic and involved
in eisosome regulation (a subdomain of the plasma mem-
brane marked for endocytosis).70 In mammalian cells,
emp70 has been considered to be involved in endosome
to Golgi trafficking.71 In our study, 51 TM9SF3 is a Golgi
integral membrane protein uncovered for the first time as
a Golgi marker not only to germ cells (Figure 1.8a and c),
but also with a ubiquitous expression in many other cell
types, such as Sertoli and Leydig cells (Figure 1.8c), epi-
didymal epithelial cells, 51 and neurons of the brain (not
shown).
DEFINING GOLGI PROTEINS LOCALIZED TO
SPERMATOCYTES: MG160, GBF1, GL54D, AND GIANTIN
Meiosis is unique to germ cells and involves one round of
DNA replication followed by two consecutive cell divisions
enabling diploid cells to form haploid cells. Meiosis serves
important cellular activities such as DNA repair, genetic
exchange, and synapsis of homologous chromosomes,
with many genes implicated in this process.8,10,72–82
In addition to changes in nuclear events, pachytene
spermatocytes undergo a dramatic increase in size dur-
ing meiosis coincident with major changes to the size and
structure of their spherical Golgi apparatus.83,84 An exam-
ple of the Golgi apparatus of spermatocytes as seen in the
EM is illustrated in Figure 1.9a.
MG160 AND GBF1
While the three proteins localized to the Golgi apparatus
of spermatogonia continue to be expressed in spermato-
cytes, additional proteins first appear during spermato-
cyte development, such as MG160 (Golgin160/MEA-2/
Golga3) and GBF1 (GTPase involved in Golgi vesicular
traffic), with the latter extending into early spermiogenesis
(Figure 1.10a).
MG160 is a coiled-coil protein and part of the Golgin
family.85 It is normally expressed in all somatic cells and
is a universal Golgi marker.86,87 The protein is known
8  Golgi apparatus regulation of differentiation

the null mutant may be speculated to be due to an essen-

tial and nonredundant role during meiosis. MG160 has
II 1 2 been implicated in tethering Golgi mini stacks together,91
3
Di and in this role it may be implicated in the doubling in
4 size of the Golgi apparatus of spermatocytes and its even-
P(XII) Mitosis 5 tual partitioning into four Golgi apparatuses with each
(A–B)
6
one destined for the four resulting haploid spermatids.83
P(VII)
Meiosis Several of the Golgi markers used in our study51 readily
P(I) (PL–II) 7 visualized the segregated Golgi fragments during meiosis
Acrosome (Figure 1.8c). This was also visualized by EM, where a dis-
Z
(1–7) 8 tinct Golgi apparatus was noted at opposite poles of the
L nucleus of a spermatocyte at stage XIV of the cycle during
Postacrosome
PL
(8–18) 9 its meiotic division (Figure 1.9b).
Hermes biogenesis GBF1 (Golgi-specific brefeldin A-resistance guanine
B (19) nucleotide exchange factor) regulates vesicular traffick-
In ing for active secretion.92 The expression of GBF1 during
A spermatogenesis marked the first evidence for a differen-
10 tial expression of this protein in any cell differentiation
pathway. GBF1 expression begins at preleptotene sper-
11 matocytes and extends into early spermiogenesis (Figure
1.10a and c). Hence, the burst of GBF1 expression in sper-
12 matocytes and early spermatids likely reflects their high
19 18 17 16 15 14 13
secretory activity. In fact, forerunners of the proacrosomic
granules first appear in zygotene primary spermatocytes
Figure 1.7 Thirty representative germ cell types are and extend into late pachytene spermatocytes,74,93–95 with
depicted as they undergo differentiation during spermato- acrosome formation advancing through the first half of
genesis. Illustrated are the mitotic spermatogonia types A, spermiogenesis.10,14
intermediate (In) and B; spermatocytes undergoing meiosis:
preleptotene (PL), leptotene (L), zygotene (Z), pachytene (P),
GL54D AND GIANTIN
diplotene (Di), and secondary spermatocytes (II). Spermatids The most abundant protein of our isolated testis germ cell
evolve through 19 steps of spermiogenesis that can be sub- Golgi fraction was a protein designated from the annota-
divided into acrosome formation (steps 1–7), postacrosome tion of UniProtKB/Swiss-Prot, as glycosyltransferase 54
events (steps 8–18) and Hermes body (cytoplasmic drop- domain-containing protein (GL54D), an MGAT4 family
let) formation (step 19). (Reproduced with permission from member also known as GNT1IP-S.96 GL54D, an integral
Mol Biol Cell: Au, CE et al., Mol Biol Cell. 2015; 26(22):4015–4032 membrane glycoprotein like TM9SF3, is a Golgi marker.
DOI:10.1091/mbc.) GL54D was uncovered for the first time at the endogenous
protein level by the mass spec characterization of our iso-
lated testis Golgi fraction.51 However, whereas TM9SF3 is
ubiquitous to all cells as a universal Golgi marker, GL54D
to interact with Golgi membranes through RAB6 and is germ cell specific, revealing a differential expression
Arf1.88 In the testis, MG160 expression demarcates the profile during spermatogenesis. GL54D is first detected
boundary between spermatogonial renewal and spermio- in pachytene spermatocytes at stage VII of the cycle with
genesis (i.e., spermatocyte differentiation) with expres- expression extending up to step 16 spermatids (Figure
sion exclusive to spermatocytes (Figure 1.10b). MG160 1.11a, b, and c). GL54D is a type II, N-linked integral mem-
has long been known to be a prominent gene product of brane terminally glycosylated glycoprotein largely facing
male germ cells89; however, during spermatogenesis it the lumina of Golgi cisternae and with sole expression in
is differentially expressed. A null mutation in the gene the testis, where it is expressed as the most abundant Golgi
abolishes protein expression in mice with a phenotype protein of germ cells.
of disrupted germ cell development during late meiosis, Giantin (GOLGB1) was the most abundant protein of
coinciding precisely with its localization to meiotic sper- the isolated testis Golgi apparatus fraction in the tethering
matocytes. Sperm development is markedly decreased docking and fusion category and at far higher concentra-
in these mice and the resultant few sperm are infertile. tions than in liver Golgi fractions.51 Like MG160, giantin is
When the gene is truncated in a transgenic mouse model a Golgin involved in the tethering and vesicular trafficking
exactly as for the MG160 null mutant, pachytene sper- functions of the Golgi apparatus.97 During spermatogen-
matocytes are depleted.90 esis, giantin has been revealed in pachytene spermatocytes
Although MG160 (membrane protein of the Golgi but its precise expression pattern during spermatogenesis
apparatus) is expressed and localized to Golgi apparatus has not been deduced.98–100 Nevertheless, the coincidence
in all somatic cells,86,87 the male germ cell specific effect of between giantin, a Golgi resident protein on the cytosolic
 GRASP55 and GPP34  9

(a) (b)

II 1 2
3 P Lu
Di S
4
P(XII) Mitosis 5
(A–B) 10 µm
P(VII) 6
Meiosis
(PL–II) (c)
P(I) 7 S
Acrosome
Z (1–7) L
8
L Postacrosome P
(8–18)
PL 9
Hermes biogenesis P
(19)
B
10 µm
In
A
10

11
TM9SF3; TMED4/p25
12 TMED7/p27
19 18 17 16 15 14 13

Figure 1.8  (a) Wheel depicting the expression profile of TMED7/p27, TMED4/p25, and TM9SF3. All three proteins span most of
spermatogenesis from spermatogonia until step 16 spermatids, with the exception of TMED7/p27 that reappears at step 19 sperma-
tids. (b) LM-IHC of a seminiferous tubule reactive for TMED7/p27 at stage VII of the cycle. Reactivity is noted in the spherical Golgi
apparatus of spermatocytes (P), early round spermatids (small arrowheads), late step 19 spermatids (curved arrows), and Sertoli
cells (S). (c) LM-IHC of tubules at stages XIV and XIII of the cycle reactive for TM9SF3. Golgi reactions are noted for pachytene sper-
matocytes (P), Sertoli cells (S), Leydig cells (L), and late spermatids (large arrowheads). At stage XIV, spermatocytes undergo meiosis
and reveal multiple Golgi bodies (boxes).

side and rims of Golgi cisternae,85 and GL54D, a luminal
Golgi protein, and with both being expressed in pachy-
tene spermatocytes, suggests that a coordination exists
between these two proteins in protein modifications
within the Golgi apparatus.
DEFINING GOLGI LOCALIZED PROTEINS IN EARLY
SPERMATIDS DURING ACROSOME FORMATION
The acrosome is a lysosomal structure that is formed by
the Golgi apparatus during early spermiogenesis with an
unprecedented specificity for adherence to the nuclear
surface of spermatids. As such it follows the contours
and modifications that occur to the shape of the head
and nucleus of spermatids during late steps of spermio-
genesis.13 At the time of fertilization, sperm undergo the
acrosome reaction, an event unique in the life of a sperma-
tozoon. Acrosomal contents released during fertilization
promote sperm penetration through the oocyte’s invest-
ments, leading to sperm-egg interaction and fusion.101
Acrosome formation is an intense period of protein
synthesis marked by the expression of a large number
of proteins localized to the Golgi apparatus as it forms
the acrosome. Some of the proteins uncovered in our
study were traditional Golgi markers, but others had
unexpected Golgi localizations.51 EM images of acro-
some formation by the Golgi apparatus are illustrated in
Figure 1.9c–e.
In addition to TMED7/p27, TMED4/p25, TM9SF3,
GBF1, and GL54D, all with widespread distributions, five
additional proteins with Golgi characteristics (Grasp55,
GPP34, Man2α1, ManIIX, and LAMAN) revealed onset of
Golgi expression in early spermatids during acrosome for-
mation (Figure 1.12a). Examples are illustrated for GRASP
55 (Figure 1.12b) and GPP34 (Figure 1.12c).
GRASP55 AND GPP34
GRASP55 (Golgi reassembly associated protein) has been
implicated in the stacking of Golgi cisternae.102–104 Its
expression first appears after meiosis with Golgi immuno-
reactivity to steps 1–7 spermatids, and with no localization
to Sertoli or Leydig cells of the testis, all of which reveal
stacked Golgi cisternae.105,106 Alternative functions to those
of cisternae stacking have been proposed for GRASP55,
such as unconventional protein secretion and Golgi rib-
bon formation.107 Unlike typical lysosomes, the acrosome
has to bind to the nuclear surface of spermatids and this
10  Golgi apparatus regulation of differentiation
N
G
N Remarkably, three normally ER-located proteins
G
S (Figures 1.12a and 1.13a).51 They included UBX domain
S (b)
(a) S protein of the testis (PDILT) (Figure 1.13c).
A
S
N tail.107 The only other protein known to have saposin
(d)
S secretory pathway to lysosomes where it is converted by
S S
A
N
(c) (e) N During acrosome formation, sulfoglycolipids are highly
Figure 1.9  EM of the Golgi apparatus of different germ
cells. (a) Golgi apparatus of a mid pachytene spermatocyte. N:
nucleus; S: stacks of Golgi cisternae. (b) Spermatocyte under-
going meiotic division revealing a Golgi apparatus (G) at
opposite poles of the nucleus (N). (c) Stack of Golgi saccules
(S) of step 1 spermatid shows several proacrosomic granules
(arrowheads) next to the nucleus (N). (d) Forming acrosome
(A) at step 4 spermatid is applied to the nucleus (N). (e) Step 7
spermatid shows a Golgi stack (S) and expanded acrosome (A)
applied to the nucleus (N). Original magnifications: 9a: 22,500×;
9b: 38,000×; 9c–e: 48,750×.
may involve unconventional secretion pathways to accom-
modate this novel function for the acrosome. Of note is the
fact that of all the five Golgi proteins indicated above, only
GRASP55 does not become expressed again during later
steps of spermiogenesis and in the forming Hermes body,
where Golgi cisternae do not form a stack (Figure 1.12a).
GPP34, now known as GOLPH3 (Golgi phophopro-
tein), has a well-defined role in the regulation of Golgi
ribbon structure,108 and this property may also be shared
with GRASP55.107 During steps 1–7 spermatids, the Golgi
apparatus is crescent in shape with the TGNs exposed to
the nuclear surface of early spermatids. This would allow
easy access of the proacrosomic granules to the nuclear
surface to which they bind. Defining the shape of the Golgi
ribbon may be in part the role of GOLPH3 and GRASP55,
but this would be limited to its expression profile during a
defined time point during spermatogenesis. GOLPH3 is not
expressed from steps 8–18 but returns at step 19 spermatids
to be included in the forming Hermes body (Figure 1.12a).
UNEXPECTED ER RESIDENT PROTEINS WITH GOLGI/
ACROSOME LOCALIZATIONS: SAPRETICULIN, PDILT,
AND UBXD8
were also Golgi localized during acrosome formation
containing 8 protein (UBXD8/FAF2), sapreticulin
(Figure 1.13b), and protein disulphide isomerase-like
Sapreticulin is a saposin B domain containing ER
luminal protein with the canonical ER targeting KDEL
domains is prosaposin, normally targeted through the
selective proteolysis to individual saposins A, B, C, and
D that are needed to solubilize different glycolipids for
degradation. Intriguingly, prosaposin is not expressed
in male germ cells.110 Hence, sapreticulin remains as the
only saposin domain expressing protein in spermatids.
expressed and localized to the Golgi apparatus.111 The
major sulfoglycolipid is seminolipid112 and it is pre-
dicted to be the potential client of sapreticulin. Hence, a
speculated role for sapreticulin is in sulfoglyolipid orga-
nization of the Golgi of spermatids during acrosome
formation.
The testis-specific PDILT performs a specialized
chaperone function by liaising with partner proteins
in disulfide-dependent complexes within germ cells
of the testis.113 The PDI family member erp44 (endo-
plasmic reticulum resident protein) is now accepted to
be Golgi localized in most cell types but whose local-
ization changes to ER depending on its cargo.114,115
A known PDILT client in germ cells is the acrosome
located membrane protein ADAM3 (a disintegrin and
metalloprotease).113,116 However, after localizing to the
Golgi and acrosome from steps 1–7 (solid brown line,
Figure  1.13a), PDILT relocalizes to the cytoplasm (ER)
where it is present from steps 8–19 (stippled brown line,
Figure 1.13c).113,117 Hence, it is the PDILT client proteins
that likely dictate its localization to specific organelles of
the secretory pathway.
The ubiquitin-like domain containing protein UBXD8
(FAF2), an ER associated degradating protein, is promi-
nent in the cytoplasm (ER) of spermatocytes (prelepto-
tene to diplotene, stippled blue line, Figure 1.13a) but
relocalizes to the Golgi during acrosome formation
(solid blue line, Figure 1.13a). In the ER of other cell
types, a function for UBXD8 in quality control to remove
newly synthesized misfolded secretory proteins has been
proposed as well as in lipid droplet formation118 and
ER-associated protein degradation (ERAD).119,120 Its role
in the Golgi and acrosome of early spermatids remains
to be determined.
 Other unexpected proteins with Golgi/acrosome localizations  11

(a) (b)
L P

1 PL
II 2 P
3
Di S
4 S
P(XII) Mitosis 5
(A–B)
P(VII) 6 10 µm
Meiosis
(PL–II) (c)
P(I) 7
Acrosome Lu
Z (1–7) 8
L Postacrosome A
(8–18) P
9 A
PL Hermes biogenesis A
(19) P
B
10 µm
In
A
10

11
GBF1
12
19 18 17 16 15 14 13 MG160

Figure 1.10  (a) Wheel depicting the expression profile of GBF1 and MG160. While both begin expression in PL spermatocytes,
MG160 is only expressed during spermatocyte development while GBF1 extends up to step 7 spermatids and then is absent until
it reappears at step 19. (b) LM-IHC of seminiferous tubules reactive for MG160. Reactive germ cells include the Golgi apparatus of
preleptotene (PL) and pachytene (P) spermatocytes. Sertoli (S) and Leydig (L) cells also show a Golgi reaction. (c) LM-IHC of GBF1 at
stage VI with Golgi reaction in pachytene spermatocytes (P) and Golgi (arrowheads) and acrosome (A) reactions in round spermatids.

OTHER UNEXPECTED PROTEINS WITH GOLGI/ somatic counterpart Hsc70,125 HSP70.2 may participate in
ACROSOME LOCALIZATIONS clathrin-coated vesicle trafficking known to be prominent
Several other proteins were unexpectedly localized to during acrosome formation.26
the Golgi apparatus and acrosome of steps 1–7 sperma- Using a different antibody obtained from Eddy,126
tids (Figures 1.12a and 1.13a). They include Annexin A6, HSP70.2 localized to spermatocytes and elongating sper-
HSP70.2 (Figure 1.13d), and FAM3C (Figure 1.13e). matids, including the sperm flagellum. The HSP70.2
Annexin A6 is a calcium-dependent membrane binding family of chaperone proteins may facilitate the intracel-
protein linked to membrane and cytoskeletal organization lular transport of proteins in elongating spermatids and
and cholesterol homeostasis.121 It may serve a function in likely supports plasma membrane remodeling and remov-
membrane trafficking122 that is highly active in acrosome ing surplus cytoplasm to maintain proper sperm head
formation. shapes.125–129
HSP70.2 (HspA2) is part of the heat shock protein A family with sequence similarity 3, member C
(HSP)70 family of molecular chaperones needed to sta- (FAM3C) (Figure 1.13e) is a protein of poorly under-
bilize otherwise labile proteins.123 The HSP70 family of stood function that is linked to GPP34 (GOLPH3) during
chaperones usually masks exposed hydrophobic residues Golgi extension and vesicular release130 and together with
on client proteins, preventing their aggregation and insol- GRASP55 and GOLPH3 may be required for maintaining
ubility. Our recent study51 represents the first localization the shape of the crescentic Golgi ribbon during acrosome
of the germ cell cytosolic chaperone HSP70.2 to the acro- formation.
some and Golgi apparatus of steps 1–7 spermatids (Figure Taken together, the recruitment of chaperones
1.13d), which we found to be antibody-specific.124 As for its (HSP70.2), protein-folding enzymes (PDILT), and ERAD
12  Golgi apparatus regulation of differentiation

(a) (b) Lu

II 1 2
3
Di
4
P(XII) Mitosis 5
(A–B)
P(VII) 6 10 µm
Meiosis
P(I) (PL–II)
7 (c)
Acrosome
Z (1–7) 8
L Postacrosome
(8–18) 9
PL Lu
Hermes biogenesis
B (19)

In 10 µm
A
10

11

12 GL54D
19 18 17 16 15 14 13

Figure 1.11  (a) Wheel depicting the expression profile of GL54D, which spans the Golgi apparatus from pachytene spermato-
cytes at stage VII until step 16 spermatids. (b) LM-IHC of seminiferous tubules reactive for GL54D at stage XIV. The upper tubule shows
Golgi reactions for pachytene spermatocytes (P), step 1 (small arrowheads), and step 14 (large arrowheads) spermatids. (c) LM-IF of
GL54D with reactions in Golgi of round (small arrowheads) and late (large arrowheads) spermatids. Lu, lumen.

quality control proteins (UBXD8) to the Golgi apparatus SEGREGATION OF GOLGI LOCALIZED PROTEINS
must reflect a requirement to assure the fidelity of sort- TO THE ACROSOME
ing and transport of proteins during the enormous bur- During acrosome formation (steps 1–7), 16 proteins
den of protein synthesis and trafficking during acrosome localized to the Golgi apparatus (Figure 1.14). Of the
biogenesis. 16, 12 (i.e., GRASP55, GBF1, ManIIX, Annexin A6,
GPP34, Man2α1, HSP70.2, Fam3C, PDILT, sapreticu-
Man2α1, ManIIX, AND LAMAN lin, UBXD8, and LAMAN) localized to both the Golgi
Man2α1 and ManIIX are both well-known Golgi manno- apparatus and developing acrosome. However, four
sidases. Man2α1 is a universal Golgi marker localizing to Golgi proteins (i.e., GL54D, TM9SF3, TMED4/p25, and
germ and Sertoli cells.100,132 ManIIX is a male germ cell spe- TMED7/p27) reacted only for the Golgi apparatus with
cific mannosidase partially overlapping with Man2α1 in its no evidence of reactivity for the acrosome (Figure 1.15).
specificity from steps 1–7 spermatids where both the Golgi Hence, a segregation of proteins exists during acrosome
and acrosome are reactive (Figure 1.12a). These sequence- formation whereby some proteins are excluded from the
related proteins likely have related functions in alpha man- acrosome but are present in the Golgi apparatus involved
nosidase trimming of high mannose containing N-linked in its formation.
oligosaccharides during acrosome formation.133–135 Acrosome formation is often explained by the matura-
LAMAN, also known as Man2β1, a major glycoprotein, tion model for Golgi biogenesis and traffic13; however, the
was also found in the Golgi apparatus and acrosome of segregation of the four Golgi markers is not consistent
steps 1–7 spermatids (Figure 1.14). LAMAN is a ubiqui- with this model. Alternative models to maturation have
tous lysosomal alpha mannosidase that cleaves alpha man- been proposed for Golgi traffic,137–140 and based on our
nosidase linkages in glycans.136 Its functional significance observations the spermatid Golgi is well suited to examine
in acrosome formation and spermatogenesis is unknown. the validity of these various models.
 Golgi role in chromatoid body and MVB formation  13

(a) (b)
Lu
A
II 1 2
3 A
Di
4
A
P(XII) Mitosis 5
(A–B) 10 µm
P(VII) Meiosis 6
(PL–II) (c)
P(I) 7 Lu
Acrosome
Z A
(1–7) 8 A
L Postacrosome
(8–18)
PL 9
Hermes biogenesis A
B (19)
10 µm
In
A
10

11 GRASP55

12 ManIIX
19 14 13 Annexin A6; Man2 1; Fam3C; GPP34;
18 17 16 15
Sapreticulin; HSP70.2; LAMAN

Figure 1.12  (a) Wheel depicting the expression profiles of several Golgi localized proteins. Spanning the Golgi apparatus
from steps 1-7 spermatids are GRASP55, ManIIX, Man2α1, Annexin A6, Fam3C, GPP34, sapreticulin, HSP70.2, and LAMAN. While
GRASP55 is limited to steps 1–7 spermatids, ManIIX reappears from steps 15–19 while the others only reappear at step 19 spermatids.
(b) and (c) LM-IHC of GRASP55 (b) and GPP34 (c) at stage V showing Golgi (small arrowheads) and acrosome (a) reactions of the step
5 spermatids.

GOLGI ROLE IN CHROMATOID BODY AND MVB
FORMATION
During early spermiogenesis, the chromatoid body is
closely associated with the Golgi apparatus. It is seen
as a spongy-looking mass of electron dense granulo-
filamentous material with areas of low electron density
infiltrated with numerous medium-sized vesicles.14,141–143
In late spermiogenesis, the chromatoid body migrates
toward the centrioles occupying the opposite pole of the
nucleus alongside the annulus of the flagellum.142,144 The
vesicles are CMPase- and NADPase-positive, suggesting
a contributing role from the Golgi apparatus.24 VPS26A/
VPS35-containing retromer vesicles have also been iden-
tified within the chromatoid body.145 Some of our Golgi
localized proteins during acrosome formation may also
have an impact on vesicles associated with the chroma-
toid body.
Spermatid development is also marked by the presence
of endocytic structures (i.e., pits, endosomes, multivesicu-
lar bodies [MVBs], and lysosomes).146,147 Another proposed
role of the spermatid Golgi apparatus is to contribute to
MVB formation.24,148 Conversely, MVBs play a role in the
processing of the Golgi-derived components to the develop-
ing acrosome. The imino sugar N-butyldeoxynojirimycin
(NB-DNJ) is an inhibitor of the N-linked oligosaccharide
processing enzymes alpha-glucosidase I and II, with the
potential to inhibit both glucosidases and glucosyltrans-
ferases. Treatment with NB-DNJ results in accumulation
of MVBs in the vicinity of the nucleus and presence of pro-
acrosomic granules that do not fuse together to form an
acrosome. Thus, acrosomal membrane proteins and cyto-
solic acrosome-associated proteins become redirected to
MVBs in affected testes.149,150
Additionally, a role for the endocytic apparatus of early
spermatids has been defined in acrosome formation.
Ubiquitin-specific peptidase 8 (USP8), a deubiquitinating
enzyme that works as a regulator of endosomal sorting and
vesicle morphology, is highly expressed in male germ cells.
USP8 interacts with the spermatid protein endosomal-
sorting complex required for transport-0 (ESCRT-0) and
microtubule structures with both contributing to acrosome
formation. USP8 links, via its MIT domain, the developing
acrosome and vesicles to microtubules, which monitor both
acrosome assembly and shaping.151
14  Golgi apparatus regulation of differentiation

(a) (b) (c)

Lu
A Lu
II 1 2 * *
3
A A
Di 4 A A
P(XII) Mitosis 5
(A–B) 10 µm 10 µm
P(VII) Meiosis 6

P(I) (PL–II) (d) (e)

7
Acrosome A
Z (1–7) 8 Lu
L Postacrosome Lu
(8–18) A
PL 9 T
Hermes biogenesis
A A
B (19)
10 µm 10 µm
In
A
10
Annexin A6; Man2 1; Fam3C; GPP34;
Sapreticulin; HSP70.2; LAMAN
11
UBXD8
12
13 PDILT
19 18 17 16 15 14

Figure 1.13  (a) Wheel depicting the expression profiles of several Golgi localized proteins. All proteins listed span the Golgi
apparatus of steps 1–7 spermatids. In addition, UBXD8 also reveals a cytoplasmic reaction in spermatocytes during meiosis while
PDILT is cytoplasmic from steps 8–19. Annexin A6, Man2α1, Fam3C, GPP34, sapreticulin, HSP70.2, and LAMAN reappear at step 19
spermatids. (b), (c), (e) LM-IHC of sapreticulin (b), PDILT (c), and Fam3C (e) at stages V of the cycle revealing Golgi (small arrowheads)
and acrosome (a) reactions of the step 5 spermatids. In (c), the cytoplasmic lobes of the late spermatids show a cytoplasmic reaction
(white asterisks). (d) LM-IF of HSP70.2 at stage VII of the cycle showing a Golgi (small arrowhead) and acrosome (A) reaction at step 7
spermatids. The tails (T) of the late spermatids in the lumen (Lu) also reveal reactivity.

VPS54, the vacuolar-sorting protein responsible for early
endocytic retrograde transport, is also detected in male germ
cells and follows the intracellular route of USP8/ESCRT-0
during acrosome formation. Spontaneous recessive mutant
mice lacking Vps54 are infertile. The mutant mouse sperma-
tids do not develop an acrosome, as the PAS-positive proac-
rosomal granules are prevented from coalescing to form the
acrosome.152 Thus, as the role of proteins related to endocy-
tosis in spermatids is mounting, some of the proteins exam-
ined in our study51 may also be related to the early endosome
pathway and roles related to acrosome biogenesis.
DATA FROM OTHER SOURCES REVEALING PROTEINS
REQUIRED FOR ACROSOME FORMATION
An important role of the Golgi apparatus of early sperma-
tids is formation of proacrosomic granules that are capable
of fusing together as they elaborate the developing acro-
some. The list of Golgi proteins critical for this process
and involved in globozoospermia (abnormal head and no
acrosome) is growing as evidenced from studies includ-
ing mutant and/or deficient mouse models (see reviews in
Foster and Gerton153 and Niu et al.154).
Early evidence of abnormalities of acrosome forma-
tion was noted for the blind-sterile (bs) autosomal reces-
sive mutation in mouse that results in male sterility.
This is caused by mutations in the Tbc1d20 gene, which
encodes a GTPase-activating protein specific for RAB1
and RAB2 small GTPase families.155 Sterile bs/bs males
exhibit reduced testis weights and few epididymal sperm
that when present reveal morphologically aberrant head
shapes. The most striking effect of the bs mutation is the
failure of acrosome formation during spermiogenesis with
disorganized proacrosomic granules being detected and
no evidence of a single acrosomal cap or fully developed
acrosome.156
The GTPase-activating protein, AGFG1, Arf-GAP
domain and FG repeats-containing protein 1 (also
known as HRB), specific for ARF proteins, shows a simi-
lar phenotype when absent.157 HRB, an Asn-Pro-Phe
motif-containing protein, associates with the cytosolic
surface of proacrosomic transport vesicles. Although
such vesicles form in spermatids that lack HRB, the
vesicles are unable to fuse, blocking early acrosome
development.157,158
 Data from other sources revealing proteins required for acrosome formation  15

GBF1 GL54D
GRASP55 TM9SF3; TMED4/p25 GRASP55
TMED7/p27 GPP34
ManIIX
UBXD8 GBF1
Annexin A6; Man2α1; Fam3C; GPP34;
Sapreticulin; HSP70.2; LAMAN PDILT Man2α1
ManIIX TM9SF3
HSP70.2 GL54D
Fam3C TMED7/p27
Sapreticulin TMED4/p25
Annexin A6
1 LAMAN
II 2 PDILT
3
Di UBXD8
4
P(XII) Mitosis 5
(A–B)
P(VII) Meiosis 6

P(I) (PL–II) 7 CE
Acrosome ER
Z (1–7) W
8 S T
L Postacrosome
(8–18) ER
9
PL Hermes biogenesis
(19) A S
B TGN
In W
A N
10

11
12
19 18 17 16 15 14 13
Figure 1.14 Wheel depicting the expression profiles
(solid lines) of Golgi localized proteins during the course of
spermatogenesis. Dashed lines represent the cytoplasmic
localizations of UBXD8 and PDILT. While some proteins show
widespread distributions, many localize to steps 1–7 sperma-
tids during their involvement in the formation of the acro-
some. Some proteins are expressed early in spermatogenesis
but then are repressed in later spermatids only to return in late
step 19 spermatids.
The Golgi-associated PDZ- and coiled-coil motif-
containing protein (GOPC) localizes to the trans-Golgi
region in early spermatids, and male mice in which GOPC
is disrupted are infertile. The primary defect is fragmenta-
tion of acrosomes in early round spermatids and abnormal
vesicles that fail to fuse and form the acrosome.159
Autophagy-related gene 7 (Atg7) is homologous to the
ubiquitin-activating enzyme E1 (Uba1) and encodes the
E1-like enzyme that is essential to the ubiquitin conju-
gation system. Germ cell specific Atg7-knockout mice
are infertile due to a defect in acrosome biogenesis. Atg7
partially regulates GOPC during acrosome biogenesis.
Proacrosomic vesicles fail to fuse into a single acrosomal
vesicle during the Golgi phase of early spermatids.160
Figure 1.15  Schematic representation of stacks (S) of Golgi
apparatus of the step 7 spermatid adjacent to the nucleus (N).
Proteins localized to both the Golgi and acrosome are enclosed
by the red bracket. Proteins restricted to Golgi apparatus are
enclosed by the green bracket. The trans Golgi network (TGN),
cis Golgi element (CE), endoplasmic reticulum (ER), and wells
(W) of spermatid Golgi apparatus are indicated. (Reproduced
with permission from Mol Biol Cell: Au, CE et al., Mol Biol Cell.
2015; 26(22):4015–4032 DOI:10.1091/mbc.)
Protein interacting with C kinase 1 (PICK1), a periph-
eral membrane protein involved in protein trafficking, is
highly expressed in round spermatids, where it localizes
to Golgi-derived proacrosomic granules. It cooperates
with other proteins such as Golgi GOPC and CK2alpha’
in acrosome biogenesis. Male mice deficient in PICK1 are
infertile and show fragmentation of acrosomes in the early
steps of spermiogenesis.160
Sirt1, a member of the sirtuin family of proteins, has
important roles in numerous biological processes. Germ
cell specific Sirt1-knockout mice revealed that spermio-
genesis was disrupted due to a defect in acrosome biogen-
esis, resulting in a phenotype similar to that observed in
human globozoospermia. Sirt1 depletion resulted in the
failure of microtubule-associated protein 1 light chain 3
(MAP1LC3/LC3) and Atg7 to be recruited to Golgi appa-
ratus-derived vesicles, and as a consequence resulted in
the failure of GOPC and PICK1 recruitment to nucleus-
associated acrosomic vesicles. Thus Sirt1 has a novel physi-
ological function in acrosome biogenesis.161
16  Golgi apparatus regulation of differentiation
Islet cell auto antigen 1-like protein (ICA1L), which
has sequence similarities to ICA69 (also known as ICA1),
has been identified as a BAR-domain binding partner
(Bin, amphiphysin, Rvs) of PICK1 that is crucial for acro-
some formation. ICA1L and PICK1 are highly expressed
in spermatids and traffic together at different steps of
spermiogenesis. ICA1L-knockout mice reveal that PICK1
expression is reduced by 80% in the testes of these mice.
Sperm from ICA1L-knockout mice have abnormalities
in the acrosome, nucleus, and flagellar mitochondrial
sheath formation. Both total and motile sperm numbers
are reduced and about half of the remaining sperm have
the characteristics of globozoospermia. These defects
ultimately result in reduced fertility of male ICA1L-
knockout mice. ICA69/ICA1L-double knockout male mice
are sterile.163
CCDC136 (coiled-coil domain containing 136), a
novel testis-specific protein, also localizes to the acro-
some of round and elongating spermatids. Ccdc136-
deficient males are infertile due to disrupted acrosome
formation. The expression levels of proteins SPACA1
(sperm acrosome membrane-associated protein 1, see
below) and PICK1 involved in acrosome formation are
also significantly downregulated in the testes of Ccdc136-
deficient mice.164
The Fads2 gene encodes an enzyme enriched in testicular
membrane phospholipids that is required for the endog-
enous synthesis of an omega-3 fatty acid, docosahexaenoic
acid (DHA).86 In Fads2-deficient spermatids, proacrosomic
vesicles fail to fuse and form a well-developed acrosome.
Furthermore, syntaxin 2 reveals no extensive colocal-
ization with acrosin in these mice. Syntaxins are a large
protein family implicated in the targeting and fusion of
intracellular transport vesicles. Absence of acrosomes in
Fads2-deficient round spermatids and misplaced syntaxin
2 suggests an essential role of DHA in proper delivery
of membrane proteins required for proacrosomic vesicle
fusion.165
Sec23 is a component of the coat protein complex II
(COPII) which promotes the formation of transport vesi-
cles from the endoplasmic reticulum. Mutations affecting
SEC23IP, a phospholipaseA1-like protein that interacts
with Sec23, also cause male subfertility by interfering
with acrosome biogenesis and leading to round-headed
spermatozoa.166
SMAP2, an ArfGTPase-activating protein detected in
the TGNs of pachytene spermatocytes up to early round
spermatids, binds to both clathrin, a protein that plays
a major role in the formation of coated vesicles, and the
clathrin assembly protein (CALM). In SMAP2-deficient
male mice, the diameter of proacrosomic vesicles budding
from the TGNs increases, TGNs are distorted, and acro-
some formation is severely impaired. Moreover, a link is
established between SMAP2, CALM, and syntaxin2 in
clathrin-coated vesicle formation derived from the TGN
and subsequent acrosome formation. Together the data
reveal complex mechanisms and interacting proteins for
acrosome formation.167
Galnt3, N-acetylgalactosaminyltransferase 3, is a mem-
ber of the GalNAc transferase gene family involved in the
initiation of mucin-type O-glycosylation. Galnt3-deficient
mice are infertile. Galnt3 protein localizes in the cis-medial
cisternae of the Golgi stacks of spermatocytes and sper-
matids in seminiferous tubules. Spermatozoa in Galnt3-
deficient mice are rare and immotile, and reveal deformed
heads. During acrosome formation, Golgi-derived proac-
rosomal vesicles of various sizes do not coalesce to form
a single acrosomal vesicle. Overall, the data reveal that
deficiency of Galnt3 results in a severe reduction of mucin-
type O–glycans in spermatids and causes impaired acro-
some formation, leading to oligoasthenoteratozoospermia
(abnormal low number of sperm and with low motility).168
Polypeptide N-acetylgalactosaminyltransferase-like  pro­­
tein 5 (GALNTL5) belongs to the pp-GalNAc-T gene fam-
ily. GALNTL5, catalyzes the first step in mucin-type
O-glycosylation of peptides in the Golgi apparatus.
Heterozygous mutation of Galntl5 causes infertility in male
mice because of immotile sperm. Mouse GALNTL5 local-
izes in the cytoplasm of round spermatids in the region
of the acrosome, and in Galntl5-deficient mutant mice an
abnormal distribution of protein loading into acrosomes is
observed along with other abnormalities.169
Several other Golgi proteins are associated with acro-
some development and interact with GOPC or HRB but
a phenotype for their absence has not as yet been defined.
They include the Golgi protein SPATA16 protein, a tes-
tis-specific protein that belongs to the tetratricopeptide
repeat-like superfamily (also known as NYD-SP12)170,171
and family with sequence similarity 170, member B
(Fam170b).172
An interesting twist is the protein MFSD14A, a sol-
ute carrier protein expressed in Sertoli cells. Its absence
is associated with transport of a solute, suggested to be
mannose, from the bloodstream that is required for sper-
miogenesis. Small vesicles that stain for glycoproteins are
abundantly found in early spermatids of mutant mice
model suggesting a defect in vesicular trafficking from the
Golgi and/or fusion with the developing acrosome.173
PROTEINS INVOLVED IN ACROSOME BINDING
TO THE NUCLEAR SURFACE
In addition to a role in acrosome formation, the early sper-
matid has developed a mechanism whereby the forming
acrosome binds to the nuclear surface opposite the trans
face of the Golgi apparatus.
Sperm acrosome associated 1 (Spaca1) is a member of
the globozoospermia-related genes. Disruption of Spaca1
leads to the disappearance of the acrosome-anchoring
scaffold, the acroplaxome, and decreased expression of
Ccdc-136 and PICK1. This coincides with the failure of
acrosomal expansion during spermiogenesis resulting in
the degeneration and disappearance of the acrosome.174

Dpy19l2, a transmembrane protein, is expressed in sper-
matids with a specific localization restricted to the inner
nuclear membrane facing the acrosome. In the absence of
Dpy19l2 the acrosome fails to be linked to the nucleus lead-
ing to a disruption of vesicular trafficking from the Golgi
apparatus.175,176
The TATA element modulatory factor (TMF), a well-
known Golgi associated protein, was highly abundant in
our isolated testis Golgi fraction.51 Also known as TMF/
ARA160, its absence in male mice led to a specific devia-
tion of the trans face of the Golgi (TGNs) away from
the nucleus of the developing early round spermatids.
Concomitantly, proacrosomic vesicles derived from the
TGNs of KO mice lacked targeting properties and did not
tether to the spermatid nuclear membrane acroplaxome.
A proposed function in round spermatids is its association
with microtubules for Golgi positioning as a requirement
for acrosome formation.176
DEFINING GOLGI PROTEINS IN SPERMATIDS
Post acrosome period: Differential expression
of SORTILIN and ManIIX
After acrosome formation, the Golgi apparatus becomes
spherical and positions itself in the cytoplasmic lobe of
late spermatids. An EM illustration of a step 12 spermatid
Golgi is seen in Figure 1.16a. Coincident with the move-
ment of the Golgi apparatus into the cytoplasmic lobe at
step 8 spermatids, Golgi-located immunogenicity of 12
proteins (i.e., GRASP55, GBF1, ManIIX, Annexin A6,
GPP34, Man2α1, HSP70.2, Fam3C, PDILT, sapreticulin,
UBXD8, and LAMAN) was below detection. In contrast,
four Golgi proteins (i.e., GL54D, TM9SF3, TMED4/p25,
and TMED7/p27) maintained reactivity into late spermio-
genesis, thus defining the identity of the Golgi apparatus
in late spermatids.
Golgi migration away from the acrosome also corre-
sponded precisely with the onset of expression of sorti-
lin in the Golgi apparatus at step 8 and continuing until
step 19 spermatids (Figure 1.17a and examples in Figure
1.17b, c). Sortilin transports lysosomal enzymes from the
Golgi apparatus to MVBs and lysosomes.178 At the end of
acrosome formation the TGNs appear to be fully utilized
and undergo fragmentation into vesicles and tubules24,28,29,
46,179 and are no longer evident in step 8 spermatids and
beyond. In early spermatids, trafficking from the TGNs
to the developing acrosome appears to utilize mannose 6
phosphate receptors as their RNA transcripts are promi-
nent.180 However, from step 8 onward, the expression of
sortilin suggests its role in trafficking that would involve
Golgi cisternae other than the TGNs.
Of note is the absence of MVBs and lysosomes from
steps 9–10 spermatids onward.14,33 Hence, sortilin in the
Golgi apparatus of late spermatids would not be involved
in lysosomal targeting but rather likely functions in
plasma membrane remodeling.
Defining Golgi proteins in spermatids  17
S
*
S
*
(a) * (b)
(c) (d)
Figure 1.16  EM images of Golgi apparatus of late sperma-
tids. (a) Golgi apparatus of a step 14 spermatid lies freely in the
cytoplasmic lobe. It is compact, spherical, and composed of
tightly apposed flattened cisternae forming stacks (S) with ER
(asterisks) close by. (b) A step 16 spermatid reveals a small iso-
lated Golgi stack of cisternae (S) with autophagosomes (curved
arrows) containing cisternae nearby. (c) Step 17 spermatid
reveals an aggregate of small flattened cisternae and associ-
ated vesicular profiles with a few isolated cisternae (arrows) at
a distance. (d) Forming Hermes body of a step 19 spermatid
reveals small flattened cisternae (arrows) that do not form a
stack but at times show close and random focal associations.
Original magnifications: 16a–d: 48,750×.
The Golgi is responsible for glycosylation180 and this
occurs during acrosome formation with expression
of Man2α1 and ManIIX. These sequence-related pro-
teins likely have related functions in alpha mannosidase
trimming of high mannose containing N-linked oli-
gosaccharides.133 The lack of detection of both of these
proteins from steps 8–14 would suggest a diminution in
Golgi mannosidase activity that in turn would be linked
to the well-known major changes in N-linked glycosyl-
ation of proteins accounting for the high mannose and
complex glycoproteins differentially expressed during
spermatogenesis.32,96
However, from steps 15–19, ManIIX expression returns
(Figure 1.17a and d), likely heralding a return of complex
sugar modification reactions on N-linked glycoproteins
with its expression being essential for spermatogenesis
and male fertility.182,183 Terminal sugar incorporation in
the Golgi apparatus has been documented in late sper-
matids for glycoprotein synthesis destined for the plasma
membrane.184
During the last steps of spermiogenesis (steps 15–19),
there is the appearance of tubulobulbar complexes.185
18  Golgi apparatus regulation of differentiation

(a) (b)
Lu

II 1 2
3 S
Di
4 Rb Rb
S
P(XII) Mitosis 5
(A–B) 10 µm
P(VII) 6
Meiosis (c)
(PL–II)
P(I) 7
Acrosome S Rb
Z (1–7) Lu
8
L Postacrosome Rb
(8–18)
9
PL Hermes biogenesis S
10 µm
B (19)
In (d)
A A
10
Rb
A

11 Rb
Lu
10 µm
12
19 18 17 16 15 14 13

Sortilin ManIIX

Figure 1.17  (a) Wheel depicting the expression profiles of ManIIX and sortilin. ManIIX spans the Golgi apparatus of steps 1–7
spermatids and steps 15–19 while sortilin is restricted to steps 8–19. (b) LM-IHC of sortilin in a tubule at stage VI of the cycle. Reactions
are noted in the Golgi apparatus (large arrowheads) of the cytoplasmic lobes of late spermatids. Sertoli cells (S) are also reactive, as
are small spherical bodies near the base of the tubule corresponding to residual bodies (Rb). (c) LM-IHC of sortilin at stage VIII shows
reactions in Golgi of Sertoli cells (S) and step 8 spermatids (arrowheads) and residual bodies (Rb). (d) LM-IHC of ManIIX. Right tubule
at stage VI shows acrosome (A) and Golgi reaction at step 6 spermatids (small arrowheads) and late spermatids (large arrowheads);
left lower tubule at late stage VIII with reactions in residual bodies (Rb).

Such complexes are long (2–3 μm), narrow (50 nm) tubu-
lar projections of the spermatid’s plasma membrane that
invaginate into Sertoli cells. They disappear just before the
release of spermatids into the lumen of the seminiferous
tubule at step 19.186 The Golgi apparatus may serve to pro-
vide plasma membrane proteins needed for formation of
these membranous complexes, and this may involve a role
for ManIIX and sortilin, both highly expressed during late
spermiogenesis.
FRAGMENTATION OF THE GOLGI APPARATUS
IN LATE SPERMATIDS
After step 15, the Golgi apparatus undergoes fragmen-
tation, vacuolation, and autophagy (Figure 1.16b), 29,34
suggesting that components of the Golgi ribbon frag-
ment into individual stacks between steps 16–19 (Figure
1.16b), with Golgi cisternae separating from the stack
and becoming liberated as distinct entities floating in
the cytoplasm (Figure 1.16c). At step 19, the cisternae
as unstacked entities congregate into a focal bulbous
dilation of cytoplasm at the neck region of the step 19
spermatid flagellum (Figure 16d), which we have termed
the Hermes body, previously named the cytoplasmic
droplet.35
ManIIX selectively reappears at step 15, coinciding
closely with Golgi fragmentation and with a marked
expression of β-COP. 35,186 These findings suggest a
marked retrograde transport of COPI vesicles from the
Golgi to the ER,187 which may be linked to Golgi frag-
mentation and cisternal unstacking.188 Nevertheless,
the expression of ManIIX and sortilin suggests that the
Golgi is still functional during these very late steps of
spermiogenesis. In addition, the Golgi apparatus after
fragmentation appears to be destined to end up in two
different locations. The individual Golgi cisternae end
up in the forming Hermes body at step 19 spermatids
for functions related to epididymal sperm. 35 Other com-
ponents of the Golgi ribbon appear in residual bodies as
 Fate of Golgi localized proteins destined for the forming Hermes body at step 19 spermatids  19

small but yet discrete entities that are especially reactive
for sortilin (Figure 1.17c) and ManIIX (Figure 1.17d).
What these entities correspond to awaits future inves-
tigation by EM.
FATE OF GOLGI LOCALIZED PROTEINS DESTINED FOR
THE FORMING HERMES BODY AT STEP 19 SPERMATIDS
At the last step of spermiogenesis, two prominent
structures appear in relation to step 19 spermatids
(Figures 1.4 and 1.5). The larger structure adjacent to the
head of the spermatid constitutes the residual body into
which the majority of unused organelles congregate and
which is phagocytosed and degraded by Sertoli cells.189,190
The other smaller structure appears as a bulge of cyto-
plasm positioned at the neck (connecting piece) region
of the flagellum; it defines the cytoplasmic droplet191
that is retained by sperm as they traverse the epididy-
mis14,34,192–194 and that has been renamed recently as the
Hermes body.35
Step 19 spermatids span two stages of the cycle (i.e.,
stages VII and VIII) with a duration of 58 hours for VII
and 30 hours for VIII (Figure 1.3).3 However, it is only at
stage VIII of the cycle that the forming Hermes body seg-
regates itself from the residual body and is seen as a dis-
tinct entity. At both of these stages, the late spermatids
are defined as step 19 spermatids, and it may be advanta-
geous to define those at stage VII as 19a and at stage VIII
as 19b, as illustrated by Clermont in Figure 1.4, which
was never published by him but which we used in an arti-
cle describing his life in research.16
Interestingly, a resurgence of protein expression
highlights the step 19 spermatids. Reactivity returns
for TMED7/p27 that persisted until step 16, as well as
for GBF1, Annexin A6, GPP34, Man2α1, LAMAN,
HSP70.2, PDILT, sapreticulin, and FAM3C, which were
shut down at step 8. ManIIX becomes absent from steps
8–14 but then reappears from steps 15–19 spermatids
(Figure 1.18a). All of these proteins localized to the
forming Hermes body. In addition, two additional Golgi
proteins became expressed for the first time at step 19
(i.e., TMED2/p24 [Figure 1.18a] and carboxypeptidase
D, a serine exopeptidase that releases C-terminal amino
acids and which is concentrated in the Golgi apparatus),
where it functions by cycling to and from the plasma
membrane195 (Figure 1.18a and b).
Although a stacked Golgi apparatus and ribbon are
no longer detectable at step 19, these proteins define the
flattened membranous elements that congregate into the
forming Hermes body.35 Interestingly, sortilin is also
expressed at step 19 spermatids, but unlike the 13 proteins

(a) (b)
Rb
Rb

II 1 2
3
Di Lu
4
P(XII) Mitosis 5 10 µm
(A–B)
P(VII) 6
Meiosis
P(I) (PL–II)
7
Acrosome
Z (1–7) 8
L Postacrosome
(8–18)
PL 9
Hermes biogenesis
B (19)
In
A
10 Carboxypeptidase D; TMED2/p24
GBF1
11 ManIIX
Annexin A6; Man2 1; Fam3C; GPP34;
12 Sapreticulin; HSP70.2; LAMAN
19 18 17 16 15 14 13 TMED7/p27
PDILT

Figure 1.18  (a) Wheel depicting several Golgi localized proteins (solid lines) showing different expression profiles during sper-
matogenesis. The dashed line for PDILT shows its cytoplasmic localization during late spermiogenesis. (b) LM-IHC of carboxypepti-
dase D shows reaction in Hermes bodies (curved arrows) and residual bodies (Rb) of step 19 spermatids.
20  Golgi apparatus regulation of differentiation
listed above, sortilin ends up exclusively in residual bod-
ies at step 19 spermatids and is not a constituent of the
Hermes body.
An important function of step 19 spermatids is
in spermatid individualization into spermatozoa by
disruption  of the syncytium formed by intercellular
bridges.196–198 It is attractive to propose the internal
membranes (Golgi cisternae and vesicles) of the form-
ing Hermes body as the source of new plasma mem-
brane to seal off each individual spermatid resulting in
spermatozoa.
The forming Hermes body of step 19 spermatids is
maintained in epididymal sperm. The protein composi-
tion of the Hermes body of epididymal sperm has been
analyzed in detail by proteomics and localization stud-
ies.34 The data suggest that the flattened cisternal mem-
branes show marked differences in their Golgi protein
composition from the Golgi apparatus of germ cells
evolving during spermatogenesis with some Golgi local-
ized proteins not entering the Hermes body (i.e., GL54D,
TMED4/p25, sortilin, UBXD8, TM9SF3, and GRASP55).
In this way, the flattened cisternae of the step 19 sper-
matids appear to have different molecular constituents
and this may be analogous to that postulated for yeast-
flattened cisternae.199,200 In addition, these cisternae may
function independent of a stacked configuration as sug-
gested for Golgi cisternae in other systems201 and with-
out the conventional setup of a continuous Golgi ribbon.
Thus, the Hermes body of epididymal sperm formed at
step 19 spermatids appears to have evolved a system of
unique flattened membranous cisternae unlike the con-
ventional stacked Golgi apparatus to carry out specific
functions for sperm maturation. 35
FATE OF GOLGI LOCALIZED PROTEINS DESTINED
FOR RESIDUAL BODIES AT STEP 19 SPERMATIDS
Before being released from the seminiferous epithelium,
the step 19 spermatid rids itself of organelles not uti-
lized during spermatid differentiation. Defined as the
residual body (Figures 1.4 and 1.5), this excess cytoplas-
mic mass contains mitochondria not used during for-
mation of the midpiece of the flagellum, lipid droplets,
and ER cisternae including remnants of the annulate
lamellae and radial body.9,14,94,189,202,203 The residual body
is embraced by processes of Sertoli cells and phagocy-
tosed within the Sertoli cell cytoplasm, where it under-
goes degradation with participation of its lysosomal
system.189
Intriguingly, in our study, all proteins found in the
step 19 forming Hermes body were also noted in resid-
ual bodies. This suggests that specific components of
the Golgi ribbon segregate themselves into these two
distinct structures. While the reaction for Golgi pro-
teins in residual bodies was usually diffuse, in only two
cases were the reactions seen as discrete clumps of reac-
tion products (i.e., sortilin) (Figure 1.17c) and ManIIX
(Figure 1.17d). In addition, the data also revealed that a
segregation of both of these proteins was taking place,
as sortilin ended up exclusively in the residual body
while ManIIX expression was also noted in the forming
Hermes body at step 19 (Figure 1.18a). These proteins
not only serve as controls for each other but also point
to key dramatic differences.
PROTEINS OF UNKNOWN FUNCTION
In the present work, we describe two abundant proteins
of unknown function as new Golgi markers (GL54D and
TM9SF3). Moreover, over 200 other proteins of unknown
function were uncovered in the isolated testis Golgi frac-
tion, each of which may now be studied in detail to eluci-
date their mechanisms during germ cell differentiation.51
DO PATTERNS OF DIFFERENTIAL EXPRESSION
OF GOLGI LOCALIZED PROTEIN EXPRESSION
COINCIDE WITH NON-GOLGI PROTEINS DURING
SPERMATOGENESIS
The data on all 20 Golgi localized proteins revealed a
marked differential expression pattern as germ cells
evolved during spermatogenesis (Figure 1.19). Hence, a
proteomics-based strategy of the isolated testis Golgi frac-
tion enabled the identification and selection of 20 proteins
whose differential Golgi localization was mapped during
spermatogenesis. Figure 1.20 depicts proteins expressed
during the period of mitosis, meiosis, acrosome, postacro-
some, and Hermes body formation.
In an earlier study we hypothesized that the flattened
cisternae of the Hermes body at step 19 spermatids were of
Golgi identity.34 One of the objectives of our study on iso-
lation of the Golgi apparatus of germ cells51 was to attempt
to validate this hypothesis. In a companion study, we iso-
lated the Hermes body of epididymal sperm and charac-
terized its protein composition by proteomics. Based on
their abundance, several selected non-Golgi proteins were
characterized and localized to the Hermes body of epidid-
ymal sperm.35 A detailed morphological analysis was also
performed of their differential expression during sper-
matogenesis. In this study, a comparison of the differential
expression between the 20 Golgi localized proteins will
be made with the non-Golgi proteins35 in order to define
possible functional correlations between these two sets of
proteins during spermatogenesis.
MATCHING PROTEIN EXPRESSION PROFILES
OF GOLGI AND NON-GOLGI PROTEINS DURING
SPERMATOGENESIS
The most abundant protein of the isolated Hermes body
fraction was glucose transporter 3 (GLUT-3). 35 Its expres-
sion was first detected in pachytene spermatocytes at
stage VII of the cycle exactly coincident with the expres-
sion of GL54D, the most abundant Golgi protein of germ
cells (Figure 1.21a). 51 The reaction product delineated the
entire plasma membrane of early germ cells (solid green
 Coincidental temporal expression of non-Golgi and Golgi proteins: Relationship with ER of spermatids  21

Annexin A6; Man2 1; Fam3C; GPP34; all the way up to step 19 spermatids (Figure 1.22a and
Carboxypeptidate D; TMED2/p24 Sapreticulin; HSP70.2; LAMAN
GRASP55 GL54D b). Golgi proteins with widespread distribution include
GBF1 TM9SF3; TMED4/p25 TMED7/p27 (Figure 1.22c), TM9SF3, and TMED4/p25, all
Sortilin TMED7/p27 of which may serve as the client for MCT1 (Figure 1.22a).
ManIIX UBXD8 The enhanced expression of a number of proteins dur-
MG160 PDILT
ing meiosis (Figure 1.23a) was remarkable. From pachy-
tene spermatocytes (stage I) until the end of meiosis, there
was prominent immunoreactivity noted for calnexin, a
chaperone participating in the folding and quality con-
trol of client proteins (Figure 1.23b) and binding immu-
II 1 2 noglobulin protein (BIP), an ER HSP70 family member
3
Di also known as glucose-regulated protein 78 or HSPA5.
4
Vesicle-associated membrane protein 3 (VAMP3), the
P(XII) Mitosis 5
GTP-binding protein RAB14 (Figure  1.23c), clathrin
(A–B)
P(VII)
Meiosis 6 heavy chain (CHC), elongation factor EF1α, the ubiq-
P(I) (PL–II) 7 uitin-directed AAA-ATPase P97, proteasome α 1-7, the
Acrosome GTPase activating protein, sec23, a coat component of
Z (1–7) COPII vesicles, and ubiquitin were also highly expressed
8
L Postacrosome during this time point in meiosis (Figure 1.23a). All of
(8–18) 9
PL
Hermes biogenesis
these proteins mark in large part to the period of MG160
B (19) expression to these cells (Figure 1.23a). Another coin-
In
cident pattern is that seen for UBXD8 as a cytoplasmic
A localized protein (Figure 1.23d) and MG160 (Figure
10 1.23a) both expressed in spermatocytes and both reveal
overlapping expression during meiosis. The meiotic
11 period represents a dramatic size increase of the Golgi
12
apparatus during spermatocyte maturation due to an
19 18 17 16 15 14 13 accumulation of trans Golgi elements83 and correspond-
ing to a significant increase in size of the Golgi apparatus
(0.5–1.0 to 2–3 μm in diameter) to produce four haploid
daughter cells as a consequence of meiosis.209 These pro-
teins may be related to this event.
Figure 1.19  Wheel depicting widespread Golgi distribu- Another similar distribution pattern (Figure 1.24a)
tions (solid lines) during spermatogenesis. Cytoplasmic local- exists for the renewed onset of expression of ManIIX from
izations for PDILT and UBXD8 are depicted by dashed lines. steps 15–19 (Figure 1.24b) with the coincident expression
of atlastin 3 (functioning in tubular ER network forma-
tion) (Figures 1.24c and d), β-COP, ERP57, and RAB14 at
these same steps of differentiation. A rationale for these
line, Figure 1.21a) with reactivity becoming spotty from coincidences of expression patterns is still unknown but
steps 8–18 spermatids (dashed green line, Figure 1.21a; indicates a partnership between non-Golgi and Golgi
and Figures 1.21b and c). Curiously, the wave of sorti- proteins.
lin expression in the Golgi apparatus at step 8 coincided
with a marked diminishment in surface located GLUT-3 COINCIDENTAL TEMPORAL EXPRESSION OF
of the plasma membrane of late spermatids (Figure NON-GOLGI AND GOLGI PROTEINS: RELATIONSHIP
1.21a). While sortilin expression is known to regulate WITH ER OF SPERMATIDS
GLUT-4 trafficking in insulin-responsive cells, 204–208 its Extensive membrane trafficking exists between the ER
role in GLUT-3 sorting and abundance is unclear but is and Golgi especially for the secretory transport of newly
predicted from the present study. Sortilin also shows a synthesized proteins, their posttranslational modifica-
coincident expression pattern with PDILT, as the latter tions including N and O glycosylation, and the quality
turns from being Golgi localized (steps 1–7) to a cyto- control of folding of newly synthesized secretory pro-
plasmic ER localization from steps 8–19 (Figure 1.21a). teins. Of the numerous Golgi proteins identified in our
Monocarboxylate transporters (MCTs) utilize proton previous work (50), only three Golgi proteins (Figure
gradients to move molecules with one carboxylate group 1.25a) (i.e., TMED7/p27, TM9SF3 [Figure 1.25b], and
such as lactate or pyruvate across the plasma membrane. TMED4/p25) showed widespread distributions across
MCT1 showed a widespread immunoreactivity decorat- spermatogenesis coincident in part with that for BIP
ing the plasma membranes of preleptotene spermatocytes (Figure 1.25c) and calnexin.
22  Golgi apparatus regulation of differentiation

Ax
Aa

TMED7/p27
TM9SF3 Ax G
TMED4/p25 Aa
C ICB TMED7/p27
UBXD8 DL
CB TM9SF3
GL54D
Testis TMED4/p25
GBF1
ICB M GL54D
Annexin A6
ManIIX
Man2a1
Sortilin
Fam3C CA
GPP34 G
AS N
Sapreticulin
PDILT Acrosome
AS
HSP70.2 (steps 1–7)
LAMAN Postacrosome
ManIIX (steps 8–18)
Grasp55

ER
S
TMED7/p27
TM9SF3 ER
TMED7/p27
TMED4/p25 GBF1
UBXD8 Annexin A6
Meiosis
GL54D Golgi Man2a1
spermatocytes (PI-II)
MG160 1318 proteins An Fam3C
GBF1 GPP34
Sapreticulin
PDILT
HSP70.2
M LAMAN
ManIIX
HB
Carboxypeptidate D
TMED7/p27 TMED2/p24
RB
TM9SF3
TMED4/p25 Ad
UBXD8
Mitosis
spermatogonia Hermes biogenesis
(step 19)

Figure 1.20  Schematic representation of key events in germ cell differentiation, including mitosis of spermatogonia, meiosis of
spermatocytes from preleptotene (PL) to secondary (II) spermatocytes, acrosome formation from steps 1–7 spermatids, postacro-
some events from steps 8–18, and Hermes body formation at step 19 spermatids. The Golgi apparatus of adult rat germ cells was
isolated and a proteomic analysis by tandem mass spectrometry identified 1318 Golgi proteins. LM-IHC identified several Golgi
proteins belonging to different classes of germ cells and appearing at different time points during germ cell differentiation. The pro-
teins highlighted in red first localized to spermatogonia, those in blue first appeared in spermatocytes, those in green to spermatids
(steps 1–7), those in orange to late spermatids, and those in purple to step 19 spermatids. Thus, the different Golgi localized proteins
reveal differential expression profiles during germ cell differentiation. Note that as each group of proteins, indicated by a specific
color, appeared during development, some maintained expression at later steps of development while others were cut short. Some
expressed in early spermatids (steps 1–7) lost expression from steps 8–18 only to reappear in step 19 spermatids. A few proteins only
localized to step 19 spermatids.
 Coincidental temporal expression of non-Golgi and Golgi proteins: Relationship with ER of spermatids  23

(a) (b)

1 T
II 2
3 Lu
Di
4
P(XII) Mitosis 5
P P
(A–B) 10 µm
P(VII) 6
Meiosis
(PL–II) (c) Lu
P(I) 7
Acrosome PM
PM
Z (1–7) 8
L Postacrosome
(8–18) PM
PL 9
Hermes biogenesis
B (19)
10 µm
In
A
10

11
Sortilin
12 GL54D
19 18 17 16 15 14 13 PDILT
GLUT-3

Figure 1.21  (a) Wheel depicting the expression profiles of GL54D, sortilin, GLUT-3, and PDILT. Both GL54D and GLUT-3 dem-
onstrate a precise onset of expression in pachytene spermatocytes at stage VII of the cycle. GL54D is Golgi localized, while GLUT-3
displays an intense reaction on the plasma membrane of germ cells. After step 7, GLUT-3 becomes spotty and remains as such until
step 19 where it shows intense reactivity in the forming Hermes body. Coincident with the spotty reaction of GLUT-3 commencing at
step 8 is the onset of expression of sortilin and the cytoplasmic localization of PDILT. (b) LM-IF of a tubule at stage VII showing intense
reactivity of GLUT-3 on the plasma membrane of pachytene spermatocytes (P), early round spermatids positioned in the center of
the tubule (arrowheads), and the tails (T) of late spermatids in the lumen (Lu). (c) LM-IHC of GLUT-3 at stage II-III reveals plasma mem-
brane (PM) reaction of round spermatids and tails of late spermatids in the lumen (Lu).

Spermiogenesis marks the presence of several specific
events in the life history of ER.210,211,212 During acrosome
formation, ER cisternae are intimately associated with
various components of the Golgi apparatus9,27,28,213,214
(Figure 1.6). Of the many ER proteins noted in our study,51
only BIP and calnexin overlapped with Golgi proteins
involved in acrosome formation and for which a func-
tional correlation may be deduced. During the later part
of spermiogenesis, the ER forms annulate lamellae and
the radial body. Annulate lamellae are focal gatherings
of short flattened membranous cisternae forming a stack
where individual cisternae are separated by a fair dis-
tance and characterized by numerous porelike structures
having a similar appearance to the pore complex of the
nuclear envelope.14,202,213–222 The radial body appears as an
aggregate of collapsed ER cisternae in the form of a spoke
wheel. It has been suggested that the radial body is a site
where ER regresses.214,223
These modified forms of ER appearing at late steps of sper-
miogenesis coincide at a time point when PDILT (its cyto-
plasmic expression), the chaperone ERP57 (ER protein 57),
UDP-glucose: glycoprotein glucosyltransferase (UGGT), a
key player in the quality control ER mechanisms, glucose-
regulated protein 170 (GRP170) and a component of the ER
chaperone network, and atlastins 1 and 3 predominate (Figure
1.26a). As the annulate lamellae reveal pores of fixed size
and shape in their stacked flattened cisternae that resemble
nuclear pores, then the presence of RanBP5, a nuclear import
factor that spans steps 12–17 spermatids (Figure 1.26a and
b) suggests that some cytosolic spermatid proteins contain
a nuclear localization signal that are transported by RanBP5
to the annulate lamellae for some as-yet-unknown function.
The granular-like localizations revealed with the antibodies
to these proteins in the cytoplasmic lobe of late spermatids
probably correspond to annulate lamellae and radial bod-
ies and suggest roles for these proteins in their structural
24  Golgi apparatus regulation of differentiation

(a) (b)
P
RS
II 1 2
3 P
Di RS
4 Lu
P(XII) Mitosis 5
(A–B) 10 µm
P(VII) 6
Meiosis
(PL–II) (c)
P(I) 7 Lu
Acrosome
Z (1–7) 8
L Postacrosome
(8–18) 9
PL
Hermes biogenesis
B (19) P
P 10 µm
In
A

10

11
12 MCT1
19 18 17 16 15 14 13 TM9SF3; TMED4/p25
TMED7/p27

Figure 1.22  (a) Wheel depicting the expression profiles of MTC1, TMED7/p27, TM9SF3, and TMED4/p25. A widespread distri-
bution is noted for all four of these proteins. (b) LM-IHC of MTC1 in a tubule at stage VII of the cycle showing reactions over the
plasma membrane of pachytene spermatocytes (P), early round spermatids (RS), and step 19 spermatids (curved arrows). (c) LM-IF
of TMED7/p27 at stage V with reactions in Golgi of round (small arrowheads) and late (large arrowheads) spermatids and pachytene
(P) spermatocytes.

integrity and functions (e.g., GRP170, Figure 1.26c). Lactate
dehydrogenase C (LDHC) (Figure 1.26d) was also seen to
correlate in its cytoplasmic localization to that of PDILT, for
which an explanation is as yet unknown.
The pan-germ cell ER markers calnexin and BIP were
exclusively restricted to ER and localized from pachytene
spermatocytes to step 19 spermatids. These two ER mark-
ers were never localized to the acrosome or Golgi apparatus
but did reveal cytoplasmic concentrations as well as focal
distinct entities at late steps of spermiogenesis presum-
ably corresponding to annulate lamellae/radial body com-
plexes. These proteins thus provide a control for UBXD8
and PDILT described above that relocated between the ER
and Golgi during spermatogenesis.
OVERLAPPING EXPRESSION PROFILES OF NON-GOLGI
AND GOLGI PROTEINS AT STEP 19 SPERMATIDS
As seen in Figure 1.27a, protein expression is extensive in
step 19 spermatids. Some non-Golgi proteins are solely
expressed at step 19 spermatids, such as annexin A1, AP1,
and AP2 (activator protein transcription factors), atlastin
2, malectin (membrane-anchored ER protein), peroxire-
doxin 1, peroxiredoxin 6, syntaxin 13 (member of the
syntaxin family with a role in mediating endosomal traf-
ficking), α-tubulin, Vps13c (member of the Vps13 family
of vacuolar protein sorting-associated proteins) (Figure
1.27b), and MCT2 (Figure 1.27c). Transferrin and albu-
min also localize to step 19 spermatids as secretory prod-
ucts of Sertoli cells that adsorb to their surface. Several
glutathione S transferase (GST) isoforms have also been
noted to be expressed only at step 19 spermatids, 224 as
was also seen for two Golgi proteins (i.e., carboxypepti-
dase D and TMED2/p24 [see above]).
Many non-Golgi and Golgi proteins while expressed
earlier during spermatogenesis are repressed for some
time only to become reexpressed again at step 19 (Figure
1.28). However, some Golgi proteins, along with RanBP5,
although expressed earlier in spermatogenesis do not end
up in step 19 spermatids (Figure 1.29). This suggests an
active but specific biosynthetic and membrane trafficking
machinery for step 19 spermatids during which time the
Hermes body is being formed, as we have speculated.35,51
It is well established that many genes required for the
development and/or function of spermatozoa are tran-
scribed much earlier during germ cell differentiation, with
an arrest of transcription by step 8 spermatids,30,225 then
 Conclusion 25

(a) (b)
Lu
*
* *
II 1 2
3 RS
Di RS
4
Mitosis P
P(XII) 5 P 10 µm
(A–B)
P(VII) Meiosis 6 (c)
P(I) (PL–II)
7
Acrosome
Z Lu
(1–7) 8
Postacrosome *
L
(8–18)
PL 9 *
Hermes biogenesis
(19) * 10 µm
B
In (d)
A
* Lu
10
A
11 *

12 * A
*
19 18 17 16 15 14 13 10 µm

MG160 Calnexin; BIP EF1α; VAMP3; p97; Proteasome α 1-7; Sec23; Ubiquitin
CHC RAB14 UBXD8

Figure 1.23  (a) Wheel depicting the expression profiles of several proteins. Golgi localized MG160 and the cytoplasmic localiza-
tion of UBXD8 match each other precisely from PL to the end of meiosis. Precise onset of expression in pachytene spermatocytes
at stage I of the cycle occurs for calnexin and BIP as well as for EF1α, VAMP3, p97, proteasome α 1-7, Sec 23, RAB14, and CHC; these
proteins overlap in their expression profiles until the end of meiosis. Their distribution profiles at later steps of germ cell differentia-
tion vary according to a given protein. (b) LM-IF of calnexin in a tubule at stage II–III of the cycle. Cytoplasmic localizations are noted
in pachytene spermatocytes (P), early round spermatids (RS) and in cytoplasmic lobes of the late spermatids (asterisks). (c) LM-IHC of
RAB14 shows intense cytoplasmic reaction in pachytene spermatocytes (asterisks) and in Hermes bodies (curved arrows). (d) LM-IHC
of UBXD8 with Golgi (arrowheads) and acrosome (A) reaction in round spermatids and cytoplasmic reaction (asterisks) in pachytene
spermatocytes.

translationally repressed, and finally translated several CONCLUSION

days after mRNA production due to a complex interplay
of RNA-binding proteins and non-coding RNA.226–230 As
a result, the differential expression of the Golgi and non-
Golgi proteins after step 8 must necessarily reflect transla-
tional regulation. Transcriptional regulation of changes in
the structure and function of organelles of the secretory
pathway is well established in somatic cells.231 Therefore, the
translation-controlled pathway for organelle modification
and morphogenesis of germ cells232 must be prominent even
at step 19, an unexplored area of investigation.
A detailed morphological analysis of the differen-
tial expression profile of the non-Golgi proteins dur-
ing spermatogenesis can be seen in Figure 1.30. A
comparison of the differential expression between the
non-Golgi proteins and Golgi localized is illustrated in
Figure 1.31.
For the first time we have identified the differential
expression pattern of proteins and molecular markers
related to the Golgi apparatus during germ cell devel-
opment as well as non-Golgi proteins. The end point of
Hermes body formation in step 19 spermatids was also
characterized by a unique set of markers. The differen-
tiation specific expression of new and established Golgi
markers during spermatogenesis may now help define a
new model system to answer the unresolved questions
in Golgi apparatus structure and function.233 Since the
resource also includes unexpected waves of changes
in expression patterns of proteins of the plasma mem-
brane, endosomes, ER, and cytosol then this also enables
a context to test the current models of Golgi appara-
tus structure and trafficking thus far unresolved by
experimentation.
26  Golgi apparatus regulation of differentiation

(a) (b)
Lu

II 1 2
3
Di
4

P(XII) Mitosis 5 10 µm
(A–B)
P(VII) 6
Meiosis (c)
(PL–II)
P(I) 7
Acrosome
Z (1–7)
8 *
L Postacrosome Lu
(8–18)
*
9
PL
Hermes biogenesis
(19) 10 µm
B
*
In (d)
A

10 Rb
Rb

11

Rb Lu
12
13
19 18 17 16 15 14 10 µm

ManIIX Atlastin-3; COP; ERP57 RAB14

Figure 1.24  (a) Wheel depicting the expression profile of the Golgi localized protein ManIIX with several non-Golgi proteins.
Remarkable similarities are noted for these proteins from steps 15–19. (b) LM-IF at stage VII of ManIIX with Golgi reactions in round
(small arrowheads) and late (large arrowheads) spermatids. (c) LM-IHC of atlastin 3 at stage VI shows cytoplasmic reaction in late
spermatids (asterisks). (d) LM-IHC of atlastin 3 at stage VII reveals reactions in residual bodies (Rb) and Hermes body (curved arrows)
of step 19 spermatids.
 Conclusion 27

(a) (b)

Lu

1 2
II
3
Di
4 P
P
P(XII) Mitosis 10 µm
5
(A–B)
P(VII) 6
Meiosis (c)
(PL–II)
P(I)
7
Acrosome
Z (1–7) ER
8 Lu

L Postacrosome
(8–18)
PL 9
Hermes biogenesis ER
(19) 10 µm
B

In
A

10

11

12
13
19 18 17 16 15 14

TM9SF3; TMED4/p25 TMED7/p27 Calnexin; BIP

Figure 1.25  (a) Wheel depicting the expression profiles of Golgi localized proteins (TMED7/p27, TM9SF3, TMED4/p25) with non-
Golgi proteins, calnexin, and BIP. While not identical, there is considerable overlap between these two sets of proteins. (b) LM-IF of
TM9SF3 reveals Golgi reactions in pachytene spermatocytes (P), round (small arrowheads), and late (large arrowheads) spermatids.
(c) LM-IHC of BIP reveals cytoplasmic reaction in round spermatids (arrows) and granular ER reactions in late spermatids.
28  Golgi apparatus regulation of differentiation

(a) (b)
Lu *

II 1 2
3 *
*
Di
4
P(XII) *
Mitosis 5
(A–B)
P(VII) Meiosis 6 10 µm

P(I) (PL–II) (c)

7
Acrosome Lu
Z (1–7) 8 ER
L Postacrosome ER
(8–18)
9
PL Hermes biogenesis
B (19)
10 µm
In
A (d)
10 Lu
* *
11 *

12
13
19 18 17 16 15 14
10 µm

UGGT Atlastin-1; GRP170; Hexokinase 1 Atlastin-3; βCOP; ERP57

RanBP5 LDHC PDILT

Figure 1.26  (a) Wheel depicting the expression profiles of several proteins. LDH and the cytoplasmic localization of PDILT over-
lap each other precisely from steps 8–19. Several proteins show somewhat overlapping profiles in late spermatids. RanBP5 also
shows localization in late spermatids (steps 12–16). (b) LM-IHC of RanBP5 in a tubule at stage II–III of the cycle. Reactivity is noted in
the cytoplasm of the late spermatids (asterisks). (c) LM-IHC of GRP170 in the cytoplasmic lobe of a late spermatid shows granular ER
reactions (arrows). (d) LM-IHC of LDHC showing cytoplasmic reaction in cytoplasm of late spermatid (asterisks).

(a) (b)
Lu
II 1 2
3
Di
4 S S
P(XII) 5
Mitosis
P(VII) (A–B) 6 10 µm
Meiosis
P(I) (PL–II) 7 (c)
Z Acrosome Lu
8
(1–7)
L
Postacrosome
(8–18) 9
PL
Hermes biogenesis
B
(19)
In 10 µm
A
10

11 Annexin A1; AP1; AP2

Atlastin-2; Malectin; Peroxiredoxin 1;
12 Peroxiredoxin 6; Syntaxin 13;
13
19 18 17 16 15 14 Transferrin; Albumin; α Tubulin;
MCT2; Vps13c

Figure 1.27  (a) Wheel depicting the expression profile of a number of proteins that are solely expressed at step 19 spermatids.
Albumin and transferrin are secreted products of Sertoli cells that adsorb to the surface of step 19 spermatids. (b) and (c) LM-IHC
of Vps13c (b) and MCT2 (c) in a tubule at stage VII of the cycle. In (b), Sertoli cells (S) are reactive and in (b) and (c) the small dilated
Hermes bodies (curved arrows) attached to the neck region of the step 19 spermatids are also reactive.
 Conclusion 29

Carboxypeptidase D; TMED2/p24 Annexin A1; AP1; AP2; Atlastin-2; EF1α; VAMP3; p97; Ubiquitin;
Malectin; Peroxiredoxin 1; Peroxiredoxin 6; Proteasome α 1-7; Sec23
Sortilin Syntaxin 13; Transferrin; Albumin; CHC
GBF1 α Tubulin; MCT2; Vps13c RAB14
UGGT
Annexin A6; Man2α1; Fam3C; GPP34; Calnexin; BIP
Sapreticulin; HSP70.2; LAMAN Atlastin-1; GRP170; Hexokinase 1
MCT1
Atlastin-3; βCOP; ERP57
ManIIX
PDILT
LDHC
TMED7/p27 GLUT-3

II 1 2
3
Di
4
P(XII) 5
Mitosis
P(VII) (A–B)
6
Meiosis
P(I) (PL–II) 7

Z Acrosome
(1–7) 8
L
Postacrosome
PL (8–18) 9
Hermes biogenesis
B (19)
In
A
10

11

12
14 13
19 18 17 16 15

Figure 1.28  Wheel depicting Golgi and non-Golgi proteins that end up in step 19 spermatids. All of these proteins localize to the
Hermes body with the exception of sortilin, which ends up exclusively in residual bodies.
30  Golgi apparatus regulation of differentiation

GRASP55 TM9SF3; TMED4/p25 Annexin A1; AP1; AP2; Atlastin-2; Calnexin; BIP
Malectin; Peroxiredoxin 1; Peroxiredoxin 6;
MG160 RanBP5 Syntaxin 13; Transferrin; Albumin; EF1α; VAMP3; p97;
α Tubulin; MCT2; Vps13c Proteasome α 1-7; Sec23; Ubiquitin
GL54D UBXD8 UGGT CHC
Atlastin-1; GRP170; Hexokinase 1 RAB14
Atlastin-3; βCOP; ERP57
UBXD8
RanBP5
LDHC PDILT

MCT1 GLUT-3
II 1 2
3
Di
4
P(XII) Mitosis 5
(A–B) II 1 2
P(VII) 6 3
Meiosis Di
(PL–II) 4
P(I) 7
P(XII) Mitosis 5
Acrosome
Z (1–7) (A–B)
8 P(VII) 6
Meiosis
L Postacrosome
P(I) (PL–II)
(8–18) 7
PL 9 Acrosome
Hermes biogenesis Z (1–7) 8
(19)
B L Postacrosome
(8–18) 9
In PL
A Hermes biogenesis
B (19)
10
In
A
11 10

12 11
14 13
19 18 17 16 15
12
19 18 17 16 15 14 13

Figure 1.29  Wheel depicting Golgi proteins that do not

end up in the Hermes body of step 19 spermatids.

Figure 1.30  Wheel summarizing the expression profile

of all the non-Golgi proteins in germ cells during the entire
course of spermatogenesis. A varied distribution profile is
noted for each protein. UBXD8 and PDILT are represented
showing their cytoplasmic localizations in germ cells but not
their Golgi localizations.
 Conclusion 31

Carboxypeptidase D; TMED2/p24 TM9SF3; TMED4/p25 LDHC

GRASP55 TMED7/p27 Calnexin; BIP
GBF1 MCT1 EF1α; VAMP3; p97; Ubiquitin;
Annexin A1; AP1; AP2; Atlastin-2; Proteasome α 1-7; Sec23
Sortilin Malectin; Peroxiredoxin 1; Peroxiredoxin 6;
Syntaxin 13; Transferrin; Albumin; CHC
ManIIX α Tubulin; MCT2; Vps13c RAB14
MG160 UGGT
UBXD8
Annexin A6; Man2α1; Fam3C; GPP34; Atlastin-1; GRP170; Hexokinase 1
Sapreticulin; HSP70.2; LAMAN PDILT
Atlastin-3; βCOP; ERP57
GL54D RanBP5 GLUT-3

II 1 2
3
Di
4
P(XII) Mitosis 5
(A-B)
P(VII) 6
Meiosis
P(I) (PL-II)
7
Acrosome
Z (1-7) 8
L Postacrosome
(8-18)
PL 9
Hermes biogenesis
B (19)
In
A
10

11

12
13
19 18 17 16 15 14

Figure 1.31  Wheel summarizing the expression profiles of all the Golgi (solid lines) and non-Golgi (dashed lines) proteins in
germ cells during spermatogenesis. A varied profile is noted for each given protein. UBXD8 and PDILT are represented showing both
their Golgi and cytoplasmic localizations.
32  Golgi apparatus regulation of differentiation
ACKNOWLEDGMENTS
We thank the Facility for Electron Microscopy Research at
McGill University (FEMR) (http://www.medicine.mcgill​
.ca/femr/) for EM services (Drs. H. Vali and Kelly Sears)
and the excellent tissue thin sections prepared by Jeannie
Mui. The EM images were taken with a Technai 12 TEM
(FEI, Hillsboro, OR), coupled to or connected to an AMT
XR80C CCD camera (Advanced Microscopy Techniques
Corp, Woburn, MA). Daniel Friedman, Kristyn Malcolm,
Zariyat Mannan, Rebecca Richard, Carl Dahlen, Nadiya
Goswami, Abigail Belasen, Maria-Teresa Eyzaguirre,
Aurore Fonderflick, Sohee Kang, Dru Perkins, Andrea
Prince, Jason Lee, Raja Talla, Rachel Adilman, and Adam
Parent contributed by assistance in LM-IHC. We are espe-
cially grateful to the investigators indicated in Table S2 of
Au et al.50 who generously provided antibodies, advice,
and guided us to commercial antibodies when available.
Supported by CIHR grant MOP 5605.
Note: This chapter is dedicated to Yves Clermont and
Charles Philippe Leblond, who formulated the stem cell
renewal theory for male germ cell differentiation: http://
www.mcgill.ca​/anatomy/stem-cell-renewal-theory.
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213. Thorne-Tjomsland G, Clermont Y, Tang XM.
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Androgen regulation
of spermatogenesis
WILLIAM H. WALKER
SPERMATOGENESIS
Male fertility is dependent on the continuous process of
spermatogenesis, the production of sperm. Spermatogenesis
occurs within the seminiferous tubules of the testis with
somatic Sertoli cells surrounding and supporting the
developing germ cells (Figure 2.1). Peritubular myoid cells
form webs that encircle the seminiferous tubules while
providing additional factors supporting spermatogen-
esis and also contract to force sperm through the tubules
toward the rete testis and the epididymis. In the intersti-
tial region between seminiferous tubules, Leydig cells pro-
duce testosterone and blood vessels provide nutrients and
growth factors from serum.
Spermatogenesis initiates with spermatogonial stem
cells located along the interior basement membrane of
the seminiferous tubule dividing to produce B spermato-
gonia (nonhuman primates and men) or undifferentiated
spermatogonia (rodents). The spermatogonia undergo
a series of mitotic divisions with incomplete cytokinesis
to form chains of developing germ cells along the base-
ment membrane. In the presence of retinoic acid signal-
ing, a final spermatogonial mitosis produces preleptotene
spermatocytes that are committed to undergo meiosis.
The preleptotene spermatocytes detach from the basement
membrane and “pass through” the blood-testis barrier
(BTB), which is required for spermatogenesis. The BTB is
a collection of adhesion junctions formed between adja-
cent supporting Sertoli cells near the basement membrane
that separates the basal portion of the tubule containing
spermatogonia from the apical (luminal) portion that con-
tains meiotic and post-meiotic germ cells. The BTB is dis-
solved above the preleptotene spermatocyte and reformed
below as the germ cell moves toward the lumen of the
tubule. After passing through the BTB, meiosis continues
through the lengthy leptotene, zygotene, pachytene, and
diplotene stages of prophase I followed by the rapid com-
pletion of meiosis I and II cell divisions that result in hap-
loid round spermatids. The spermatids undergo extensive
differentiation (spermiogenesis) including condensation
of the nucleus and DNA, termination of transcription,
elimination of much of the cytoplasm, acrosome forma-
tion, cell elongation, and development of a flagellum.
Spermatogenesis is completed with the release of mature
elongated spermatids as spermatozoa (spermiation) into
the lumen of the tubule (reviewed in Sharpe1).
40
2
TESTOSTERONE FUNCTIONS
Testosterone is required to maintain spermatogenesis.1,2
Testosterone deprivation studies performed in rodents
established that the steroid hormone is required for
germ cells to progress beyond meiosis and for the release
of mature spermatids (reviewed in Sharpe1). Decreased
intratesticular testosterone levels or elimination of
androgen receptor (AR, NR3C4), the receptor for tes-
tosterone and other androgens, disrupts four major pro-
cesses in the testis that are required for spermatogenesis
and male fertility. First, the BTB cannot be maintained
in the absence of testosterone signaling, resulting in the
loss of meiotic and postmeiotic germ cells. 3,4 Second,
meiosis cannot be completed. Third, round spermatids
are prematurely released as they are differentiating into
elongated spermatids. 5–7 Fourth, mature spermatozoa
cannot be released, resulting in the phagocytosis of the
germ cells by Sertoli cells.7 Although these physiologi-
cal effects of testosterone have been known for decades,
it is only during the past few years that there has been
some better understanding of the molecular mecha-
nisms by which testosterone acts.
ANDROGEN RECEPTOR FUNCTIONAL DOMAINS
The effects of testosterone and other androgens are medi-
ated by AR, a 110 kDa protein member of the steroid hor-
mone receptor family encoded by a single gene on the
X chromosome. AR has numerous well-­characterized
structurally and functionally distinct domains (reviewed
in Heinlein and Chang8 and Lonergan and Tindall9)
including the poorly conserved N-terminal domain
(NTD) encoded by exon 1 that contains a proline-
rich region (PRR) required for interactions with Src
kinase10,11 (Figure  2.2). There is also a highly conserved
DNA-binding domain (DBD) including two zinc fingers
encoded by exons 2 and 3 that recognize specific DNA
sequences called androgen response elements (AREs).
A ligand-binding domain (LBD) also contains a nuclear
export sequence that is encoded by exons 4 through 8.
Another portion of exon 4 encodes a short hinge region
that separates the LBD from the DBD and contains a
ligand-dependent nuclear localization signal (NLS).
Domains encoding transcription activation functions
are located in the N terminus (AF-1, AF5) and in the LBD
(AF-2).
 Androgen receptor functional domains  41

Seminiferous tubule

PTM cell

Spermatogonial GDNF Pachytene Round

spermatocyte spermatid
stem cell
GDNF Elongated
spermatid
Leptotene Mature
spermatocyte spermatozoa

Testosterone Sertoli cell

Leydig cells Testo BTB
stero
ne

Testoste
rone

Spermatogonia

Figure 2.1  Somatic cells supporting spermatogenesis. Leydig cells in the interstitial space between seminiferous tubules pro-
vide testosterone to themselves, Sertoli, and pertitubular myoid (PTM) cells. The PTM and Sertoli cells produce glial cell-derived neu-
rotrophic factor (GDNF) to support the self-renewal of spermatogonial stem cells. The blood-testis barrier (BTB) is shown between
adjacent Sertoli cells. The progression of germ cell development from spermatogonial stem cell through mature spermatozoa is
shown.

AR gene

5′ Exon 1 2 3 4 5 6 7 8 3′

AR protein
NTD DBD LBD

1 PRR 537 625 669 919

Hinge
NLS
AF-1 AF-5 AF-2

Figure 2.2  Androgen receptor (AR) structure. The eight exons of human AR are shown. Exon 1 encodes the amino terminal
domain (NTD) that includes the proline rich region (PRR) that interacts with Src kinase. Exons 2 and 3 encode the DNA binding
domain (DBD) and exons 4 through 8 encode the hinge region and ligand binding domain (LBL). Within the hinge region is the
nuclear localization signal (NLS). Transcription activation domains 1, 2, and 5 (AF-1, AF-2, AF-5) are shown. Amino acid residues that
separate domains are shown below the AR protein.
42  Androgen regulation of spermatogenesis
TESTOSTERONE PRODUCTION
The major sites of testosterone production in men are the
Leydig cells located in the interstitial regions of the tes-
tis. Testosterone is produced in response to luteinizing
hormone binding to its receptor on Leydig cells, which
causes the induction of the steroidigenic machinery that is
responsible for testosterone production and secretion. Due
to the intratesticular location of the Leydig cells, testos-
terone concentrations in the testes of men (340 to 2000 nM)
are  25- to 125-fold greater than that in serum (8.75 to
35  mM). Testosterone is similarly elevated in the rodent
testis and is the major androgen in the testis, exceeding the
levels of dihydrotestosterone (DHT) by 15- to 40-fold.12–16
The bioavailable levels of testosterone in the testis far
exceed that required for function, as the binding affinity of
AR for testosterone is approximately 1 nM and regulation
of gene expression via AR binding requires 1 to 10 nM.17
The physiological importance of high testosterone levels in
the testis is not well understood, but it is known that high
testosterone levels are required to maintain spermatogen-
esis because sperm counts decrease at a logarithmic rate as
testosterone levels fall below 70 nM in the rodent.18
TESTOSTERONE SIGNALING PATHWAYS
Testosterone has been shown to act via two pathways: the
classical and the nonclassical.19,20 In the classical pathway,
testosterone diffuses through the plasma membrane and
binds AR that is sequestered by heat shock proteins in the
cytoplasm. A conformational change in AR causes the
receptor to be released from the heat shock proteins. AR
then translocates to the nucleus where it binds to AREs
in gene promoter regions, recruits coregulator proteins,
and regulates gene transcription. Activation of the clas-
sical pathway requires at least 30 to 45 minutes to detect
the first increases in gene transcription, with protein-­
mediated effects occurring even later.21
In the nonclassical pathway (also called nongenomic)
characterized in Sertoli cells, testosterone stimulation ini-
tiates cellular events within seconds that can be observed
within minutes. Thus far, two rapidly activated nonclas-
sical pathways have been described in Sertoli cells. In the
first pathway, testosterone stimulation of cultured Sertoli
cells isolated from rats transiently increases AR localiza-
tion to the region near the plasma membrane and trig-
gers the direct association of the proline-rich region of
AR (amino acids 352–359) and the SH3 domain of Src
tyrosine kinase.22,23 Studies employing Sertoli cells from
AR-deficient testicular feminized (tfm) rats or siRNA
against AR in wild-type Sertoli cells determined that
AR was essential to drive this nonclassical testosterone
pathway. Testosterone-mediated activation of Src causes
the phosphorylation and stimulation of the EGF recep-
tor (EGFR).22 Stimulation of EGFR is required to acti-
vate the MAP kinase cascade (Raf, MEK, ERK) that in
turn activates p90Rsk kinase to phosphorylate the CREB
transcription factor.22 It is likely that there are numerous
other downstream targets of the kinases activated by the
nonclassical pathway. Stimulation of cultured Sertoli cells
with levels of testosterone (10–250 nM) that are similar to
or lower than those found in the testis (200–800 nM)1,13,14
activated the nonclassical pathway, including the phos-
phorylation of ERK and CREB, and increased the tran-
scription of CREB-regulated genes, including CREB itself,
LDH-A and EGR.24 Thus, the nonclassical pathway also
regulates gene expression downstream of kinase activa-
tion. Together, these findings indicate that the nonclassical
pathway has rapid effects such that increased phosphor-
ylation and activation of Src, ERK, and CREB can be
detected within 1 minute with gene expression regulated
at later time points. Moreover, the pathway has prolonged
effects through increased protein phosphorylation that
can be sustained for at least 12 hours and via long-term
gene expression changes.24
Recently, the first in vivo assays of nonclassical testos-
terone signaling were performed for Sertoli cells within rat
testes. Adult rats were treated with a GnRH antagonist to
decrease intratesticular testosterone levels. After 7 hours,
testicular testosterone concentrations fell to 23% of con-
trol and the levels of phosphorylated ERK kinase (p-ERK)
in stage VII seminiferous tubule cross sections decreased
to 34% of controls. Subsequent injections of testosterone
increased intratesticular testosterone to 125% of controls
and restored p-ERK levels in stage VII tubules to control
levels within 1 hour.25 The rapid testosterone-mediated
increase in p-ERK levels is not likely to result from clas-
sical testosterone signaling because accumulation of tes-
tosterone in the testis would require some latent period
and testosterone-mediated gene expression and protein
production requires more than 45 minutes.21 These studies
provided the first evidence that nonclassical testosterone
signaling occurs in vivo.
Both the nonclassical and classical signaling pathways
are required to maintain spermatogenesis. Adenovirus
constructs expressing either a peptide that inhibits AR-Src
kinase interaction (nonclassical inhibitor) or a modi-
fied AR that acts as a dominant negative inhibitor of
AR-mediated transcription (classical inhibitor) were
delivered into the seminiferous tubules and Sertoli cells
of mice. Both inhibitors disrupted the integrity of the
BTB. In addition, the nonclassical inhibitor resulted in
the loss of spermatocytes in adult mice. Similar studies of
prepubertal mice indicated that the nonclassical inhibitor
blocked the progression of meiosis and the production of
spermatocytes. In contrast, the major effect of the classical
inhibitor was the premature release of meiotic and post-
meiotic germ cells. The combined effects of both inhibitors
resulted in a synergistic disruption of spermatogenesis.
Together, these studies indicate that both signaling path-
ways contribute to the maintenance of spermatogenesis.25
A second rapid-acting nonclassical pathway has been
described in which testosterone causes depolarization of
the Sertoli cell within 30 seconds due to the closing of
K+ATP channels via G protein-mediated activation of phos-
pholipase C. As a result, there is rapid influx of Ca 2+ and
the activation of intracellular signaling factors (reviewed
in Loss et al.26). This second nonclassical pathway has only

been described in immature Sertoli cells. It is not known
whether AR is required to activate the pathway or whether
the pathway contributes to Sertoli cell function in mature
Sertoli cells. Other studies performed in the Sertoli cell
line 93RS2 that lacks AR showed that testosterone acted
via the Zn2+ transporter ZIP9 to activate ERK1/2, CREB
and ATF1, stimulate Claudin 1 and Claudin 5 expression,
and enhance tight junction formation between Sertoli
cells.27 Thus far, it has not been determined whether the
ZIP9-mediated signaling pathway is relevant to Sertoli
cells present in vivo that express AR.
CELL- AND TEMPORAL-SPECIFIC EXPRESSION OF AR
In the adult testis, AR is expressed in peritubular myoid,
Leydig, Sertoli, arteriole smooth muscle, and vascu-
lar endothelial cells. In peritubular myoid cells AR is
expressed from the fetal period through adulthood in men
and rodents.28–31 Fetal and adult Leydig cells express AR at
constant levels.32 Sertoli cells in rodents first express AR
3 to 5 days after birth.33–36 In men, AR is expressed at low
levels in Sertoli cells beginning about 5 months after birth
and remains weak until four years of age.28–31 In the adult
rodent, AR levels are low in Sertoli cells throughout most
of the cycle of the seminiferous epithelium except during
stages VI–VIII when AR levels peak.34,37,38 AR expres-
sion in men is also stage-specific with the highest levels
of AR expressed during stage III of the six stages.39 The
stage-specific peaks in AR expression that are observed
in Sertoli cells of the rodent correspond with the initia-
tion of testosterone-dependent processes that are essential
for spermatogenesis, including the formation of special-
ized adhesion junctions with elongating spermatids and
the release of mature sperm that occur in stage VIII. The
higher levels of AR also coincide with the appearance of
preleptotene spermatocytes and the initiation of meiosis
during stages VII–VIII. Thus, it is possible that testosterone-
mediated paracrine signals from Sertoli cells to prelepto-
tene spermatocytes may be responsible for the production
of factors that are required later to complete meiosis.
There is less information available regarding the poten-
tial importance of limiting AR levels and testosterone
signaling in Sertoli cells during much of the remaining
stages of cycle of the seminiferous epithelium. One study
of a transgenic mouse model in which high levels of AR
were expressed in Sertoli cells from the promoter of the
androgen-binding protein gene caused reduced spermato-
genesis because Sertoli cells matured prematurely, halted
proliferation, and the fewer Sertoli cells present could
only support smaller populations of germ cells. However,
no information was provided about any direct effects of
increased AR expression on processes that were required
for spermatogenesis in adults.40,41
DOES TESTOSTERONE LIMIT THE NUMBER
OF GONOCYTES?
There is some controversy regarding whether testoster-
one regulates the migration and number of gonocyte (also
known as prospermatogonia) germ cells in the fetal testis.
Does testosterone limit the number of gonocytes?  43
Evidence supporting the idea that AR actions do not influ-
ence gonocyte development was found in mice having the
androgen receptor knocked out in all cells (ARKO mice).
In this model, gonocyte migration to the basement mem-
brane of seminiferous tubules was not altered. Also, gono-
cyte transformation into spermatogonia stem cells and
the expansion of spermatogonia was not dependent on
AR based on the finding that wild-type and ARKO mice
had similar numbers of germ cells from E 17.5 through
P10.42 The idea that testosterone signaling via AR does not
regulate gonocyte development was supported by another
study of 1-day-old mice in which gonocytes are the only
germ cells present. At this age, the number of germ cells,
Sertoli cells, and Leydig cells were not altered 1 day after
birth in ARKO mice.43 A different study that assessed the
number of germ cells in 2-day-old mice found that the
number of gonocytes present in ARKO mice exceeded
those present in wild-type mice.44 However, the apparent
difference in ARKO versus wild-type germ cell numbers at
1 and 2 days after birth may be accounted for by the meth-
ods by which the cells were counted. Specifically, for the
2-day-old mice only, the relative nuclear volumes of germ
cells normalized to the nuclear volumes of Sertoli cells was
used to compare gonocyte numbers. Using this method,
the apparent increase in germ cells could be accounted for
by normalizing to the nuclear volume of Sertoli cells.
In contrast to the ARKO mouse studies, the Habert
group showed that tfm mice that are androgen-insensitive
due to global mutation of the androgen receptor had 40%
more gonocytes at E17.5. The increase in gonocytes was
associated with an increased percentage of gonocytes that
were proliferating at E15.5 but not at later stages of devel-
opment. In addition, the number of gonocytes in testis
explants from wild-type mice was decreased by treatment
with DHT.45 These data argue that testosterone acts to
limit the number of gonocytes during fetal development
by yet-to-be-defined mechanisms.
Previous studies showed that AR was not present in fetal
germ cells.46 In contrast, immunoreactive AR was found
in gonocyte nuclei at E 15.5 and AR transcripts were pres-
ent in gonocytes purified from E15.5 and E17.5 mouse tes-
tis. Furthermore, a functional assay of AR responsiveness
suggests that an AR is active in fetal gonocytes at these
times.45,47 These observations raise an important question:
Does testosterone act through AR in fetal gonocytes to
limit their proliferation, and if so, then why is there not an
increase in the number of gonocytes in ARKO mice?
Although it is possible that AR could be expressed in
gonocytes, and there are periodic reports of androgen
receptors being expressed in developing mammalian germ
cells,48–52 there is general consensus that AR is either not
present or not essential in germ cells after birth. Three
functional studies indicate that if expressed, AR is not
required in germ cells to complete spermatogenesis. First,
spermatogonial stem cells isolated from tfm mice were able
to complete spermatogenesis after transplantation into
wild-type mice.53 Second, spermatogenesis is not affected
in knockout mice in which AR expression is eliminated
44  Androgen regulation of spermatogenesis
beginning in early meiosis.54 Third, chimeric mice hav-
ing both AR-defective and wild-type germ cells produced
pups from the AR defective germ cells.55 These findings
reinforce the prevailing idea that germ cells respond to
testosterone signaling only indirectly via paracrine and
justacrine signals from somatic cells.
TESTOSTERONE SUPPORTS THE EXPANSION
OF SPERMATOGONIA
The expansion of germ cells within the first days after
birth is affected by androgen signaling because by PD5,
when only spermatogonial stem cells and the first differ-
entiating spermatogonia are present, germ cell numbers
were reduced in ARKO mice by approximately 60%.43 In
contrast, testosterone does not appear to alter the numbers
of spermatogonia that are present later in the developing
and adult mouse because testosterone treatment initiated
at day 21 and continuing for 6 weeks did not increase the
number of spermatogonia in hpg mice that lack gonado-
tropins and thus have low endogenous levels of testos-
terone.56 These apparently contradictory results could be
explained by testosterone not having an effect on the pro-
liferation and survival of differentiating spermatogonia,
whereas testosterone-mediated signals soon after birth
may be required to support the population of spermatogo-
nial stem cells that are expanding at that time.
Peritubular cell-specific AR knockout (PTM-ARKO)
mice display a progressive decrease in spermatogonia
and marked reductions in spermatocytes and spermatids
that cause the mice to become azoospermic.57 A poten-
tial mediator of peritubular signals required to maintain
spermatogonia is glial cell-derived neurotrophic factor
(GDNF). GDNF acts through the GFRA1 and RET core-
ceptor complex to support the proliferation and mainte-
nance of spermatogonial stem cells and undifferentiated
spermatogonia.58–61 A relatively small window of optimal
GDNF production is required to maintain spermato-
genesis as shown by the collapse of spermatogenesis due
to global mutation of one GDNF allele or by constitu-
tive expression of GDNF after the introduction of a Gdnf
transgene.62 GDNF is a growth factor that is expressed
by Sertoli cells63–65 and peritubular cells.66,67 Testosterone
was recently found to induce the production of GDNF
by peritubular cells. In cultures of enriched peritubular
cells, testosterone stimulation increased the expression of
GDNF mRNA and protein. Further studies showed that
testosterone stimulation of cocultures of peritubular cells
and undifferentiated spermatogonia increased the pro-
liferation of the spermatogonia. Finally, the number and
size of germ cell colonies observed after transplantation
into recipient mice was increased after coculture of undif-
ferentiated spermatogonia with peritubular cells in the
presence of testosterone.66,67 Together, these data raise the
possibility that in PTM-ARKO mice, GDNF production
by peritubular cells is decreased due to the lack of testos-
terone signaling, resulting in insufficient GDNF signaling
activity for stem cell self-renewal, which could explain the
progressive loss of spermatogonia in this mouse model.
Other studies of PTM-ARKO mice determined that
although Sertoli cell numbers, maturation markers, and
the expression of AR were not affected, the expression of
some AR-regulated genes in Sertoli cells was decreased
and the size of the seminiferous epithelium lumen was
reduced. These last observations suggest that there are sig-
nals from the PTM cell to the Sertoli cell that control gene
expression and the production of seminiferous tubule
fluid that carries nutrients to germ cells and developing
sperm.57
It is interesting to note that the number of spermato-
gonia was similar in Sertoli cell AR knockout (SCARKO)
and wild-type mice, suggesting that AR actions in Sertoli
cells do not have a large effect on the maintenance of sperm­
togonial stem germ cells or their conversion to spermato-
gonia. In Sertoli cells, there is no evidence yet that Gdnf
expression is regulated by AR. Instead, follicle-stimulating
hormone (FSH) is a major hormonal stimulator of Gdnf
RNA expression in these cells.68,69
There is now evidence that testosterone signaling in
Sertoli cells can indirectly regulate gene expression in
spermatogonia. The introduction of adenovirus vectors
expressing androgen receptor into the Sertoli cells of
SCARKO mice resulted in the activation of the ZBTB16
(PLZF) and c-Kit genes that in testis are only expressed in
spermatogonia and support the expansion of undifferen-
tiated and differentiated spermatogonia, respectively.25 It
also was found that the nonclassical and classical pathways
provide different signals to spermatogonia because only
activation of the classical pathway permitted the reestab-
lishment of ZBTB16 transcription, whereas activation of
classical or nonclassical signaling resulted in increased
expression of the c-Kit gene.
TESTOSTERONE AND AR ARE REQUIRED
TO COMPLETE MEIOSIS
There is significant loss of spermatocytes and sperma-
tids in rodents, especially in stages VII–VIII of the cycle
of the seminiferous epithelium after reducing intrates-
ticular testosterone levels by hypophysectomy or GnRH
antagonist treatment as well as in hpg mice that lack
gonadotropins. However, the loss of these germ cells can
be reversed under these conditions by testosterone treat-
ment (reviewed in O’Shaughnessy70). AR is required for
testosterone support of meiosis because spermatocytes
numbers are reduced and very few round spermatids are
produced in ARKO and tfm mice.43,71 Germ cell meiosis
requires AR activity in Sertoli cells as shown by the pro-
gressive loss of primary spermatocytes between stages
VI–VII in SCARKO mice.72 The stage-specific loss of
germ cells in SCARKO mice matches the stages in which
AR expression levels normally peaks in Sertoli cells,
implicating AR-mediated signals from Sertoli cells as
being essential for maintaining meiosis. In support of this
idea, SCARKO mice have meiosis arrested in the pachy-
tene or diplotene stage of prophase I such that meiosis I
cell division does not occur and few haploid spermatids
are produced.54,72 Together, these findings indicate that
 Testosterone is required to maintain the blood-testis barrier  45

testosterone and AR in Sertoli cells are required for the from Sertoli cells was blocked. The unreleased elongated
pachytene spermatocytes to enter into meiotic divisions spermatids were observed to be degenerating between
that will produce round spermatids and for the survival Sertoli cells or were phagocytized by Sertoli cells and
of the spermatocytes. transported to the basement membrane for recycling.7,74,75
These hypomorph SCARKO mouse results match those
Meiotic processes and factors regulated by testosterone: In the
from earlier testosterone suppression models that caused
absence of testosterone signaling, male germ cells enter
the premature detachment of step 8 round spermatids.6,76
into meiosis I and progress through the leptotene and
The premature release of spermatids in the absence of
zygotene stages of meiosis to initiate the pairing of the
testosterone signaling may be explained by the changes in
chromosomes (synapsis) and the production of double-
the Sertoli-spermatid adhesion complex that occur dur-
strand DNA breaks that are required for later recombi-
ing the transition to elongated spermatids. During stage
nation events and the formation of chiasmata that are
8 when round spermatids initiate their elongation process,
needed for separation of the chromosomes. However,
the adhesion complexes between Sertoli cells and round
during the pachytene stage of meiosis, synapsis is not
spermatids are transformed from predominately desmo-
completed and double-strand breaks are not repaired,
some type connections to the specialized ectoplasmic
which inhibits recombination and desynapsis such that
specialization (ES) adherans junction that is unique to the
85% of spermatocytes had univalent chromosomes.73
testis.77 It is possible that the expression or localization of
These events result in the induction of cell cycle check-
proteins needed for the formation of the ES and elongated
point responses that lead to cell cycle delay or arrest
spermatid attachment may be dependent on AR signal-
and the apoptosis of the germ cells. The triggering of
ing in Sertoli cells because suppression of intratesticular
the checkpoint responses explains the halting of mei-
testosterone decreases the levels of proteins present at ES
osis during pachytene and the apoptosis of pachytene
junctions, including β1-integrin, p-FAK, and c-Src via the
spermatocytes.
ERK signaling pathway.78
One explanation for cell cycle arrest and apoptosis in
The paradox of how testosterone can be essential for
the absence of testosterone signaling is the loss of proteins
both the adhesion of elongating spermatids and the release
that are essential for the completion of meiosis. In sper-
of mature elongated spermatids (sperm) appears to be
matocytes of SCARKO mice, expression was decreased
resolved. The ES at the site of spermiation is disassembled
for RAD51 and DMC1 proteins that are required for the
and the components are relocated and recycled for use at
resolution of double-strand DNA breaks, recombina-
the site of new spermatid adhesion sites by 22 hours before
tion, and chromosome separation. There is also decreased
the final release of sperm during stage VII.79 Thus, the ES
expression of other proteins (TEX15, BRCA1, BRCA2, and
is not present when the sperm are released. Instead, sperm
PALB2) that regulate the loading of RAD51 and DMC1
adhesion to Sertoli cells is maintained via the tubulobul-
onto the sites of double-strand breaks.73 Thus far, it is not
bar and focal adhesion-related disengagement complexes.
known whether testosterone signals from Sertoli cells first
Testosterone signaling supports the disengagement of
act during the pachytene stage of meiosis on the RAD51,
sperm from these complexes.75 The O’Donnell group
DMRT, and associated proteins or earlier in leptotene and
provided evidence that only the disengagement complex
zygotene stages to permit correct chromosome synapsis,
remains associated with sperm that fail to be released in
which is essential for the later events in meiosis. The mech-
the absence of testosterone. This complex retains α6β1
anisms by which testosterone and AR-mediated signals
integrin and tyrosine-phosphorylated FAK from the ES.75
are delivered to the meiotic cells are not well understood.
Notably, FAK can be phosphorylated in response to non-
However, increased signaling by members of the EGF-
classical testosterone signaling and by Src kinase that is
EGFR family of growth factors in the testis was detected
activated by the same pathway. Furthermore, inhibition
in SCARKO mice and may be one mechanism by which
of Src kinase activity resulted in lower levels of sperm
decreased AR signaling may alter signals to germ cells.73
release.80 The idea that testosterone-mediated phosphory-
lation of components of the disengagement complex enable
TESTOSTERONE IS REQUIRED FOR SPERMATID sperm release also is consistent with findings that activa-
ATTACHMENT AND RELEASE OF SPERM tion of nonclassical signaling target ERK kinase occurs at
Most SCARKO mouse models do not provide informa- the time and location of sperm release in cultured semi-
tion about testosterone support of spermiogenesis because niferous tubules.81
spermatogenesis is arrested in meiosis. However, the
hypomorph SCARKO model from Braun’s group that TESTOSTERONE IS REQUIRED TO MAINTAIN
displays partial AR activity provided important informa- THE BLOOD-TESTIS BARRIER
tion about testosterone-regulated events during sperma- Testosterone is required for maintaining the BTB that
tid maturation. This model showed that progression of consists of numerous cellular junctions between Sertoli
round spermatids to elongated spermatids is sensitive to cells.3,4 The BTB is present near the basement membrane
the reduction in AR activity in Sertoli cells such that the of the seminiferous tubule and separates the basal com-
round spermatids are released prematurely. As well, ter- partment that has access to the circulatory system from
minal differentiation and release of elongated spermatids the luminal compartment that is dependent on the Sertoli
46  Androgen regulation of spermatogenesis
cell to provide nutrients and growth factors. All germ
cell development beyond the leptotene stage of meiosis
occurs in the specialized environment beyond the BTB.
Spermatogenesis in rodents is halted after structural dis-
ruption of the BTB.82,83 However, the extent to which BTB
disruptions affect fertility in men is less well understood.
Studies of cultured Sertoli cells showed that testosterone
increased tight junction function as measured by transepi-
thelial resistance (TER).84–86 Testosterone also stimulated
the expression of mRNA encoding a major tight junction
protein N-cadherin and stimulated the localization of
Claudin 11 to Sertoli-Sertoli contact sites.86,87 Follow-up
studies indicated that testosterone signaling increases
steady-state levels of clatherin and clatherin association
with tight junction proteins occludin and Claudin 11. In
addition, testosterone facilitates the transcytosis and recy-
cling of tight junction proteins that are associated with the
dissolution of the BTB as spermatocytes transit through
the barrier allowing the rapid reformation of the BTB that
occurs below the spermatocytes.88,89
Claudin 3 is an adhesion protein that is induced by
androgen and localizes to newly forming tight junctions at
the BTB.4 However, Claudin 3 deficient mice retain fertility,
suggesting that other androgen-regulated junctional pro-
teins are sufficient to maintain the BTB.90 Claudin 11 and
occludin, major transmembrane components of the BTB,
were found to be downregulated in SCARKO mice.3,91,92
The mRNAs encoding Tjp1, Tjp2 iso1, Tjp2 iso2, and Tjp2
iso3 that link the transmembrane tight junction proteins
to the cytoskeleton are also downregulated in SCARKO
mice, suggesting the possibility that these genes are reg-
ulated by testosterone. These results are consistent with
the finding that in SCARKO mice the BTB is formed but
formation is delayed and the barrier is incomplete.3 Rats
injected with flutamide to block AR actions had decreased
expression of the Cx43 gap junction mRNA and protein
and the loss of Cx43 localization to the BTB, whereas the
tight junction associated ZO-1 protein was mislocalized
away from the cell surface to the Sertoli cell cytoplasm.93
DHT treatment of hpg mice restored the expression and
localization of Claudin 3, Cx-43, and Claudin 11 to the
BTB.94
Inhibition of either classical or nonclassical testoster-
one signaling disrupted the BTB in vivo as injection of
mouse testis seminiferous tubules with adenovirus con-
structs expressing inhibitors of either pathway resulted
in decreased expression of Claudin 11 at the BTB and
increased transit of a biotin tracer through the BTB and
into the lumen of the tubules.25 The finding that the non-
classical testosterone signaling pathway is required for
maintaining the BTB is consistent with previous studies
showing that ERK kinase activity and Src family kinases
components of the nonclassical pathway are required for
maintaining the BTB.95–97 The finding that classical testos-
terone signaling is required to maintain the BTB is sup-
ported by previous studies showing that AR induces the
expression of genes encoding BTB-associated proteins
including N-cadherin, Claudin 11, Occludin, Gelsolin,
Jam3, and Claudin 3.3,4,91 However, presently it is not
known whether the expression of genes encoding BTB
proteins is enhanced by classical or nonclassical signaling
in vivo.
TESTIS GENE EXPRESSION IS REGULATED
BY TESTOSTERONE
Altered testosterone signaling and SCARKO models:  Surveys
of gene expression in various rodent models have been
performed in the presence and absence of testosterone
signaling. The various models include neonatal wild-
type or adult hpg mice injected with testosterone pro-
pionate for 4 to 24 hours,94,98,99 comparisons of normal
vs tfm mice,100 AR hypomorph mice,101 and four other
SCARKO models.3,91,92,102 These studies of testosterone-
and AR-regulated gene expression have all employed
isolation of mRNAs from the whole testis. One common
theme for nearly all of the models was the finding that
a greater percentage of genes were downregulated by
AR and testosterone. Nevertheless, these studies deter-
mined that testosterone signaling increased the expres-
sion of genes encoding the BTB proteins Claudin 3 and
Claudin 11,99 genes associated with vitamin A metabo-
lism, solute transport, or cytoskeletal function,100 as
well as genes involved in metabolic processes and signal
transduction.103 The SCARKO mouse models identi-
fied AR repressed genes associated with MAP kinase
and serine endoprotease activity plus upregulated genes
needed for signal transduction, cell adhesion, Ca2+ bind-
ing, Ca2+-dependent phospholipid binding, and serine
protease inhibition.92 Also, in SCARKO models there
was differential expression of genes encoding proteases,
cell adhesion proteins, cytoskeletal elements, extra-
cellular matrix components,3 tight junction proteins,
basement membrane components, tissue remodeling
factors, paracrine factors for the support of germ cells,
transport proteins,91 as well as cell adhesion, nuclear
localization, and meiosis.102
RiboTag mouse models:  An important advance in the
identification of cell-specific changes in AR-regulated
mRNAs is the use of transgenic mice expressing the
RiboTag, which is the ribosomal protein S6 having an
HA epitope tag. The expression of the RiboTag allows
for immunoprecipitation with antisera directed against
the HA epitope that will isolate ribosomes and any
associated RNA. Conditional expression of the RiboTag
using a cell-specific cre recombinase allows the isolation
of the population of RNAs in the process of translation
from individual cell types (the translome). The initial
RiboTag strategy using a Sertoli cell-specific cre recom-
binase combined with microarray analysis did not
reveal an extensive list of AR-regulated mRNAs specific
to Sertoli cells perhaps due the short time period of tes-
tosterone stimulation (4 hours).104
A different RiboTag approach was used in which mRNA
expression was compared in 10-day-old mice express-
ing the RiboTag in an otherwise wild-type background

(SCRIBO mice) versus a SCARKO background
(SCARIBO mice). Analysis of the ribosome-­associated
RNAs by RNA-seq yielded the most extensive list
of AR-regulated genes in the testis to date. Without
first isolating mRNAs from ribosomes of specific cell
types, RNA-seq analysis identified 938 AR-regulated
genes from all testis cells in 10-day-old mice with
slightly more genes being stimulated than inhibited
by AR. About 20% (187) of the genes were differen-
tially expressed by twofold or more. The use of the
RiboTag strategy to isolate mRNAs associated with
the ribosomes of Sertoli cells identified 1090 mRNAs
that were differentially expressed in response to the
loss of AR. The AR-regulated genes enriched in Sertoli
cells included those that encoded proteins associated
with calcium ion, actin, or cytoskeletal protein bind-
ing, proteins active at the plasma membrane, adhesion
functions including cell-cell junction and anchoring
junctions, as well as proteins located at the stereocil-
ium. AR was found to be more likely to repress (59%)
than activate (41%) Sertoli cell enriched gene expres-
sion. Of the 1090 Sertoli cell enriched AR-regulated
genes found using the RiboTag strategy, 55% escaped
detection in the less specific whole testis SCARKO vs
wild-type (SCARIBO vs SCRIBO) comparison and
38% were differently expressed by two-fold or more.
This result indicates that the RiboTag strategy has
greater sensitivity than a total testis approach for assay-
ing differential gene expression. However, as with other
surveys of AR-regulated genes in the testis, few genes
known to be essential for fertility were identified in the
list of AR-regulated genes. Nevertheless, several of the
identified genes were known to contribute to processes
important for male fertility, including Tgfb2, which
is involved in SC barrier formation and maintenance;
Nr5a2, an activator of steroidogenic enzymes; and
Timp1, an inhibitor of matrix metalloproteinases. It is
possible that repeating the survey using mature mice
would reveal additional AR-regulated genes that are
required for fertility.
The RiboTag approach also found 445 mRNAs that are dif-
ferentially expressed in the absence of AR in Sertoli cells
but are enriched in other cell types. These non-Sertoli
cell mRNAs encoded proteins involved in binding to
adenyl ribonucleotides, adenyl nucleotides, nucleosides,
and ATP, which may suggest that androgen signaling in
SCs leads them to instruct other testicular cells to alter
their nucleic acid and energy metabolism. The use of
similar RiboTag strategies could be used in the future to
identify genes in germ cells at specific stages of develop-
ment that are regulated by testosterone and AR actions
in Sertoli and other somatic cells of the testis.
Of the major spermatogenesis processes regulated by
testosterone, including spermatogonia proliferation,
BTB formation, meiosis, spermatid attachment, and
spermiation, thus far AR-regulated gene expression
can be shown to be directly linked to the production
of proteins (Claudin 11, Occludin) that are essential
Summary 47
to maintain Sertoli cell tight junctions and the BTB.105
Although AR-regulated genes contribute to the other
testosterone-mediated processes, few are known to
be essential. It is possible that the combined effects of
AR-regulated gene expression may be required to sup-
port specific stages of spermatogenesis. However, fur-
ther studies are required to identify the mechanisms by
which testosterone and AR support specific processes
required for spermatogenesis.
FUTURE CHALLENGES
Cell-specific AR knockout mouse models have provided
important phenotypic information regarding the func-
tions of testosterone in the testis, including testosterone-
mediated actions within targeted cells and effects on
neighboring cells. The next goal will be to understand
how testosterone-mediated signals are transferred to other
cells. For example, results from SCARKO mice suggest that
AR-mediated EGF signals originating from Sertoli cells
must be limited to permit meiosis progression through
prophase I.73 However, it is not known how testosterone
acts via AR to decrease EGF signaling to spermatocytes or
which stage of meiosis must be protected from EGF. It is
likely that additional AR-regulated paracrine signals from
Sertoli and other cells remain to be identified. How these
signals act to support meiosis and other germ cell pro-
cesses will need to be determined. Once these questions
are addressed, we will then need to understand the intra-
cellular signaling events and cellular responses that occur
after the paracrine signals are received. After these issues
are understood, the information may be used to identify
potential sources of defects causing male infertility or tar-
gets for contraception strategies.
SUMMARY
Testosterone-mediated alterations in the expression,
phosphorylation, and localization of proteins associ-
ated with adhesion complexes in Sertoli cells are essen-
tial for the maintenance and continuous reestablishment
of the BTB, the adhesion of elongating spermatids, and
the release of mature sperm. Additional, less well char-
acterized paracrine and juxtacrine signals from Sertoli
cells are required for spermatocytes to complete meiosis.
Although testosterone is essential for many processes that
are required to complete spermatogenesis, it is also useful
to consider critical spermatogenesis processes for which
testosterone signaling is not required. At present, avail-
able evidence suggests that testosterone is not essential for
(1) the survival and proliferation of spermatogonial stem
cells required to continually replenish germ cells, (2) the
proliferation of differentiating spermatogonia needed
to expand the population of germ cells, (3) the entry
into meiosis, and (4) the cellular restructuring required
for differentiation of elongating spermatids. Decades of
research has brought us to a good understanding of the
spermatogenesis process that are and are not regulated by
testosterone. We hope that future studies will provide a
better understanding of the intracellular and extracellular
48  Androgen regulation of spermatogenesis
signaling strategies that are used by testosterone to sup-
port spermatogenesis.
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65. Johnston DS, Olivas E, DiCandeloro P, Wright WW.
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66. Chen LY, Brown PR, Willis WB, Eddy EM. Peritubular
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67. Chen LY, Willis WD, Eddy EM. Targeting the Gdnf
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68. Ding LJ, Yan GJ, Ge QY et al. FSH acts on the pro-
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69. Lamberti D, Vicini E. Promoter analysis of the gene
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70. O’Shaughnessy PJ. Hormonal control of germ cell
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71. Johnston H, Baker PJ, Abel M et al. Regulation
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72. De Gendt K, Swinnen JV, Saunders PT et al. A Sertoli
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73. Chen SR, Hao XX, Zhang Y et al. Androgen receptor
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74. Saito K, O’Donnell L, McLachlan RI, Robertson DM
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76. Cameron DF, Muffly KE, Nazian SJ. Reduced testos-
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78. Wong CH, Xia W, Lee NP et al. Regulation of ecto-
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79. O’Donnell, L, Nicholls PK, O’Bryan MK, McLachlan
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81. Chapin RE, Wine RN, Harris MW, Borchers CH,
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86. Kaitu’u-Lino TJ, Sluka P, Foo CF, Stanton PG.
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89. Yan HH, Mruk DD, Lee WM, Cheng CY. Blood-testis
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91. Wang RS, Yeh S, Chen LM et al. Androgen recep-
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Testicular immunoregulation
The role of Tyro3, Axl, and Mer receptor tyrosine
kinases and pattern recognition receptors
FEI WANG, QIAN JIANG, and DAISHU HAN
INTRODUCTION
Mammalian spermatogenesis is a unique male germ cell
differentiation process occurring in the testis that requires
a special microenvironment. From an immunological per-
spective, the testis possesses a remarkable immunoprivi-
leged environment essential for the protection of germ
cells from detrimental immune responses and adopts an
effective innate local defense mechanism against micro-
bial infection.1,2 Disruption of the testicular immune
homeostasis may impair spermatogenesis and result in
male infertility.
Spermatogenesis is completed during puberty, a long
time after the establishment of immune self-tolerance.
Therefore, late-stage male germ cells synthesize a large
number of immunogenetic molecules that are strang-
ers to the immune system. However, these germ cells are
immunologically tolerated in the testis under physiologi-
cal conditions because of its immunoprivileged environ-
ment. Multiple mechanisms are involved in the regulation
of this testicular immunoprivileged environment. The role
of Tyro3, Axl, and Mer (TAM) receptor tyrosine kinases in
regulating immune privilege in the testis has been investi-
gated recently and a review of TAM functions in the testis
is provided in this chapter.
Although the testis is an immunoprivileged organ, a
broad spectrum of microorganisms, including viruses,
bacteria, and parasites, may infect the testis via circula-
tory dissemination or the ascending genitourinary tract.
However, microbial infection usually recovers without a
significant systemic immune response, suggesting that the
testis is well-equipped with an effective innate defense sys-
tem against microbial infection. Various leukocytes can
be found in the interstitial spaces of the testis, and these
immune cells may play roles in counteracting invading
microorganisms.3 Recent studies have demonstrated that
tissue-specific cells, including somatic and germ cells, are
also equipped with innate immune machinery.4 Sertoli,
Leydig, and male germ cells abundantly express vari-
ous pattern recognition receptors (PRRs), which initiate
innate immune responses in the testicular cells. This chap-
ter also describes PRR-initiated innate immune responses
in testicular cells and their effects on testicular function.
TAM receptor tyrosine kinases
Receptor tyrosine kinases (RTKs) belong to a superfam-
ily of transmembrane proteins with diverse extracellular
52
3
regions and a common highly conserved intracellular
tyrosine kinase domain.5 Different subfamilies of RTKs
are categorized based on their sequence identities and
extracellular structural similarities, and the members
of a subfamily often bind to common or similar ligands.
TAM receptors belong to one subfamily of RTKs. Two
highly similar vitamin-K–dependent proteins, the prod-
uct of growth-arrest–specific gene 6 (Gas6) and a negative
regulator of blood coagulation, protein S (ProS), are two
common ligands of TAM receptors.6 Gas6 and ProS are
composed of N-terminal gamma-carboxylated glutamic
acid (GLA) followed by four epidermal growth-factor
(EGF)-like and C-terminal sex-hormone-binding globu-
lin (SHBG)-like domains (Figure 3.1). TAM receptors have
a similar structure, composed of two immunoglobulin
(Ig)-like domains in N-terminal and two fibronectin type
III (FNIII) domains in extracellular regions, followed by
a transmembrane domain and an intracellular protein
tyrosine kinase (TK) domain at C-terminal. The SHBG-
like domains of ligands interact with the Ig-like domains
of TAM receptors, initiating signaling pathways through
the activation of intracellular TK. Studies using  gene-
knockout mice have provided direct insights into the
physiological functions of TAM receptors. TAM signaling
pathways regulate several important biological processes,
including immune responses, phagocytosis of apoptotic
cells, and cell proliferation.7
Role of TAM receptors in regulating immune
homeostasis
Genetic mutations in TAM receptors result in chronic
inflammatory and autoimmune diseases in mice, sug-
gesting that TAM signaling plays an important role in
maintaining immune homeostasis.8 TAM signaling
contributes to immune homeostasis by regulating two
biological processes: the inhibition of innate immune
responses and the promotion of phagocytic clearance of
apoptotic cells.9
Innate immune responses are initiated by micro-
bial pathogens through the activation of PRRs. Toll-
like receptors (TLRs) are the most well-characterized
PRRs10 and TLR signaling in innate immune cells, such
as dendritic cells (DCs) and macrophages, leads to the
production of numerous cytokines and chemokines,
recruiting and activating leukocytes to counteract
invading microorganisms. The TLR-initiated innate

N TAM receptors in the testis
GLA domain
Ligands:
Gas6, ProS
EGF-like domains
N
SHBG-like domain
C
Ig domains
TAM receptors:
Tyro3, Axl, Mer FN III domains
Cell membrane
TK domain
C sperm. Evident defective spermatogenesis initially appears
Immune Phagocytosis
Cell proliferation
responses of apototic cells
Figure 3.1  Schematic structure of TAM receptors and their
ligands. TAM receptors contain three members: Tyro3, Axl, and
Mer. TAM receptors are composed of two Ig-like domains at
N-terminal followed by two extracellular fibronectin type III
(FN III) domains, a transmembrane domain, and an intracellu-
lar protein tyrosine kinase domain at C-terminal. The product
of growth arrest specific gene 6 (Gas6) and protein S (ProS)
are ligands of TAM receptors. Gas6 and ProS share a common
structure composed of an N-terminal gamma-carboxylated
glutamic acid (GLA) domain, four epidermal growth factor
(EGF)-like domains, and a C-terminal sex hormone binding
globulin (SHBG)-like domain. The SHBG-like domain of ligand
binds to the Ig domain of TAM receptors, thereby activating
TAM receptors. TAM receptor activation initiates intracellu-
lar signaling to regulate several important cellular processes
such as proliferation, uptake of apoptotic cells, and immune
responses.
immune response also directs the adaptive immune
response against microbial infection.11 Although TLR
activation in innate immune cells is critical in the
host defense against microbial infection, TLR-initiated
innate immune responses must be tightly regulated, as
unrestrained TLR signaling generates a chronic inflam-
matory condition that may be harmful to the host itself.
Therefore, the negative regulation of TLR signaling is
essential for immune homeostasis.12 TAM receptors are
important negative regulators of the innate immune
response and play a critical role in maintaining immune
homeostasis.13
Introduction 53
The role of TAM receptors in the testis was initially rec-
ognized using TAM triple knockout (TAM−/−) mice.14
Although mutations of individual members or any com-
bination of two receptors do not affect mouse fertility,
TAM−/− male mice are sterile because of spermatogenesis
degeneration. To investigate the mechanisms by which
TAM receptors regulate spermatogenesis, TAM receptor
and Gas6 expression in the mouse testis has been exam-
ined.15 All three TAM receptors are expressed in Sertoli
cells. Axl and Mer, but not Tyro3, are expressed in Leydig
cells, and Gas6 is exclusively expressed in Leydig cells.
By contrast, neither TAM receptors nor their ligands are
expressed in male germ cells. The distribution of the TAM
system in the testis suggests that impaired spermatogen-
esis in TAM−/− mice is not germ cell autonomous, and the
degeneration of TAM−/− somatic cell functions contributes
to the impairment of spermatogenesis.
Notably, TAM−/− male mice fulfill the first wave of
spermatogenesis, producing a few aberrant elongated
5 weeks postnatally, following the onset of sexual matura-
tion.16 As TAM−/− mice age, male germ cells are progres-
sively lost, beginning from elongated spermatids to round
spermatids, spermatocytes, and spermatogonia. TAM−/−
mice have a normal testosterone level, suggesting that
defective spermatogenesis is attributable to impairment of
Sertoli cell functions.
Role of TAM receptors in the phagocytosis of apoptotic
germ cells
Sertoli cells, the only somatic cells in the seminifer-
ous epithelium, play a critical role in spermatogenesis,
and one Sertoli cell function is the phagocytic removal
of apoptotic germ cells and residual bodies. More than
70% of male germ cells undergo apoptosis during sper-
matogenesis. Moreover, the most cytoplasmic portions of
elongated spermatids form residual bodies and are shed
before sperm are released from the seminiferous epithe-
lium. Timely phagocytic removal of apoptotic germ cells
and residual bodies is critical for maintaining seminifer-
ous epithelial homeostasis.17 The apoptotic germ cells and
residual bodies are recycled after phagocytosis by Sertoli
cells as a source of energy, suggesting that apoptotic germ
cells and residual bodies are more than mere wastes.18
Notably, the TAM/Gas6 system plays an important role
in regulating the phagocytosis of apoptotic germ cells
by Sertoli cells.19 All three TAM receptors recognize and
tether apoptotic cells through Gas6 in a redundant man-
ner, whereas Mer is responsible for triggering phagocy-
totic signaling (Figure 3.2).
From an immunological perspective, phagocytic
removal of apoptotic cells by phagocytes is a critical mech-
anism in the elimination of endogenous autoantigens for
the prevention of an autoimmune response.20 Impairment
of apoptotic cell clearance is associated with autoimmune
diseases.21 Late-stage male germ cells are formed after the
54  Testicular immunoregulation

Sperm

Residual body

Apoptotic germ cell

ATP
Breakdown

Recycle Lysosome

TAM
Residual body
Phagolysosome
DAMPs TRIF Phagosome
TLRs TAM
MyD88 TAM
SOCS1/3
Microbes
MAPKs NF-κB IRFs SOCS1/3 Apoptotic germ cell

AP-1

TAM
AP-1
P NF-κBP P
IRFs ???
TNF-α TAM Gas6
IL-1
IL-6 IFN-α/β
......

Figure 3.2  Functions of TAM receptors in Sertoli cells. Two major functions of TAM receptors in Sertoli cells have been defined.
(1) TAM receptors mediate phagocytosis of residual bodies and apoptotic germ cells by Sertoli cells. The phagosomes are fused
with lysosome to form phagolysosome. The residual bodies and apoptotic cells in the phagolysosome are degraded and recycled
as an energy source for Sertoli cells. (2) TAM receptors negatively regulate toll-like receptor (TLR) signaling pathways. TAM signaling
induces the expression of suppressor of cytokine signaling proteins (SOCS1/3), which inhibit TLR signaling pathways.

establishment of immune self-tolerance and synthesize
a large number of autoantigens that are immunogenetic.
Phagocytic removal of apoptotic germ cells and residual
bodies by Sertoli cells can prevent immunogenetic autoan-
tigens from inducing immune responses. Therefore, TAM
receptors may contribute to testicular immune homeosta-
sis by promoting the removal of germ cell autoantigens.
Moreover, damaged germ cells also induce the production
of inflammatory cytokines in Sertoli cells through the
activation of TLR2 and TLR4.22 Therefore, apoptotic germ
cells and residual bodies, if not removed in time, may
release endogenous TLR ligands and trigger an inflam-
matory response through TLR activation (Figure 3.2). The
most well-characterized endogenous TLR ligands include
high-mobility group box 1 (HMGB1) and several heat
shock proteins (HSPs), which induce endogenous inflam-
mation through the activation of TLR2 and TLR4.23,24
Notably, HMGB1 and HSPs are abundantly expressed in
male germ cells25,26 and HMGB1 is involved in testicu-
lar inflammation.27 The role of endogenous TLR ligands
in testicular endogenous inflammation remains largely
unknown.
TAM inhibition of innate immune responses
in testicular cells
TLR-initiated innate immune responses in the testis
were initially found in mouse Sertoli cells. 28,29 Given that
the TAM/Gas6 system is a negative regulator of TLR sig-
naling,13 it is reasonable to speculate that TAM recep-
tors inhibit TLR-initiated innate immune responses in
Sertoli cells because TAM are abundantly expressed in
these cells. The mechanisms by which TAM receptors
inhibit TLR signaling in Sertoli cells have been investi-
gated. 30 TAM−/− Sertoli cells display excessive activation
of TLR3 signaling characterized by the overexpression
of immunoregulatory cytokines. The TAM/Gas6 system

upregulates the suppression of cytokine signaling, which
in turn inhibits TLR-driven expression of cytokines in
Sertoli cells (Figure 3.2). Several TLRs are also expressed
in mouse Leydig cells and can be activated by their
respective ligands. 31 Axl and Mer are also expressed in
Leydig cells and inhibit TLR-initiated innate immune
responses.
TLR-initiated innate immune responses in Sertoli and
Leydig cells may contribute to testicular defense against
microbial infection. However, the innate immune response
in testicular cells needs to be tightly controlled by the
negative regulators because an excessive innate immune
response may disrupt testicular immunoprivileged status
and impair spermatogenesis. Accordingly, TAM−/− male
mice spontaneously develop autoimmune orchitis charac-
terized by progressive degeneration of germ cells accom-
panying leukocyte infiltration into the testis, upregulation
of inflammatory cytokines, disruption of blood-testis
barrier (BTB) integrity, and generation of autoantibodies
against male germ cells.32 Notably, autoimmune orchitis
in TAM−/− mice develops progressively after the onset of
sexual maturation, and during this period a large number
of residual bodies are formed. We speculate that impair-
ment in the phagocytic ability of TAM−/− Sertoli cells
slows the timely removal of residual bodies. These residual
bodies then release endogenous ligands to activate TLRs
in Sertoli cells. Excessive activation of TLR signaling in
TAM−/− Sertoli cells leads to the production of high levels
of inflammatory cytokines. High levels of tumor necrosis
factor alpha (TNF-α), a major inflammatory cytokine, can
alter BTB permeability,33 resulting in the release of auto-
antigens into the interstitial spaces to induce an autoim-
mune response. A high level of TNF-α also induces germ
cell apoptosis,34 which may amplify endogenous innate
immune responses.
The role of TAM receptors in immune tolerance to male
germ cells
Local immunosuppressive mechanisms are important
for maintaining the immunoprivileged status of the tes-
tis. Systemic immune tolerance to autoantigens also con-
tributes to its immunoprivileged environment. Although
local immunosuppression in the testis has been inten-
sively investigated, the mechanisms underlying the sys-
temic immune tolerance to germ cell antigens are largely
unknown. Considering that TAM receptors inhibit auto-
immune disease,35 they may regulate systemic immune
tolerance to male germ cell autoantigens. This hypothesis
has been investigated using an experimental autoimmune
orchitis (EAO) model36 in rodent animals, a testis-specific
autoimmune inflammation model used for the investi-
gation of the pathomechanisms of testicular autoimmu-
nity.37 EAO can be induced in animals by immunization
with an injection of allogeneic testicular homogenates,
male germ cell lysates, or viable male germ cells.38 EAO
usually develops after three immunizations and is char-
acterized by generation of autoantibodies against germ
cell antigens, infiltration of leukocytes into the testis, and
The innate defense system in the testis  55
damage to the seminiferous epithelium. Using the EAO
model, we found that Axl and Mer receptors coopera-
tively regulated systemic immune tolerance to male germ
cell antigens.36 Although Axl and Mer double knockout
(AM−/−) male mice were fertile and had no signs of auto-
immune orchitis, they were susceptible to EAO induc-
tion and AM−/− mice developed severe EAO after a single
immunization with germ cell antigens.36 Mild EAO was
observed in Axl−/− and Mer−/− mice after similar immuni-
zation. By contrast, a single immunization did not induce
EAO in Tyro3−/− and wild-type (WT) mice. These observa-
tions suggest that Axl and Mer, but not Tyro3, cooperatively
regulate systemic immune tolerance to male germ cell
antigens. Accordingly, antigen-presenting cells, includ-
ing dendritic cells and macrophages, express Axl and Mer
but not Tyro3,13 and the mechanisms by which Axl and
Mer regulate systemic immune tolerance to germ cell anti-
gens are worthy of further investigation. TAM−/− devel-
oped autoimmune orchitis spontaneously,32 suggesting
that Tyro3 is also involved in the maintenance of testicu-
lar immunoprivileged status. Considering that Tyro3 is
exclusively expressed in Sertoli cells,15 Tyro3 may locally
regulate the testicular immunoprivileged environment via
modulation of Sertoli cell function, although this remains
to be clarified. TAM receptors are also essential for tissue
homeostasis in other immunoprivileged organs, such as
the brain and eye, confirming that TAM receptors play a
universal role in the regulation of immune privilege.39
THE INNATE DEFENSE SYSTEM IN THE TESTIS
Various immune cells, mainly macrophages in testicular
interstitial spaces, are believed to be the frontline against
the arrival of microbial pathogens from circulating blood.
However, testicular macrophages display anti-inflammatory
phenotypes in favor of the immunoprivileged environ-
ment.40 Therefore, it is doubtful whether testicular mac-
rophages play a crucial role in counteracting microbial
pathogens.41 Recent studies have revealed that tissue-
specific testicular cells, including Leydig, Sertoli, and
germ cells, are equipped with innate immune machinery
and are probably involved in testicular defense against
microbial infections.
PRRs
Recognition of microbial pathogens by PRRs is the first
step in the initiation of immune responses.42 PRRs recog-
nize the conserved molecular structures that are broadly
shared by microbial pathogens; namely, pathogen-associated
molecular patterns (PAMPs). Upon PAMP recognition,
PRRs initiate signaling pathways that execute the first line
of host defense against invading microbes. Further, PRR-
initiated immune responses direct adaptive immunity, the
second line of host defense.11
Several PRR families have been identified (Figure 3.3).
TLRs, retinoic acid-inducible gene (RIG-I)-like receptors
(RLRs), and cytosolic DNA sensors are the most well-
characterized PRR families43 and these PRRs recognize
a broad spectrum of PAMPs, thereby initiating signaling
56  Testicular immunoregulation

TLR4

MyD88 TRIF RNA virus

DNA virus

Endosome
TLR7, 8 dsRNA
, 9 TLR3

MyD88 DNA
RIG-I/MDA5
TRIF
TRAF3
IRAKs
IPS-1 DNA sensors
TRAF6 Mitochondrion
Bacterium

IRAKs

TRAF6 STING

TBK1
NEMO MAPKs Endoplasmic reticulum
IKKα IKKβ
MyD88

ISG15
TLR1, 2, 5, 6

AP-1 OAS1 IFNR

IκB IRF3 MX1
NF-κB
......

IFN-α, IFN-β
Nucleus

P P P
NF-κB AP-1 IRF3

IFN-α
IFN-β

TNF-α, IL-1β, IL-6, MCP-1.....

Figure 3.3  PRR-initiated innate immune signaling pathways. TLRs are localized on cellular and endosomal membranes. TLRs
initiate MyD88- and/or TRIF-dependent pathways. MyD88-dependent pathway induces the production of the proinflammatory
cytokines, including TNF-α, IL-1β, IL-6, and MCP-1, through the activation of NF-κB and AP-1. TRIF-dependent pathway induces the
production of the proinflammatory cytokines and IFN-α/IFN-β through the activation of NF-κB and IRF3. RIG-I and MDA5 sense
viral dsRNA, thus initiating an IPS-1-dependent pathway. Cytosolic DNA sensors recognize viral and bacterial DNA, thus initiating a
STING-dependent pathway. Both IPS-1- and STING-dependent pathways induce IFN-α and IFN-β production through IRF3 activa-
tion. The IPS-1-dependent pathway also activates NF-κB and induces proinflammatory cytokine production. IFN-α and IFN-β induce
the expression of the antiviral proteins, including ISG15, OAS1, and MX1, in autocrine and paracrine manners. Abbreviations: AP-1,
activator protein 1; dsRNA, double-strained RNA; IKK, IκB kinase; IPS-1, IFN-β promoter stimulator 1; IRAK, interleukin-1 receptor
associated kinases; IRF3, interferon regulatory factor3; ISG-15, IFN-stimulating gene 15; MAPKs, mitogen-activated protein kinases;
MCP-1, monocyte chemotactic protein 1; MDA5, melanoma differentiation-associated protein 5; MX1, MX GTPase 1; MyD88, myeloid
differentiation protein 88; NEMO, NF-κB-essential modulator; OAS1, 2’5’-digodenylate synthetase 1; RIG-I, retinoic acid-inducible
gene I; STING, stimulator of IFN gene; TBK1, TANK-binding kinase 1; TLR, toll-like receptor; TRAF, tumor necrosis factor receptor-
associated factor; TRIF, toll/IL-1R domain-containing adaptor inducing IFN-β.

pathways that lead to the secretion of immunoregulatory
cytokines. These cytokines activate immune cells and
directly counteract invading pathogens. To date, 13 TLRs
have been identified in mammals.44 RLRs include two
functional members, RIG-I and melanoma differentiation-
associated protein 5 (MDA5), both of which recognize
double-stranded (ds) RNA produced during viral replica-
tion, thus coordinating antiviral responses via the induc-
tion of type 1 IFNs.45 The study of cytosolic DNA sensors
has progressed rapidly in recent years. Several types of
cytosolic DNA sensors, including DNA-dependent activa-
tor of IFN regulatory factor (DAI), IFN-inducible protein
16 (p204 in mouse), and cGMP-AMP synthase (cGAS),
have been intensively investigated.46 These DNA sensors
recognize viral DNA and initiate antiviral responses.
PRR signaling pathways
PRRs initiate innate immune responses through several
signaling pathways (Figure 3.3). TLRs are type 1 trans-
membrane proteins that may be localized on plasma or
endosomal membranes. Most TLRs exclusively initiate the
myeloid differentiation protein 88 (MyD88)-dependent
pathways, with the exception of TLR3 and TLR4. TLR3
exclusively initiates the Toll/IL-1R-domain-containing
adaptor-inducing IFN-β (TRIF)-dependent pathway,
whereas TLR4 activation triggers both MyD88- and TRIF-
dependent pathways.47 The MyD88-dependent pathway
predominantly induces the secretion of proinflamma-
tory cytokines and chemokines through the activation
of nuclear factor kappa B (NF-κB). The TRIF-dependent
pathway activates NF-κB and IFN regulatory factor 3
(IRF3), thereby leading to the induction of proinflam-
matory cytokines and type 1 IFNs (IFN-α and IFN-β).
These cytokines promote the recruitment and activation
of leukocytes and the induction of IFN-inducible antiviral
proteins, thereby counteracting invading microbial patho-
gens. Moreover, TLR signaling facilitates the maturation
of antigen-presenting cells, thereby directing adaptive
immunity. RIG-I and MDA5 are cytosolic RNA sensors
and recognize the dsRNA formed by various viruses dur-
ing replication. RIG-I and MDA5 activation triggers sig-
naling using an adaptor IFN-β promoter stimulator-1
(IPS-1) that is localized to mitochondria.48 The cytosolic
DNA sensor signaling pathway requires the stimulator
of IFN gene (STING) as a common adaptor localized to
the endoplasmic reticulum.49 IPS-1-dependent signal-
ing results in the activation of IRF3 and NF-κB, which
induces the expression of type 1 IFNs and proinflamma-
tory cytokines, whereas the STING-dependent pathway
predominantly induces type 1 IFN production through
the activation of IRF3.
TLR-initiated innate immune responses in Sertoli
cells
The role of TLRs in the testis was initially investigated in
mouse Sertoli cells, with Riccioli et al. demonstrating that
TLR2, TLR4, TLR5, and TLR6 were expressed in mouse
Sertoli cells.28 Stimulation of Sertoli cells with TLR2
The innate defense system in the testis  57
and TLR5 agonists induces the expression of chemokine
MCP-1 and adhesion molecule ICAM-1. These two mol-
ecules are responsible for the migration of monocytes
and lymphocytes from blood vessels to inflamed tissues.50
Therefore, the activation of TLR2 and TLR5 in Sertoli
cells may promote leukocyte recruitment in response to
microbial infection. We demonstrate that mouse Sertoli
cells abundantly express TLR2, TLR3, TLR4, and TLR5,
and these TLRs are activated by their ligands and induce
the expression of proinflammatory cytokines and type
1 IFNs.29 These results suggest that TLR-initiated innate
immune responses in Sertoli cells directly counteract
microbes. Interestingly, TLR3 activation promotes phago-
cytosis of apoptotic germ cells by Sertoli cells,29 suggesting
that TLR3 may play a role in regulating tissue homeosta-
sis. Another study confirmed that TLR3 induced antiviral
immune responses in mouse Sertoli cells, showing that
TLR3 ligand induced the expression of proinflammatory
cytokines and IFNs through the activation of IRF3 and
NF-κB.51 In addition to TLRs, cytosolic RNA and DNA
sensors have been detected in Sertoli cells at relatively low
levels compared with Leydig cells.52,53 The potential roles
of cytosolic RNA and DNA sensors in testicular defense
against viral infection are worthy of investigation.
PRR-initiated innate immune responses in Leydig
cells
Early studies demonstrated that rat Leydig cells express
IFNs and antiviral proteins.54,55 These observations imply
that rat Leydig cells possess antiviral capacities. However,
in response to viral infection, human Leydig cells express
relatively low levels of IFNs and antiviral proteins com-
pared with rat Leydig cells,56 suggesting that the innate
antiviral response in the human testis may be weaker than
that in the rat testis. This finding may explain why natu-
ral viral orchitis is frequently observed in humans but not
in murine animals. Dissection of the mechanisms under-
lying the different antiviral defense systems of murine
and human testes may provide novel strategies for the
development of preventive and therapeutic approaches
against viral infection in the testis. Recent studies on PRR-
initiated innate immune responses in mouse Leydig cells
have expanded our understanding of the innate defense
system of the mouse testis. Mouse Leydig cells abundantly
express TLR2, TLR3, and TLR4.31 TLR3 and TLR4 can be
activated by their respective ligands in Leydig cells, initi-
ating innate immune responses inducing the production
of proinflammatory cytokines and type 1 IFNs. Moreover,
the activation of TLR3 and TLR4 suppresses testosterone
synthesis in Leydig cells. This suppression is an indirect
effect of TLR-induced proinflammatory cytokines because
several cytokines, including IL-1β, IL-6, and TNF-α,
inhibit steroidogenesis.57 Therefore, the innate immune
responses in testicular cells may have detrimental conse-
quences for spermatogenesis.
Viral infection in the male genital system is of consid-
erable concern, not only because of its detrimental effect
on male fertility, but also the possibility of pathogen
58  Testicular immunoregulation
transmission to sexual partners and the fetus.58 Various
viruses, such as mumps virus (MuV), human immuno-
deficiency virus (HIV), and hepatitis virus, can infect the
testis and impair testicular function. The innate antiviral
response in the testis is of particular importance because
viral infection may lead to testicular dysfunction and male
infertility. Major testicular cells, including somatic and
germ cells, produce IFNs in response to viral infection.
IFNs induce the expression of various antiviral proteins
in testicular cells, suggesting that the testis possesses its
own antiviral defense system.59 The PRR-initiated innate
antiviral response in Leydig cells has been recently inves-
tigated in mice. In addition to TLR3, which recognizes
dsRNA and induces an innate immune response,31 mouse
Leydig cells also express RIG-I and MDA5.52 The acti-
vation of RIG-I and MDA5 with dsRNA in Leydig cells
triggers the IPS-1-dependent pathway to induce proin-
flammatory cytokine and type 1 IFN production through
NF-κB and IRF3 activation. Moreover, major antiviral
proteins, including IFN-stimulating gene 15 (ISG15),
2’5’-oligodenylate synthetase1 (OAS1), and Mx GTPase1
(MX1), are upregulated in Leydig cells. ISG15, OAS1, and
MX1 can amplify antiviral signaling, degrade viral RNA,
and block viral gene transcription, respectively, thereby
limiting viral replication within infected cells. Notably,
RIG-I and MDA5 signaling in Leydig cells inhibits tes-
tosterone synthesis, indicating that RNA-triggered innate
antiviral response perturbs testicular function.52
DNA sensor signaling has also been demonstrated
in Leydig cells. p204 and its signaling adaptor, STING,
are constitutively expressed in mouse Leydig cells53 and
p204 activation induces the expression of type 1 IFNs
and antiviral proteins via a STING-dependent pathway.
p204 signaling does not significantly induce proinflam-
matory cytokine production in Leydig cells. Notably, the
p204-initiated antiviral response in Leydig cells does not
affect testosterone synthesis. In agreement with these
findings, RNA viruses, such as MuV and HIV, usually
induce orchitis and perturb male fertility, whereas DNA
viruses rarely cause orchitis.58 Based on these observa-
tions, we speculate that the induction of DNA sensor
signaling is an ideal strategy for enhancing testicular
defense against viral infection and protecting testicular
function.
PRR-initiated innate immune responses in male
germ cells
During puberty, more than 90% of testicular cells are germ
cells and reside within the seminiferous tubules. Microbial
pathogens from the blood circulation and the ascending
genital tract can reach male germ cells localized to both
sides of the BTB. Male germ cells at certain stages produce
IFNs after stimulation with Sendai virus60 and spermato-
gonia constitutively express IFN-α, IFN-γ, and antiviral
proteins.54,55 These early observations suggest that male
germ cells possess antiviral activities. Additionally, PRR-
initiated innate immune responses in male germ cells have
been recently revealed.
TLR3 is expressed in mouse male germ cells.61
Spermatogonia and spermatocytes constitutively express
TLR3, and TLR3 ligands induce the production of pro-
inflammatory cytokines and type 1 IFNs. These germ
cells also express antiviral proteins via TLR3 signaling.
Considering that spermatogonia and spermatocytes are
located on both sides of the BTB, TLR3-initiated antiviral
response in male germ cells may contribute to testicular
defense against viral infections from the circulating blood
and the ascending genital tract. TLR11 is abundantly
expressed in late-stage male germ cells, predominantly in
round spermatids.62 TLR11 is recognized by Toxoplasma
gondii and uropathogenic Escherichia coli (UPEC), thereby
initiating innate immune responses against these patho-
gens in mice.63,64 However, functional TLR11 is absent in
humans.65 Different TLR11 in mice and humans may be
a reason why T. gondii and UPEC cause pathogenesis in
humans but not in murine animals. T. gondii and UPEC
induce MCP-1, IL-12, IFN-γ, TNF-α, and IL-6 expression
in mouse germ cells, suggesting that male germ cells are
involved in testicular defense against T. gondii and UPEC
infections.62 Round spermatids also express MDA5, sug-
gesting that late-stage male germ cells are also equipped
with innate antiviral machinery.52 The innate immune
responses in the late stages of male germ cells appear to
be particularly important for testicular defense against
microbial infections from the ascending genital tract
within the seminiferous tubules, as these compartments
are separated from the immune components in the inter-
stitial spaces by the BTB.
Although in vitro studies have demonstrated that cer-
tain PRRs initiate innate immune responses in male
germ cells, the response grades are relatively low com-
pared with those in testicular somatic cells.61,62 However,
male germ cells can effectively resist microbial infec-
tions. Microorganisms rarely replicate in male germ cells,
which suggests that these cells possess other mechanisms
to counteract microbial infections in addition to PRR-
initiated innate immune responses. The abundant antimi-
crobial defensins in the genital tract may play a role in the
defense against microbial infections.66 The contribution
of male germ cells to testicular defense against microbial
infections is worthy of further investigation.
TLR signaling in testicular macrophages
Testicular macrophages are major populations of immune
cells in the testicular interstitial spaces and represent
approximately 20% of total interstitial cells. These macro-
phages are believed to constitute the frontline of testicular
innate defense against microbial infections.67 However,
testicular macrophages display immunosuppressive phe-
notypes by predominantly producing anti-inflammatory
cytokines.40 Interestingly, TLR signaling is inhibited by
UPEC infection in testicular macrophages.68 Treatment
of testicular macrophages with UPEC does not induce
secretion of proinflammatory cytokines because of block-
age of the MyD88-dependent signaling pathway. In con-
trast, UPEC predominantly induces the expression of

type 1 IFNs and chemokines through the activation of the
TRIF-dependent signaling pathway.68 Therefore, testicu-
lar macrophages display an antiviral response but prevent
proinflammatory cytokine secretion.41 The mechanisms
underlying the subdued inflammatory response of tes-
ticular macrophages have been recently investigated.69
In comparison with peritoneal macrophages, testicular
macrophages express relatively low levels of TLR pathway-
related  genes. TLR4 ligand does not activate NF-κB
because of a lack of IκBα degradation by ubiquitination in
testicular macrophages. However, TLR3 and TLR4 ligands
indeed induce low levels of proinflammatory cytokines
independent of NF-κB activation. The levels of proinflam-
matory cytokines produced by testicular macrophages
are much lower than those produced by peritoneal mac-
rophages. Moreover, testicular macrophages produce high
levels of IL-10, an anti-inflammatory cytokine, in response
to TLR4 activation, confirming that testicular macro-
phages favor testicular immune privilege.69 Therefore, the
role of testicular macrophages in the testicular defense
against microbial infections needs redefining.
ORCHITIS
The mammalian testis possesses a unique immune envi-
ronment because of its immunoprivileged status and
locally effective innate defense system.2 The maintenance
of immune homeostasis is essential for normal testicular
function. However, certain pathological conditions, such
as microbial infection, physical trauma, testicular tumors,
and even environmental toxicants, may disrupt testicu-
lar immune homeostasis and this may lead to orchitis, an
etiological factor in male infertility.70 Orchitis can be clas-
sified as infectious or autoimmune.
Infectious orchitis is caused by microbial infection.
In particular, a broad spectrum of viruses cause orchi-
tis and usually impair male fertility. 58 The well-known
viral orchitis is caused by MuV. MuV orchitis in prepu-
berty is usually transient and fully recovers in about 2
weeks. However, MuV orchitis in adult men frequently
cause infertility.71 Viral orchitis can also be caused
by HIV infection, and HIV orchitis severely impairs
testicular function.72 Moreover, some other viruses,
including hepatitis C virus (HCV), coxsackievirus,
influenza virus, and dengue virus, may induce orchi-
tis. 58 Although certain viruses, such as MuV, HIV, and
HCV, can be detected in the testis of infected individ-
uals, viral titers are usually very low and it is unclear
whether these viruses directly perturb testicular func-
tion. Vaccination with inactivated MuV may occasion-
ally lead to orchitis, suggesting that MuV orchitis can
be indirectly caused by systemic immune-mediated
mechanisms.73 The mechanisms by which viral infec-
tion induces orchitis remains to be clarified. It should
also be noted that RNA viral infection more frequently
induces orchitis than DNA viral infection, and the
underlying mechanism of this is worthy of further
investigation. Recent studies have shown that DNA
Concluding remarks  59
virus sensor signaling neither induces proinflamma-
tory cytokine production nor inhibits testosterone syn-
thesis in Leydig cells. 53 In contrast, RNA virus sensor
signaling significantly induces proinflammatory cyto-
kine production and inhibits testosterone synthesis. 52
We speculate that downregulation of testosterone and
upregulation of proinflammatory cytokines in Leydig
cells in response to RNA viruses contributes to detri-
mental consequences in the testis.
In addition to viruses, certain bacterial and parasitic
infections may also lead to testicular dysfunction. Sexually
transmitted bacteria, most frequently Neisseria gonor-
rhoeae and Chlamydia trachomatis, usually cause orchi-
tis and epididymitis,74 impairing male fertility. Bacterial
infections are generally susceptible to antibiotic treatment
but may lead to infertility if not treated in time. T. gondii
can be transmitted to humans from cats and is associated
with impairment of testicular function and male fertility,75
although there is no evidence that T. gondii directly infects
the testis.
Autoimmune orchitis is chronic inflammation in the
testis characterized by the generation of autoantibodies
against male germ cells and the impairment of spermato-
genesis. Autoimmune orchitis may lead to male steril-
ity.37 Pathomechanisms underlying autoimmune orchitis
have been investigated using EAO models.38
CONCLUDING REMARKS
The mammalian testis is a remarkable immunoprivi-
leged site essential for protecting immunogenic germ
cells from detrimental defense responses. However,
the testis adopts a local innate defense system against
microbial infection. Testicular immunoregulation is a
broad field worthy of further investigation, in which
several aspects need prioritizing in future research, as
follows. (1) Mechanisms underlying systemic immune
tolerance to male germ cells are largely unknown. In
this context, the role of TAM receptors in the regula-
tion of immune tolerance is worth in-depth investiga-
tion. (2) TLR2 and TLR4 cooperatively mediated EAO
induction. Endogenous TLR ligands in male germ cells
remain to be identified. (3) Although various PRRs ini-
tiate innate immune responses in testicular cells, the
roles of these PRRs in counteracting natural microbes
in the testis have yet to be elucidated. (4) Male germ
cells display an active defense system against micro-
bial infections. However, PRR-initiated innate immune
responses are relatively weak in germ cells compared
with somatic cells. Other mechanisms against micro-
bial infection in male germ cells remain to be identified.
(5) Systemic immune-response-mediated testicular dys-
function is worth clarifying. Inflammatory cytokine
storm in response to microbial infection may result in
testicular dysfunction. Identification of key cytokines
that impair testicular function may aid the develop-
ment of therapeutic strategies for immunological tes-
ticular dysfunction.
60  Testicular immunoregulation
ACKNOWLEDGMENTS
This work was supported by the Major State Basic Research
Project of China (Nos. 2015CB943001, 2016YFA0101001)
and CAMS Initiative for Innovative Medicine (Nos.
2017-IZM-B&R-06, 2017-IZM-3-007).
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Inflammation and spermatogenesis
MARIA SUSANA THEAS, PATRICIA VERÓNICA JACOBO, CECILIA VALERIA PÉREZ,
VANESA ANABELLA GUAZZONE, and LIVIA LUSTIG
HUMAN ORCHITIS
The testis is an immunologically privileged site with a
unique immunosuppressor microenvironment able to tol-
erate haploid germ cell antigens that appear at puberty and
to easily accept tissue grafts. The existence of the blood-
testis barrier (BTB), the secretion of immunosuppressor
mediators by somatic cells (mainly Sertoli cells), and the
immunomodulatory function of regulatory T (Treg) cells
contribute to immunoprivilege.
However, this immunosuppressed microenvironment
does not prevent the testis from developing inflammatory
and immune reactions in response to different stressors
such as pathogens or tissue damage.
Infection and inflammation are widely accepted as
important etiological factors of subfertility or infertility.
Prevalence rates up to 15% have been reported in patients
examined in infertility clinics.1 The most frequent orchitis
or epididymo-orchitis induced by specific pathogens occur
by retrograde ascent of different urethral bacterial patho-
gens (Escherichia coli, Chlamydia trachomatis, and Neisseria
gonorrhea, among others) or systemic viral infections. The
best-known orchitogenic viruses in humans are mumps
virus and human immunodeficiency virus (HIV). Mumps
virus induces innate immune responses in mice through
retinoic acid-inducible gene signaling and TLR2 expressed
by Sertoli and Leydig cells, which results in the produc-
tion of proinflammatory cytokines and chemokines.2
Both viruses are able to replicate in Leydig cells in vitro,
inhibiting testosterone.3,4 Acquired immune deficiency
syndrome patients often suffer from oligozoospermia or
azoospermia and hypogonadism.5 Immunosuppression
is also a potential mechanism for HIV persistence in the
testis.6
Orchitis is characterized by impairment of seminiferous
tubules (STs) presenting germ cell sloughing and degener-
ation associated with interstitial and peritubular immune
infiltrates of T cells and macrophages that may eventually
invade STs. Granulomas may also occur in severe orchitis.
Increase of Th1 and Th17 proinflammatory cytokines have
been demonstrated in human orchitis associated with
oligozoospermia and fibrosis contributing to transient
or permanent subfertility or infertility.1,7,8 Mast cells and
peritubular cells are involved in testicular fibrosis mainly
through increased production of profibrotic cytokines.9
Lymphomonocyte infiltration is also frequent in
pathologies other than infection in which the testicular
4
parenchyma is damaged, such as neoplasia, trauma, toxic
agents, and cryptorchidia.10–13 Continuous release of sper-
matic antigens from impaired ST induces disruption of
tolerance and induction of an autoimmune inflamma-
tory response.14 Orchitis may also have a genetic basis as a
manifestation of polyglandular autoimmune syndrome. In
particular, this rare autosomal recessive disease is caused
by mutation of the AIRE gene or is related to a defect in
Treg cell function.15,16
ANTIBODIES TO SPERMATIC ANTIGENS
Although cellular immune response is the main patho-
genic mechanism in autoimmune orchitis, the presence
of antisperm antibodies (ASAs) and immune complex
deposits at the seminiferous tubular walls also contribute
to testicular inflammation.17
ASAs may be detected in serum, sperm, seminal plasma,
and also in follicular fluid or cervicovaginal secretions in
women. Patients with a clinical history of testis inflam-
mation associated with damage frequently present high
ASAs levels in spermatozoa and seminal plasma. As well,
40%–70% of vasectomized men and patients with con-
genital absence of the vas deferens18 develop antibodies to
sperm antigens. ASAs IgG type present in seminal plasma
interacts with sperm plasma membrane antigens, affect-
ing sperm motility or inducing sperm agglutination,19
while ASAs IgA type detected in the sperm head interferes
mainly with the fertilization process. The detection of
autoantibodies against protein disulfide isomerase ER60,
expressed in the developing acrosome, has been proposed
as a tool for diagnosis of infertility associated to testicular
inflammation.20
EXPERIMENTAL MODELS OF AUTOIMMUNE ORCHITIS
Experimental models of orchitis contributed significantly
to understanding of testis immunoregulation and inter-
actions among immune, somatic, and germ cells. Acute
orchitis can be induced by injection of lipopolysaccha-
ride21 or human chorionic gonadotrophin.22 However,
the experimental model of chronic autoimmune orchi-
tis (EAO) better mimics human orchitis associated with
infertility. The classical model of EAO has been induced
in guinea pig, mice, and rats using as immunogen a tes-
ticular homogenate obtained from adult animals in adju-
vants.23–25 Sperm cells without adjuvants have also been
used to induce EAO.26 With the utilization of a proteomic
63
64  Inflammation and spermatogenesis

approach with sera of rats and mice with EAO or by inject-
ing recombinant proteins, several immune-dominant
autoantigens have been identified in orchitis. Zonadhesin,
expressed on the outer acrosomal membrane of sperma-
tids and sperm and ODF2 present in the outer dense fibers
of sperm flagellum are testis-specific, and other nonorgan-
specific antigens such as heat shock protein 70 or RNA
binding protein H1 have also been detected.27–29
Other models of chronic EAO can be induced by
manipulation of the immune system, such as thymectomy
in 3-day-old mice, which induces autoimmune diseases
in several organs including the testis30 or after infection
of the testis with viruses such as Sendai virus (closely
related to mumps virus) or myxoma virus.31,32 Unilateral
infection with bacteria (Listeria monocytogenes)21 induces
autoimmune orchitis in the contralateral testis.33 In this
last model, orchitis was also passively transferred to unin-
fected recipients with CD4+ T cells from infected donors.34
PATHOGENESIS OF AUTOIMMUNE ORCHITIS
We will analyze the main features of the immune
response induced in rats immunized with testicular
homogenate in Freund’s complete adjuvant and Bordetella
pertussis as coadjuvant.25 Focal orchitis develops 50 days
after the first immunization and severe orchitis after 80
days. Focal orchitis is characterized by multiple foci of
STs with different degrees of germ cell sloughing asso-
ciated with discrete peritubular immune cell infiltrates,
mainly of dendritic cells (DCs), macrophages, and lym-
phocytes. In severe orchitis, aspermatogenesis occurs
in most of the STs in which only spermatogonia, a few
basal spermatocytes, and Sertoli cells are present (Figure
4.1). Abundant immune cell infiltrates are present in the
interstitium, but unlike EAO in mice or human orchitis,
the inflammatory cells are not detected inside damaged
STs. Most are atrophic and the number of apoptotic post-
meiotic germ cells increases. Granulomas are frequently

(a1) 100 µm (a2) 50 µm (a3) 20 µm

(b1) 100 µm (b2) 50 µm (b3) 20 µm

*
*

(c1) 100 µm (c2) 50 µm (c3) 20 µm

Figure 4.1  Microphotographs of testis sections. (a) Normal rat showing seminiferous tubules (ST) with normal spermatogen-
esis and no signs of interstitial inflammation. (b) Rat immunized with testis homogenate and adjuvants showing a focus of ST with
germ cell loss or aspermatogenesis (*) intermingled with normal ST and mild interstitial immune cell infiltrates (focal orchitis).
(c) Rat immunized with testis homogenate and adjuvants showing severe orchitis. The whole section presents aspermatogenic ST (*)
associated with interstitial and perivascular cell infiltrates of macrophages and lymphocytes. Hematoxylin eosine
 Pathogenesis of autoimmune orchitis  65

observed. Proinflammatory mediators, secreted mainly
by immune cells, increase in the testis of EAO rats, gener-
ating an inflammatory microenvironment responsible for
impairment of testicular function.
Blood-testis barrier
BTB is a key player in the maintenance of testicular
immunoprivilege. Its structure, which involves specialized
cell junctions between adjacent Sertoli cells close to the base-
ment membrane, prevents entry of leukocytes and antibodies
into seminiferous tubules and limits interaction among germ
cell autoantigens and the immune system. During testicular
inflammation, BTB structure and function are impaired
(Figure 4.2). An increase in BTB permeability together
with a reduction in expression of the tight junction mol-
ecule occludin and the gap junction molecule connexin-43
were detected in the testis of EAO rats. Reduced adhe-
sion ability of germ cells and the increased expression of
adherent junction proteins, N-cadherin and α-catenin,
in Sertoli cells are also associated with germ cell slough-
ing.35,36 Several inflammatory mediators and cytokines,
increased during testicular inflammation, are described as
modulators of junction dynamics and BTB function. IL-6
disrupts the Sertoli cell tight junction barrier in vitro via
the p38 MAPK signaling pathway and by inhibiting deg-
radation of BTB constitutive proteins.37 In vivo, IL-6 was

N-cadherin
Connexin 43

Claudin 11

Occludin
α-catenin
β-catenin

p120
ZO-1
Actin

IL17R

IL6R

TNFR1
(a) IL-17
IL-6
TNF-α
NO

(b)

Figure 4.2  Schematic drawing illustrating BTB under normal (a) and inflammatory conditions (b). (a) BTB consists of coexisting
integral membrane proteins of the adherens junctions (cadherins), gap junctions (connexins), and tight junctions (claudins, occludin)
between adjacent Sertoli cells. Each integral membrane protein is associated with its corresponding adaptors (ZOs, catenins, and
p120) that link them to the actin cytoskeleton. (b) Proinflammatory cytokines (IL-6, TNF-α, IL-17) and nitric oxide (NO) contribute to
BTB impairment. Decreased occludin and connexin-43 expression, delocalization of claudin-11, and upregulation of N-cadherin and
α-catenin are also shown.
66  Inflammation and spermatogenesis
shown to induce a reduction in occludin expression after
local administration to adult rat testes.38 Tumor necrosis
factor alpha (TNF-α) and transforming growth factor beta
(TGF-β) can alter testicular tight junction function by
accelerating endocytosis of integral membrane proteins.38
IL-1α induces BTB disruption but exerts its effect on the
Sertoli cell actin cytoskeleton network without affecting
levels of junction proteins.39 IL-17 disrupts the Sertoli cell
barrier both in vitro and in vivo, decreasing the expres-
sion of occludin.40 As well, nitric oxide (NO) has been
shown to regulate testicular adherens and tight junction
dynamics.41,42
Immune cells
DCs are specialized sentinel cells that bridge the innate
and adaptive immune system. DCs are present in the tes-
ticular interstitium and their numbers strongly correlate
with development of tissue damage in the inflamed tes-
tes.43 DCs from normal and orchitic testis express simi-
lar intensity levels of major histocompatibility complex
class II and costimulatory molecules (CD80 and CD86).
However, a significantly higher mRNA expression of IL-12
and chemokine receptor CCR7 was detected in isolated
testicular DCs from rats with EAO compared to normal
rats.44 While IL-12 supports differentiation of the Th1 sub-
set of helper T cells, the upregulation of CCR7 is required
for entry and homing of DCs into lymph nodes (LNs) in
order to present antigens to antigen-specific T cells. In fact,
during the development of orchitis, testicular-draining
LNs express the CCR7 ligand, CCL19, and present a sig-
nificant increase in the percentage of DCs compared to
testicular-nondraining LNs. Functional analyses show
that DCs isolated from testis and testicular-draining LN
from rats undergoing orchitis significantly enhanced pro-
liferation of naïve T cells. This supports the notion that
DCs from chronically inflamed testis are immunogenic,
present antigens to T cells, and stimulate an autoimmune
response against testicular antigens, thus causing impair-
ment of spermatogenesis and immunological infertil-
ity 44,45 (Figure 4.3).
Macrophages also play the role of antigen-presenting
cells and release inflammatory cytokines and chemokines
that contribute to EAO development. In fact, in vivo deple-
tion of these cells with clodronate-containing liposomes
in rats undergoing EAO decreased incidence and severity
of testicular damage. Testicular macrophage population
is heterogeneous; it includes resident macrophages (ED2+
cells), inflammatory monocytes recently arrived from cir-
culation (ED1+ cells), and double-positive (ED1+ ED2+)
subpopulation, the major contributor to the macrophage
number increase in EAO.46 Inflammatory chemokines
CCL2, CCL3, and CCL4 regulate macrophage recruit-
ment within the interstitium via CCR2, CCR1, and CCR5
receptors.47–49
The existence of scarce CD4+ and CD8+ T lympho-
cytes and natural killer cells in normal testis of human
and rodents has been established. 34,50,51 In testis of rats
with EAO a significant increase in the number of CD4+
and CD8+ cells, including pathogenic Th1, Th17 subsets,
and Foxp3+ T cells, occurs. 51 Th1- and Th17-type immune
response was characterized by secretion of TNF-α and
interferon gamma (IFN-γ) (Th1 cytokines) and IL-17
and IL-23 (Th17 cytokines) were observed in the testis
throughout the two phases of EAO. The number of CD4+
T cells producing TNF-α, IFN-γ, or IL-17 peaked dur-
ing the focal phase of EAO; however, these cells declined
during the severe phase of the disease. CD8+ T cells were
also found to express Th1 and Th17 cytokines along the
course of the disease, with these cells predominating
in the severe phase of EAO. Testicular IL-17 and IL-23
content increased in EAO with maximum levels in the
severe phase, suggesting their contribution to mainte-
nance of chronic testicular inflammation.52 The highest
number of CD4+ T cells producing TNF-α and IFN-γ
at EAO onset in rats is consistent with the major role
attributed to these cells in the development of murine
EAO. 53–56
CD4+ Foxp3+ Treg cells also accumulated in the testis
with EAO, reaching a peak at the focal phase, then dras-
tically declining during the severe phase of the disease.
In contrast, the number of CD8+Foxp3+ Treg cells that
increased in the focal phase remained stable during the
severe phase of EAO.51 Along EAO development, testicular-
draining LNs, but not non-draining LNs, are strategically
enriched for antigen-specific CD4+CD25+Foxp3+ Treg
cells. We reported that CD4+ Treg cells expressing high
levels of CD25 derived from testicular-draining LNs of
both normal and EAO rats suppress T cell proliferation
in vitro, exhibit an antigen-experienced status, and also
express TGF-β, a suppressor of effector T cells. However,
Treg cells derived from testicular-draining LNs of rats
with EAO exhibit a stronger antigen-specific prolifera-
tive response and suppressive capacity compared to cells
derived from normal rats.57 Our results showed that Treg
cells with the potential to inhibit autoimmune response
against germ cells are present in the testis and in testis-
draining LNs. However, these cells fail to effectively con-
trol testicular inflammation and tissue destruction since
multiple mechanisms may disturb local immune regula-
tion: (1) inadequate ratio effector/Treg cells (2) intrinsic
functional defects in Treg cell populations, (3) patho­
genic  Th17 cells resistant to Treg cell regulation, and
(4)  impairment of Treg cell function by inflammatory
milieu. We infer that these last two mechanisms are cen-
tral to understanding why inflammation progresses in the
testis despite the presence of increased numbers of func-
tional Treg cells.
Testosterone levels were found to be decreased in serum
of rats in the early phase of EAO.58 Fijak et al.59 reported
that testosterone supplementation effectively reduced dis-
ease development in rats with EAO exerting a systemic
effect by inhibiting proinflammatory Th1-specific cyto-
kine production in testicular-draining LNs and by induc-
ing suppressive Treg cells in the testis.
 Pathogenesis of autoimmune orchitis  67

Immunization Testicular
Normal testis
site draining lymph node draining lymph node

Treg
ST
Th17
L

CD8

Th1
DC

Chemokines
IFN-γ
TNF-α
Ag TGF-β
Mφ IL-6
IL-12
IL-17
Orchitic testis

Figure 4.3  Schematic drawing illustrating some features of experimental autoimmune orchitis induction. During the immuni-
zation period, the autoimmune response initiates in the immunization site draining lymph nodes (LN) where dendritic cells (DCs)
and macrophages capture and present the antigen (Ag). The presentation of self-Ag induces activation of specific autoreactive T
cells. Subsequently, DC-sensitized lymphocytes migrate by chemokines to the testis where they contribute to testicular inflamma-
tion thereby causing tissue damage. Spermatic antigens released to the interstitium during the course of the disease are processed
by inflammatory testicular DCs that migrate to testicular-draining LN and activate T lymphocytes. Under inflammatory conditions
with increased levels of cytokines, immature testicular DC overcome immune tolerance (privilege). This view is supported by the
finding that testicular DC in EAO showed increased CCR7 expression, whereas testicular-draining LN express the CCR7 ligand CCL19,
a chemokine involved in DC traffic. Progressive amplification of the autoimmune response finally leading to chronification of EAO
may result from continuous antigen presentation to lymphocyte by DCs in testicular-draining LN and in the testis. L: Leydig cell. ST:
seminiferous tubules ϕ: macrophages.

Germ cell apoptosis mediators compared to normal testis. Specifically, inflammatory

Spermatogonial stem cells reside on the basement mem-
brane of STs in intimate contact with Sertoli cells and
with factors generated in the interstitium. In this complex
microenvironment (niche) stem cells are able to self-renew
and differentiate into committed progenitors that sustain
spermatogenesis.
Under inflammatory conditions spermatocytes and
spermatids are the main target of immunological attack;
these cells die by apoptosis through external and mito-
chondrial pathways, whereas basal germ cells are pro-
tected from death by overexpressing Bcl-260 (Figure 4.4).
Immune cells play an active role in promoting germ
cell death. In EAO, an increased number of macrophages
and a rise in nitric oxide synthase (NOS) activity and
expression generate a threefold increase in NO content
macrophages upregulate expression of inducible (iNOS)
and constitutive NOS isoforms, whereas resident mac-
rophages, less sensitive to inflammatory microenviron-
ment stimuli, upregulate only iNOS.61 NO released from
EAO macrophages activates external and mitochondrial
apoptotic pathways and also contributes to support high
intratesticular testosterone content.62 Administration
of Nω-Nitro-L-arginine methyl ester hydrochloride
(L-NAME), a competitive inhibitor of NOS, to rats
undergoing EAO decreased incidence and severity of the
disease by preventing testicular NO production and pro-
tecting germ cells from apoptosis.61
Testicular macrophages from rats with orchitis
release a high amount of TNF-α and in vitro neutral-
ization of this cytokine with etanercept (Embrel®)
68  Inflammation and spermatogenesis

Sertoli
Apoptosis cell

↓ Bcl-2
Cytochrome c
Postmeiotic
↑ Bax
germ cells
Caspase 9
activation
Sertoli NO
cell Caspase 8 Caspase 3
activation activation

NO
Apoptosis
NO

NO
NO
NO

SURVIVAL Premeiotic
Bcl-2 germ cells
Bcl-2
Bcl-2

IL-6 IL-6R Myoid cells

TNF-α TNFR1
Fas L Fas R
Nitric oxide

Figure 4.4  Schematic drawing illustrating molecules and pathways involved in germ cell apoptosis in orchitis. In EAO, spermato-
cytes and spermatids upregulate death receptors TNFR1 and Fas. TNF-α produced by interstitial macrophages activates caspase 8,
which in turn activates executioner apoptotic caspase 3. FasL shed from interstitial lymphocytes or expressed by neighboring germ
cells trigger FasR activation in a paracrine or autocrine pathway, inducing apoptotic death. NO released by interstitial macrophages
activates both external pathway (caspase 8 activation) and mitochondrial pathway, promoting release of cytochrome c from mito-
chondria and caspase 9 activation followed by caspase 3 execution of apoptosis. Release of cytochrome c from mitochondria is
facilitated by increased Bax/Bcl-2 ratio in the mitochondria. Cytokines such as IL-6 and IL-17 contribute to germ cell death. Whereas
postmeiotic germ cells present in the adluminal compartment die by apoptosis, basal premeiotic germ cells are protected by upreg-
ulation of the antiapoptotic Bcl-2 protein.

reverts the effect of TNF-α on germ cell apoptosis. The
fact that 40% of apoptotic germ cells express TNFR1
in EAO testis suggests the contribution of other pro-
apoptotic factors in the induction of germ cell death.63
During the severe phase of EAO the number of IL-6R+
germ cells and IL-6 production by inflammatory mac-
rophages increases. This cytokine is able to induce germ
cell death in vitro.64 This apoptotic effect may be the
result of direct activation of apoptotic pathways, or
more likely, IL-6 may generate a microenvironment
permissive to the effect of proapoptotic factors such as
TNF-α or IFN-γ.65–67 IL-6 injected intratesticularly into
normal adult rats induces focal testicular inflammation
associated with germ cell sloughing. 35 Similarly, in vivo
administration of IL-17 induces recruitment of inflam-
matory cells into the testicular interstitium, impairment
of BTB function, and indirectly, germ cell apoptosis.40
Membrane-bound Fas L (mFasL), like other members
of the TNF family of death ligands, can be processed by
proteolytic shedding to an active soluble form (sFasL).68
Testicular macrophages do not express membrane or intra-
cellular FasL; however, testicular CD4+ and CD8+ T cells
expressing mFasL may be the source of the increased sFasL
present in the extracellular milieu in severe orchitis.69 FasL
Strep (a Strep-Tag molecule fused to three monomers of
the extracellular domain of FasL) injected in rats can enter

Table 4.1  Factors used to reduce inflammation in experimental autoimmune orchitis.
Agent
Deoxyspergualin
Mn Ab to IFN-γ
Testosterone
Melanocyte stimulating hormone, TGF- β
Secretory leukocyte
protease inhibitor (SLPI)
Ethyl piruvate Blocks high mobility group box protein 1’s inflammatory action76
Intermedin Antioxidant capacity and proinflammatory cytokine production77
L-NAME
Galectin-1
seminiferous tubules. The fact that this molecule induces
germ cell Fas activation in vitro, triggering apoptosis,
highlights sFasL as a key molecule involved in germ cell
death. In EAO, apoptotic spermatocytes and spermatids
express Fas or FasL or coexpress both molecules, indicat-
ing involvement of the Fas-FasL system in germ cell death
in an autocrine and/or paracrine way.70
TREATMENT OF ORCHITIS
Symptomatic treatment, bed rest, and antibiotics for
specific bacterial infections and eventually IFN-α2B in
mumps orchitis are used to reduce testicular pain and
inflammation in patients with acute orchitis.
No specific treatment exists for chronic orchitis.
Experimentally, several drugs have been used to reduce
testicular inflammation (Table 4.1). Corticoids have been
used in the past in infertile patients with high levels of
ASAs. However, at present, intracytoplasmic sperm injec-
tion represents the chosen treatment for infertility of these
patients.
PERSPECTIVES
Infertility affects 16%–20% of couples in reproductive
age,13 and inflammation of the male reproductive tract is
considered an important etiological factor. Diagnosis of
human autoimmune orchitis associated with subfertility
or infertility, a chronic inflammatory pathology, is cur-
rently possible only by histopathological evaluation of tes-
tis biopsy, an invasive procedure. Although detection of
ASAs as well as immune complexes is helpful, there is a
need to find reliable peripheral markers of testis inflam-
mation. Deeper understanding of mechanisms by which
pathogenic immune cells and Tregs interact in testis
under inflammation as well as identification of mediators
involved in germ cell damage and spermatogenic arrest
will facilitate exploration of specific therapies to reduce
inflammation and reinitiate spermatogenesis. In patholo-
gies like orchitis and spermatogenic arrest, regeneration of
spermatogenesis seems to be possible since spermatogonia
remain within the STs despite the loss of postmeiotic germ
cells.79,80
References 69
Action
Inhibits lymphocyte IFN gamma production71
Antiviral and immune-modulatory capacity72
Stimulates differentiation of Treg cells73
Anti-inflammatory action74
Immunosuppressor75
Competitive inhibitor of NOS62
Anti-inflammatory action78
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Junctional adhesion molecule (JAM)
family
Recent findings and their role and regulation
in spermatogenesis
KUN HUANG and WING-YEE LUI
INTRODUCTION
Extensive and timely restructuring of cell junctions takes
place at the interface between adjacent Sertoli cells and
between Sertoli cells and germ cells during spermatogen-
esis. Restructuring of junctions at the blood-testis barrier
(BTB) allows the transit of germ cells across the BTB at
stage VIII so the germ cells enter the apical compartment
for further development. After entering the apical com-
partment, developing germ cells remain attached to the
seminiferous epithelium (Sertoli cells) for physical support
and at the same time have to keep migrating toward the
tubular lumen for further differentiation. Therefore, cell
junctions formed at the BTB and between Sertoli germ cell
interface undergo dynamic restructuring. Such restruc-
turing can be achieved by the timely and spatial changes
of structural proteins such as occludin, claudin, and junc-
tional adhesion molecules (JAMs), peripheral proteins
such as zonula occludens and catenin, as well as signal-
ing molecules including JNK and p38 kinase. JAM is the
family of Ca2+-independent cell adhesion molecules found
at both the BTB and Sertoli germ cell interface. In this
review, we discuss the role of classical JAMs and coxsacki-
evirus and adenovirus receptor (CAR) in the testis as well
as their regulatory mechanisms. We highlight some recent
findings of classical JAMs and CAR in other tissue models
and hope that this information can serve as the blueprint
for planning new studies to delineate the unknown func-
tions in the testis and the regulation of JAMs and CAR
during spermatogenesis.
GENERAL PROPERTIES OF JAM FAMILY MEMBERS
Molecular structure of classical JAMs and CAR
Proteins of the JAM family are type I glycoprotein belong-
ing to the immunoglobulin superfamily (IgSF). The JAM
family consists of seven members including three classical
members (JAM-A, JAM-B, and JAM-C) and four nonclas-
sical members (CAR, endothelial cell-selective adhesion
molecule [ESAM], JAM-L, and JAM-4).1–10 Like other
junction protein families, the nomenclature of JAM pro-
teins is diverse because JAMs have been identified by sev-
eral research groups. For instance, VE-JAM and human
JAM-2 are the aliases of JAM-B. Efforts were made in 2003
to standardize the nomenclature of the classical JAMs
5
and designate them as JAM-A, JAM-B, and JAM-C.11 All
JAM family proteins are characterized by a V-type and a
C2-type immunoglobulin-like domain in their extracel-
lular domain followed by a transmembrane segment and
a cytoplasmic tail with a PDZ-binding motif (Phe-Leu-
Val) (Figure 5.1). The three classical JAMs share up to 32%
of amino acid sequence identity12 and also share ~15%
sequence homology with the nonclassical JAM members.
The nonclassical JAMs are diverged from the classical JAM
by their cytoplasmic tails.13 The cytoplasmic tails of the
classical JAMs are relatively conserved in length and motif
composition.14 PDZ-containing proteins such as ZO-1,
AF-6, CASK, PAR-3, and MUPP-1 bind onto the PDZ
motif of JAM-A15–18 while ZO-1 and PAR-3 bind to JAM-B
and JAM-C.19 ZO-1, MUPP-1, and LNX2 bind to CAR.20–22
Interaction of PDZ motif with various PDZ-containing
proteins is involved in intracellular signal transduction
and an array of cellular functions including cell polar-
ity, cell migration, and barrier permeability (Table 5.1). In
the extracellular domain, a tripeptide motif (Arg-Val-Glu
for JAM-A, Arg-Leu-Glu for JAM-B, and Arg-Ile-Glu for
JAM-C) in the V-type domain acts as a dimerization motif
and another tripeptide linker sequence (Val-Leu-Val) con-
nects the two immunoglobulin domains to impose a bent
conformation (Figure 5.1).23–25
Homophilic and heterophilic trans-interaction
Crystal structures have shown that two JAM-A molecules
on the same plasma membrane form cis-dimer via the
dimerization motif in the V-type Ig domain.23 Based on
the bent conformation and the positioning of the dimer-
ization motif, Kostrewa et al. have proposed that cis-dimer
appears like a U shape on the cell surface and forms a two-
dimensional network by trans-interaction of their extra-
cellular domain with cis-dimer on apposing membrane.23
Having the conserved dimerization motif between the
classical JAMs, JAM-B and JAM-C might dimerize in a
similar manner as JAM-A for the establishment of adhe-
sive interaction.23 These motifs may also be involved in het-
erophilic cis-dimer between the classical JAM members.26
Trans-homophilic interactions of JAM-A have been con-
firmed using recombinant soluble JAM-A (rsJAM-A) and
anti-JAM-A Fab BV11 (a monoclonal antibody targeting
73
74  Junctional adhesion molecule (JAM) family

cis/trans-Interaction
trans-hetero-dimer e.g. JAM-A/αLβ2
cis-dimer trans-homo-dimer e.g. JAM-B/-C JAM-A/αVβ1
Cytosol
PAR-3
ZO-1

αLb2
JAM-A (LFA-1)
JAM-C JAM-a
β α
Extracellular region
α β
Dimerization JAM-A
motif V-type domain JAM-B
αVb1
Tripeptide linker
C-type domain

PAR-3 PAR-3
Type II ZO-1 ZO-1
PDZ binding motif AF-6
Monomer of CASK
Classical JAMs MUPP-1
(JAM-A, -B, -C)
and CAR

Figure 5.1  Molecular structures of JAMs and their modes of interactions. Classical JAMs and CARs share structural homology
with two extracellular Ig-like domains, a transmembrane domain and a cytoplasmic tail. Two JAMs on the same plasma membrane
form cis-dimers followed by the formation of trans-interaction between cis-dimer on the plasma membrane of an apposing cell,
resulting in the formation of trans-homo-dimer or trans-heterodimer. JAMs also interact with integrin.

Table 5.1  Classical JAMs and CAR.

Extracellular Intracellular Tissue
Member partner partner Function distribution Knockout mouse phenotype
JAM-A αVβ3 ZO-1, AF-6 Reovirus attachment Blood, bone marrow, Colonic injury46
αLβ2 PAR-3 Barrier permeability brain, heart, intestine, Enhanced thrombotic function
CASK Cell polarity kidney, liver, lung, of platelets75
MUPP-1 Leukocyte transmigration pancreas, skin, Hepatocyte apoptosis and
Angiogenesis spleen, testis attenuation of neutrophil
infiltration76
Shape alterations in corneal
epithelial47
Male subfertility36,61
JAM-B JAM-C ZO-1, PAR-3 Barrier permeability Heart, kidney, lymph Normal mouse development62
α4β1 Leukocyte migration nodes, intestine,
testis
JAM-C JAM-B ZO-1, PAR-3 Cell-cell adhesion Brain, bone marrow, Growth retardation and
CAR Cell polarity heart, lung, liver, pulmonary dysfunction77
αMβ2 Leukocyte migration kidney, spleen, testis, Severe hydrocephalus78
αXβ2 Barrier permeability nerve Male-specific infertility37
αVβ3 Angiogenesis
Nerve conduction
Spermatogenesis
CAR JAM-C ZO-1, LNX2, Coxsackievirus and Brain, kidney, liver, Dilated intestine and atrophy of
JAM-L MUPP-1, MAGI-1 adenovirus entry lung, intestine, skin, the exocrine pancreas and
Leukocyte migration testis, pancreas, atrioventricular block79
Barrier permeability cornea
Spermatogenesis

specifically to rsJAM-A dimers but not monmers).27 It was
found that anti-JAM-A Fab BV11 inhibits the binding of
fluid-phase rsJAM-A to immobilized rsJAM-A.27 Trans-
homophilic interactions of JAM-C have also been confirmed
by a similar approach. A dose-dependent binding of Fc
fusion protein of rsJAM-C to immobilized rs-JAM-C was
reported.25 Surface plasmon resonsance analysis has fur-
ther confirmed the specificity of the homophilic interac-
tion of JAM-C.25
Apart from homophilic interaction, classical JAMs can
form trans-heterophilic interaction with the JAM family.
Among the classical JAMs, JAM-B/JAM-C heterophilic
interactions have been reported,6 heterophilic interactions
of JAM-A with classical JAMs have not yet been detected.
JAM-B/JAM-C heterophilic interaction is achieved
through the dimerization motifs in the V domain, while
the C domain is believed to be important for stabilization
of the interaction.28 Dynamic light scattering (DLS) analy-
sis and analytical ultracentrifugation (AUC) have revealed
that JAM-B/JAM-C heterodimer is a preferential inter-
action compared with JAM-C homodimer formation.28
This heterophilic interaction enables specific properties of
intercellular junction complexes that play important roles
in leukocyte adhesion and migration.28
Trans-heterophilic interactions are not restricted to the
classical JAMs; the classical JAMs interact heterophilically
with nonclassical JAMs and various integrins (Table 5.1).
For instance, JAM-A can interact with β2 of LFA-1 (αLβ2)
and αVβ3.29,30 JAM-B can interact with integrin α4β131
while JAM-C can interact with many heterophilic inter-
acting partners such as CAR and various integrins includ-
ing αVβ3, αΜβ1, and αXβ2.28,32–34 A broad spectrum of
trans-interaction of JAMs within their family members
and with other transmembrane proteins is a notable and
unique feature that supports JAMs to exert a wide range of
biological functions ranging from cell adhesion, polarity,
and leukocyte migration.
Tissue distribution and localization in the testis
Classical JAMs and CAR are present in many tissues
including brain, heart, lung, skin, and testis (Table 5.1).
Classical JAMs are expressed in epithelial cells, endothe-
lial cells, and leukocytes, while CAR is expressed mainly
in epithelial cells, endothelial cells, and cardiomyocytes.
As well as being involved in leukocyte transmigration,
the JAM family is also involved in an array of cellular
functions such as cell adhesion and polarity. Both classical
JAMs and CAR can be found in the testis and are crucial for
spermatogenesis. JAM-A is highly expressed in Sertoli cells
and can be found at the BTB.35 Immunohistochemistry
staining has indicated that JAM-A expression is stage-
dependent. JAM-A expression reaches its highest at stages
IX–XIV and its lowest at stages IV–VI.35 Germ cells express
a moderate level of JAM-A and are localized on the cell sur-
face of spermatozoa.36 Unlike JAM-A, JAM-B is only pres-
ent in Sertoli cells.37 It is mainly localized at the BTB and
the apical ectoplasmic specialization (ES) between Sertoli
cells and germ cells. JAM-C is solely expressed by germ
General properties of JAM family members  75
cells. Immunofluorescence staining has shown that JAM-C
is widely distributed on the germ cell surface and a portion
of JAM-C protein is concentrated in the anterior area of
round spermatogenic cells.37 As spermiogenesis proceeds,
JAM-C is confined to the head region of the elongated
spermatids at the junctional plaques and colocalized with
JAM-B expressed in Sertoli cells.37 Heterophilic interaction
of JAM-B/JAM-C between Sertoli cells and germ cells at
the apical ES provides a structural interlock for the attach-
ment of elongated spermatids on the seminiferous epithe-
lium prior to spermiation.37 CAR is present in Sertoli cells
and all types of germ cells at different developmental stages
from spermatogonia to spermatozoa.32,38 CAR is localized
at the BTB and the apical ES.32,38 Preferential expression of
JAM-A, JAM-B, JAM-C, and CAR in different cell types
and compartments of the seminiferous epithelium con-
tribute to the heterogeneity and specific properties of the
intercellular junction complexes.
Functions of classical JAMs and CAR
JAM family proteins not only function as key molecules
for leukocyte transmigration, but also are involved in vari-
ous cellular functions and physiological processes such as
barrier function, cell adhesion, cell polarity, and pathogen-
esis (Table 5.1). For leukocyte transmigration, readers are
strongly encouraged to read Weber et al.,26 Wang et al.,39
and Bazzoni40 for a more comprehensive view of the topic.
In this chapter, we focus our discussion on the findings
of classical JAMs and CAR on barrier function, cell adhe-
sion, and cell polarity in other epithelia, and highlight the
recent advances in the studies of classical JAMs and CAR
in the testis.
Barrier function
Studies in various epithelial cells and endothelial cells
have confirmed that classical JAMs and CAR are the
key molecules that regulate barrier permeability. Several
lines of evidence in epithelial and endothelial cell models
have confirmed that JAMs are found in the region of the
tight junction (TJ) when JAMs are transfected. However,
transfected JAMs are localized at distinct subcompart-
ments of the TJs.41 Exogenous JAM-A in Madin-Darby
canine kidney (MDCK) cells has been found to colo-
calize partially with ZO-1 at the TJs, while JAM-B is
found to be diffuse at the lateral membrane. Transfected
JAM-C is found to colocalize perfectly at the TJs.41
Distinct subcompartmental localization of JAMs at the
site of TJs reflects the heterogeneity of JAM members in
nature. Furthermore, the differential expression of JAMs
in different organs are crucial for the establishment of
­t issue-specific TJs/AJs. For instance, the BTB in the testis
constituted by TJs and basal ES is classified as one of the
tightest BTBs where JAM-A and JAM-B are localized.37
In contrast to the BTB, high endothelial venules (HEVs),
which support high levels of lymphocyte extravasation
from the blood, express high levels of JAM-B and JAM-C
but not JAM-A.41 In the seminiferous epithelium, the
apical ES does not coexist with TJs, where only JAM-B
76  Junctional adhesion molecule (JAM) family
and JAM-C are present to form heterophilic interaction
between Sertoli cells and the elongated spermatids.37
Several in vitro studies have demonstrated that JAM-A
homodimers are critical for epithelial barrier function.42–44
For instance, antibodies against JAM-A have shown to
inhibit the recovery of the barrier function after TJ dis-
ruption in T84 and SK-CO15 epithelial cells.42,43 siRNA
knockdown of JAM-A increases the barrier permeability
as shown by reduced transepithelial resistance (TER) and
increased FITC-dextran flux and also alters the epithelial
cell morphology via Rap1-mediated regulation of β2 integ-
rin.44 Antibody blockade of JAM-A not only affects epithe-
lial TJ barrier function, but disrupts corneal endothelial
barrier function and results in corneal swelling.45 In addi-
tion, mice having JAM-A knockout display an increase in
intestinal mucosal and corneal epithelial permeability.46–48
Collectively, both in vitro and in vivo data strongly sug-
gest a central role of JAM-A in the regulation of the barrier
function. In contrast, JAM-C seems to exert the opposite
effect on barrier permeability. Inhibition of JAM-C causes
a significant reduction in endothelial permeability in vitro
and in vivo.49 Although classical JAMs share similar struc-
ture, they differentially regulate barrier permeability.
CAR is found to colocalize with TJ proteins such as
occludin and ZO-1 in various tissues such as kidney and
brain as well as cultured epithelial cells.50 In vitro studies
have revealed that CAR mediates homotypic interaction.20
Overexpression of CAR in airway epithelial cells increases
TER, while soluble CAR or anti-CAR antibody cause TJ
disruption in airway epithelial cells and MDCK cells.20,51
In addition, yeast two-hybrid analyses have revealed that
cytoplasmic domain of CAR directly interacts with vari-
ous TJ-associated molecules such as MUPP-1, MAGI-1,
and TAPP1.21,52 These data further support the idea that
CAR is a crucial player in barrier function.
In the testis, JAM-A, JAM-B, and CAR are found at
the site of the BTB.35,37 Knockdown of CAR by siRNA
perturbs the Sertoli cell TJ barrier function as evidenced
by the decrease in TER, while overexpression of CAR in
Sertoli cells exerts the opposite effect.53 More importantly,
CAR not only functions as a TJ structural protein but also
acts as a regulatory protein in the seminiferous epithe-
lium. The siRNA knockdown of CAR induces endocytosis
of occludin via modulation of the phosphorylation status
of occludin, resulting in TJ disruption.53
Cell adhesion
Studies have clearly demonstrated that heterophilic inter-
actions among JAM members or between JAMs and
integrins contribute to cell adhesion. JAM-B and JAM-C
undergo heterophilic interaction in cell-cell contacts and
this interaction is important to facilitate the adhesion of
T natural killer and dendritic cells to the endothelium.54
JAM-B also modulates JAM-C/αMβ2 integrin-mediated
adhesion of cultured leukocytes to the lymph node section
by controlling the accessibility of JAM-C to αMβ2 inte-
grin.28 Apart from classical JAMs, studies performed in
nonpolarized cells have revealed that CAR is localized to
the site of cell-cell contact but not to tight junctions, which
suggests that CAR may function as an adherens junction
protein.55
In the testis, JAM-B/JAM-C heterophilic interaction is a
crucial adhesive structure between Sertoli cells and elon-
gated spermatids at the apical ES of the seminiferous epi-
thelium prior to spermiation.37 JAM-B/JAM-C interlocks
not only function as junction complexes, but also act as a
platform for actin bundling and are involved in the assem-
bly of cell polarity complexes that are crucial for spermatid
differentiation.37
Cell polarity
Polarity is a fundamental property of all eukaryotic cells
and is crucial to many aspects such as cell differentia-
tion, cell migration, and morphogenesis.56 The establish-
ment of apicobasal polarity and proper formation of tight
junctions requires the interaction of cell polarity proteins
and junction proteins.56 Par3/Par6/aPKC complex is a
cell polarity complex in which Par3 and Par6 are linked
through aPKC. Studies have shown that Par3 directly
associates with JAM-A.16,57 JAM-A is colocalized with
ZO-1 and E-cadherin to form the spotlike adherens junc-
tion at the initial cell-cell contact during junction forma-
tion. Par3 and aPKC are found after formation of spotlike
adherens junctions.19,58,59 The association between JAM-A
and Par3 provides a way for anchoring the Par3/Par6/
aPKC complex at TJs, resulting in the establishment of a
fully polarized epithelial cell.57 Subsequent studies have
further confirmed that JAM-B and JAM-C, but not CAR,
strongly associate with Par3, thus suggesting that classical
JAMs are involved in the establishment of cell polarity.19
However, in the testis, JAM-C localization in sperma-
tids shows a little overlap with Par337 By coimmunopre-
cipitation, JAM-C is found to associate with Par6, Cdc42,
PKCλ, and PATJ,37 suggesting that JAM-C can interact
with two polarity protein complexes, CRB3/Pals1/PATJ
and Par3/Par6/aPKC. Subsequent studies have confirmed
that JAM-C forms a stable complex with Pals1 and Par6 at
the apical ES to confer adhesion and polarity.60 The inter-
action of Src kinase with Pals1 and Par6 can destabilize
JAM-C/Pals1/Par6 complex and results in detachment of
spermatids from the seminiferous epithelium.60
Knockout phenotypes in male reproductive system
Through loss-of-function studies, the physiological roles
of JAMs have been identified (Table 5.1). A wide range
of physiological processes such as spermatogenesis and
leukocyte transmigration requires temporal and spatial
expression as well as the participation of JAMs. JAMs are
also involved in various diseases including viral infection
and cancer development. This review focuses on the role
of JAMs in male reproductive functions, and therefore we
will put more emphasis on knockout mice displaying male
reproductive defects.
Among classical JAMs and CAR models, knockout of
JAM-A and JAM-C exhibit notable abnormality in sper-
matogenesis. Detailed investigation of localization of

JAM-A has found that JAM-A is present in spermatids and
mature spermatozoa and epididymal cauda sperms.36 Male
JAM-A knockout mice produce progeny with altered sex
ratio coupled with reduced litter size.61 They also exhibit
sperm motility defects such as a significant reduction of
the percentage of motile spermatozoa and progressive and
hyperactivated motility.36 Sperms from JAM-A knockout
mice also display abnormal morphology in the mitochon-
drial sheaths and in the flagella.36
JAM plays a role in the recruitment of cell polarity com-
plex for the establishment of polarity.16,57 Loss of JAM-C
hinders the recruitment of Par6/Cdc42/PKCλ complex to
the anterior region of spermatid head for the polarization
of round spermatids, resulting in male infertility.37 Testis
from JAM-C knockout mouse is significantly reduced in
size and has no elongated spermatids in the seminiferous
tubules.37 It is known that JAM-B is the heterophilic inter-
acting partner of JAM-C at the apical ES of the seminif-
erous epithelium. However, JAM-B-deficient mice show
normal reproductive function without any detectable
sperm abnormality.62 Still, we could not rule out the func-
tional role of JAM-B in spermatogenesis since the fertil-
ity tests were observed using mice at age 8 weeks old62;
it remains unknown if abnormalities of spermatogenesis
might appear in aging JAM-B-null mice as was reported
in occludin knockout.63 It is also possible that other JAMs
may complement one another in some ways due to func-
tional redundancy.
Regulation of JAMs and CAR in the testis
Deciphering the regulation of JAMs and CAR definitely
helps gain a better understanding of spermatogenesis and
pathogenesis. The following section reviews some of the
recent studies that are pertinent to the regulation of JAMs
and CAR.
Both in vitro and in vivo studies have shown that JAMs
and CAR can be regulated by hormones such as angioten-
sin and cytokines such as transforming growth factor-βs
(TGF-βs). For instance, angiotensin II upregulates the
expression of JAM-A and contributes to the progression
of hypertension.64 The JAM-A-associated hypertension
can be attenuated by the use of blockers targeting ANGII
signaling.64 Knockdown of TGF-β receptors and canoni-
cal Smad signaling causes the upregulation of the JAM-A
level and inhibits cell invasion in MDA-MB-231 cells.65
Studies have shown that TGF-β1 significantly inhibits
JAM-A gene transcription via the activation of Smads
and promotes JAM-A protein degradation via the activa-
tion of p54 JNK signaling.65 In the testis, TGF-β3 regu-
lates JAM-B expression to facilitate the disassembly of the
BTB and the apical ES.66 TGF-β3 regulates cell junction
restructuring via ERK1/2- and p54 JNK-mediated JAM-B
mRNA destabilization and Smad-dependent JAM-B pro-
tein degradation.66 In addition, TGF-β3 increases in the
kinetics of JAM-A and occludin endocytosis and blockade
of TβR1 can abolish TGF-β3-induced JAM-A endocyto-
sis.67 IL-1α promotes JAM-B expression by facilitating the
binding of positive transcription factors to the cis-acting
Future perspectives and concluding remarks  77
motifs, which leads to an additive effect on Sp1- and NRSF-
mediated JAM-B transactivation, while TGF-β2 inhibits
JAM-B transcription by the activation of Smad proteins.68
Interferon-gamma (IFNγ) and tumor necrosis factor alpha
(TNF-α) are two major cytokines that are elevated dur-
ing testicular inflammation and cause reduced fertility.
Studies in our laboratory have revealed that IFNγ+TNF-α
disrupt testicular cell adhesion by exerting a synergistic
effect on CAR downregulation.69 IFNγ+TNF-α treatment
inhibits the binding of the basal transcription factors and
promotes the binding of negative regulators including
NFkB subunits and Sp1 to the CAR promoter region.69
IFNγ+TNF-α also upregulates CAR protein degradation
via ubiquitin-proteasome and NFkB pathways.69 In endo-
thelial cells, IFNγ+TNF-α and basic fibroblast growth fac-
tor (bFGF) can induce the redistribution of JAM-A from
the TJs toward the luminal surface of the endothelium.30,70
Taken collectively, hormones and cytokines are key regu-
lators to modulate the expression and availability of JAMs
and CAR in both epithelial and endothelial cells.
MicroRNAs (miRNAs) are small noncoding RNAs
that control gene expression at both transcriptional and
translational levels and have been identified to regulate
the expression of JAMs. For instance, in breast cancer,
miR-145 is downregulated and miR-495 is upregulated
and they both target JAM-A.71,72 Overexpression of miR-
145 downregulates JAM-A and causes the restructuring
of the actin cytoskeleton, resulting in decreased motility
and invasiveness,71 while inhibition of JAM-A by miR-495
promotes breast cancer cell migration.72 Efforts should be
made to examine if JAMs and CAR in the testis can be
regulated by miRNA.
Proteolytic cleavage of JAMs has been reported in endo-
thelial models. The process itself as well as the cleaved
fragments are crucial for the control of the JAM func-
tion. For instance, a significant increase in disintegrin-/
ADAM17-mediated JAM-A cleavage has been reported in
inflamed vascular endothelium.73 Soluble JAM-A has been
shown to reduce the transendothelial migration of neutro-
phils in vitro and decreases the infiltration of neutrophil
in vivo.73 Soluble JAM-C is found to be elevated in the
serum of patients with rheumatoid arthritis and is capa-
ble of inducing angiogenesis.74 Like JAM-A, the cleavage
of JAM-C is mediated partially by disintegrin, ADAM10,
and ADAM17.74 Up to now, no studies have been done to
examine if proteolytic cleavage of JAMs occurs in the tes-
tis, let alone its regulatory effects on spermatogeneis.
FUTURE PERSPECTIVES AND CONCLUDING REMARKS
There is no doubt that the JAM family is a group of impor-
tant cell adhesion proteins involved in the process of germ
cell development and differentiation. Some studies have
been performed to investigate the regulation of JAMs
and CAR during spermatogenesis, such as the regulatory
mechanisms involved in the timely expression of JAMs
and CAR in normal or pathological conditions. Further
studies are warranted to address the many questions that
remain unknown and unaddressed. For example, how
78  Junctional adhesion molecule (JAM) family
does the JAM protein complex interact and regulate the
dynamics of junction restructuring during spermatogen-
esis? Are sJAMs or sCAR present in the testis, and if so,
what are their functions? What is the mechanism and
signaling involved in cleavage of JAMs? What regulatory
mechanisms are involved in recycling and degradation of
JAMs and CAR in the testis? Are miRNAs present and
involved in the regulation of JAMs and CAR in the testis?
The answers may provide important information on how
junction restructuring between adjacent Sertoli cells and/
or between Sertoli cells and germ cells via the JAM family
proteins occurs.
ACKNOWLEDGMENT
The work was funded by Hong Kong Research Grants
Council (HKU774213) and HKU/CRCG Seed Funding for
Basic Research.
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Sertoli cell immune regulation
within the testis
GURVINDER KAUR, KANDIS WRIGHT, ROBIN HANNAH GREER, KARL MUELLER,
ALLAN HAYNES, and JANNETTE M. DUFOUR
INTRODUCTION
It is estimated that 8%–15% of couples worldwide are
infertile. Of these, 40%–60% are infertile due to a male-
related infertility problem.1,2 Causes include trauma, infec-
tion, varicoceles, and environmental toxins.3 Associated
inflammation can evoke an immune response to auto-
antigenic germ cells.4 Consistently, in developed nations
sperm autoantibodies have been detected in 5%–12% of
male infertility cases.5 Under normal circumstances the
unique immune privilege environment of the testis pro-
tects the germ cells from these autoimmune reactions.
Sertoli cells (SCs) play a central role in creation and main-
tenance of this unique environment, which is essential for
the immune protection of autoantigenic germ cells. This
chapter will focus on the unique immunoregulatory abili-
ties of the SCs necessary for immune protection of germ
cells.
TESTIS IMMUNE PRIVILEGE
The testis is the central male reproductive organ; as such,
it is the site of androgen production and spermatogenesis.
Morphologically, the testis is comprised of the seminifer-
ous tubules separated by the interstitial space. The semi-
niferous tubules consist of more than 80% of the mass of
the adult testis and are composed of SCs and developing
germ cells surrounded by single (rodents) or multiple
(nonhuman primates and humans) layers of peritubu-
lar myoid cells. The interstitium consists of Leydig cells
(cells that produce androgens), macrophages, blood ves-
sels, lymphocytes, and fibroblasts (Figure 6.1). During
spermatogenesis, which takes place in the seminifer-
ous tubules, the undifferentiated spermatogonial cells
undergo three phases of development: mitosis (prolifera-
tion, where spermatogonia undergo self-renewal or dif-
ferentiation), meiosis (spermatocyte DNA recombination,
reduction, and division), and spermiogenesis (spermatid
differentiation), to generate highly specialized spermato-
zoa. As the germ cells progress through development, they
begin to express novel antigens that first appear after the
establishment of systemic self-tolerance. Despite the pres-
ence of new surface and intracellular proteins, an immune
response is generally not elicited against these germ cells6,7
due to the immune privileged environment within the tes-
tis. The importance of immunological tolerance to the
spermatocytes and spermatids was demonstrated by the
lysis of these cells after injection of sera collected from
rodents immunized with whole semen6,7 and is further
6
supported by models of autoimmune orchitis where loss of
spermatogenesis resulting in infertility is due to an auto-
immune reaction against the germ cells.8,9 Testis immune
privilege is clinically relevant as humans can also develop
autoimmune orchitis.4
Previously it was believed that the unique physiologi-
cal conditions of the testis, such as the lower temperature
of the scrotum, impaired lymphatic drainage or high zinc
concentrations were responsible for testis immune privi-
lege (Figure 6.2) (reviewed in Kaur et al.10). However, when
the testis was positioned in the abdominal cavity at nor-
mal core body temperature, it maintained the ability to
protect transplanted cells. Similarly, studies of testicular
lymphatic drainage revealed a fully functional lymphatic
system. The testis has elevated levels of zinc. Given that
high levels of zinc can suppress allograft rejection, it was
thought that this could be responsible for testis immune
privilege. However, transplantation studies demonstrated
that zinc was not the key component. Pancreatic islets,
which contain high levels of zinc, are rejected when trans-
planted as allografts or xenografts, and when parathy-
roid allografts were transplanted into the prostate, which
contains the highest zinc concentration of all organs, the
parathyroid grafts were similarly rejected (reviewed in
Kaur et al.10).
Other studies examining the main cellular compo-
nents of the testis point to SCs as important players
in testis immune privilege (Figure 6.2). Foreign tissue
transplanted in testes depleted of germ cells, either
by cold testicular ischemia, placement of testes in the
abdominal cavity, or irradiation, continue to enjoy
prolonged graft survival (reviewed in Kaur et al.10).
Furthermore, depletion of Leydig cells still resulted in
the immune protection of allografts transplanted into
the testes (reviewed in Kaur et al.10), although there
is evidence that Leydig cells (by secreting androgens)
and germ cells, while not essential, contribute to tes-
tis immune privilege. Additionally, the immune cells
located within testis and testicular draining lymph
nodes are of an altered phenotype (M2 macrophages,
immature dendritic cells, and regulatory T cells) and
contribute to the unique testicular immune environ-
ment (Figure 6.2). However, immune cells need to be
guided by external signals to take on and maintain
this regulatory phenotype, suggesting that other cells
within the testis, such as SCs, are actively involved in
creating this immune privileged environment.
81
 (a)
Lumen (b)
Spermatid

Round
spermatid SC Adluminal
compartment

Spermatocyte
Tight junction
SC
Spermatogonia nucleus Basal
compartment
Peritubular
myoid cell
T cell DC Blood vessel

Leydig cell Interstitium

MΦ
50 µm
82  Sertoli cell immune regulation within the testis

(c) (d)

50 µm 50 µm

Figure 6.1  (a) Schematic illustration of a cross section of a seminiferous tubule. Testis interstitium contains Leydig cells, macrophages (MΦ), blood vessels, lymphocytes, and dendritic
cells (DC). The seminiferous epithelium is surrounded by peritubular myoid cells and is composed of SCs and maturing germ cells. Tight junctions between adjacent SCs divide the tubules
into basal and adluminal compartments. (b–d) Testes were collected from a 51-year-old male (b) or 6–8-week old BALB/c mice (c and d), paraffin embedded and sectioned. Tissue sec-
tions were immunostained for Wilms’ tumor 1 to detect SCs (brown color, c) or smooth muscle alpha actin that detects peritubular myoid cells and blood vessels (brown color, b and d).
Sections were counterstained with hematoxylin for cell nuclei (blue color, b–d). All animal experiments were performed in accordance with guidelines of the National Institutes of Health
and Institute for Laboratory Animal Research Care and Use of Laboratory Animals, and TTUHSC Institutional Animal Care and Use Committee approved protocols. For the human sample,
the study was approved by the Institutional Review Board. An informed consent was obtained from the patient.
 Blood-testis barrier or SC barrier  83

What is responsible for testis immune privilege?

Impaired lymphatic Lower temperature? Zinc? Blood testis barrier (BTB)?

drainage?
Within minutes, dye and cells injected Allogeniec parathyroid tissue was Transplanted parathyroid tissue Allogeniec or xenogeniec skin and
into the testis drained to lymph nodes. rejected following transplantation into survived in the testis with a high [Zn], parathyroid tissue survived post
the subcutaneous ear (hypothermic), but was rejected in the prostate despite transplantation into the testis
but survived post transplantation in the having the highest [Zn]. interstitum (outside the BTB).
1. Dye or cells cyrptorchid testis (in the abdomen at
2. Lymphatic 1. Parathyroid cells 1. Parathyroid cells
drainage body temperature).
Skin cells
1. Parathyroid cells Scrotol testis Interstitum
Prostate BTB
Epididymis

Dsp LCs
Cyrptorchid
Ear
Scrotol testis testis T-cell
( [Zn]) ( [Zn])
Testis

( Temp) M
( Temp) (Scrotol temp)

DC 2. CELL
3. Lymph nodes 2. REJECTION 3. SURVIVAL 2. SURVIVAL 3. REJECTION SURVIVAL

NO NO NOT REQUIRED NOT SOLE FACTOR

Germ cells (GCs) and

Leydig cells (LCs)? Other immune cells? Sertoli cells (SCs)?
spermatogenesis
Transplanted islet or parathyroid tissue The human testis contains SCs survive long term post allo- and
Islet allografts survived transplantation
survived in both cryptorchid and macrophages, immature dendritic cells, xeno- transplantation without immune
into the testis despite LC depletion
irradiated testis lacking and T-cells. Under certain suppression and they can protect other
with dimethansulphonate (EDP) or
spermatogenesis and GCs. circumstances T-regulatory cells (Treg) co-transplanted cells (islets,
leuprolide (GnRH analong inhibiting
testosterone production). may also be present in the testis hepatocytes, etc.) from tissue rejection.
1. Parathyroid cells
interstitium.
or islet cells
1. EDP or leuprolide Cyrptorchid 1. Islet cells 2. Isolated 3. Combined
Irratiated testis BTB T-cell
testis SCs
LCs
SC
2. Islet SC SC
SC LCs Treg +
cells
Transplant
SC under the
SC No spermatogenesis
M kidney
or GCs
capsule

3. CELL 2. SURVIVAL 3. SURVIVAL DC 1. CELL 2. and 3. CELL

SURVIVAL REJECTION SURVIVAL
NOT REQUIRED NO LIKELY INVOLVED YES

Figure 6.2  Schematic illustration of potential testis immune privilege mechanisms. Lymphatic drainage, scrotal temperature,
zinc concentration, Leydig cells, germ cells, spermatogenesis, and the BTB/SCB are not solely responsible for testis immune privilege,
while SCs are critical in immune privilege. Temperature (temp); zinc concentration ([Zn]); developing spermatogonia (Dsp); macro-
phages (MΦ); dendritic cells (DCs); dimethanulphonate (EDP); gonadotropin-releasing hormone (GnRH); T-regulatory cells (Tregs).

BLOOD-TESTIS BARRIER OR SC BARRIER
SCs extend from the outer edge of the seminiferous tubules
to the lumen and completely surround the majority of the
developing germ cells. Tight junctions located along the
basal region between adjacent SCs form a physical barrier
that separates the seminiferous tubules into basal (con-
taining spermatogonia and preleptotene spermatocytes)
and adluminal (containing most spermatocytes and sper-
matids) compartments (Figure 6.1). This barrier, known
as the blood-testis barrier (BTB) or SC barrier (SCB),
allows SCs to control and regulate the milieu required for
the development of the germ cells by separating the cells
within the adluminal compartment from the blood supply
and through expression of transporters to control passage
of numerous factors.11 SC ablation studies demonstrated
that SCs are critical for germ cell survival, maintenance of
spermatogenesis and the spermatogonial stem cell niche,
as well as survival of adult Leydig cells.12–14 When SCs
were depleted after a single injection of diphtheria toxin
into mice expressing diphtheria toxin receptor specifi-
cally in SCs, there was a rapid loss of germ cells, and by
10 days postinjection no germ cells were detected in the
testis,13,14 indicating loss of spermatogenesis and the stem
cell niche.12,13
In addition to controlling the transport of factors neces-
sary for germ cell development, the BTB/SCB prevents the
passage of antibodies into the adluminal compartment of
the seminiferous epithelium15,16 and in combination with
the peritubular myoid cell layer (Figure 6.1), which forms a
semipermeable barrier, inhibits the entry of immune cells
into the seminiferous epithelium.17–19 SC ablation studies
found that SCs are necessary for peritubular myoid cell
fate and establishment of the tubule architecture prior
to puberty.12,13 Depletion of SCs in the embryo or in the
neonate resulted in loss of tubule architecture, the absence
of smooth muscle actin, and a decrease in other myoid
cell markers, suggesting dedifferentiation of peritubular
myoid cells. In contrast, if SCs were depleted at puberty or
84  Sertoli cell immune regulation within the testis
in the adult, the basic tubule architecture was maintained
and smooth muscle actin expressing peritubular myoid
cells were still present, although expression of myoid cell
functional markers was decreased.13,20 Analysis of the
integrity of the BTB/SCB after depletion of SCs in adults
demonstrated that SCs are necessary to prevent entry of
molecules into the tubules while the remaining peritu-
bular myoid cells were sufficient to inhibit immune cell
infiltration.
Given that the BTB/SCB prevents entry of antibodies
and immune cells from entering the adluminal compart-
ment of the seminiferous tubules and that the majority
of the autoantigenic advanced germ cells are sequestered
behind the BTB/SCB, historically testis immune privilege
was attributed solely to the presence of the BTB/SCB. The
BTB/SCB consists of tight junction proteins that include
members of the claudin family, occludin, zonula occludens
(ZOs), junctional adhesion molecules (JAMs), and tricel-
lulin.21 In addition to the tight junction proteins, special-
ized adherens junctions (basal ectoplasmic specializations
and basal tubulobulbar complex), desmosomes, and gap
junctions also contribute to the functionality of the BTB/
SC barrier.21
Of all the tight junction proteins, occludin and claudin
11 are critical for barrier integrity as male mice lacking
occludin or claudin 11 are infertile.22 Histological analy-
sis of testes from the occludin knockout mice revealed
normal testicular morphology, containing germ cells, in
young animals (6 weeks old), but as the animals aged (40–
60 weeks old) atrophied tubules devoid of germ cells were
observed.22 Similarly, a single injection of synthetic occlu-
din peptide resulted in disruption of the BTB/SCB, loss
of elongated spermatids (day 8 postinjection), and sper-
matocytes and spermatids (day 27 postinjection) in rats.23
While the immune response was not measured in this
study it is interesting to note that the treatment was revers-
ible and despite disruption of the BTB/SCB for several
weeks, normal spermatogenesis was observed by 68 days
postinjection. Consistently, in mice treated with a mutant
occludin peptide, the integrity of the BTB/SCB was dis-
rupted, demonstrated by permeability to inulin, resulting
in germ cell loss. Nevertheless, antisperm antibodies were
not detected in serum.24 Claudin 11 knockout mice are
sterile and their seminiferous tubules contain degenerat-
ing germ cells. Despite the lack of tight junctions in these
mice, an immune response is not generated as testicular
autoantibodies were not detected in the serum or within
the adluminal compartment of the seminiferous tubules
and CD4 T cell infiltrate was not detected within the tes-
tis.25 Interestingly, in humans and guinea pigs, SC tight
junctions lack occludin,26 suggesting occludin may not be
critical for the integrity of human or guinea pig BTB/SCB.
In testicular biopsies of men with impaired spermato-
genesis the spatial organization of claudin 11 was altered
and increased claudin 11 expression was detected.27–31 For
instance, 82% of control subject’s seminiferous tubules
featured a filamentous claudin 11 staining pattern that
was restricted to the basal aspect of the tubule. In contrast,
the majority of the tubules from meiotic arrest (55%) and
SC-only syndrome (61%) subjects had a punctate claudin
11 staining pattern that was spread throughout the basal
and adluminal aspects of the tubules.29
The importance of the other components of the BTB/
SCB is not clear. ZO-1 and -2 knockout mice are embry-
onic lethal and claudin-5 knockout mice die within
10  days of birth.32,33 In humans ZO-1 has been found to
colocalize with claudin 11 and in tubules with carcinoma
in situ ZO-1 and -2 expression is decreased.28,34 Deletion
of JAM-A results in subfertility in mice, although this is
due to a defect in sperm motility as they have normal tes-
tis morphology.35 Mice with JAM-B gene disruption are
fertile with normal testicular morphology,36 while ZO-3
knockout mice have no apparent phenotype.33 For claudin
3, it was initially concluded that the increased permeabil-
ity of the BTB/SCB and germ cell autoantibodies in SC
androgen receptor knockout (SCARKO) mice was due to
decreased claudin 3 expression.37,38 However, it has since
been shown that claudin 3 knockout mice are fertile and
have an intact barrier function.39 The importance of ZOs,
JAM-A and –B, and claudin-3 and -5 in human spermato-
genesis is not well studied.
Experimental autoimmune orchitis (EAO) studies sup-
port the immunological importance of the BTB/SCB. The
BTB at the rete testis is incomplete40–43 and leaky to anti-
bodies,44 making this region susceptible to the development
of autoimmune orchitis. Under normal circumstances
spermatozoa traverse through this region in immense
numbers and yet an immune response is not generated
against these germ cells. However, under certain condi-
tions an immune response can be triggered. For instance,
immunization of mice with syngeneic testicular germ cells
(without adjuvant) or adoptive transfer of autoreactive
CD4 T cells leads to EAO. Analysis of the testis revealed
that lymphocyte infiltration started in the rete testis with
lymphocyte migration into the epithelium and epithelial
degeneration followed by infiltration into the rest of the
testis and loss of the germ cells.45,46 Overall, it can be con-
cluded that the BTB/SCB is critical for spermatogenesis by
creating a controlled environment for germ cell develop-
ment. While the importance of the tight junction proteins
in protection of germ cells from an autoimmune response
is less clear, sequestering the majority of the autoantigenic
germ cells behind the BTB/SCB is important to decrease
antigen exposure and prevent an immune response.
MOVEMENT OF GERM CELLS ACROSS THE BTB/SCB
If sequestering of germ cells behind the barrier is nec-
essary, how do germ cells cross the BTB/SCB to enter
adluminal compartment without exposing the advanced
germ cells? The BTB/SCB undergoes extensive restructur-
ing at late stage VIII–early stage IX to allow the transit
of preleptotene/leptotene spermatocytes from the basal to
adluminal compartment. The movement of preleptotene/
leptotene spermatocytes across the BTB/SCB never elicits
an autoimmune reaction, suggesting that restructuring of
the BTB/SCB is a controlled process. The exact mechanism

by which this transit takes place is largely unknown. Several
theories for migration of preleptotene/leptotene spermato-
cytes across the BTB/SCB without compromising its integ-
rity have been suggested. (1) The “zipper theory” proposes
that occludin fibrils positioned above the preleptotene/
leptotene spermatocytes are dissolved and new occludin
fibrils are reformed under these spermatocytes, resulting
in their migration across the BTB/SCB.47 (2) The “repeti-
tive removal of membrane segments theory” suggests that
the stress created by upward migration of germ cells alters
the SC junctional complexes, resulting in proliferation of
junctional complexes to form intercellular pockets around
the developing germ cells (except elongated spermatids).
These intercellular pockets, containing developing germ
cells, are sealed at both ends by tight junctions, adherens,
and gap junctions and traverse through the seminiferous
epithelium (see Mruk and Cheng,21 Pelletier48). (3) The
“junctional restructuring theory” suggest that germ cell
movement consists of intermittent phases of tight junc-
tion disassembly and assembly, tightly controlled by cyto-
kines that are released by SCs and/or germ cells, which
allows passage of developing germ cells from the basal to
adluminal compartment (see Mruk and Cheng21). (4) The
“intermediate cellular compartment theory” supports the
existence of a third transient compartment, containing
leptotene spermatocytes, in between basal and adlumi-
nal compartments.49 Once new tight junctions are formed
near the basal end of the transient compartment the old
tight junctions are dissolved. The junctional restricting
and intermediate cellular compartment theories gained
popularity due to recent evidence derived from biochemi-
cal studies.21,50 For instance, whole seminiferous tubules
dissected from adult mice were immunostained for clau-
din 11 and imaged by confocal microscopy to visualize the
three-dimensional organization of the SC tight junctions.
Analysis revealed the presence of claudin 11 on both the
basal and apical sides of the migrating spermatocytes,
resulting in the enclosure of leptotene spermatocytes in an
intermediate compartment.50
TESTIS IMMUNE PRIVILEGE: NOT MERELY DEPENDENT
ON THE BTB/SCB
Reducing antigen exposure by sequestering the advanced
germ cells behind the BTB/SCB is important to prevent
an immune response to the advanced germ cells. However,
the whole testis, not just the adluminal compartment of
the seminiferous tubules, is immune privileged as dem-
onstrated by the studies described below. Under normal
circumstances preleptotene spermatocytes fail to gener-
ate an immune response even though they are located
within the basal compartment of the seminiferous epi-
thelium, outside of the BTB/SCB, and it has been shown
previously that they express antigens that can evoke an
immune response.51 In seasonal breeders the BTB/SCB
breaks down during the nonbreeding cycle repeatedly
exposing the meiotic spermatocytes, and yet, an immune
response does not develop.52 In a recent study BTB/SCB
restructuring was analyzed in rats treated with the GnRH
Immune regulation by Sertoli cells  85
antagonist acyline to suppress spermatogenesis followed
by testosterone treatment to reinitiate spermatogenesis.
Using permeability tracers of different sizes (0.6, 70, and
150 kDa) to emulate factors that could cross the BTB/SC,53
it was demonstrated that meiosis occurs when the BTB/
SCB is permeable to factors up to 70 kDa (such as growth
factors, cytokines, and hormones) but not 150 kDa (such
as antibodies).
Clinically fine needle biopsies are routinely performed
to obtain human sperm for in vitro fertilization or to
analyze testis samples. Even though this procedure can
cause local injury to the seminiferous epithelium, it does
not normally lead to autoimmune orchitis.54 Consistently,
traumatic injury by puncturing the testis or epididymis
multiple times with a needle, resulting in migration of
germ cells into the interstitial space, causes immune cell
infiltration and granuloma formation in the epididymis,
which does not occur in the testis.55 Moreover, injections
of testicular germ cells, spermatozoa, or Bordetella pertus-
singens into the testis interstitial space does not result in
granuloma formation or immune cell infiltrate while sim-
ilar experiments performed in the epididymis leads to in
an increase in inflammatory cells and granuloma forma-
tion within the epididymal interstitium.55,56
Transplantation studies further demonstrate that the
whole testis rather than just the adluminal compartment
of the testis is immune privileged. For instance, trans-
plantation of allogeneic or xenogeneic pancreatic islets
or parathyroid grafts, and allogeneic insulinomas, skin
grafts, or pituitary grafts, into the testis prolonged their
survival compared to other nonimmune privileged sites,
even though the transplanted tissue or cells were located
in the testis interstitium (outside the BTB/SC barrier)
(Figure 6.2) (extensively reviewed in Kaur et al.10). In
addition, successful syngeneic or allogeneic spermatogo-
nial stem cell transplantation resulted in sperm produc-
tion in chickens, fish, and large animals without the use
of immune suppression (reviewed in Honaramooz and
Yang57). Interestingly, transplantation of germ cells did not
evoke an inflammatory immune response even though the
germ cells colonized the basal compartment of the semi-
niferous tubules (reviewed in Kaur et al.,10 Honaramooz
and Yang57). Collectively these studies demonstrate that
germ cells and other cells transplanted into the testis can
be exposed to the immune system without evoking an
immune response, suggesting that other mechanism(s)/
factor(s) work in collaboration with the BTB/SCB to create
a tolerogenic environment in the testis.
IMMUNE REGULATION BY SERTOLI CELLS
Transplantation studies provide evidence that SCs mod-
ify the immune response to create an immune privileged
environment for their survival and also the survival of
cotransplanted cells. SCs survive as allografts or xenografts
without the use of chronic immune suppression when
transplanted outside the testis (Figure 6.2). Additionally,
SCs provide immune protection to cografted cells such
as pancreatic islets to treat diabetes, adrenal chromaffin
86  Sertoli cell immune regulation within the testis

T-cell

Cell mediated Immunoregulatory Sertoli

M
cytotoxicity factors cell Immune tolerant
Apoptosis Apoptosis and environment
DC antibodies complement inhibitors
complement

Figure 6.3  Generalized Sertoli cell modulation of the immune response. Immune-mediated cytotoxicity occurs when antigen
presenting cells (APCs) (macrophages or dendritic cells), B cells, and T cells are activated against the autoimmunogenic germ cells.
SCs express immunoregulatory factors, apoptosis inhibitors, and complement inhibitors to protect cells from detrimental immune
responses and convert the cytotoxic immune cells to be more regulatory. This creates an immune-tolerant environment within the
testis. Pink circles represent SC surface factors. Purple circles represent SC secreted factors. Green circles represent SC intracellular
factors. Macrophages (MΦ); dendritic cells (DCs).

cells, or dopaminergic neurons for neurodegenerative dis-
eases and hepatocytes for liver disease when transplanted
across immunological barriers as allografts or xenografts
(reviewed in Kaur et al.58). These studies suggest modula-
tion of the immune response by SCs plays a key role in
testis immune privilege.
A fully functional immune response is required for pro-
tecting human beings from deadly pathogens and foreign
antigens while at the same time it also has the potential to
negatively impact male reproduction. Thus, regulation of
the immune system is required for successful spermato-
genesis in the testis. Fundamentally, an immune response
can be divided into humoral and cell-mediated responses.
The cellular components of the humoral and cell-mediated
immune response mainly include B cells, NK cells, T cells,
and antigen-presenting cells (APCs, such as dendritic
cells [DCs] and macrophages). Studies investigating the
immune privilege mechanism of SCs in vitro and in vivo
(SC transplantation studies) clearly indicate that SCs have
the capability to modify both the humoral and cell medi-
ated immune responses (Figure 6.3). Here, we will spe-
cifically discuss the factor(s)/mechanism(s) by which SCs
regulate immune cells.
Humoral immune response
In response to nonself antigens, B cells produce antibod-
ies (IgM and IgG) that bind to these antigens and coat the
target cells. This results in killing of the target cell through
the following pathways (Figure 6.4a). Antibodies acti-
vate the complement cascade leading to formation of the
membrane attach complex (MAC) on the cell membrane
of the target cells, which leads to complement mediated
cell lysis. Additionally, complement cascade activation
causes the release of anaphylatoxins, specifically C4a, C3a,
and C5a, which mediate acute inflammation by attracting
immune cells. Lastly, natural killer (NK) cells can initi-
ate antibody-dependent cellular cytotoxicity by binding to
the IgG1 or IgG3 coated cells through their low affinity
Fcy receptor. After binding, NK cells kill the target cells
through several different mechanisms: (1) exocytosis of
cytoplasmic granules containing perforin and granzymes,
(2) releasing proinflammatory cytokines, or (3) TNF fam-
ily death receptor signaling. Both the uptake of perforin
and granzymes and TNF family death receptor signaling
causes apoptosis of target cells.
SCs are resistant to complement mediated cell lysis
since they survive both when transplanted as discordant
xenografts,59,60 which by definition indicates the presence
of antibodies directed against transplanted cells, and when
subjected to an in vitro human antibody/­ complement
mediated killing assay.61 For example, neonatal pig SCs
(NPSCs) survive throughout the study (at least 42, 90,
and 30 days) when transplanted into mice, rats, and dogs,
respectively.59,60 No immune suppression was used in
rodent recipients while a short course (10 days) of immu-
nosuppression was used in dogs. Recently, it was shown
that NPSCs survive throughout the study (40 days) despite
the presence and deposition of preformed antibodies,
while neonatal pig islets (NPIs) were rejected when trans-
planted as xenografts underneath the kidney capsule of
rats.62 SCs express several complement inhibitors includ-
ing membrane cofactor protein (MCP; CD46), decay
accelerating factor (DAF; CD55), SERPING1, clusterin,
and CD59.63–66 Comparison of the levels of these comple-
ment inhibitors revealed that NPSCs express MCP and
DAF at significantly higher levels than the rejected NPIs,62
suggesting these inhibitors could explain how SCs escape
complement mediated cell lysis (Figure 6.4a).
In rodent and human testis, substantial numbers of
cells expressing NK cell markers are detected and a sig-
nificant proportion of these cells are NKT cells.67 Further
analysis of NKT cells from rat testis revealed that under
both unstimulated and phorbol 12-myristate 13-acetate
(PMA)/ionomycin-stimulated conditions, the NKT cells
produce interleukin (IL)-10,67 suggesting these cells have
a tolerogenic phenotype in the testis. Studies investigating
the direct effect of SCs on NK cells are limited. In vitro
analysis revealed that rat SC secreted factors inhibit rodent
and human NK cell activity but fail to suppress the lysis of
YAC-1 cells (mouse lymphoma cell line sensitive to lysis
by NK cells) by rat NK cells.68 In vivo, despite the deposi-
tion of IgG antibody, NK cell infiltration was not detected
 (a) Humoral immune response
Secreted factors
SERPING1 Generation of
TGF-β (Immunoregulatory factor)
MCP anaphylatoxins
C1 (C4a, C3a, C5a) Thrombospondin 1&2 (Enzymes that active TGF-β)
Target complex DAF
NK-cell Activin A (Activates alternative macrophages)
cell death Galectin-1 (Immunoregulatory factor)
? C4b
C3 C2a Clusterin CCL7 (Immunoregulatory cytokine and chemokine)
Convertase C4b CD59
C2a
CXCL1
SC C5 C3b
Cell SERPING1 (Complement inhibitor)
Convertase (Apoptosis inhibitor)
MAC lysis SERPINA3n
(C5b-9) SERPINB9
?
PGE2 (Anti-inflammatory and T-cell inhibitor)
B-cell PGI2 (Anti-inflammatory)
Antibodies Target cell lysis Intracellular factors
by complement
activation IDO (T-cell inactivation and immune suppression)
PLA2G4A (Anti-inflammatory prostanoid production)
(b) Cell-mediated immune response PLA2G6
PTGES (Anti-inflammatory prostaglandin production)
ivin
Regulatory MΦ
P 1&2, Act
PTGIS
TGF-β, TS in-1, IDO and DC
A, G al e ct
Surface factors
T-cell MCP (CD46) (C3 and C5 convertase inhibitor)
SC
TGF-β DAF (CD55)
,
Galec TSP 1&2
tin-1, Indirect Clusterin (MAC inhibitor)
IDO
n CD59
Target cell A3 Direc
t
PIN 9 B7-H1 (T-cell negative regulatory receptor)
death SER PINB
Regulatory T-cells
SER
(apoptosis) (tregs)

Figure 6.4  Factors expressed by Sertoli cells that modulate the humoral and cell-mediated immune responses. SCs express various intracellular, cell membrane, and secreted factors
that modulate and maintain a tolerogenic environment. (a) Humoral immune response: SCs inhibit complement mediated cell lysis both directly and indirectly. In the smaller box, antibod-
ies (IgG and IgM) bind to the target cell. Binding of the C1q complex to these antibodies results in the formation of the C1 complex, which activates the complement cascade by generating
C3 convertase. C3 is recruited to the C3 convertase and together forms the C5 convertase, which cumulates factors (C5-C9) to produce the membrane attack complex (MAC) on the target
cell surface. Formation of both the C3 and C5 convertases generates anaphylatoxins that increase inflammation, chemotaxis, and immune cell recruitment. SCs inhibit the complement
cascade by expressing the complement inhibitors (pink dots, green text). SCs can inhibit B cell proliferation through unknown factors (black dots), thereby preventing the production of
antibodies and activation of complement cascade indirectly. SCs can also inhibit NK cells. Red arrows represent immune response pathways and green lines represent SC inhibition of
these pathways. (b) Cell-mediated immune response: SCs inhibit T cell mediated apoptosis directly by secreting apoptosis inhibitors (SERPINA3n and SERPINB9; yellow dots) or indirectly by
inhibiting T cell proliferation. Factors expressed by SCs such as transforming growth factor (TGF)-β, thrombospondin (TSP)-1 and -2, activin A, galectin-1, and indoleamine 2,3-dioxygenase
(IDO) are implicated in inducing regulatory antigen presenting cells (APCs) and Tregs. SCs can induce Tregs directly or indirectly through regulatory APCs. Collectively this tolerance results
in survival of the target tissue. Together these factors are crucial in maintaining testicular immune privilege and protecting autoimmunogenic germ cells. SC factors are listed in the upper
right box. Chemokine ligand 7 (CCL7); chemokine ligand 1 (CXCL1); serpin family G member 1 (SERPING1); prostaglandin (PGE2); prostacyclin (PGI2); phospholipase A2 Group IVA (PLA2g4a);
phospholipase A2 Group VI (PLA2g6); prostaglandin E synthase (PTGES); prostaglandin 12 synthase (PTGIST); membrane cofactor protein (MCP); and decay accelerating factor (DAF).
Immune regulation by Sertoli cells  87
88  Sertoli cell immune regulation within the testis
in surviving SC allografts or xenografts or rejecting NPI
xenografts, suggesting that NK cells play a minimal role in
SC graft survival or NPI graft rejection at the kidney site
(unpublished data, Dufour lab, 2012).
Cell mediated immune response
T cells and APCs are the main constituents of the cell-
mediated immune response. T cells stimulated by APCs
kill tissue via apoptosis mediated by the Fas-FasL or perforin-
granzyme B pathways. Additionally, antigen-primed, CD4
T cells activate macrophages creating a proinflammatory
environment that promotes cell death. Several in vitro
and in vivo studies suggest that SCs modulate the overall
immune response (Figure 6.4b). Transplanted SCs have the
capability to escape cell death via apoptosis as they express
SERPINA3N and SERPINB9,69–71 both inhibitors of the
granzyme B apoptosis pathway. While controversial, it has
also been reported that SCs can inhibit the Fas-FasL path-
way (reviewed in Mital et al.72). In vitro experiments using
rodent SC conditioned media (SCCM) demonstrated that
SCs are capable of inhibiting the proliferation of acti-
vated lymphocytes (B and T cells).73–75 Treatment with
SCCM significantly reduced the IL-2 production and IL-2
responsiveness by lymphocytes.73,74 Moreover, SCs express
several immunoregualtory factors, such as ­ galectin-1,
indoleamine-pyrrole 2,3-dioxygenase (IDO), prosta-
glandin, transforming growth factor (TGF)-β),76–80 and
have the capability to modulate the immune response in
the testis or when transplanted ectopically. For instance,
testicular macrophages and DCs express MHC-II and
costimulatory molecules, implying these cells can mount
an effective immune response. Yet upon lipopolysaccha-
ride stimulation, rodent testicular macrophages express
low levels of proinflammatory cytokines and high levels
of the anti-inflammatory cytokine, IL-10 (reviewed in
Winnall and Hedger81). Similarly, DCs isolated from the
testis or testicular lymph nodes are unable to induce T cell
proliferation.82
There is evidence that SCs induce tolerogenic APCs.
Coculture of immature DCs with SCs or SCCM resulted
in downregulation of MHC-II and costimulatory mol-
ecule (CD80, CD83, and CD86) expression on DCs.83
Additionally, these DCs displayed reduced phagocytic
activity, expressed low levels of proinflammatory cyto-
kines (IL-12p70 and tumor necrosis factor-α) and high
levels of anti-inflammatory cytokines (IL-10 and TGF-β).
These SC conditioned DCs were tolerogenic as they inhib-
ited T cell proliferation and induced CD4+CD25+Foxp3+
regulatory T cells (CD4 Tregs). Galectin-1, expressed by
SCs, was critical as knockdown of galectin-1 in SCs using
siRNA abrogated the generation of tolerogenic DCs. It
has also been demonstrated in vitro that SCs can directly
induce CD4 Tregs.84,85 Similarly, transplantation of por-
cine SCs resulted in diabetes prevention and reversion
in 88% and 81% of the non-obese diabetic (NOD) mice,
respectively.77 This effect was associated with the induc-
tion of a regulatory environment by SCs as evidenced
by generation of anti-inflammatory cytokines and CD4
Tregs. Furthermore, IDO expression by SCs was critical as
SCs silenced for IDO expression failed to protect the NOD
mice from diabetes development.77 Collectively, these data
demonstrate that SC have the capability to generate reg-
ulatory APCs and T cells, which could explain how SCs
protect autoantigenic germ cells (Figure 6.4b).
POTENTIAL DOWNSIDE OF IMMUNE PRIVILEGE
Immune privilege has the possibility to be detrimental,
as the immune system functions to protect the body from
infections and cancerous transformations, and immune
modulation may pose an increased risk of these events.
For example, immune privilege may play an important
role in the disease progression of human immunodefi-
ciency virus (HIV). It has been proposed that the testis
may act as an HIV sanctuary site and protect the virus
during antiretroviral therapy.86 The potential for testis
immune privilege to be used by viral pathogens may not
be unique to HIV, and there is some evidence that Ebola
and Zika viruses may also use the testis as a reservoir. One
study found that all nine males tested had Ebola-virus–
positive semen 3 months after onset of illness, half were
positive 4–6 months after the onset of illness, and one
quarter were positive 7–9 months after onset of illness.87
In another study it was reported that Ebola was present in
the semen of survivors in Liberia for 18 months after infec-
tion.88 Moreover, transmission of the Zika virus through
the semen from sexual contact has been reported.89
Despite testis being an immune privileged tissue, infec-
tions within the testis are rare67 and genitourinary tract
infections (with the exception of viral pathogens) seldom
involve the testis,90,91 suggesting that testis immune privi-
lege is not a permanent phenomenon and can be broken
down if it threatens the existence of the host. On the same
note, the rate of cancer in the testis is not different than in
other sites.67 Primary testicular cancer is infrequent, with
germ cell tumors (GCTs) accounting for 1% of malignan-
cies in males in the United States.92 The low incidence of
infections and cancer has been attributed to activation of
the innate and adaptive immune responses.4 Innate immu-
nity is initiated by recognition of pathogen-associated
molecular patterns (found on bacteria, virus fungus, and
protozoa) by pattern recognition receptors such as Toll-
like receptors (TLRs). So far, 10 and 13 TLRs have been
identified in human and rodents, respectively.93 Several
studies have demonstrated that SCs and testicular mac-
rophages express a majority of these TLRs and respond
to TLR activation by initiating inflammation, thereby
clearing pathogens and protecting the host (reviewed in
Hedger,94 Meinhardt and Hedger95). Interestingly, GCTs,
particularly seminomas (Figure 6.5), have characteris-
tic immune cell infiltrates that consist mainly of mac-
rophages and T lymphocytes that correlate with a better
prognosis. At the same time there is a lack of suppressor T
cells in GCTs, perhaps reflecting a gradual breakdown of
testicular immune privilege. Thus active immune surveil-
lance leads to immune infiltrate, breakdown of the BTB/
SCB, and loss of immune privilege, resulting in a good
 References 89

(a) (b)

200 µm 200 µm
(c) (d)

200 µm 50 µm

Figure 6.5  Testicular germ cell tumor. (a) and (b) Hematoxylin and eosin staining of testicular tissue sections collected from
a 36-year-old male. The patient was diagnosed with classic seminoma, Leydig cell hyperplasia, and intratubular germ cell tumor.
(c) and (d) Testicular tissue section collected from the patient was immunostained for monocyte/macrophages using CD14 as a
marker (brown color; Cell Marque, Rocklin, CA). Cell nuclei were counterstained with hematoxylin (blue color, [c] and [d]). This study
was approved by the Institutional Review Board. An informed consent was obtained from the patient.

prognosis.34,96 However, similar to viral pathogens, if a
lymphoma metastasizes, the testis can act as a reservoir
for this tissue and increase risk of relapse. For instance,
the testis is a major site for relapse of acute lymphoblastic
leukemia4; however, due to advances in modern medicine,
testicular relapse is now a rare event.97
Overall, SCs continuously guide the immune cells
rather than just constantly suppressing the immune
response. Thus, if a foreign pathogen threatens the exis-
tence of the host, SCs abandon their motherly duties and
initiate inflammation, resulting in disruption of immune
privilege and spermatogenesis. For instance, Chlamydia
has been detected in urethral or urine samples from
11%–35% of men with epididymo-orchitis, and acute
epididymo-orchitis is associated with decreased sperm
counts and decreased sperm motility.98
SUMMARY
Testis immune privilege is necessary to prevent an auto-
immune response to the developing germ cells. SCs play
an important role in maintaining this immune privileged
environment by sequestering the majority of the germ cells
behind the BTB/SCB (adluminal compartment), thereby
preventing the immune cells from gaining access to these
cells and modifying the immune response by expressing
several immunomodulatory factors and inducing regula-
tory cells. However, a delicate balance between immune
privilege and inflammation is necessary to prevent infec-
tions and cancer while maintaining male fertility.
ACKNOWLEDGMENTS
This work was supported in part by NIAID grant AI109398
and funding from The Jasper L. and Jack Denton Wilson
Foundation. The authors wish to acknowledge the con-
tribution of the Texas Tech University Health Sciences
Center Clinical Research Institute for their assistance with
this research.
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The mitotic phase of spermatogenesis
Recent advances and perspectives
KIN LAM FOK and HSIAO CHANG CHAN
INTRODUCTION
In healthy men, millions of sperm are produced daily in
the testis through a process known as spermatogenesis,
owing to the tremendous replenishing power of sper-
matogonia, a heterogeneous population consisting of both
spermatogonial stem cells (SSCs) and committed progeni-
tors.1 The spermatogonia need to undergo three phases—
the mitotic phase, the meiotic phase, and the postmeiotic
differentiation phase—to give rise to spermatozoa. In the
mitotic phase, spermatogonia undergo a series of mitosis to
expand the number of daughter cells. These daughter cells
have two fates: (1) self-renewal to maintain the stem cell
pool, and (2) committed to differentiation and give rise to
primary spermatocytes that enter the meiotic phase. Since
the primary spermatocytes will only undergo two rounds
of meiotic division whereas the spermatids undergo dif-
ferentiation without division, the mitotic phase is consid-
ered the foundation that provides a steady supply of a large
number of committed progenies while maintaining the
stem cell pool to sustain spermatogenesis. Perturbation of
the homeostasis between spermatogonial self-renewal and
differentiation has detrimental effects on spermatogenesis.
In the past two decades, a number of valuable tools for
the study of spermatogonia have been developed. These
include the germ cell transplantation technique that
allows the assessment of stem cell activity by observ-
ing the regeneration of spermatogenesis, 2 the in vitro
propagation of spermatogonia that facilitates functional
studies by pharmacological and genetic manipulations, 3,4
and the fluorescent protein-mediated lineage tracing in
transgenic mice models that monitors the migration and
dynamics of live cells in real time.5 In particular, the in
vitro propagation of spermatogonia has recapitulated
the mitosis phase of spermatogenesis. In the presence
of a cocktail of growth factors and supporting feeder
cells or extracellular matrix proteins, primary culture
of spermatogonia can be maintained and expanded in
vitro. The cultured spermatogonia remain heterogeneous
and the stem cell activity is maintained in a subpopu-
lation of cells. Using these valuable tools, a plethora of
critical regulators on the self-renewal and differentia-
tion of spermatogonia has been identified.6–10 However,
the mitotic phase of spermatogenesis is peculiarly com-
plicated, involving the regulation by extrinsic factors
such as paracrine signaling and cell-niche interaction,
intrinsic factors such as specific genes and proteins, and
the interplay between the two. Nevertheless, the current
knowledge appears to represent only a few pieces of the
94
7
puzzle from the whole picture. In this chapter, we will
reveal the key concepts and recent findings with a par-
ticular focus on the emerging regulators of the mitotic
phase of spermatogenesis.
ORIGIN AND DIFFERENT SUBPOPULATIONS
OF SPERMATOGONIA
Spermatogonia are originated from gonocytes, also
known as pre- or prospermatogonia. Gonocytes are
positioned within the lumen of the seminiferous tubules
before birth. Shortly after birth, they proliferate, dif-
ferentiate, and migrate from the lumen to the basement
membrane of the seminiferous tubules where a specialized
microenvironment or niche is formed.11,12 Gonocytes give
rise to two spermatogonial populations, the undifferenti-
ated spermatogonia and differentiating spermatogonia.
The differentiating spermatogonia proceed to the meiotic
process and migrate from the basement membrane to the
adluminal compartment to initiate the first wave of sper-
matogenesis in the juvenile while the undifferentiated
spermatogonia self-renew and support the steady state of
spermatogenesis in the adult.13 In the mouse, the undif-
ferentiated spermatogonia consist of type A spermatogo-
nia (Aundiff ) that can be further classified into Asingle (As),
Apaired (Apr), and Aaligned (Aal) spermatogonia while the
differentiating spermatogonia are made up of type A1–4,
intermediate, and type B spermatogonia. The mitotic divi-
sion of spermatogonia undergoes distinctive incomplete
cytokinesis where the daughter cells were interconnected
with the intercellular cytoplasmic bridge, also known as
the syncytial state.14 The first mitotic division of As sper-
matogonia will give rise to Apr spermatogonia. Subsequent
rounds of mitosis will give rise to Aal spermatogonia of
4–32 cells in length. The spermatogonial stem cells (SSCs)
mainly reside in the As spermatogonia. Interestingly, the
syncytial state spermatogonia undergo stochastic frag-
mentation and give rise to As spermatogonia, indicating
that the hierarchy of Aundiff is reversible and stochastic14
(Figure 7.1).
A number of markers have been identified for different
spermatogonial populations. GFRα1 is mainly expressed
in As spermatogonia and its expression is decreased grad-
ually in Apr and Aal spermatogonia. In contrast, Ngn3 is
weakly expressed in or absent from As spermatogonia
while its expression increases in Apr and Aal spermatogo-
nia.15 Expression of c-Kit is only observed in Aal and differ-
entiating spermatogonia.16 Thus far, a specific marker for
SSC has not been identified. Nonetheless, the expression

Differentiated
As Apr
Undifferencitated
PAX7
ID4
GFRα1
NGN3
PLZF
Complete mitosis
Figure 7.1  Spermatogonial subpopulations in the mitotic phase of mouse spermatogenesis. Schematic diagram showing the
interconversion between As,Apr, and Aal spermatogonia through cell division and syncytial fragmentation among the Aundiff popula-
tion (bottom panel). These subpopulations differentiate to form the differentiated spermatogonia (upper panel). The corresponding
marker genes expression among the Aundiff is also shown in the bottom panel.
of Id4 or Pax7 marks the majority of SSCs that reside in As
spermatogonia17,18 (Figure 7.1), and the stem cell capacity
gradually decreases as spermatogonia advance from As to
Aal stages.19,20 While these markers are useful for labeling
different spermatogonial subpopulations, the functional
activity of SSCs is defined by the ability to colonize the
seminiferous tubules and regenerate spermatogenesis
after transplantation.21,22
THE NICHE OF SPERMATOGONIA
Spermatogonia reside in the basal compartment of the
seminiferous tubules (i.e., the compartment between
the tight junctions of neighboring Sertoli cells, also
known as the blood-testis barrier (BTB), and the base-
ment membrane of the seminiferous tubules.1 Within the
basal compartment, the Aundiff preferentially localized to
a specialized niche that plays essential roles in modulat-
ing the self-renewal, differentiation, and migration. Aundiff
migrates to their niche during the establishment from
gonocyte and leaves it when they differentiate.5 In a physi-
ological condition, the BTB is formed after Aundiff arrives at
the basement membrane due to the immaturity of Sertoli
cells in neonatal testis.23
The spermatogonial niche comprises of various cell
types and extracellular matrix (ECM) proteins. The Sertoli
cells and the ECM of the basement membrane are in clos-
est proximity with Aundiff in the niche. Aundiff directly inter-
act with the ECM through cell adhesion molecules. Apart
from the Sertoli cells, other somatic cells, including the
Leydig cells, peritubular myoid cells, immune cells such as
The niche of spermatogonia  95
c-KIT positive
Aal–4 Aal (8–32 cells)
Aal (3, 5 or 6 cells)
Incomplete mitosis Syncytial fragmentation
macrophages,24 and probably the neighboring spermatogo-
nia also contribute to the niche through paracrine signaling25
(see section “Autocrine/paracrine regulation of spermatogo-
nia”) (Figure 7.2). Moreover, by chasing the location of Aundiff
using time-lapse imaging in vivo, it was found that Aundiff
is biasedly localized to the vascular network. Alteration in
vasculature leads to the rearrangement of Aundiff,5 suggest-
ing that the vasculature pattern may act as a backbone of the
spermatogonial niche in the seminiferous tubules.
Apart from the vasculature niche in the convoluted
seminiferous tubules, a recent study has identified a sec-
ond niche of GFRα1+ Aundiff at the tubuli rectus26 (Figure
7.2), a straight seminiferous tubule that leads the convo-
luted seminiferous tubule to the rete testis. This region
comprises a valvelike structure set up by a group of modi-
fied Sertoli cells (also known as Sertoli valve) that retained
proliferative capacity in adult life as well as a stable number
of GFRα1+ Aundiff but not more differentiated germ cells.
Since proliferating cells often show differential expression
of tight junction molecules compared to nonproliferating
cells,27 the question as to whether the BTB is functional
in the Sertoli valve niche remains elusive. This region also
expresses a higher level of the glial-derived neurotrophic
factor (GDNF), a Sertoli cell-secreted factor essential for
the self-renewal and maintenance of SSCs, compared to
that in the convoluted seminiferous tubules.26 Therefore,
the Sertoli valve niche is postulated to serve as a reserve
pool of SSCs in the testis. However, the stem cell activity in
the GFRα1+ Aundiff population of this niche awaits further
investigation.
96  The mitotic phase of spermatogenesis

Sertoli valve niche

Nondividing Proliferating
Sertoli cell Sertoli cell Testis

TJs ? GDNF Seminiferous

tubules Rete
As Apr testis
Basement membrane
Blood
Myoid cells stream
GDNF-
Leydig cells positive

Vasculature niche
Nondividing Spermatocytes
Sertoli cell
Epididymis
Nondividing
As TJs Sertoli cell
Apr Aal

Basement membrane
Myoid cells

Leydig cells Blood

vessel
Macrophage

Figure 7.2  Spermatogonial niches in mouse testis. Schematic diagram showing the localization of the Sertoli valve niche near the
rete testis and the vasculature niche at the seminiferous tubules (right panel). The somatic cells within the Sertoli-valve niche include
Sertoli cells (both proliferating and nonproliferating), Leydig cells, and peritubular myoid cells whereas the As and Apr spermatogo-
nia are the only germ cells in this niche (upper left panel). Compared to the vasculature niche, the Sertoli-valve niche expresses a
higher level of GDNF and the tight junctions (TJs) may be absent (as indicated by ? in the upper left panel) due to the proliferation of
Sertoli cells. The Sertoli cells in the vasculature niche are nonproliferative and form the blood-testis barrier with neighbor Sertoli cells
through TJs. Blood vessel and macrophage also contribute to the vasculature niche (bottom left panel). Differentiated spermatogo-
nia (denoted in purple) are also found in the vasculature niche but not in the Sertoli valve niche.

INTERACTION BETWEEN SPERMATOGONIA interact with laminin, β1-integrin is expressed in Aundiff

AND THE NICHE
Differentiating spermatogonia are not found at the sper-
matogonial niche,5 suggesting that spermatogonial differ-
entiation may be associated with cell migration to allow
the dissociation of committed progenies from the niche.
On the other hand, a minority of As spermatogonia may
be formed by syncytial fragmentation of Aal spermatogo-
nia.14 These As spermatogonia need to be associated with
the niche in order to receive support for self-renewal.
These studies highlighted the importance of the dynamic
interaction between spermatogonia and the niche.
Similarly, after transplantation of SSCs into the seminifer-
ous tubules, the transplanted cells need to migrate across
the BTB and reach the niche in order to regenerate sper-
matogenesis. The migration of SSCs to the spermatogonial
niche is described as homing. Although homing of trans-
planted SSCs does not occur in physiological condition,
this model provides a unique opportunity for the study of
spermatogonial migration.
Similar to the migration process in other cell types, the
migration of spermatogonia involves the modulation of
cell adhesion and the interaction with ECM (Figure 7.3).
An important ECM protein in the spermatogonial niche
is laminin. It is abundantly expressed at the basement
membrane of the seminiferous tubules and supports the
in vitro propagation of spermatogonia in the absence of
a feeder layer.4 Among the cell adhesion molecules that
and antibody for β1-integrin has been used for the enrich-
ment of SSCs.28 β1-integrin plays an indispensable role in
the homing of SSCs. Gene knockout or neutralizing anti-
body treatment that deplete functional β1-integrin in pri-
mary spermatogonial culture decreases the attachment to
laminin and reduces the homing of SSCs after transplanta-
tion.29 A subsequent study has demonstrated that Rac1, a
downstream target of integrin receptor, is required for the
migration of SSCs across the BTB through regulating the
level of cell adhesion molecule claudins.23 Intriguingly,
the Rac1-mediated migration is not required for homing of
transplanted SSCs in neonatal testis where BTB is absent,23
suggesting the migration during gonocyte to spermatogo-
nia transition may be regulated by an independent pathway.
Apart from the cell adhesion molecules, the differentiat-
ing spermatogonia also express a range of matrix metallo-
proteinases (MMP) that are capable of degrading different
ECM proteins.30,31 The expression of MMP-2, 7, 9, 23, and
CD147, a recognized regulator of MMPs activity and cell
survival, increase along with spermatogonial develop-
ment in neonatal mice. Depleting CD147 in a differentiat-
ing spermatogonia cell line GC1-spg inhibits the activity
of MMP2 and suppresses cell migration.30 Intriguingly,
in contrast to its recognized function in the survival of
GC2-spd spermatocyte cell line through interaction with
its partner TRAF2 that modulates the NFkB signaling,32,33
CD147 is dispensable for the maintenance of GC1-spg due
 Autocrine/paracrine regulation of spermatogonia  97

Self-renewal and maintenance Differentiation

Sertoli cell Spermatocyte Sertoli cell Spermatocyte

TJs TJs
GDNF
FGF2
GFR 1, KITL RA
c-Ret FGFR1 As BMP4 RARs
Wnt5a c-KIT
As Nodal FGF8
Apr Aal Vitamin
CSF-1
Laminin-containing basement membrane Laminin-containing basement membrane

Blood Blood
Leydig cells
Leydig vessel vessel
cells Macrophage Macrophage

Homing and interaction with the niche

Sertoli cell Spermatocyte

CXCL12
CXCR4 TJs
Apr CD147 Aal
As
MMP2
1-integrin
Laminin-containing basement membrane

Leydig cells Blood

vessel
Macrophage

Figure 7.3  Factors involved in different biological processes in the mitotic phase of spermatogenesis. Schematic diagram
showing the factors (highlighted in red) involved in the homing of SSC and the interaction between the niche and different sper-
matogonial population (bottom panel), the self-renewal of SSC and the maintenance of spermatogonia (upper left panel), and the
spermatogonial differentiation (upper right panel). Aundiff (c-Kit–) is denoted in green whereas differentiating spermatognia (c-Kit+)
and primary spermatocyte are denoted in purple.

to the absence of TRAF2 expression.32,33 This suggests a
primary role of CD147 in spermatogonial migration but
not cell survival.
A recent study comparing the transcriptome of Aundiff
and differentiating spermatogonia during spermatogonial
development has shed light on the molecular mechanism
underlying the migration of spermatogonia. The results
showed that c-Kit+ differentiating spermatogonia express
a higher level of genes involved in epithelial-mesenchymal
transition (EMT), a process well-established to be involved
in cell migration compared to that of Id4+ As spermatogo-
nia.34 Moreover, genes involved in cell adhesion and ECM
demonstrated dynamic methylation pattern in As versus
that in differentiating spermatogonia.34 These results sug-
gest that the differentiating spermatogonia possess higher
migration ability that may be attributed to the differential
interaction with ECM of the niche.
AUTOCRINE/PARACRINE REGULATION
OF SPERMATOGONIA
The niche where spermatogonia reside contains cyto-
kines and growth factors that regulate the self-renewal,
differentiation, and migration of spermatogonia in a para-
crine manner35 (Figure 7.3). Two best-characterized growth
factors required for self-renewal are GDNF and fibroblast
growth factor 2 (FGF2, also known as bFGF) secreted by
the Sertoli cells. FGF2 may bind to the four known recep-
tors FGFR1-4 expressed in spermatogonia,36 whereas
GDNF binds to the GFRα1-c-Ret coreceptor on sper-
matogonia. Both factors promote the self-renewal of SSC
independently by the activation of AKT and MAP2K1/2
pathway.37 FGF2 is also expressed by the Leydig cells and
differentiating germ cells that may contribute to the para-
crine regulation in vivo.37 Activation of signaling pathways
by GDNF and FGF2 induces the expression of transcrip-
tion factors, Plzf, Bcl6b, and Etv5, that play critical roles
in SSC self-renewal.38,39 The Leydig cells also contribute to
the paracrine regulation of spermatogonial self-renewal by
secreting colony-stimulating factor 1 (CSF-1) that stimu-
lates the self-renewal of SSC with modest effect on overall
proliferation of spermatogonia.40
Recent studies have demonstrated that a pan-Wnt
antagonist significantly reduces the SSC activity in cul-
ture, suggesting the involvement of Wnt signaling cascade
98  The mitotic phase of spermatogenesis
in modulating SSC self-renewal.41 Subsequent studies have
shown that Wnt5a directly promotes SSC self-renewal
through β-catenin-independent pathway whereas Wnt3a
indirectly modulates SSC self-renewal by promoting the
expansion of committed progenitors through β-catenin-
dependent pathway.42 Wnt6 is another Wnt ligand uniquely
expressed in Sertoli cells that may contribute to the prolif-
eration of Aundiff.43 Apart from the positive regulation on
Aundiff, Wnt4 secreted by the Sertoli cells induces cell death
and decreases the stem cell activity of cultured spermato-
gonia.44 These results suggest that the Sertoli-cell-secreted
Wnt ligands are pivotal paracrine signals that differen-
tially orchestrate the maintenance of Aundiff.
Paracrine signaling in the niche also regulates sper-
matogonial differentiation. Retinoic acid (RA), an active
metabolite of vitamin A, is a well-established spermatogo-
nial differentiation inducer.45,46 In the testis, the biogenesis
and catabolism of RA are regulated by the expression of
CYP26B1 in Sertoli cells and RALDH2 in germ cells.47 RA
generated by the Sertoli cells binds to the nuclear RA recep-
tors (RARs) in spermatogonia and transcriptionally acti-
vates or represses gene expression through direct binding
to the RA response element (RARE). Thus far, three RARs
(RARα, RARβ, and RARγ) have been identified. Knockout
of either one of these RARs disrupts spermatogenesis at
different stages, indicating their importance in spermato-
genesis.48–50 RA treatment in cultured spermatogonia or in
mice in vivo both upregulates the expression of transcrip-
tion factors Stra8 and Sall4 that are implicated in sper-
matogonial differentiation.51–53 RA also downregulates
Oct4 and Plzf, the transcription factors that are involved
in spermatogonial self-renewal.45 Recent studies have
shown that ectopic overexpression of RARγ in spermato-
gonia primes spermatogonial differentiation,20 whereas
inactivation of RA signaling by expression of a dominant
negative RAR abolishes spermatogonial differentiation.54
Our recent study has also shown that in a spermatogonial
cell line C18-4,55 RARα, but not RARβ and RARγ, tran-
scriptionally activates a membrane channel protein CFTR
(our unpublished data), which has been recently shown
to be involved in regulating the differentiation of embry-
onic stem cells.56 These results suggest differential roles of
RARs in mediating the effect of RA through both direct
and indirect activation of spermatogonial differentiation
regulators.
Besides the biogenesis of RA, the Sertoli cells also secrete
the stem cell factor (also known as c-kit ligand KITL) at
the basal membrane at a particular stage of the cycle of the
seminiferous epithelium when it is known to interact with
differentiating type A spermatogonia.57 The stem cell fac-
tor is required for the differentiation of Aundiff and mainte-
nance of differentiating spermatogonia by binding to the
c-Kit receptor expressed on the differentiating spermato-
gonia.58 The expression of c-Kit per se is also under para-
crine regulation from Sertoli cells. Bone morphogenetic
protein 4 (BMP4) secreted by the Sertoli cells in neonatal
testis exerts a mitogenic effect and induces c-Kit expres-
sion in cultured spermatogonia.59 It also acts cooperatively
with RA and exacerbates the spermatogonial differen-
tiation.60 Moreover, it impairs the maintenance of SSC in
primary spermatogonial culture.61 Interestingly, BMP4 is
also expressed in spermatogonia in adult testis,59 suggest-
ing the potential involvement of spermatogonial cells in
paracrine regulation.
Paracrine factors may have dual roles. For example,
apart from its well-characterized effect on the self-renewal
of SSCs, GDNF also regulates SSC homing by acting as a
chemotactic factor.62 Treatment of GDNF in cultured sper-
matogonia increases their migration beneath the Sertoli
cells through the BTB. Expression of a dominant nega-
tive GDNF receptor reduces the colonization of SSCs after
transplantation.62 GDNF also increases the expression of
chemokine receptor type 4 (CXCR4), which binds to the
Sertoli-cell-secreted chemokine (C-X-C motif) ligand 12
(CXCL12). CXCL12 treatment increases the migration of
cultured spermatogonia and enhances the colonization
of transplanted SSCs.62–64 Overexpression of CXCL12 in
Sertoli cells in vivo also enhances the homing of SSCs.
These results suggest the involvement of paracrine signal-
ing in regulating the migration of spermatogonia.
In contrast with the paracrine growth factors, little is
known about the autocrine regulation in spermatogo-
nia. In 2009, He et al. identified Nodal, a member of the
transforming growth factor-beta superfamily, as the first
autocrine factor that promotes the proliferation of sper-
matogonial stem/progenitor cells.65 A recent study has
shown that FGF8 is another autocrine factor that acts
through FGFR1 to elicit signaling required for the main-
tenance of Aundiff.66 FGF8 is expressed in spermatogenic
cells in a seminiferous tubules cycle-dependent manner
while its receptor, FGFR1, is expressed in Aundiff and Sertoli
cells. Overexpression of FGF8 leads to the accumulation
of Aundiff. Knockout of both FGF8 or FGFR1 reduces the
number of Aundiff. More importantly, conditional knock-
out of FGFR1 in Aundiff blunted the accumulation of Aundiff
induced by FGF8 overexpression, suggesting that the
action of FGF8-FGFR1 signaling is cell-autonomous.66
These studies suggest that spermatogonia are also sub-
jected to autocrine regulation.
Recently, emerging players in the autocrine/­paracrine
regulation of spermatogonia have been identified. Sahin
et  al. have established a spermatogenesis ­ regeneration
model by first depleting spermatogenic cells except Aundiff
using irradiation followed by the induction of spermatogo-
nial differentiation by administration of ­gonadotrophin-​
releasing hormone agonist. Using this model, they observed
that the Desert Hedgehog ligand Dhh, the Hedgehog recep-
tor Ptc 2, and the receptor downstream signaling mole-
cules Gli1 and Gli2 are expressed in Aundiff, suggesting the
involvement of Hedgehog signaling in the autocrine regu-
lation of spermatogonia.67 Apart from the involvement of
cytokines and growth factors, a recent study has suggested
the involvement of purinergic signaling in the a­ utocrine/
paracrine regulation of spermatogonia.68 Purinergic sig-
naling is mediated by the binding of extracellular nucle-
otide or nucleoside such as ATP to the P2 receptor. Two
 Ubiquitin proteasome system in the regulation of spermatogonia  99
subclasses of P2 receptors are metabotropic P2Y receptors
(P2YRs) and ionotropic P2X receptors (P2XRs) that acti-
vate G-protein–coupled signaling and nucleotide-gated ion
channels, respectively.69 By employing electrophysiological
techniques, Flect et al. have shown that spermatogonia are
sensitive to a board concentration range of extracellular
ATP. They have further demonstrated two modes of ATP
response: high-affinity (10-µM extracellular ATP) and low
affinity (>300-µM extracellular ATP), which are mediated
by P2X4 and P2X7 receptors, respectively.69 Further study
is required to demonstrate the physiological role of puri-
nergic signaling in the autocrine/paracrine regulation in
spermatogonia.
UBIQUITIN PROTEASOME SYSTEM IN THE REGULATION
OF SPERMATOGONIA
Stem cell homoeostasis often requires a precise orches-
tration of protein networks. Therefore, it is expected that
the ubiquitin-proteasome system (UPS), which is capable
of regulating protein turnover and activity, could play
important roles in this process. The UPS begins with
ubiquitination, a process where ubiquitin, an evolution-
ary conserved small peptide, is covalently conjugated to
the target proteins. This process includes three different
enzymes: E1 activating enzyme, E2 conjugating enzyme,
and E3 ubiquitin ligase. The ubiquitin is first activated by
the E1 enzyme at the cost of ATP. The activated ubiquitin
is then transferred to the E2 enzyme, and in the presence
of a E3 ligase the ubiquitin will be conjugated to target
protein in a substrate-specific manner.70,71 Ubiquitination
can be a single-round or multiple-round event that leads to
monoubiquitination or polyubiquitination, respectively.
In a polyubiquitination event, the incoming or distal ubiq-
uitin is piled up to the previous or proximal ubiquitin at
one of the seven lysine residues (K) on the ubiquitin pep-
tide. Polyubiquitination of K48 chain is often recognized
by the proteasome that leads to the degradation of the
target protein. Other types of ubiquitination can be rec-
ognized as posttranslation modifications that regulate the
localization and activity of target protein.72 Ubiquitination
can be reversed by deubiquitinating enzymes (DUBs)
that provide a path for recycling of ubiquitin after deg-
radation or for modification of ubiquitin chain on target
proteins.73 Emerging evidence has suggested the involve-
ment of functionally versatile UPS in the mitotic phase of
spermatogenesis.74
The requirement of protein degradation or UPS in the
regulation of spermatogonia came from two studies that
employed Rattus and Drosophila as model systems. The
study in Drosophila has used an unbiased large-scale
RNAi screening approach to identify genes involved in
male germline stem cells maintenance and differentia-
tion. Complex analysis of the screening result has revealed
the genes involved in protein synthesis and degradation,75
suggesting the involvement of UPS in the process. The
study in Rattus has shown that the RA-induced differen-
tiation in gonocytes can be blocked by proteasome inhibi-
tors lactacystin or bortezomib, indicating the requirement
of proteasome activity during gonocyte differentiation.76
Gene array analysis comparing the expression profile of
UPS genes in gonocytes and spermatogonia isolated from
neonatal testis has identified 205 UPS genes. Among them,
28 genes showed differential expression in gonocyte ver-
sus spermatogonia. Intriguingly, genes encoding ubiqui-
tinating enzymes have been demonstrated to have higher
expression in the gonocytes whereas those encoding for
deubiquitinating enzymes have higher expression in sper-
matogonia,76 suggesting the involvement of UPS in the
establishment of spermatogonia.
In the mitotic division of spermatogonia, it has been
proposed that both symmetric and asymmetric division
occurs. Symmetric division allows the stem cell to become
two stem cells or two committed progenies while asym-
metric division allows the stem cell to give rise to a stem cell
and a committed progeny.9 The asymmetric segregation of
cell fate determinant could be one plausible mechanism
for determining the identity of progenies.9 The deubiq-
uitinating enzyme UCH-L1 is expressed in spermatogo-
nia and asymmetrically distributed to the two progenies
in the asymmetric division of spermatogonia. A higher
level of UCH-L1 is observed in Plzf+ Aundiff spermatogonia
whereas c-Kit+ differentiating spermatogonia inherit low/
undetectable level of UCH-L1.77 This result suggests that
UCH-L1 may be involved in regulating the self-renewal
and differentiation of spermatogonia.
The functional role of UPS in the mitotic phase of sper-
matogenesis can be highlighted by the E3 ligases involved
in Skp1-Cullin-F-box (SCF) complex. The SCF complex
is one of the major ubiquitin ligases complex required for
cell cycle progression.78 The F-box protein component of
the SCF complex is an E3 ligase that binds to the complex
and controls the substrate recognition. Various F-box pro-
teins can bind to the SCF complex, and this variability
allows the SCF complex to target a range of substrates.79
Skp2 is an F-box protein required for S phase progression
by target degradation of cyclin-dependent kinase inhibitor
p27.80 A study has shown that knockout of Skp2 reduces
fertility, which is associated with the progressive loss of
spermatogonia and an increase in apoptosis. More impor-
tantly, double knockout of Skp2 and p27 partially restored
the number of germ cells and the fertility outcome, sug-
gesting that Skp2 is required for the maintenance of sper-
matogonia by targeting p27.81
β-TrCP is another well characterized E3 ligase of the SCF
complex that targets a number of the cell cycle and apopto-
sis regulators. There are two paralogs: β-TrCP1 (also known
as BTRC) and β-TrCP2 (also known as Fbxw11). The two
paralogs possess indistinguishable biochemical properties
but are varied in their expression. β-TrCP1 demonstrated a
high expression in the spermatocytes and a median expres-
sion in the spermatogonia. In contrast, β-TrCP2 showed a
median expression in spermatogonia and low expression in
the spermatocytes.82 Of note, the two paralogs showed non-
redundant roles in the testis. Knockout of β-TrCP1 leads
to impairment in meiotic progression and male subfertil-
ity.83 Intriguingly, loss of total β-TrCP activity by inducible
100  The mitotic phase of spermatogenesis
knockdown of β-TrCP2 in β-TrCP1 knockout mice leads
to an ectopic localization of spermatogonia associated with
impaired cell junctions. This phenotype is attributed to
the stabilization of Snail1, a bona fide substrate of β-TrCP
involved in regulating the transcription of cell adhesion
molecules, since knockdown of Snail1 restored the local-
ization of spermatogonia and rescued the entire spermato-
genesis. These results suggest that β-TrCP regulates the cell
adhesion of spermatogonia by targeting Snail1.
Recently, another F-box protein FBXW7 has been shown
to be expressed specifically in Aundiff and regulate the self-
renewal of SSC. Knockout of FBXW7 leads to accumula-
tion of Aundiff and increases the colonization of transplanted
SSC while its overexpression compromises SSC activity.
Moreover, loss of FBXW7 is associated with an increase
of MYC protein. Double knockdown of MYC and MYCN
mimics the effect of FBXW7 overexpression and obliterate
SSC activity.84 These results suggest that FBXW7 negatively
regulates SSC self-renewal by targeting MYC.
Apart from the SCF complex, recent studies showed
that E3 ligase HUWE1, which is an established tumor sup-
pressor known to target p53 and MYCN,85–87 is expressed
in both spermatogonia and spermatocytes.88 HUWE1
protein isolated from testicular lysate can ubiquitinate
core histone proteins.89 Knockout of HUWE1 specifi-
cally in neonatal germ cells perturbed the transition from
gonocytes to spermatogonia (our unpublished data). These
results suggest that HUWE1 positively regulates the estab-
lishment and maintenance of spermatogonia. Together,
these studies suggest the pivotal roles of UPS in regulating
the self-renewal and maintenance of spermatogonia.
NONCODING RNA IN THE REGULATION
OF SPERMATOGONIA
The previous sections have revealed the importance
of protein as intrinsic factors in regulating the mitotic
phase of spermatogenesis. However, the involvement of
genes/nucleic acid should not be overlooked. Mammalian
genome analyses show that the protein-coding genes
only contribute to 5%–10% of the genome. However, the
remaining >90% of the genome are not junk DNA. Rather,
these regions are actively transcribed into RNA, which is
classified as noncoding RNA (ncRNA). Depending on the
size/nucleotide (nt) length, ncRNA can be further divided
into long (lncRNA; >200 nt) or short (<200 nt) ncRNA.
Short ncRNA, such as PIWI-interacting RNA (piRNA),
plays important roles in silencing retrotransposon and
safeguarding the germline genome during meiotic pro-
gression.90 MicroRNA (miRNA) is another class of short
ncRNA that is well reported to regulate gene expres-
sion posttranscriptionally by binding to the untranslated
region of the mRNA. lncRNAs are expressed from a wide
variety of genomic locations. They can be expressed from
both autosomes and sex chromosomes in either sense or
antisense direction. They can be either intergenic or in
association with a gene at the promoter, exon, intron, or
the untranslated region. Given their broad spectrum of
expression, lncRNAs play versatile roles in a range of cellu-
lar processes. lncRNAs can act as baits to attract the bind-
ing of transcription factors and impair their interaction
with DNA and hence their functions. lncRNAs can also
act as decoys for RNA species. The binding of lncRNA to
mRNA can regulate their degradation and nuclear export.
The binding of lncRNA to miRNA can perturb their bind-
ing to bona fide mRNA targets and thus inhibit their activ-
ity. As well, the binding of lncRNA to genomic DNA has
also been shown to recruit epigenetic modifiers that mod-
ulate epigenetic events such as methylation and histone
modification. Finally, lncRNA acts as a precursor that after
further processing by RNase will give rise to miRNA. This
property provides a reservoir of miRNA without transcrip-
tion.91 Intriguingly, emerging evidence has suggested that
both the miRNA and lncRNA are involved in regulating
the mitotic phase of spermatogenesis.
Using high throughput sequencing techniques, Niu et al.
have compared the miRNA expression in Thy1+ and Thy- tes-
ticular population in 6-day-old mice as well as in cultured
spermatogonia. The Thy1+ population is enriched in Aundiff
while the majority of the Thy1– population is comprised of
somatic cells. Analysis of the sequencing results showed
that ~50% of the annotated miRNAs in available databases
were expressed in these samples, suggesting that miRNA
is the predominant short ncRNA species in neonatal mice
testis.92 Further characterization by comparing the miRNA
profile in Thy1+ and Thy1– population has identified 139 dif-
ferentially expressed miRNAs, of which 84 were upregu-
lated and 55 were downregulated. They further identified
miR-21, an antiapoptotic factor, as one of the differentially
expressed miRNA under direct transcriptional control of
the GDNF signaling pathway. Moreover, inhibition of miR-
21 by antagomir oligonucleotide increases apoptosis and
decreases the SSC number and activity.92 These results sug-
gest an important role of miR-21 in SSC self-renewal.
He et al. have compared miRNA expression in GFRα1+
and GFRα1– population by microarray. They have identi-
fied miR-20 and miR-106a as two miRNAs preferentially
expressed in GFRα1+ spermatogonia and in spermatogo-
nial cell line C18-4. Transfection of miRNA mimics for
these two miRNAs can each promote the proliferation/
self-renewal of SSCs as reflected by the increased BrdU
uptake, the increased expression of proliferation marker
PCNA and self-renewal marker Plzf, and the increased SSC
activity as measured by transplantation assay. Consistent
with the gain-of-function model, antagomir against these
two miRNAs can each inhibit SSC numbers and activity.
Further studies have identified Stat3 and Ccnd1 as the
downstream targets of miR-20 and 106a that contribute to
SSC self-renewal.93
Yang et al. have demonstrated the important roles of
miR-221 and 222 in maintaining SSC stemness by sup-
pressing the translation of c-Kit. In hematopoietic stem
cells, protein expression of c-Kit is regulated by miR-221
and 222. Similarly, the expression of miR-221 and 222
is observed in c-Kit– spermatogonia and upregulated

by GDNF, FGF2, and CSF-1. On the other hand, RA
decreases the expression of miR-221 and 222 in spermato-
gonial culture. More importantly, inhibition of miR-221
or 222 decreases SSC activity while overexpression of
either one perturbs spermatogonial differentiation.94 RA
also represses the expression of other miRNAs. Tong et al.
showed that the miRNAs from two clusters miR17-92 and
its paralog cluster miR-106b-25 are both downregulated
by RA in spermatogonial culture. Intriguingly, knock-
out of miR17-92 cluster upregulates the expression of
miRNAs from the miR-106b-25 cluster and leads to mild
defects in spermatogenesis.95 Huszar and Payne showed
that expression of miR-146 is markedly decreased by
RA. Overexpression of miR-146 inhibits the RA-induced
expression of spermatogonial differentiation mark-
ers c-Kit, Stra8, and Sohlh1 while inhibition of miR-146
upregulates them.96 Chen et al. showed that the expression
of miR202-3p was elevated by GDNF but decreased by
RA. Knockout of miR202-3p decreases SSC activity and
induces differentiation into meiotic cells by inhibiting the
translation of RNA binding protein Rbfox2.97 Together,
these results suggest that miRNAs regulate both sper-
matogonial self-renewal and differentiation.
A bulk of nonannotated lncRNAs is differentially
expressed in the hallmark stages of testis develop-
ment,98–100  suggesting a hidden layer of molecular
regulation in spermatogenesis.101 Compared with the
spermatocyte and spermatid, the spermatogonia express
the largest number of lncRNAs. Microarray study showed
that among the 24,316 lncRNAs, 17,354 of them are
expressed in spermatogonial populations.102 Despite the
abundant expression in spermatogonia, the functions of
these lncRNAs in the mitotic phase of spermatogenesis
remain largely unknown. Nonetheless, emerging evi-
dence suggests that these lncRNAs play important roles
in spermatogonia. The meiotic recombination hot spot
locus (mrhl) is a lncRNA identified during the analysis
of meiotic recombination.103 Intriguingly, subsequent
silencing studies in a differentiating spermatogonial cell
line GC1-spg showed that mrhl regulates the expression
of genes involved in cell adhesion and differentiation and
negatively regulates Wnt signaling through interaction
with p68/Ddx5 RNA helicase.104,105 Reciprocally, treat-
ment of GC1-spg cells with Wnt3a downregulates the
expression of mrhl,106 suggesting a negative feedback loop
between mrhl and Wnt signaling. lncRNA033862 is a
lncRNA expressed in the antisense direction that encom-
passes exon 9 and the intronic sequence of the Gfra1
gene. Its expression is tightly dependent on GDNF.107
lncRNA033862 acts as an antisense transcript that binds
to Gfra1 mRNA according to the sequence complementar-
ity and positively regulates the level of Gfra1. Knockdown
of lncRNA033862 triggers apoptosis and decreases the
stemness of cultured spermatogonia as revealed by trans-
plantation.107 These studies suggest the functional impor-
tance of lncRNAs in the regulation of spermatogonial
self-renewal and differentiation.
Metabolism of spermatogonia  101
METABOLISM OF SPERMATOGONIA
Spermatogonia is one of the most active stem cell popu-
lations in adults. In order to sustain their cellular activi-
ties, they require a metabolic machinery that converts
the available nutrients in the microenvironment into
a sufficient amount of energy. Therefore, it is expected
that the metabolism affects the maintenance of sper-
matogonia. Intriguingly, recent studies suggest that the
metabolism of spermatogonia also affects the fate deci-
sion. The mammalian target of rapamycin (mTOR), an
atypical serine/threonine kinase, is a master regula-
tor of cell growth and metabolism.108 mTOR interacts
with different protein components to form two protein
complexes, mTORC1 and mTORC2, that have differ-
ent upstream input and downstream output.108 Among
these two complexes, the activity of mTORC1 is pre-
cisely regulated in order to maintain the homoeostasis of
spermatogonial self-renewal and differentiation. Plzf, a
transcription factor essential for the self-renewal of sper-
matogonia, 39 has been shown to induce the expression of
Redd1, which suppresses mTORC1 activity. Knockout of
Plzf elevates mTORC1 activity that increases cell growth
but decreases self-renewal.109 A recent study has further
shown that elevated mTORC1 activity is observed in
spermatogonia primed for differentiation.110 Knockout
of mTORC1 inhibitor Tsc2 in the whole spermatogo-
nial population promotes differentiation and leads to
germline degeneration whereas knockout of Tsc2 in dif-
ferentiating spermatogonia does not compromise the
spermatogonial pool.110 These results suggest that the
activity of mTORC1 is tightly regulated to maintain
the fate decision of spermatogonia.
The self-renewal of many types of stem cells relies on
anaerobic glycolysis.111 Similarly, glycolysis also enhances
the self-renewal of spermatogonia. The glycolytic activity
in spermatogonia is coupled with the level of MYC/MYCN
protein. Loss of MYC/MYCN diminishes glycolysis and
limits spermatogonial self-renewal. Chemical induction of
glycolysis promotes spermatogonial self-renewal induced
by growth factors in vitro.112 These results suggest that
spermatogonia use glucose as the major energy source. In
fact, Aundiff is preferentially located near the blood stream
where glucose is readily available.
Cellular metabolism produces reactive oxygen species
(ROS) as a byproduct of energy production. Emerging evi-
dence suggests the involvement of ROS in regulating stem
cell homoeostasis.113 Excessive ROS can induce DNA dam-
age that leads to apoptosis or senescence. Therefore, stem
cells possess antioxidant activity to safeguard genome
integrity. Intriguingly, while excessive ROS also leads to
apoptosis, completely removing the ROS species by scav-
enger treatment abolishes spermatogonial self-renewal.114
Moreover, knockout of ROS-producing enzymes, NADPH
oxidase 1 (Nox1) or 3 (Nox3), reduces spermatogonial
self-renewal.114,115 These results suggest that a fine bal-
ance in the level of ROS is required for the maintenance
of spermatogonia.
102  The mitotic phase of spermatogenesis
PERSPECTIVES
In this chapter, we have presented the key concepts and the
recent findings on the mitotic phase of spermatogenesis,
describing the sophisticated networks of nucleic acids and
proteins that act extrinsically or intrinsically in orches-
trating the homeostasis between the self-renewal and
differentiation of a heterogeneous population of spermato-
gonia. However, key questions regarding the biological
properties of spermatogonia remain to be answered. For
example, regarding the fate decision in spermatogonia,
while the asymmetric distribution of the fate determinant
is observed during division, the syncytial state spermato-
gonia undergo stochastic fragmentation that includes a
chain of cells rather than one daughter cell. Whether the
stochastic fragmentation also involves asymmetric segre-
gation of determinants or these are independent mecha-
nisms remain unresolved. Moreover, the determinants,
regardless of nucleic acid or protein, remain to be char-
acterized. Regarding the spermatogonial niche, both the
vasculature niche in convoluted seminiferous tubules and
the Sertoli valve niche adjacent to the rete testis have not
been completely characterized. The question as to whether
As spermatogonia residing in these niches are similar
remain to be tested. With the advances in characterizing
markers for spermatogonial subpopulations, different sub-
populations can be isolated for single-cell omics analyses.
Therefore, we anticipate the discovery of novel regulators
at a higher resolution. Functional studies of these emerg-
ing regulators will be the keys toward deciphering the
whole picture of the mitotic phase of spermatogenesis.
ACKNOWLEDGMENTS
This study was supported in part by National 973 proj-
ects (2013CB967401 and 2013CB967404), and GRF/
RGC grant  (CUHK 14127316 and 14129316). We thank
Dr. Ruan Yechun (Hong Kong Polytechnic University) for
the artwork.
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Genetics of mammalian meiosis
FANG YANG and P. JEREMY WANG
INTRODUCTION
In sexually reproducing organisms, diploid germ cells
undergo meiotic cell divisions to produce haploid gametes.
Meiosis occurs in both sexes. After fertilization, the diploid
state is restored in the resulting embryos. Meiosis comprises
one round of DNA replication followed by two successive
rounds of cell divisions. After DNA replication, each chro-
mosome consists of two sister chromatids. The chromatids
from the homologous chromosomes are called nonsister
chromatids. During meiosis I, homologous chromosomes
pair, synapse, recombine, and segregate. Recombination
occurs between nonsister chromatids of the homologous
chromosomes. Meiotic recombination, a hallmark of meio-
sis, is critical for faithful segregation of homologous chromo-
somes during the first meiotic cell division. Unlike meiosis
I, meiosis II involves segregation of sister chromatids and
thus is similar to mitosis. Because of meiotic recombina-
tion and random chromosome segregation, meiosis shuffles
genetic information and thus increases genetic diversity in
the offspring, which provides a constant source of genetic
variations for selection during evolution. Errors in meio-
sis are leading causes of genetic diseases including aneu-
ploidy (such as Down syndrome, Klinefelter syndrome, and
Turner syndrome), pregnancy loss, and infertility. Studies
from a variety of model organisms (yeast, fly, worm, plant,
and mouse) have elucidated many conserved and divergent
molecular processes in the regulation of meiosis. Genetic
and cell biological studies have identified a large number of
proteins that are specifically involved in meiosis. This chap-
ter examines the major advances in the study of mamma-
lian meiosis.
MEIOTIC INITIATION
The timing of meiotic initiation differs between sexes. In
males, spermatogonia (diploid germ cells) enter meiosis at
puberty, whereas in females, oogonia enter meiosis shortly
after sex determination at the embryonic stage. Retinoid
signaling regulates the timing of meiotic entry in mice.1,2
Retinoic acid induces the expression of Stra8. Stra8 is
required for meiotic entry in female germ cells at the fetal
stage and in male germ cells at puberty.3,4 Rec8, encod-
ing a meiosis-specific cohesin, is induced during premei-
otic S phase.5 The expression of Rec8 is Stra8-independent.
Retinoic acid induces the expression of both Stra8 and
Rec8, demonstrating that retinoic acid activates at least
two molecular pathways in meiosis.6 Fetal male germ cells
do not enter meiosis but instead enter mitotic arrest. It
turns out that the enzyme CYP26B1 degrades retinoids in
fetal male germ cells to prevent precocious meiotic entry
until puberty.2 FGF9 further inhibits meiotic initiation in
106
8
fetal male germ cells.7 Therefore, retinoic acid and FGF9
antagonistically regulate the sex-specific timing of meiotic
entry.
MAX, a partner of MYC family proteins, suppresses the
expression of meiosis genes in embryonic stem cells and
germline stem cells.8 Reduction of Max causes precocious
meiotic entry in these stem cells in a Stra8-dependent
manner, as evidenced by meiotic-like cytological changes,
but not in fibroblasts. MAX is a component of an atypical
polycomb repressive complex 1 (PRC1.6). Therefore, MAX
represses meiosis genes in stem cells partly through the
PRC1.6 complex.
MEIOC is expressed at the onset of meiosis and pres-
ent throughout the meiotic prophase I.9,10 In mice, germ
cells lacking MEIOC initiate meiosis but proceed to meta-
phase precociously without completing chromosomal
synapsis and meiotic recombination. MEIOC interacts
with YTHDC2, an RNA helicase with a YTH domain.
YTHDC2 is also required for meiosis.11 Both MEIOC and
YTHDC2 bind to RNA transcripts. However, the identity
of the associated transcripts differs between two studies.9,10
In the study by Abby et al., MEIOC/YTHDC2 binds to and
protects meiosis-specific transcripts upon meiotic entry.9
However, Soh et al. reported that MEIOC/YTHDC2 binds
to mitotic cell cycle-associated transcripts but not meiosis-​
specific transcripts.10 Soh et al. hypothesizes that the
MEIOC/YTHDC2 complex represses mitosis-associated
transcripts to facilitate mitosis to meiosis transition and
to promote a meiotic cell cycle program. Despite this dis-
agreement, the MEIOC/YTHDC2 complex is required to
maintain the lengthy meiotic prophase I program.
TELOMERE-LED MEIOTIC CHROMOSOME MOVEMENT
During the meiotic prophase I, chromosomes undergo
rapid movement. Chromosome movement facilitates pair-
ing of homologous chromosomes and resolves entangle-
ments between nonhomologs. Chromosome movement is
led by telomeres and driven by the cytoplasmic microtu-
bules.12,13 Chromosomal ends (telomeres) are attached to
the nuclear envelope through the SUN1-KASH5 nuclear
membrane proteins (Figure 8.1 and Table 8.1). SUN1, an
inner nuclear membrane protein, interacts with KASH5,
an outer nuclear membrane protein.14 KASH5 binds to
cytoplasmic dynein proteins, which are microtubule-
based motors.15 Three meiosis-specific proteins, MAJIN,
TERB1, and TERB2, form a meiosis-specific telomere
cap.16 TERB1 binds to telomeres by forming a heterocom-
plex with TRF1, a canonical telomere protein, and also
interacts with SUN1.17 MAJIN is a DNA-binding trans-
membrane protein. Therefore, MAJIN-TERB1/2 complex
 Chromosomal synapsis  107

Chromosomal synapsis

Chromosome movement SC/cohesins

KASH5, SUN1, MAJIN, SYCP1, SYCP2, SYCP3, SYCE1,
Meiotic entry
TERB1, TERB2, SPDYA, SYCE2, SYCE3, TEX12, REC8,
STRA8, CYP26B1 CDK2, Lamin C2 RAD21L, SMC1B, STAG3
DAZL, MAX
Meiotic recombination

Hotspot DSB formation DSB repair Crossover

PRDM9 SPO11, TOPOVIBL, DMC1, MND1, HOP2, MLH1, MLH3, RNF212
HORMAD1, IHO1, MEIOB, SPATA22, HEI10, CNTD1, MSH4,
REC114, MEI1, MEI4 MCMDC2, TEX15, MSH5, TEX11, EXO1,
TRIP13 HFM1, MUS81, BLM,
FANCJ

Additional meiosis proteins: DMRTB1, FMRP, HSPA2, MDC1, MEIOC, NXF2, RNF8, SCAI, SCML2, SETX, SOHLH2,
TET1, TEX14, TEX19, TH2A/TH2B, YTHDC2, ZRP318

Figure 8.1  A summary of proteins involved in the regulation of mammalian meiosis. The function of these proteins in meio-
sis have been demonstrated by studies of knockout mice. Additional meiosis proteins include proteins that cannot be definitely
placed into these categories or span more than one category. Description of genes is shown in Table 8.1. Most proteins in the piRNA
pathway are essential for male meiosis but are not shown here. The piRNA pathway proteins have been reviewed elsewhere.118,119
Developmentally essential genes with meiotic defects in germ cell specific conditional knockout mice are not listed.

physically links telomeres to the nuclear envelope.16 At
the leptotene-zygotene transition, telomeres are clustered
at one pole of the nucleus (referred to as bouquet forma-
tion) to ensure accurate homolog pairing and synapsis.
Both Speedy A (SPDYA) and CDK2 localize to telomeres
in meiotic germ cells.18–20 Speedy A is a noncanonical acti-
vator of cyclin-dependent kinases (CDK) and binds to
CDK2. CDK2 phosphorylates SUN1 in vitro. Therefore,
the Speedy A-CDK2 binding regulates the initial telomere-​
nuclear envelope attachment. Loss of any of these proteins
(SUN1, KASH5, MAJIN, TERB1, TERB2, Speedy A, and
CDK2) causes failures in chromosome movement, homo-
log pairing, and chromosomal synapsis, demonstrating
the requirement of telomere-led bouquet formation for
meiosis.
CHROMOSOMAL SYNAPSIS
During the extended prophase of meiosis I, homologous
chromosomes are physically juxtaposed in a process
called synapsis. The synaptonemal complex (SC) provides
the physical connection between homologous chromo-
somes during synapsis.21,22 The synaptonemal complex is
a 200-nm-wide tripartite protein structure consisting of
two lateral elements (LEs) and one central element (CE).
One LE is formed along the sister chromatids. Transverse
elements (TFs) connect the lateral elements and the central
element. Under transmission electron microscopy, the tri-
partite structure of SC is shown by the two electron-dense
LEs and one electron-dense CE in between.23 The LEs and
CE of the SC cannot be resolved as individual structures
under conventional light microscopy. Superresolution
imaging has not only revealed the tripartite structure
but also the organization of SC as a twisted helical struc-
ture.24 Immunoelectron microscopy and superresolution
imaging further demonstrate that the CE is a bilayered
helical structure.24,25
The prophase I is further divided into substages largely
based on the morphology of the synaptonemal complex:
leptotene, zygotene, pachytene, and diplotene. At the lep-
totene stage, synaptonemal complex proteins form axial
elements (AEs) along chromosomal axes but synapsis is
not yet formed. At the zygotene stage, chromosomal syn-
apsis is initiated but incomplete. At this stage, CE and TF
components connect the two AEs and thus only localize to
the synapsed regions. AEs are called LEs in synapsis. The
subsequent pachytene stage is characterized by full synap-
sis along the entire length of all pairs of homologous chro-
mosomes except for the X-Y chromosomes in male germ
cells. At the diplotene stage, CE and TF components begin
to disassemble from SCs to cause desynapsis of homolo-
gous chromosomes.
The tripartite structure of SC is highly conserved across
species from yeast to human. However, the SC proteins are
not conserved at the protein sequence level across species
but all contain the coiled-coil secondary structure, which
is a protein–protein interaction domain.21,22 Synaptonemal
complex proteins have been identified (Figure 8.1 and
Table 8.1). In mammals, SYCP2 and SYCP3 are LE com-
ponents.26–29 Four CE components are known: SYCE1,30
SYCE2,31 SYCE3,32 and TEX12.33 SYCP1 constitutes the
transverse filaments, with the N- and C-termini located at
CE and LEs, respectively.34 Crystal structures of several SC
proteins have been reported: C-terminal coiled-coil domain
of SYCP1,35 SYCP3,36 and SYCE3.37 Based on these structure
studies, SYCP1 C-terminal region and SYCE3 form homodi-
mers, respectively, whereas SYCP3 forms a tetramer. In addi-
tion, SYCP3 tetramer interacts with DNA and self-assembles
into filamentous structures that resemble LEs.36 In genetic
108  Genetics of mammalian meiosis

Table 8.1  Mouse genes involved in meiosis. Table 8.1 (Continued)  Mouse genes involved in
meiosis.
Gene
symbol Gene
(alias) Description symbol
Blm Bloom syndrome RecQ-like helicase (alias) Description
cdk2 Cyclin-dependent kinase 2 Rad21l RAD21 cohesin complex component like 1
Cntd1 Cyclin N-terminal domain containing 1 Rec114 REC114 meiotic recombination protein
CYP26B1 Cytochrome P450 family 26 subfamily B Rec8 REC8 meiotic recombination protein
member 1 Rnf8 Ring finger protein 8
Dazl Deleted in azoospermia like Rnf212 Ring finger protein 212
Dmc1 DNA meiotic recombinase 1 Scai Suppressor of cancer cell invasion
Dmrtb1 DMRT-like family B with proline-rich Scml2 Scm polycomb group protein like 2
(Dmrt6) C-terminal, 1 Setx Senataxin
Exo1 Exonuclease 1 Smc1b Structural maintenance of chromosomes 1B
Brip1 (FancJ) BRCA1 interacting protein C-terminal Sohlh2 Spermatogenesis and oogenesis specific
helicase 1 basic helix-loop-helix 2
Fmr1 (Fmrp) Fragile X mental retardation syndrome 1 Spata22 Spermatogenesis associated 22
Ccnb1ip1 Cyclin B1 interacting protein 1 Spdya Speedy/RINGO cell cycle regulator family
(Hei10) member A
Hfm1 HFM1, ATP-dependent DNA helicase Spo11 SPO11 meiotic protein covalently bound to
homolog DSB
Psmc3ip Proteasome (prosome, macropain) 26S Stag3 Stromal antigen 3
(Hop2) subunit, ATPase 3, interacting protein Stra8 Stimulated by retinoic acid gene 8
Hormad1 HORMA domain containing 1 Sun1 Sad1 and UNC84 domain containing 1
Hspa2 Heat shock protein family A (Hsp70) Syce1 Synaptonemal complex central element
member 2 protein 1
Iho1 Interactor of HORMAD1 Syce2 Synaptonemal complex central element
Ccdc155 Coiled-coil domain containing 155 protein 2
(Kash5) Syce3 Synaptonemal complex central element
Lmna (Lamin Lamin A protein 3
C2) Sycp1 Synaptonemal complex protein 1
Majin Membrane anchored junction protein Sycp2 Synaptonemal complex protein 2
Max MYC associated factor X Sycp3 Synaptonemal complex protein 3
Mcmdc2 Minichromosome maintenance domain Terb1 Telomere repeat binding bouquet formation
containing 2 protein 1
Mdc1 Mediator of DNA damage checkpoint 1 Terb2 Telomere repeat binding bouquet formation
Mei1 Meiotic double-stranded break formation protein 2
protein 1 Tet1 tet methylcytosine dioxygenase 1
Mei4 Meiotic double-stranded break formation Tex11 Testis expressed gene 11
protein 4 Tex12 Testis expressed gene 12
Meiob Meiosis specific with OB domains Tex14 Testis expressed gene 14
Meioc Meiosis specific with coiled-coil domain Tex15 Testis expressed gene 15
Mlh1 mutL homolog 1 Tex19 Testis expressed gene 19
Mlh3 mutL homolog 3 Hist1h2aa Histone cluster 1, H2aa
Mnd1 Meiotic nuclear divisions 1 (Th2a)
Msh4 mutS homolog 4 Hist1h2ba Histone cluster 1, H2ba
Msh5 mutS homolog 5 (Th2b)
Mus81 MUS81 structure-specific endonuclease TopoVIbl TopoVI DNA topoisomerase subunit B like
subunit Trip13 Thyroid hormone receptor interactor 13
Nxf2 Nuclear RNA export factor 2 Ythdc2 YTH domain containing 2
Prdm9 PR domain containing 9 Zfp318 Zinc finger protein 318
(Continued )

studies, loss of any of SC proteins results in a failure in the SC
assembly and thus a block or defect in meiotic progression.
In addition to SC proteins, cohesins are essential com-
ponents of the axial/lateral elements. The cohesin complex
is required for sister chromatid cohesion in both mitosis
and meiosis. The mammalian mitotic cohesin complex
consists of two structural maintenance of chromosome
(SMC) proteins (SMC1 and SMC3) and two non-SMC
subunits (RAD21 and STAG1 or STAG2). Four meiosis-
specific paralogues of mitotic cohesins have been identi-
fied in mammals: REC8,5,38 RAD21L,39–41 SMC1B,42 and
STAG3.43 REC8 and RAD21L are homologous to RAD21.
SMC1B bears sequence similarity with SMC1. STAG3
replaces STAG1/STAG2 in meiotic cohesin complexes.
All these meiosis-specific cohesin proteins localize to the
axial elements. Strikingly, absence of REC8 leads to syn-
apsis between sister chromatids, suggesting that one of the
main functions of REC8 is to limit synapsis to homologous
chromosomes.38,44 Genetic and cell biological analyses
have shown that these meiosis-specific cohesins regulate
the axial element formation, axis length, sister chromatid
cohesion, and meiotic recombination.45,46
While the identification and characterization of these
meiosis-specific structural proteins have elucidated their
role in SC formation, the mechanism regulating SC dis-
assembly is poorly understood. In budding yeast, polo-
like kinase Cdc5 promotes SC disassembly at the end of
the pachytene stage.47 In mouse spermatocytes, polo-like
kinase 1 (PLK1) localizes to the SC.48 PLK1 phosphory-
lates SYCP1 and TEX12. Treatment of spermatocytes with
the PLK1 inhibitor prevents removal of SYCP1 and TEX12
from the SC.48 In addition, PLK1 promotes dissociation
of REC8 and RAD21L from SC.39 Although a Plk1 mouse
mutant is needed to address its in vivo requirement, the
PLK1 function in SC disassembly appears to be conserved
between yeast and mouse.
The dynamic association of the cohesin complex with
chromatin during the mitotic cell cycle is controlled by
a number of regulatory proteins in the loading, mainte-
nance, and release of cohesins: NIPBL, Sororin, and WAPL.
While NIPBL is a cohesin loading factor, WAPL is a cohe-
sin releasing factor. WAPL interacts with PDS5, a cohesin-
binding protein, to remove cohesins. Sororin maintains
cohesion by antagonizing WAPL. Mechanistically, Sororin
binds to PDS5B to prevent association of PDS5B with
WAPL and thus inhibit the activity of WAPL.49 In meiotic
germ cells, all these factors (NIPBL, Sororin, PDS5, and
WAPL) localize to the synaptonemal complex, suggesting
the conserved functions of these regulatory factors in both
mitosis and meiosis.50–52 Furthermore, in meiosis, NIMA-
like kinase-1 (NEK1) phosphorylates protein phosphatase
1 gamma (PP1γ), which dephosphorylates WAPL to facili-
tate cohesin removal.52
Chromosomal synapsis is sexually dimorphic.53 In
female meiosis, the two X chromosomes, like autosomes,
are fully synapsed. However, in male meiosis, the X and
Y chromosomes only form synapsis in the pseudoauto-
somal regions, leaving the majority of sex chromosomes
Meiotic recombination  109
unsynapsed. Sex chromosomes are subjected to silenc-
ing at the pachytene and diplotene stages in a phenom-
enon termed meiotic sex chromatin silencing (MSCI).
Transcriptional silencing of sex chromosomes is a special
case of a general meiotic silencing response to chromo-
somal unsynapsis. Much progress has been made in under-
standing the regulation and function of MSCI. For further
interest in MSCI, please refer to a review by Turner.54
MEIOTIC RECOMBINATION
In many species including mouse, chromosomal synapsis
and meiotic recombination are interdependent. These two
processes are tightly coordinated. The synaptonemal com-
plex provides a structural framework for meiotic recom-
bination. Meiotic recombination initiates and stabilizes
chromosomal synapsis. Meiotic recombination leads to
reciprocal exchange of genetic material between homolo-
gous chromosomes (more specifically, between nonsister
chromatids). In addition, formation of chiasmata resulting
from recombination is essential for faithful segregation
of homologous chromosomes during the first meiotic cell
division. Meiotic recombination involves a large number
of proteins at several distinct stages, which are discussed
separately.
Programmed meiotic DSB formation
Meiotic recombination begins with the formation of DNA
double-strand breaks (DSBs). In mouse, about 300 DSBs
are generated in each meiotic germ cell. Formation of
DSBs is catalyzed by SPO11, a homologue of TopoVI DNA
topoisomerase subunit A (TopoVIA).55–57 The activity of
SPO11 requires TOPOVIBL, which displays ­ structural
similarity to the TopoVI DNA topoisomerase subunit B.58
TOPOVIBL interacts with SPO11 and is required for
DSB formation during meiosis. Additional proteins are
required for DSB formation: MEI1,59 MEI4,60 REC114,60
HORMAD1,61,62 and IHO1.63 MEI4 and REC114 interact
directly.60 HORMAD1 localizes to unsynapsed chromo-
somal axis and recruits IHO1 through direct interaction.
IHO1 recruits MEI4-REC114 to unsynapsed chromosomal
axis. The formation of this complex (HORMAD1-IHO1-
REC114-MEI4) activates the SPO11-TOPOVIBL com-
plex.63 MEI1 is also important for recruitment of MEI4.64
In conclusion, meiotic DSB formation is genetically pro-
grammed and meticulously executed (Figure 8.1).
DSB repair
Formation of DSBs entails a DNA damage response, lead-
ing to phosphorylation of histone H2AX by ATM.65,66
Following generation of DSBs, SPO11 is covalently
attached to the 5’ strands of both ends of the DSB. DSB
breaks undergo end resection to produce 3’ single-stranded
DNA (ssDNA) ends, releasing SPO11-oligonucleotide
complexes.67 RPA, an ssDNA-binding complex, coats the
ssDNA to protect it from degradation and prevent sec-
ondary structure formation.68 RAD51 and DMC1 are
also ssDNA-binding proteins and exhibit recombinase
activities. RAD51/DMC1 recombinase is loaded onto
110  Genetics of mammalian meiosis
ssDNA, displaces RPA, and directs strand invasion into
the duplex of the homologous chromosomes, resulting in
the formation of displacement loop (D-loop).69 DMC1 is
a meiosis-specific paralogue of RAD51.70 DMC1 favors
repair of meiotic DSBs through a nonsister chromatid
rather than a sister chromatid. The MND1/HOP2 het-
erodimer interacts with RAD51/DMC1 and stimulates
their recombinase activity to generate the D-loop recom-
bination intermediates.71 MCMDC2, a member of the
minichromosome maintenance (MCM) helicase family,
is required for RAD51- and DMC1-directed strand inva-
sion or stabilization of recombination ­intermediates.72,73
MEIOB is a ­meiosis-specific ssDNA-binding ­protein.74,75
MEIOB interacts and forms a heterodimer with SPATA22,
which is also a meiosis-specific protein.74,76 The MEIOB/
SPATA22 complex interacts with the RPA complex and
localizes to DSB sites.77 The MEIOB/SPATA22 complex
has been hypothesized to function in the second end
capture and maintenance of RAD51 foci.74,78 Second end
capture results in the formation of the recombination
intermediate–double Holliday junction (dHJ). Most pro-
teins involved in meiotic DSB repair form foci at the DSB
sites on meiotic chromosomes. Since meiotic recombina-
tion occurs in both male and female germ cells, loss of any
of these recombination-related proteins leads to meiotic
arrest and thus sterility in both sexes.
Crossover formation
Double Holliday junctions are resolved into either cross-
overs or noncrossovers. In mouse meiotic germ cells,
a subset of DSBs are turned into ~25 crossovers per cell
and the remaining DSBs result in noncrossovers. Each
pair of homologous chromosomes must have at least one
crossover, since crossover is required for proper chromo-
some segregation during the first meiotic cell division.
MSH4-MSH5 stabilizes dHJ structures and promotes
crossover formation (Figure 8.1).79 TEX11 promotes cross-
over formation and interacts with the synaptonemal com-
plex protein SYCP2.80 MLH1-MLH3 is an endonuclease
that is required for resolution of dHJs.81,82 MSH2-MSH3
stimulates the endonuclease activity of MLH1-MLH3.83
Exonuclease 1 (EXO1) is required for formation of chi-
asmata even though the number of MLH1 foci is normal
in its absence.84 The nuclease-dead Exo1 mutant mice are
fertile, implying that EXO1 plays a structural but not cat-
alytic role in meiosis.85 SUMO ligase RNF212 and ubiq-
uitin ligase HEI10 play antagonistic roles in crossover
designation.86 RNF212 and HEI10 mediate localization of
SUMO and ubiquitin to the synaptonemal complex, which
in turn recruits the proteasome.87 The proteasome regu-
lates homolog pairing and crossover formation in yeast.88
Cyclin-related protein CNTD1 regulates the association of
HEI10, RNF212, and MSH4 with the recombination inter-
mediates.89 Interestingly, HEI10, RNF212, and TEX11 are
dosage-sensitive regulators of crossover formation, sug-
gesting that they are rate-limiting factors.86,90 Importantly,
RNF212 and TEX11 regulate genome-wide recombination
rates.90,91 The majority of crossovers are formed through
the MLH1/MLH3-dependent class I pathway. The class
II pathway is minor and independent of MLH1/MLH3
but requires MUS81.92 These two crossover pathways are
coordinated because the class I pathway is upregulated as
shown by the increased accumulation of MLH1 foci in the
absence of MUS81.92
Meiotic recombination hotspot
Meiotic recombination occurs more frequently at cer-
tain genomic locations (so-called hotspots) than oth-
ers.93 The major advance in the study of recombination
hotspot is the identification of PRDM9. PRDM9 is a main
determinant of meiotic recombination hotspots in mam-
mals.94–96 PRDM9 binds to the 13-mer hotspot consensus
DNA motif through its array of DNA-binding zinc fin-
gers. PRDM9 catalyzes both trimethylation of lysine 4
of histone H3 (H3K4me3)97 and trimethylation of lysine
36 of histone H3 (H3K36me3).98 Meiotic recombination
hotspots are marked by both H3K4me3 and H3K36me3
modifications.99 H4K44 acetylation (H4K44ac) marks
meiotic recombination hotspots in budding yeast but its
relationship with mammalian hotspots is unknown.100
PRDM9 directs recombination away from functional
genomic elements such as gene promoters.101 The Prdm9
gene is rapidly evolving, especially in the zinc-finger cod-
ing region.102 This rapid evolution is driven by the inherent
“self-destruct” property of recombination hotspots.95 A
recombination hotspot is favored for DSB formation and
thus is more likely to be replaced with a coldspot allele
through homologous recombination. The 13-mer hotspot
consensus motif varies in sequences among species. As
a new hotspot consensus emerges, the zinc-finger region
of PRMD9 evolves to bind to the new hotspots and vice
versa. For example, humanizing the zinc-finger domain
of PRDM9 in mice changes the positions of DSB hotspots
in mouse.103 Prmd9 is the only known mammalian spe-
ciation gene and is responsible for the hybrid sterility
between mouse subspecies.104 How are H3K4me3-marked
hotspots recognized for DSB formation? Recent studies
provide insights into the connection of the hotspot to DSB.
PRMD9 binding leads to nucleosome depletion at the
hotspot, a possible permissive environment for DSB for-
mation.105 PRDM9 interacts with CXXC1, which in turn
interacts with IHO1.106 IHO1 is a part of the protein com-
plex that activates SPO11. In addition to CXXC1, PRDM9
interacts with EWSR1, EHMT2, and CDYL.107 In testis,
PRDM9 is complexed with REC8, SYCP1, and SYCP3.107
Therefore, the epigenetic and chromatin states specify the
location of DSBs and PRDM9 is recruited through asso-
ciation with cohesin and SC.
IN VITRO MEIOSIS
In vitro spermatogenesis has been pursued for nearly
a century. Many studies reported in vitro production of
haploid male germ cells or oocytes from embryonic stem
cells. The common drawbacks are the extremely low effi-
ciency and the lack of proof of gamete function. One of
the critical barriers is progression through meiosis in
 References 111

Table 8.2  Meiosis-specific genes implicated in human infertility.

Gene Description Clinical phenotype Mutation Reference
MEIOB Meiosis specific with OB Meiotic arrest (NOA) Missense 120

domains
MSH4 mutS homolog 4 POF Splice site 121

PSMC3IP PSMC3 interacting protein Ovarian dysgenesis Deletion of one residue 122

(aka HOP2) (Glu201)

SYCE1 Synaptonemal complex POF Nonsense 123

central element protein 1 Meiotic arrest (NOA) Splice site 124

SYCP3 Synaptonemal complex NOA Heterozygous a for frame- 125

protein 3 shift mutation (1 bp deletion)

STAG3 Stromal antigen 3 POF Frame-shift deletion 126

TEX11 Testis expressed gene 11 Meiotic arrest (NOA) Deletion, nonsense, splice 90,117

site, missense
TEX14 Testis expressed gene 14 Meiotic arrest (NOA) Frame-shift deletion 120

TEX15 Testis expressed gene 15 NOA Nonsense 127,128

Abbreviations: NOA, nonobstructive azoospermia; POF, premature ovarian failure.

vitro.108 The prophase of meiosis I in mouse spans 14 days,
8 days out of which are spent on the pachytene stage.109 The
pachytene stage is critical since crossovers are matured at
the mid-late pachytene stage. In previous testicular organ
cultures, germ cells were not able to progress beyond the
pachytene stage.110 A mouse testicular organ culture sys-
tem is successful for in vitro production of functional
sperm.111,112 The crucial factor for success in this system is
the use of serum-free culture media. In this organ culture
system, mouse testis tissue is used, thus preserving the
three-dimensional architecture of the testicular tubules.
Using this organ culture system, sperm are also derived
from mouse spermatogonial stem cells.112,113 In a cocul-
ture system with neonatal testicular somatic cells, primor-
dial germ cell-like cells derived from ES cells in vitro are
induced to complete meiosis to generate haploid spermatid-
like cells.114 In this system, cultures were sequentially
treated with retinoic acid, hormones, and other factors.114
Seminiferous tubules can be reconstructed from neonatal
testicular cells and cultured in vitro to support germ cell
differentiation but only up to the stage of pachytene.115
Retinoic acid is sufficient to induce mouse spermatogonial
stem cells to enter meiosis and proceed up to the zygo-
tene stage.116 These in vitro spermatogenesis systems will
facilitate mechanistic studies of meiosis. Optimization of
the existing in vitro meiosis systems and development of
new in vitro systems will likely revolutionize treatment
of human infertility.
IMPLICATIONS IN HUMAN INFERTILITY
Genetic studies in mouse and other model organisms have
identified many meiosis-specific proteins discussed here
and elucidated the molecular mechanisms underlying their
functions. These functional studies provide a strong ratio-
nale for genetic studies in humans. Mutations in meiosis-
specific genes are expected to be causes of human infertility.
To date, human genetic studies have identified causative
mutations in nine meiosis-specific genes (Table 8.2). These
mutations cause either azoospermia in men or prema-
ture ovarian failure in women. Most of these mutations
were found in the offspring of consanguineous marriage
and identified by exome sequencing of affected individu-
als. Homozygous mutations in MEIOB, PSMC3IP, STAG3,
SYCE1, TEX14, and TEX15 were identified in the offspring
of consanguineous couples (Table 8.2). Sequencing of
sporadic cases of infertile men reveals that mutations in
TEX11, an X-linked meiosis-specific gene, cause infertility
in ~1% of nonobstructive azoospermia.90,117 In the era of
precision medicine, future genomic sequencing of infertile
patients is expected to identify causative mutations in more
meiosis-specific genes. Such information will be valuable
for diagnosis and genetic counseling in the treatment of
infertility in the upcoming era of precision medicine.
ACKNOWLEDGMENTS
We would like to thank all our colleagues whose con-
tributions in this topic have not been cited due to space
limitation. This work is supported by National Institutes
of Health/National Institute of General Medical Sciences
(R35GM118052 to P.J.W.).
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Roles of membrane and nuclear
estrogen receptors in spermatogenesis
PAUL S. COOKE, MANJUNATHA K. NANJAPPA, SERGEI G. TEVOSIAN,
and REX A. HESS
INTRODUCTION
Androgens are essential for spermatogenesis, and exten-
sive progress has been made in understanding androgen
signaling in different testicular cell types. In contrast,
estrogen’s roles in the testis and spermatogenesis are less
clear. Estrogens are present systemically in males and tes-
ticular estrogen concentrations are higher than peripheral
concentrations due to testicular estrogen production. In
addition, estrogen receptors are widely distributed in the
male reproductive tract. Despite extensive literature show-
ing deleterious effects of perinatal estrogen administration
on the testis and spermatogenesis, estrogen’s role in the
normal testis and spermatogenesis, especially in humans,
remains less clear.
Before considering testicular estrogen effects, we briefly
review androgen effects on various testicular cell types to
contrast the discrepancy in information regarding the two
areas. Androgens are obligatory for spermatogenesis, and
testicular androgen effects are mediated through many cell
types. Sertoli cells are a major androgen target and adult
Sertoli cells express abundant androgen receptor (AR), but
Sertoli cells during development lack AR and only begin to
show slight AR immunostaining at about day 12 in mouse
testis.1 In a conditional knockout of AR in Sertoli cells,2
spermatogenesis proceeded only up to meiosis, indicating
an obligatory dependence for androgen signaling through
Sertoli cells for all but the earliest spermatogenic stages. In
these mice, there are increased adult Sertoli cell numbers,
indicating that signaling through AR normally inhibits
Sertoli cell proliferation.3
In contrast to somatic cells, developing testicular germ
cells lack AR and germ cell-specific AR knockout mice
are fertile with normal spermatogenesis.4 Thus, andro-
gen actions on spermatogenesis are mediated indirectly
through effects on Sertoli cells and other cells that have
paracrine effects on germ cells.
Peritubular myoid cells are separated from spermato-
gonial stem cells (SSCs) within the seminiferous tubule by
only the basement membrane of the seminiferous epithe-
lium. This suggests they may communicate with SSCs as
well as other germ and somatic cells. A peritubular cell-
specific AR knockout showed progressive spermatogonial
loss beginning at weaning, resulting in disorganized semi-
niferous epithelium and adult infertility5 and supporting
the idea that androgen actions through peritubular cells
regulate spermatogenesis.
116
9
Other cells also contribute to the SSC niche through
production of growth factors that affect SSC proliferation
and differentiation. For example, SSCs are preferentially
located near vascular elements outside the seminiferous
tubule,6 suggesting that testicular vasculature and/or asso-
ciated Leydig cells also regulate SSCs. Furthermore, auto-
crine actions of androgen in Leydig cells are required for
normal testicular function.7
Similar to androgens, estrogens affect male reproduc-
tion but the mechanisms of these effects are unclear.
Estrogens are produced in the testis and estrogen recep-
tors (ERs) are present in many testicular cell types, but
gaps remain in understanding estrogen’s role in spermato-
genesis. This chapter reviews testicular estrogen signaling
and also discusses how a lack of estrogen signaling affects
other male reproductive organs, resulting in secondary
impairments in spermatogenesis and fertility.
ESTROGEN SOURCES IN MALES
Estrogen regulation of male reproduction is now well rec-
ognized. Estrogens were first reported in males (stallions)
more than 80 years ago.8 Decades later, radioimmuno-
assay methodologies established that men and males of
other species had measureable circulating 17β-estradiol
(E2) concentrations. Testosterone (T) concentrations in
peripheral blood of men are typically two orders of mag-
nitude greater than E2 concentrations, indicating the pri-
macy of T compared to E2 for male reproduction. However,
E2 concentrations of approximately 30–200 pmol/L have
been reported in men,9 which are several-fold less than E2
concentrations reported in women during certain phases
of the menstrual cycle but greater than those for ovariec-
tomized female rats, rats in diestrus, and postmenopausal
women.
Ovaries are the major source of circulating estrogens in
females. However, in males, testes produce only about 20%
of circulating estrogens, with the remainder produced
by adipose, adrenals, brain, skin, bone, and other organs
expressing aromatase (Cyp19a1), a ubiquitous NADPH-
dependent cytochrome P450 reductase enzyme.10 Local
testicular estrogen production leads to estrogen concen-
trations in testis and seminiferous tubule fluid that exceed
circulating systemic levels. In monkeys, E2 concentra-
tions of 40–145 pmol/L have been reported in peripheral
blood.11 Concentrations in the testicular vein and rete tes-
tis (RT) are even higher (up to 640 pmol/L), illustrating
 Estrogen sources in males  117

both testicular E2 production and the potential for tes-
ticular cells to be exposed to high E2 concentrations.11
Although data are not available on E2 concentrations in
the testicular vein and RT of men, there are reports of
E2 concentrations in human semen of up to 520 pmol/L
(reviewed in Cooke et al.12). Similar increases in testicular
vein E2 vs. systemic E2 concentrations have been observed
in other species (reviewed in Cooke et al.12). Thus, cells of
the testis and other organs that come into direct contact
with seminiferous tubule fluid and the developing ejacu-
late (RT, efferent ductules, epididymis, ductus deferens,
urethra) can be exposed to high estrogen concentrations.
In the testis, aromatase is necessary for estrogen pro-
duction and is expressed in Leydig (Figure 9.1) and imma-
ture Sertoli cells.13,14 Sertoli cells were initially considered
the sole source of aromatase/estrogen in seminiferous epi-
thelia, but aromatase was identified in round spermatids
and in elongating and late spermatids in the early 1990s.15
Germ cell aromatase expression, originally described in
rodents,15 was subsequently reported in men as well as
other mammalian and avian species.12 Germ cells appear
to be the major testicular source or aromatase and thus
testicular E2.16 There are reports of epididymal aromatase
expression, which could contribute estrogen to the devel-
oping ejaculate. Thus, in many species estrogens produced
in testis and epididymis can signal through estrogen
receptor 1 (ESR1) in the male tract.
ER expression in male reproductive organs
Various types of ERs, including ESR1, estrogen receptor 2
(ESR2), and G protein-coupled estrogen receptor (GPER,
Testis
Lymphatics
PM
ESR1
GPER
Blood
? T
T E2
Arom ? AC
SC
LC ESR1
ESR1 ESR2
ESR2 GPER
GPER
originally known as G protein-coupled receptor 30) are
expressed in testis and other male reproductive organs.
Although most ESR1 and ESR2 are cytoplasmic/nuclear,
a small fraction of ESR1 and ESR2, as well as all of GPER,
are localized in the cell membrane, where these membrane
receptors mediate rapid estrogen actions.
Although there are species-specific differences, ESR1
expression has typically been reported in Leydig and peri-
tubular cells in mice and other mammals.17 Expression
of ESR1 mRNA and protein expression in Sertoli cells
has been reported, but it has not been universally con-
firmed.18–20 Testicular ESR2 expression is more wide-
spread, with expression in Leydig, peritubular, germ, and
Sertoli cells.21,22 Efferent ductules express both ESR1 and
ESR2, with ESR1 being especially abundant. Likewise,
epididymis and ductus deferens express ESR1 and ESR2,
but as in other organs, expression patterns are unique and
regionally variable.
The pattern of ER expression differs in primates com-
pared to other animals (reviewed in Cooke et al.12) fur-
ther complicating understanding ER’s role in the testis and
male tract. For example, ESR1 expression was minimal in
primate compared to rodent testis. Varying patterns of
ESR1 and ESR2 expression in other species have also been
reported (reviewed in Cooke et al.12), which in addition to
actual differences in ER expression may reflect differences
in antibody specificity, fixation, and animal ages as well as
species variations.
Expression of GPER in various testicular and other
reproductive cell types, such as Sertoli cells, germ cells,
epididymis, and sperm, is well documented. Consistent
Efferent ducts
ESR1
ESR2
ESR2 GPER
GPER
GC ? NC CC
Arom
E2 E2
BC
Epididymis
ESR1
ESR2
GPER
CL

Figure 9.1  Estrogen production and estrogen receptors in the male reproductive tract. In testis, Leydig cells and germ cells
(including sperm) express aromatase (pink) and convert testosterone (T) to 17β-estradiol (E2). ESR1 expression is low in testicular
cells, but ESR2 expression is higher and more ubiquitous. Both nuclear and membrane ERs are expressed throughout the male tract.
Specific targets for testicular E2 have not been established, but germ cell synthesis of E2 continues in the lumen of the reproductive
tract, where E2 affects the efferent ductules and epididymis as fluid transits through. The major target appears to be the efferent
ductules, where ESR1 concentrations are threefold greater than any other male or female tissue and both ciliated and nonciliated
cells are E2 targets. LC, Leydig cell; PM, peritubular myoid; SC, Sertoli cell; GC, germ cell; NC, nonciliated cell; CC, ciliated cell; AC, api-
cal cell (narrow); BC, basal cell; CL, clear cell; Arom, P450 aromatase.
118  Roles of membrane and nuclear estrogen receptors in spermatogenesis
with this, estrogen actions mediated partially or entirely
through GPER are known.22–26
Effects of early estrogen treatment in males
Beginning in the 1970s, early treatment of mice with the
potent synthetic estrogen diethylstilbestrol (DES) was
used to produce adult reproductive abnormalities. These
included impaired spermatogenesis and other testicu-
lar abnormalities, cryptorchid testes, epididymal cysts,
inflammatory orchitis and epididymitis, and accessory
organ abnormalities, frequently accompanied by infertil-
ity.27 Recent studies suggested that Sertoli cells may be a
major target for early DES effects on spermatogenesis.28
Studies using DES in animal models were partially driven
by reports of a rare cancer in young women whose moth-
ers had taken DES during pregnancy.29
ROLE OF E2/ESR1 IN MALE REPRODUCTION
Loss of ESR1 causes extensive male reproductive
defects
Despite E2 and ER presence in males and known deleteri-
ous effects of early DES treatment, it was unclear for years
whether E2/ER signaling was important in normal male
reproduction or reproductive pathologies. Over the past 20
years, development of transgenic mouse models in which
E2/ESR1 signaling was disrupted revealed that estrogens
are critical for normal development and function of male
reproductive and nonreproductive organs.
The first steroid hormone receptor knockout d ­ eveloped
was the Esr1 knockout (Esr1KO) mouse.30 This mouse
and its subsequent iterations31 provided important infor-
mation regarding E2/ESR1 signaling in male and female
reproduction. The infertility originally reported in
Esr1KO males32 was subsequently shown to result in large
part from impaired efferent ductule and epididymal func-
tion.33,34 Specifically, impaired fluid resorption and pro-
nounced luminal dilation in efferent ductules caused fluid
accumulation in RT and seminiferous tubules.33,34 Mice
lacking aromatase and thus estrogens (aromatase knock-
out, Cyp19KO) showed progressive infertility with degen-
eration and apoptosis of round spermatids and Leydig
cell hyperplasia,35 confirming that impaired E2/ESR1
signaling led to reproductive abnormalities and infertil-
ity. However, Cyp19KO mice do not totally replicate the
Esr1KO phenotype. This suggests ligand-independent ER
activation by dietary phytoestrogens or androgen metab-
olites may be sufficient to mitigate or delay pathological
changes in Cyp19KO males. Although not conclusive, clin-
ical literature describing men lacking ESR1 or aromatase
(reviewed in Cooke et al.12) largely confirmed results from
mouse models.
Loss of membrane ESR1 expression causes male
reproductive abnormalities
Expression of ESR1 is predominately cytoplasmic and
nuclear. However, approximately 5% of ESR1 localizes to
cell membranes.36,37 Although literature describing steroid
hormone effects too rapid to be mediated through classical
nuclear receptors goes back to the 1950s, for years the iden-
tity of these membrane receptors and their mechanism of
action remained unknown. A crucial step forward was the
demonstration that transfecting a plasmid encoding ESR1
into ESR1-deficient cells led to expression of both nuclear
and membrane ESR1.38,39 Furthermore, ESR1 antibodies
showed both nuclear and cell membrane staining.39 These
findings led to the conclusion that membrane estrogen
receptors40 were identical to classical nuclear ESR1.
These findings raised questions concerning how ESR1
localized to cell membranes and signaled. Membrane
localization of ESR1 was found to require ESR1 pal-
mitoylation at cysteine 451 in mice,41 which conferred
hydrophobicity and allowed cell membrane localization.
Extensive work with various steroid hormone membrane
receptors established that they activate the phosphati-
dylinositol-3-kinase (PI3K) and mitogen-activated protein
kinase (MAPK) pathways41 and have other actions such as
increasing cAMP and cytosolic Ca++.
Understanding the mechanism by which membrane
ESR1 (mESR1) localized to the membrane led to work
involving cells that expressed an altered ESR1 in which
alanine was substituted for cysteine 451 (C451A) in mouse
ESR1. Unlike cysteine, alanine cannot be palmitoylated.
This precludes cell membrane localization of ESR1 and
leads to a lack of mESR1 despite continued nuclear ESR1
(nESR1) expression.37
Recently, this altered ESR1 molecule was used to
develop transgenic nuclear-only estrogen receptor 1
(NOER) mice.41,42 Mice with the C451A mutation in ESR1
express normal amounts of functional nESR1, but mESR1
was essentially eliminated because ESR1 was not palmi-
toylated. The lack of mESR1 signaling resulted in female
infertility and other reproductive abnormalities.41,42 We
recently reported that male reproductive function was
also altered dramatically in NOER mice.43 Reproductive
abnormalities in NOER males include enlarged RT and
dilated efferent ductules with reduced epithelial height.
In addition, there were increased numbers of abnormal
seminiferous tubules and degeneration in the seminifer-
ous tubule epithelium. Sperm production was decreased
85% by 8 months of age and sperm motility was also dras-
tically reduced. Strikingly, over 95% of cauda epididymal
sperm had structural abnormalities. These abnormalities
were absent in sperm maturing in the seminiferous epithe-
lium but were present when sperm reached the RT, indi-
cating that sperm abnormalities arise following exposure
to altered fluid environments in the seminiferous tubule
lumen and downstream. The NOER males also showed a
progressive infertility. These findings revealed that loss of
mESR1, despite functioning nESR1, caused severe struc-
tural and functional sperm abnormalities and infertility.
Changes in NOER males were similar to those in Esr1KO
males, and indicate that mESR1 expression is necessary
for male43 as well as female41,42 fertility.
In both NOER and Esr1KO males, impaired efferent
ductule function leads to fluid accumulation and back-
pressure, causing testicular and seminiferous epithelial

abnormalities. However, a critical question that remains is
whether lack of normal E2 signaling causes direct testicu-
lar effects that contribute to Esr1KO and NOER testicular
phenotypes, or whether changes in organs such as efferent
ductules are entirely responsible for Esr1KO and NOER
testicular phenotypes.
Maintenance of epithelial morphological organization
and function in efferent ductules and fluid reabsorption in
the caput epididymis are ESR1-dependent, and a molecular
mechanism to explain estrogen’s role in fluid reabsorption
and regulation of bicarbonate and pH balance in the male
reproductive tract was proposed.44 Briefly, Esr1KO efferent
ducts have reduced expression of proteins regulating fluid-
ion dynamics, including the sodium/hydrogen exchanger
3 (NHE3; SLC9A3), carbonic anhydrase II (CAR2), and
two water channels, aquaporin 1 and 9 (AQP1 and AQP9).
RNA expression analysis in Esr1KO mice demonstrated
that ESR1 has a primary effect on Sls9a3 transcription
while decreased expression of Car2, Aqp1, and Aqp9 could
be secondary to efferent ductule epithelium morphologi-
cal defects.44 It was also determined that ESR1 is expressed
in the proximal epididymis and has a similar effect there.
Thus, dysregulation of proteins that regulate pH balance
results in a failure of Esr1KO epididymis to properly acid-
ify the luminal milieu and causes defects in sperm intra-
cellular pH, maturation and motility.45
In agreement with previous data, we observed a strong
(~14-fold) downregulation of Slc9a3 RNA in NOER epidid-
ymis (Figure 9.2, Tevosian et al., unpublished) confirming
that mRNA signaling is required for Slc9a3 expression.
Aqp1 was also downregulated while expression of the
ubiquitous Aqp4 was unchanged. Carbonic anhydrase 14
(Car14, CA-XIV), the primary carbonic anhydrase present
on the luminal surface in the proximal epididymis, was
unchanged, and expression of Na + / HCO3− cotransporter
SLC4A4 was slightly elevated (Figure 9.2). Collectively,
these findings suggest that, similar to Esr1KO mice,
reduced expression of Slc9A3 and Aqp1 could be involved
in NOER male reproductive abnormalities.
5
Fold-change relative to control
4 effects of xenoestrogens such as DES on male reproduc-
3 reproductive tracts, one important question is whether
2
1 reproduction.
0 1990s suggested that sperm counts in men had dropped
4 3
4a 9a r1
Slc Slc Ca
Figure 9.2  Quantitative PCR analysis of gene expression
(Slc4a4, Slc9a3, Car14, Wnt4, Aqp1, and Aqp4) in adult NOER epi-
didymis. Results are mean ± SEM of the fold-change relative to
the value in the WT control (set to 1) from at least n = 3 replicates.
Role of E2/ESR1 in male reproduction  119
While pH changes may contribute to sperm defects in
NOER mice,43 we also noted decreased Wnt4 expression
(Tevosian et al., unpublished). This is in agreement with
previous results that showed Wnt signaling is required for
epididymal sperm maturation (Koch et al.45; reviewed in
De Robertis and Ploper46), but the epididymal role of Wnt4
has not been explored.
Knockouts of ESR2 or GPER
Despite widespread GPER expression in male reproduc-
tive tracts, male fertility was not impaired in the four
GPER knockouts reported (reviewed in Cooke et al.12),
suggesting that GPER is dispensible for fertility. Similar
to GPER, ESR2 is widely distributed in the testis and
male tract, but initial Esr2 knockouts (Esr2KOs) lacked
the dramatic male reproductive defects of Esr1KO mice,47
although changes in Leydig cell numbers and germ cells
were reported. Critically, Esr2KO mice were fertile,31
confirming that ESR1 is the functionally dominant ER
in males, although important ESR2 actions in the semi-
niferous epithelium were recently described.48 Although
a new Esr2KO mouse developed to avoid complications
arising from alternatively spliced ESR2 transcripts in the
original Esr2KO mouse showed no apparent reproductive/
sperm changes, these mice were reported to have mating
defects,49 although this remains controversial. Lack of
severe reproductive abnormalities in Esr2KO males sug-
gests that ESR2, like GPER, has male reproductive actions,
but may not be essential for male reproductive function.
This has led to work in this area preferentially focusing on
ESR1 rather than GPER or ESR2.
Can environmental estrogens impact human
testicular development?
Transgenic mice have allowed normal E2 functions in
males to be identified. This work has also shown that
disrupted E2 signaling secondarily affects spermatogen-
esis, as described above. There is also extensive litera-
ture documenting effects of early DES exposure on male
reproductive tract development in animal models and
humans.27–29 Over the past two decades, extensive work
has documented estrogenic effects of many man-made
and natural chemicals in the environment. Given known
tion and the critical role of estrogen signaling in male
exposure of men and males of other species to estrogenic
environmental chemicals significantly impacts male
Work by Niels Skakkebaek’s laboratory in the early
4 4 1 4 50% over the previous half-century.50 This had worrisome
nt Aq
p
Aq
p
W
implications for human fertility and led to studies world-
wide to further test this hypothesis. Subsequent work
largely confirmed the original observations, although
regional variations exist.51
Shortly after the suggestion of declining human sperm
counts, Skakkebaek and Richard Sharpe published a
120  Roles of membrane and nuclear estrogen receptors in spermatogenesis
seminal paper expanding the initial hypothesis and offer-
ing a potential mechanistic explanation.52 These authors
noted that in addition to falling sperm counts, reproduc-
tive problems such as cryptorchidism, hypospadias, and
testicular cancer had increased in men. This suggested an
environmental cause for these reproductive problems, an
idea supported by strikingly increased reproductive disor-
ders in Danish compared to Finnish men despite minimal
racial/ethnic differences in these two populations.
Based on these observations, they proposed the testicu-
lar dysgenesis syndrome (TDS) theory, which postulated
that abnormal early testis development was causing fall-
ing sperm counts and testicular pathologies. Furthermore,
these authors suggested that these abnormalities could
involve Leydig and/or Sertoli cells and that environmental
estrogen exposure in male fetuses to environmental estro-
gens could contribute to subsequent testicular abnormali-
ties and decreased sperm production.
The TDS theory was problematical to test experimen-
tally in humans, although long-term administration
of estrogen to adult males produces adverse testicular
changes.53 Rat models showed that prenatal exposure to
environmental chemicals such as phthalates induced
abnormalities consistent with the TDS theory (increased
cryptorchidism and hypospadias, impaired spermatogen-
esis).54 These studies also suggested that impaired andro-
gen production might be one factor driving TDS.55
In contrast, a recent report that human fetal testes xeno-
grafted into nude mice did not show reduced T produc-
tion in response to DES administered to the host mouse56
failed to provide data supportive of the TDS hypothesis.
Furthermore, this same study suggested that human
Leydig cells lacked full-length ESR1 and thus would not
be direct targets of environmental estrogen exposure.
Although some earlier work reported that ESR1 was absent
in human testis, other groups reported ESR1 expression
in Leydig and germ cells.57,58 More than two decades after
original reports on falling human sperm counts and the
TDS hypothesis, the role of environmental estrogens in
various reproductive pathologies remains unresolved.
In summary, ESR1 is present in low concentrations in
human testis and varies significantly with age.59 Other spe-
cies show greater ESR1 expression. In addition to direct
estrogen effects on several testicular cell types, the potential
for cross-talk between AR and ESR1 and 2 also increases
the complexity of this topic. This is illustrated by work with
Sertoli cell lines in vitro, where low concentrations of E2
increase ESR2 but decrease AR, while T increased AR but
had no effect on ESR2.60 In efferent ductules, E2 induces
loss of ESR1 as well as AR but has no effect on ESR2; in
contrast, T increases ESR1 and AR but has little effect on
ESR2.61 Thus, the balance of estrogen/androgen and their
respective receptors appears to play a greater role in the
overall male reproductive physiology, which depends not
only on absolute levels of ER expression but also on devel-
opmental stage. In addition to direct effects on spermato-
genesis, extensive literature documents estrogen effects on
organs outside the testis, and loss of this E2/ESR1 signaling
can produce secondary changes in the testis and spermato-
genesis. This field has undergone a total transformation in
the past quarter-century, and while estrogen’s role in both
the testis and other reproductive organs is now established,
many critical questions remain in this evolving area.
ACKNOWLEDGMENTS
This work was supported by a New Florida Scholar Boost
Award, a grant from the University of Florida, and NIH
grant HD087528 to PSC.
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Regulation of fertility and infertility
in humans
NAHID PUNJANI, RYAN FLANNIGAN, and PETER N. SCHLEGEL
INTRODUCTION
Regulation of male fertility begins with the creation of
primordial germ cells (PGCs) during embryonic devel-
opment. These PGCs enter a quiescent state and eventu-
ally differentiate into spermatogonia between birth and
6 months of age. It is not until puberty where the pro-
cess of spermatogenesis occurs to produce spermatozoa
through a highly coordinated and evolutionarily con-
served process. This is the basis on which male fertility
can be achieved. The spermatozoa must then be capable
of fertilizing the oocyte and transmitting its genetic mate-
rial to create a zygote and enable appropriate embryonic
and fetal development. Many intrinsic and extrinsic fac-
tors affect spermatogenesis and fertility. In this chapter, we
will provide a brief overview of the history and epidemiol-
ogy of male fertility, discuss the process and regulation of
spermatogenesis and spermiogenesis, and discuss clinical
requirements, conditions, and treatments surrounding
male fertility.
History of fertility
Fertility requires gamete production in both male and
females, fertilization, and successful pregnancy to term.
Male gamete production occurs via spermatogenesis, ulti-
mately producing functionally competent spermatozoa.
Many anatomical and physiological factors are required
for spermatogenesis, including seminiferous tubules con-
taining both somatic cells and germ cells that eventually
give rise to spermatozoa. Spermatozoa were first identi-
fied to have an essential role for fertility in the 1600s, as
proposed by van Leeuwenhoek.1 Sertoli cells are integral
in coordinating spermatogenesis and the production of
sperm was later discovered by Enrico Sertoli in 1865.2
Spermatogenesis was identified to be under hormonal
regulation in the twentieth century when testosterone was
identified to influence sperm production3 as well as the
hypothalamic-pituitary-gonadal axis. This axis was rec-
ognized to play a pivotal role regulating spermatogenesis
via two key hormones—the follicle-stimulating hormone
(FSH) and luteinizing hormone (LH)—and was discov-
ered in the early 1900s.4 Over time, as forms of infertil-
ity have been recognized, corresponding treatments have
evolved as well, including the in vitro fertilization (IVF)
procedure first performed in humans in the 1970s.5
Epidemiology of pregnancy and infertility
Presently, in the United States, there are approximately
102.1 pregnancies per 1000 women aged 15–44, the lowest
10
level in 12 years and second lowest in the last 30 years.6
The rates of infertility are difficult to assess but there are
approximately 7%–18% of couples in the United States
who fail to conceive within 12 months of attempting.7,8
Internationally, an estimated 60–80 million couples
according to World Health Organization (WHO) suffer-
ing from infertility worldwide. These rates of infertility do
not appear to have changed in recent decades9,10; however,
male reproductive health has been suggested to be decreas-
ing among European and Western men over the past few
decades.11–14 Male factor infertility is present in 50% of
these cases and affects 10% of North American men.10,15
These men have most often been characterized by semen
parameters reflecting reduced concentration (oligozoo-
spermia or azoospermia), poor motility (asthenozoosper-
mia), and abnormal morphology (teratozoospermia).10
Interestingly, recent data and studies have suggested that
sperm counts are actually decreasing worldwide.16 While
this has previously been somewhat controversial, recent
data have shown that since the 1970s, there has been a
50%–60% decline in North America, Europe, Australia,
and New Zealand.17 The etiology of this decline remains
unclear and is an area for further study.
Spermatogenesis and spermiogenesis
During embryologic development, PGCs arise from the
extraembryonic tissues adjacent to the yolk sac. Migration
to the gonadal ridge occurs at 3 to 5 weeks, and differen-
tiation to gonocytes occurs via inducible factors mediated
by Sertoli cells (SCs).18,19 Gonocytes remain mitotically
inactive until birth, where they differentiate into sper-
matogonia (SPG) within the seminiferous tubules over
the following 6 months. This is mediated by a number of
factors, including stromal cell derived factor 1 (SDF-1),
activator protein-2 (AP-2), stem cell factor (SCF), growth
and differentiation factor 3 (GDF3), retinoic acid, estra-
diol, and DNA methylation changes.20–29 SPGs remain qui-
escent until 5–7 years of age, where proliferation occurs
followed by a combination of proliferation and differentia-
tion during puberty with the onset of spermatogenesis.30,31
Spermatogenesis occurs in the seminiferous tubules
of the testes. Several cells are involved in the production
of spermatozoa, including somatic cells (Leydig cells,
Sertoli cells, and peritubular myoid cells [PTMCs]), as
well as germ cells (spermatogonial stem cells [SSCs], type
A dark and pale spematogonia, type B spermatogonia,
primary and secondary spermatocytes, round and elon-
gating spermatids, and spermatozoa). This process begins
123
124  Regulation of fertility and infertility in humans
with the proliferation and differentiation of SSCs.32 SSCs
first undergo a proliferative phase located adjacent to the
basement membrane of seminiferous tubules.33 Here,
there is initial division of SSC that produces two cells, one
of which functions to renew the SSC population of cells
(Adark spermatogonia) and the other (Apale spermato-
gonia) commits to a path of differentiation transforming
into a type B spermatogonia.34 This process is mediated
by SC factors, including glial-derived neurotrophic fac-
tor (GDNF) that promotes proliferation and self-renewal,
while bone morphogenetic protein 4 (BMP4), retinoic acid,
and Notch1/Jagged2 signaling promote differentiation.35–37
This B spermatogonia undergoes a series of mitotic events
to give rise to preleptotene spermatocytes (phase of pri-
mary spermatocytes). These spermatocytes subsequently
enter meiosis.34 These preleptotene spermatocytes detach
from the basement membrane, cross the blood-testis bar-
rier (BTB) to the adluminal compartment, and undergo
the remaining phases of prophase I and subsequent com-
pletion of meiosis I forming secondary spermatocytes.
Secondary spermatocytes then undergo meiosis II to form
round spermatids.38
Spermiogenesis is the process where round spermatids
develop into a final product, spermatozoa that has an acro-
some on the anterior surface of the head (enzyme storage
for penetration), as well as a midpiece, and tail.39 These
cells lack cytoplasm and associated organelles.39 During
spermiogenesis, spermatids undergo acrosomal develop-
ment from the golgi apparatus, nuclear condensation, tail
elongation from the centriole, and an ultimate reduction
of cytoplasm eventually transforming from round sper-
matids to elongating spermatids and finally into sper-
matozoa.40 Spermatozoa are then ready for release from
the apex of the SC into the lumen of the tubule once they
are mature. During this process the SCs engulf excess
cytoplasm and in many instances phagocytize develop-
ing germ cells.40 Peritubular myoid cells located on the
interstitial surface of the basement membrane encircling
the seminiferous tubules function to contract and propel
sperm toward the rete testis and epididymis.38 PTMCs
also have additional roles including communication with
SCs and expression of GDNF supporting spermatogenesis
and SSCs.41,42 Furthermore, PTMCs may have a role in
immunity, interacting with immune cells such as macro-
phages (majority), dendritic cells, lymphocytes, and mast
cells located in the interstitium.43
Once in the epididymis, sperm undergo biochemical,
physiological, and morphological changes during epidid-
ymal transit where they acquire motility and the ability
to undergo fertilization.44 As sperm travel from proximal
(caput) to distal (cauda) in the epididymis, their capac-
ity for motility is enhanced by low pH and modulated by
concentrations of sodium (low) and potassium (high).45
During this transit there are numerous changes to the
sperm head, such as a decrease in length:width ratio and
reduction in cytoplasmic volume.45 Emission then occurs
with transport of sperm from distal epididymal and vas
deferens to the ejaculatory ducts and posterior urethra
where it combines with other secretions to form the ejacu-
late and is expelled through the urethral meatus during
ejaculation.46
Role of SCs
SCs are somatic cells of the testis and are critical to the
process of spermatogenesis.47 During spermatogenesis,
SCs function differently depending on the stage of the
maturing germ cell and spermatogenic cycle.48 SCs secrete
numerous factors into the tubular fluid to interact with
developing germ cells. These include androgen bind-
ing protein, GDNF, Transforming Growth Factor-alpha
(TGF-α), Transforming Growth Factor- beta (TGF-β),
transferrin, Interleukin-1alpha (IL-1α), ceruloplasmin,
plasminogen activator, insulin-like growth factor, inhibin
B, and anti-Mullerian hormone.29,35,49–56 The number of
SCs is an important determinant of the number of sper-
matozoon created.47 Each SC can support only a finite
number of germ cells in the order of 50–70 cells. FSH is the
primary hormonal regulator of SC function.57 Numerous
studies have illustrated that suppression of FSH will cause
reduced SC function and spermatogenesis production but
is rescued upon readministration.57 FSH has also been
shown to increase mitotic and meiotic DNA synthesis
in SPG and preleptotene spermatocytes.32 Evidence also
exists that thyroid stimulating hormone may influence SC
function.58 SCs along with germ cells play a significant role
in determination of testicular size, and SC replication may
continue through the pubertal period.59 The number of
SCs in adult men ranges from 25–900 × 10^6 per testis.60
Therefore, the efficiency of sperm production is closely
tied to the number and function of SCs.57 SCs form tight
junctions among themselves, forming two compartments
and the blood-testis barrier.38 This barrier is essential
for progression of germ cells through meiosis and sper-
miogenesis.38 The inner lumen, where there is secretion
of seminiferous tubule fluid, is essential for nutrient and
juxtacrine signaling to germ cells.61 SC response to FSH
then subsequently begins to decline during early puberty
and responsiveness to testosterone begins to increase.62
FSH signaling is regulated by inhibin, which is produced
by SCs acting centrally at the hypothalamus to negatively
regulate FSH production; activin, which is produced by the
SCs acts centrally to positively regulate FSH production.63
Role of Leydig cells
Leydig cells are polygonal cells found adjacent to semi-
niferous tubules in the interstitial space of the testis
and primarily produce testosterone.64 Leydig cells are
primarily stimulated by LH; however, FSH can also
indirectly regulate Leydig cells via effects on the SCs.64
Other stimulators of Leydig cells include gonadotropin-
releasing hormone, melatonin, epidermal growth factor,
insulin-like growth factor 1, atrial natriuretic peptide,
ghrelin, and gamma-aminobutyric acid (GABA).65–72
Leydig cell function is central to spermatogenesis because
testosterone is a primary driver of spermatogenesis. Tes­
tosterone produced by Leydig cells has the ability to

initiate spermatogenesis in humans and functions to
maintain qualitative levels but not quantitative levels
of sperm production.73 Leydig cells do not store testos-
terone but rapidly secrete it into the interstitial space,
capillaries, and seminiferous tubules where it stimulates
both SCs and germ cells. 38 Testosterone concentrations
are 100–1000 times greater intratesticular compared
to systemic levels.74 Testosterone stimulates differen-
tiation of type A SPG to type B SPG,75 proliferation of
SPG,76,77 survival of spermatocytes and spermatids via
antiapoptotic mechanisms,78 and helps to facilitate sper-
miogenesis.75,79–81 Testosterone negatively feeds back to
downregulate central LH production.82 In addition to
testosterone, Leydig cells have been shown to release
substances that activate EGF receptors, a known catalyst
for spermatogenesis.83
Hormonal regulation
Hormonal regulation of spermatogenesis occurs primar-
ily through intratesticular testosterone production. This
occurs via the hypothalamic-pituitary-testis axis where
the hypothalamus releases gonadotropin releasing hor-
mone (GnRH), stimulating the anterior pituitary via the
hypophyseal portal system into the adenohypophysis to
release FSH and LH. FSH subsequently stimulates SCs
to drive spermatogenesis and LH stimulates Leydig cells
to stimulate testosterone production.84,85
Additional hormones such as inhibin are produced by
SCs, and in conjunction with testosterone from Leydig
cells function to negatively regulate GnRH production in
the hypothalamus. Inhibin is a glycoprotein heterodimer
secreted from the base of SCs into interstitial fluid and into
seminiferous tubule fluid via FSH stimulation of SCs.86
This process overall is stimulated by androgens.63 Inhibin
also acts to inhibit activin, another glycoprotein heterodi-
mer also released by SCs, which normally enhances FSH
synthesis and secretion.87 It is the intricate balance of these
hormones that regulate spermatogenesis and maintain
homeostasis.
These hormonal pathways also permit opportunities
for manipulation in the presence of deficiencies and/or
abnormalities. In the presence of testosterone deficiency,
clinicians must remember to avoid treatment of men
desiring fertility with exogenous testosterone, since exog-
enous testosterone will negatively feedback and inhibit LH
production and subsequent intratesticular testosterone
production, impairing spermatogenesis.88 Medications
such as selective estrogen receptor modulators (SERMs)
(i.e., clomiphene citrate) may be used to block negative
feedback of estrogen on the hypothalamic-pituitary-axis
and ultimately increase testosterone production and pro-
mote spermatogenesis.89 Aromatase inhibitors (AIs), (i.e.,
anastrozole) decrease conversion of peripheral testoster-
one to estrogen, thus increasing circulating testosterone
and decreasing estrogen levels. Other medications, such
as recombinant FSH, human menopausal gonadotropins
(hMGs), human chorionic gonadotropin (hCG) or exog-
enous GnRH have been used in the treatment of infertility
Azoospermia 125
in men with secondary hypogonadism, also known as cen-
tral hypogonadism.89–91
Requirements for fertilization
Fertilization is the process whereby there is an interac-
tion between sperm and an egg to form a zygote, which
continues to grow forming an embryo and then a fetus.92
Oocytes are stored in female primordial follicles, where
they then mature, forming an extracellular matrix known
as the zona pellucida, which secretes glycoproteins, fol-
lowed by release of eggs during ovulation that travel to
the ampulla of the oviduct to await fertilization.92 Human
sperm produced in seminiferous tubules are released dur-
ing ejaculation during intercourse, and after deposition
in the vaginal canal travel to and through cervical mucus
via the cervical os toward the uterus and fallopian tubes.93
Throughout this process, sperm capacitation occurs in
preparation for the acrosome reaction.93 Upon completion
of the acrosome reaction, the spermatozoon can penetrate
the zona pellucida of the oocyte and eventual fusion to
form a zygote.
Natural fertilization from a male perspective requires
adequate sperm parameters in order to complete the pro-
cess described above. Precise thresholds for the lowest
sperm parameters possible to achieve fertilization are not
known. However, the World Health Organization (WHO)
has created normal values based on a population of men
who have previously demonstrated fertility. These values
are based on a properly collected sample, obtained after
2–7 days of sexual abstinence.94 Table 10.1 reports the
WHO guidelines values that represent the lowest 5th per-
centile per each respective parameter among a cohort of
men that have previously demonstrated fertility.95
AZOOSPERMIA
Azoospermia is the absence of sperm in the ejaculate after
2 centrifugations of semen, and can be regarded as nonob-
structive azoospermia (NOA) or obstructive azoospermia
(OA). OA represents 40% of cases.96,97 NOA can be divided
into pretesticular and testicular. Pretesticular azoosper-
mia is often caused by an inadequate production of FSH
and LH secondary to a central deficiency in the pituitary
or hypothalamus. Etiologies of pretesticular NOA include
Table 10.1  World Health Organization 2010 semen
analysis lower limits of normal per respective parameter.
WHO 2010 cutoff
Semen analysis parameter (5th percentile)95
Volume (mL) 1.5
Concentration (million sperm/mL) 15
Total sperm count (million) 39
Total motility (%) 40
Progressive motility (%) 32
Normal morphology (%) 4
Vitality (%) 58
Leukocyte count (million/mL) <1.0
126  Regulation of fertility and infertility in humans
congenital disease such as Kallman syndrome, or acquired
through exogenous androgens and surgical extirpation
or radiation of the pituitary.98 Testicular NOA occurs
due to impaired spermatogenesis despite adequately pro-
duced FSH. Etiologies include genetic conditions such
as Klinefelter syndrome, Y-chromosome microdeletion
(AZFa, AZFb, AZFc), or acquired through infection, che-
motherapy, or radiation.99 However, nearly 70% of NOA
cases remain idiopathic without a defined explanation.
Impaired spermatogenesis may present with complete
absence of germ cells termed Sertoli cell only (SCO), matu-
ration arrest where germ cells stop differentiation beyond
primary spermatocytes (early) or secondary spermato-
cytes (late), or hypospermatogenesis where very limited
spermatogenesis occurs. OA is due to mechanical obstruc-
tion such as congenital bilateral absence of the vas deferens
(CBAVD) due to a defect in the cystic fibrosis transmem-
brane conductance regulator (CFTR) gene, seminal vesical
atresia, ejaculatory duct cysts or obstruction, vasectomy,
or idiopathic- or infectious-related primary epididymal
obstruction. Other posttesticular etiologies include ejacu-
latory dysfunction or failure of emission due to retroperi-
toneal lymph node dissection (RPLND), pelvic surgery,
spinal cord injury, or diabetes.99,100
Treatment: Sperm retrieval for OA includes microsur-
gical epididymal sperm aspiration (MESA) and percutane-
ous epididymal sperm aspiration (PESA). MESA utilizes
a scrotal incision whereby the testis is delivered and the
epididymis is observed using an operating microscope.
Dilated epididymal tubules are aspirated at the caput of the
testis.101 This is the gold standard surgical sperm retrieval
technique for OA, with success rates of 96%–100%, yield-
ing 15–95 million sperm.102,103 PESA is performed trans-
cutaneously with a needle inserted directly into caput
epididymis and sperm is aspirated, with success rates of
61%–96%.104,105 Surgical sperm retrieval for NOA occurs
via microdissection testicular sperm extraction (micro-
TESE). This is performed through delivering the testis,
making a tunical incision, bivalving the testis, and exam-
ining for the seminiferous tubules under an operating
microscope for dilated tubules. MicroTESE has successful
sperm retrieval rates on average of 45%–63%.106 Patients
with testicular NOA may also be treated with hormonal
therapy such as clomiphene citrate or aromatase inhibitors
in the setting of low testosterone or high estradiol levels,
respectively.107 Patients with central NOA require either
GnRH, hCG, recombinant FSH, or hMG to stimulate
spermatogenesis.
Oligozoospermia
Oligozoospermia is defined as low sperm concentration
(<15 million/mL semen) in the ejaculate.108 Etiologies can
be related to primary testicular failure (idiopathic sper-
matogenic failure, testicular damage such as varicocele,
drugs and toxins, chromosomal abnormalities, and genet-
ics), mixed primary and secondary failure (nonrepro-
ductive illness), and secondary testicular failure (partial
GnRH deficiency or partial gonadotropin deficiency).107
Generally, natural fertility is poor with oligozoospermia
and therefore treatment options include correcting the
underlying cause, vitamins and antioxidants, varicocele
treatment if present, and lifestyle modification such as
smoking cessation and avoidance of drugs or excessive
alcohol.107 Assisted reproductive techniques may be neces-
sary in some instances where natural fertility is not suc-
cessful despite treatment of modifiable factors.109
Asthenozoospermia
Sperm motility is accomplished via microtubular struc-
tures powered by ATP via dynein arms.110 Their move-
ment is explained by the 9+2 configuration with motility
maturation in the epididymis.111 Asthenozoospermia is
defined as less than 40% motility or less than 32% of pro-
gressively motile sperm based on the 2010 WHO semen
analysis parameters.112 A recent meta-analysis determined
that the combination of total sperm number and motil-
ity, termed total motile count (TMC), is more predictive
of spontaneous pregnancy rate than either parameter
independently.113
Etiologies of asthenozoospermia include varicocele,
prolonged periods of anejaculation, leukocytospermia,
genital infections, oxidative stress, antisperm antibod-
ies, metabolic disorders affecting ATP production, partial
ejaculatory duct obstruction, partial epididymal obstruc-
tion or sperm (USDs).114,115 Antisperm antibodies (ASAs)
have been found in 5%–15% of infertile men versus low
rates of 1%–2% in fertile men and are often caused by
trauma, infection, malignancy, vasectomy, or sources of
inflammation.116 ASAs are variable in that they can act
against different immunoglobulins with the majority
against immunoglobulin G (IgG) and immunoglobulin A
(IgA) but also immunoglobulin M (IgM).117 ASAs interfere
with various elements of sperm including the head (inter-
fering with acrosome reaction), tail (alter sperm motil-
ity), and midpiece.116 These patients can be treated with
Assisted reproductive techniques (ARTs) with reasonable
success as meta-analyses have demonstrated that ASAs are
not related to pregnancy rates with IVF and intracytoplas-
mic sperm injection (ICSI).118 USDs may occur in axone-
mal or periaxonemal structures of the flagellum.115 These
typically occur in men with viable but severely astheno-
zoospermic sperm with motility less than 10%. Primary
immotile-cilia syndrome is an uncommon autosomal
recessive disorder with altered ciliary structure, known as
Kartagener’s syndrome, diagnosed by transmission elec-
tron microscopy illustrating dynein arm absence.119 Other
defects include the axonemal 9+0 defect lacking the cen-
tral microtubular structure that can be associated with
autosomal dominant polycystic kidney disease.115 USDs
may be treated with IVF/ICSI, although the efficiency of
ICSI appears to be lower in men with USDs. Treatment
options for asthenozoospermia should focus on any
identifiable etiology such as varicocele repair, transure-
thral resection of ejaculatory ducts if partial obstruction,
removal of external environmental stressor, and regular
intercourse. Medical treatments are limited; however,

studies examining L-carnitine have been explored in an
attempt to improve sperm motility rates with positive
results.120 If infertility persists, assisted reproductive tech-
niques should be considered.
Teratozoospermia
Normal sperm morphology is a controversial require-
ment for fertility due to its subjectivity in its assessment
and reproducibility.121 Kruger et al. have emphasized the
importance of standardized criteria, which includes a nor-
mal sperm possessing a head with a smooth, oval configu-
ration with well-defined acrosome (40%–70%), length of
head to be 5–6 μm, and no visible defect in the neck, mid-
piece, or tail.122 Teratozoospermia is the term used to iden-
tify abnormal sperm morphology that inhibits the ability
for normal fertilization. Etiologies of teratozoospermia
may include genetic disorders leading to globoozospermia,
macrocephaly, decapitated sperm syndrome, and fibrous
sheath dysplasia, and potentially environmental factors
such as tobacco, cannabis, temperature, obesity, testicular
cancer, varicocele, or infection. However, the origin of the
majority of defects remains unclear.123 Treatment options
include correcting underlying etiologies if possible or use
of ARTs such as IVF/ICSI to select for morphologically
normal sperm.123
Acrosome reaction and capacitation
Successful sperm fertilization of an egg relies on numer-
ous sperm parameters, described above, with the goal of
penetrating the zona pellucida and fusion with an oocyte
membrane, which is known as the acrosome reaction.124
Capacitation is the process that permits the acrosome
reaction to occur and creates biochemical modifications to
the acrosome of the sperm head, allowing penetration of
the outer layer, and changes in the tail permitting greater
mobility. These are ultimately facilitated by removal of
sterols, glycoproteins, and increased permeability and
influx of calcium.125 The acrosome is structurally located
on the anterior half of the sperm head. Acrosome reaction
involves the penetration of the corona radiata followed
by the release of hyaluronidase and exposure of acrosin,
which then digests the zona pellucida of the oocyte.126
These reactions are required for fertilization and defects
in these steps have negative implications on fertility.
DNA fragmentation
Sperm DNA fragmentation has emerged as another con-
dition associated with infertility and numerous assays
have been developed to assess this, with the theory that
the extent of DNA fragmentation is inversely related to
sperm quality.127 Numerous techniques have been devel-
oped to assess DNA fragmentation, each with their own
advantages and disadvantages, and include sperm chro-
matin dispersion (SCD), sperm chromatin structure assay
(SCSA), terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling (TUNEL), single-cell gel electro-
phoresis (COMET), DNA ladder, DNA-break detection,
Genetic factors  127
and fluorescence in situ hybridization (FISH).128 Studies
have shown comparable correlations in DNA measure
damage between these assays.127 The results of these assays
have been promising as a recent meta-analysis revealed
that couples are more likely to achieve pregnancy if DNA
fragmentation index was low, illustrating its significant
predictive value for pregnancy success.129 Since only neat
semen DNA fragmentation best predicts IVF/ICSI suc-
cess, it is not clear if “selection” of undamaged sperm from
an abnormal sample improves the chance of pregnancy for
men with abnormal sperm DNA fragmentation. Patients
with varicoceles have increased levels of DNA fragmen-
tation.130 Treatment options include varicocele repair,
antioxidants, and reduction of sexual abstinence.131–133 Of
note, sperm directly retrieved from the testis reliably have
lower sperm DNA fragmentation than that in the ejacu-
late of men with abnormal sperm DNA fragmentation.
Antioxidants are naturally found in seminal fluid and play
a key role due to the lack of cytoplasmic fluid in sperm,
which illustrates that antioxidant supplementation could
play a key role in protecting against DNA damage and frag-
mentation.134 A recent review of literature illustrated that
antioxidant therapy also improved outcomes for patients
undergoing ICSI with elevated DNA fragmentation levels,
and which had improved clinical pregnancy and implan-
tation rates.135 Antioxidants that have been studied include
vitamin C, vitamin E, glutathione, L-carnitine, coenzyme
q10, vitamin B9, selenium, and vitamin B12, which have
shown varying levels of success, suggesting that these sub-
stances may serve to curb DNA fragmentation, but further
adequately powered prospective studies are required.135
GENETIC FACTORS
Genetic factors are identifiable in 15%–30% of male infertil-
ity, while the majority (70%) remain idiopathic and under
investigation.136 Klinefelter syndrome (KS) is the most com-
mon genetic cause of male infertility with a prevalence 10%
in men with NOA and 5% in men with severe oligozoosper-
mia and an incidence of 0.1%–0.2% in the general popula-
tion.137–139 It is characterized by a karyotype of 47, XXY in
80% of cases and mosaic patterns in the remaining 20%.140,141
Clinically, men with KS have a variable phenotype but are
generally characterized as tall, with gynecomastia, gynoid
hips, cognitive abnormalities, sparse hair distribution, and
having progressive hyalinization-associated testicular fail-
ure resulting in infertility, primary hypogonadism, and
small firm testes.142 Another sex chromosome anomaly
includes 47,XYY, which carries an incidence of 0.1%. These
men will have variable sperm concentrations but typically
present with severe oligozoospermia or azoospermia.143
Y-chromosome microdeletions are characterized by 1 of 3
clinically relevant and described deletions, AZFa, AZFb,
and AZFc. These deletions occur within the azoosper-
mia factor (AZF) region of the euchromatin long arm of
the Y chromosome.144,145 This locus holds 14 protein cod-
ing genes that are required for spermatogenesis. AZFa and
AZFb deletions result in azoospermia and complete dele-
tions do not have any reports of successful surgical sperm
128  Regulation of fertility and infertility in humans
retrieval.146 AZFc deletions result in severe oligozoospermia
in 70% of men with total sperm concentrations typically less
than 1 million sperm per milliliter and azoospermia in the
remainder of men.147 Kallman syndrome is characterized
by hypogonadotropic hypogonadism and anosmia. Other
clinical features include cleft palate abnormalities, cryptor-
chidism, unilateral renal agenesis, neurogenic deafness and
infertility due to lack of GnRH production and subsequent
gonadotropins, testosterone, and spermatogenesis.148 This
may be the result from abnormalities in the KAL1 or FGFR1
gene.149 Mutations in the testis-expressed 11 (TEX11) gene
may lead to maturation arrest and azoospermia.150 TEX11 is
located on the X chromosome Xq13.2 and encodes a protein
that functions to repair double-strand DNA breaks, homol-
ogous chromosome synapses, and recombination.151 CFTR
gene mutations may lead to CBAVD. The most commonly
mutated alleles implicated in CBAVD include F508del
(17%), R117H (3%), and 5T (25%). This carries a prevalence
of 1% among infertile men and 25% of men with primary
obstructive azoospermia. These men require surgical sperm
retrieval from the epididymis for use with assisted repro-
ductive techniques. They also require genetic counseling,
abdominal ultrasound to assess for renal agenesis, and
CFTR testing of their partner to assess risk of cystic fibrosis
in their offspring.152
More recent research has been uncovering regulatory
mechanisms of spermatogenesis exerted by small and long
noncoding RNAs. Small RNAs comprise three different
classes of RNA: microRNA (miRNA), small interfering
RNA (siRNA), and PIWI-interacting RNA (piRNA).153
piRNAs play a vital role in spermatogenesis during both
PGC development and during meiotic prophase 1, termed
prepachytene piRNAs, and pachytene piRNAs, respec-
tively. PiRNAs function through repression of transposons
and elimination of mRNAs during spermatid forma-
tion.154–160 Further research is required to identify mutated
and aberrant functioning piRNAs among infertile males.
Numerous microRNA, however, have repeatedly demon-
strated important roles during spermatogenesis, including
proliferation of PGCs, meiotic prophase I, and spermatid
elongation.161–164 MicroRNA function via associating with
argonaute proteins (AGO) and silencing translation of
mRNA targets. Several differentially expressed microRNAs
have been identified in men with NOA and are summa-
rized by Neto et al. 2016. These include downregulation
of miR-34 family, miR-122, miR-181a, miR-146b, miR-
513a-5p, miR-509-5p, miR-274b, and miR-202-5p, and
upregulation of miR19b, Let-7a, miR-429, miR-141, and
miR-7-1-3p.29 Long noncoding RNA (lncRNA) interact
with chromatin binding proteins, RNA binding proteins,
and mRNA protection or degradation, and act as a miRNA
decoy and function to activate or repress translation.165
Expression of lncRNAs begins during early pachytene
spermatocytes, increases in late pachytene spermatocytes,
and tapers off in spermatids. Several lncRNAs have been
identified to play an important role in spermatogenesis
and male sex development,166,167 but further research is
needed to identify candidate lncRNAs in male fertility.
VARICOCELES
Varicoceles are abnormally dilated internal spermatic
veins in the scrotum. Clinically relevant varicoceles are
those that can be palpated with Valsalva (grade 1), pal-
pated without Valsalva (grade 2), or visualized through
the scrotal wall at rest (grade 3). They occur in 15% of the
general population and 35% of men presenting with infer-
tility. They are also the most common cause of second-
ary infertility, accounting for 70%–80% of these men.168
Varicoceles may lead to increased sperm DNA frag-
mentation,169 decreased sperm concentration, motility,
and abnormal morphology independently or altogether,
which is termed oligoastheoteratozoospermia (OAT).170
Varicocele repairs have been associated with improved
semen analysis parameters,171 DNA fragmentation,172 nat-
ural pregnancy rates, increased fertilization during IVF173
and intrauterine insemination (IUI),174 and decreased
miscarriage rates.175 Several theories exist regarding the
mechanism of varicoceles on male infertility; however,
increased scrotal temperature and oxidative stress are
thought to be central to the mechanisms. This may con-
tribute to germ cell apoptosis, impaired Leydig cell func-
tion, and testosterone production,176,177 as well as damage
to the BTB with associated autoimmunity and impaired
cleavage of sperm cytoplasm.178–180 Seminal proteomes also
differ in men with varicoceles; expression of semenogelins
I and II are increased and are thought to inhibit sperm
motility and prevent premature sperm hyperactivation
and capacitation.181,182 As such, varicocele repair for pal-
pable varicoceles may be of benefit to the infertile male.
However, it is important to consider both male and female
factors, such as age, when choosing the appropriate course
of therapy since the effect of varicocele repair takes 3–6
months to see potential improvements.
Fertility as a function of age
In our present-day society, patients are seeking fertility
later in life, and this impacts outcomes both for females
and males with respect to fertility potential.183 Fertility
as a function of age is commonly assessed, especially in
the female population. Women are born with a set num-
ber of primordial follicles.184 As a consequence, increased
age among women results in increased rates of infertility
particularly after age 35.185 Men, on the other hand, con-
tinue active sperm production throughout life but do lose
function with age.184 However, albeit controversial, some
studies have identified an association of increased paternal
age with increased sperm DNA fragmentation, epigenetic
changes, reduced fertility beyond late 30s, and decreased
ART successes.186 Additionally, paternal age greater than
45 is associated with risk of late fetal death.187 Increased
paternal age has also been associated with cleft lip and pal-
ate and some childhood malignancies such as retinoblas-
toma188 and childhood acute lymphoblastic leukaemia.189
Schizophrenia, bipolar affective disorder, and autism
have also been associated with increased paternal age.186
Effects of advanced paternal age are thoroughly reviewed
in Sharma et al. 2015 for further reading.186

OBESITY AND DIABETES MELLITUS
Obesity is in epidemic proportions, particularly in North
America, where the prevalence is 35% based on the defini-
tion of a body mass index (BMI) >30.190 Obesity has a neg-
ative association with sperm concentration, total sperm
count,191–193 morphology, motility, and DNA fragmenta-
tion.194–198 A central hypothesis for the effect of obesity
on abnormal sperm production includes the state of high
estradiol noted in obese men due to peripheral conversion
of testosterone to estrogen through aromatase enzymes
in adipose tissue.199,200 Estradiol negatively regulates the
hypothalamus-pituitary-testis axis, serving to decrease
LH and FSH production, resulting in less testosterone
production and less SC stimulation. Evidence to support
this includes low levels of inhibin B in obese men. 201,202
Furthermore, ERα receptors are found in Leydig cells and
SCs, and ERβ receptors are present in nearly all intersti-
tial and tubular cells of the testis.203–206 Other factors likely
contributing to impaired semen production include sed-
entary lifestyle, diet, and epigenetic changes to the germ
cells.191,207
The incidence of infertility among diabetic men is much
higher than the average population and ranges from 35%
to 51%. Men with diabetes mellitus (DM) are found to have
decreased testosterone levels, increased sperm DNA frag-
mentation, and variable reports on semen analysis param-
eters, epididymal damage and impaired sperm transport,
neuropathy and abnormalities of emission, increased oxi-
dative stress and associated damage to sperm nuclear and
mitochondrial DNA, and potential intratesticular autoim-
mune damage.208
MEDICATIONS
Several medications that are used among men in repro-
ductive years may negatively impact spermatogenesis and
fertility. This topic is reviewed in depth by Samplaski and
Nangia 2015.209 Hormonal medications such as exogenous
testosterone, anabolic steroids, and androgen deprivation
suppress endogenous testosterone production, resulting
in suppression of spermatogenesis.210 Medications used to
treat lower urinary tract symptoms such as alpha blockers
may cause impaired emission or retrograde ejaculation,
reduced sperm counts and motility,211,212 while 5 alpha-
reductase inhibitors result in reduced sperm counts among
5% of men, and rarely, sexual dysfunction.213,214 Psychiatric-
related medications such as selective 5-hydroxytryptamine
reuptake inhibitors (i.e., SSRIs, SNRIs, TCAs, MAOIs)
result in sperm DNA fragmentation and erectile and ejac-
ulatory dysfunction, likely as a consequence of impaired
sperm transport;215–218 lithium has been shown to reduce
sperm viability.219 Antihypertensives such as beta block-
ers may lead to sexual dysfunction and impaired sperm
motility;220,221 diuretics may also cause sexual dysfunction,
impaired sperm motility and concentration;222,223 calcium
channel blockers result in decreased sperm concentration,
motility, and acrosome reaction.224–226 Antibiotics have
varying effects: nitrofurantoin has been shown to lead to
spermatogenic arrest and reduced sperm concentration,227
Environmental chemicals  129
macrolides result in impaired sperm motility and via-
bility,228,229 aminoglycosides lead to impaired sperm
motility,230,231 subtilosin results in impaired sperm motil-
ity,232 and ketoconazole results in impaired testosterone
production.209,233
Chemotherapeutic agents generally have significantly
negative effects on germ cells, Leydig cells, and SCs,
resulting in temporary or permanently impaired sper-
matogenesis;234 this topic is reviewed in depth by Wallace
et al. 2005.235 High-risk agents include cyclophosphamide,
ifosfamide, chlorambucil, melphalan, busulfan, dacarba-
zine, procarbazine, and nitrogen-mustard, vincristine,
procarbazine, prednisone (MOPP). Intermediate risk
agents include carboplatin, cisplatin, doxorubicin, and
adriamycin, bleomycin, vinblastine, dacarbazine (ABVD),
and bleomycin, etoposide, cisplatin (BEP). Low-risk agents
include methotrexate, vincristine, vinblastine, dactinomy-
cine, mercaptopurine, and bleomycin. Imatinib has been
demonstrated to induce Leydig cell dysfunction with
subsequently impaired testosterone production.235,236 It
is essential for clinicians to discuss fertility preservation
with men and couples prior to undergoing chemotherapy
to afford the ability for sperm cryopreservation as many
men may suffer from irreversible infertility following
therapy.
Fertility and pollution
There are many reported factors affecting male infertil-
ity, which include cigarette smoking, alcohol consump-
tion, and environmental hazards.237,238 Air pollution is
an increasing problem in large populations living in large
urban settings and its effects on fertility are apparent but
remain unclear.239 Studies have demonstrated that in both
animal and human studies, air pollution may alter game-
togenesis and therefore alter reproductive ability.240 Other
studies have shown increases in miscarriage rate in IVF.239
Numerous components of air pollution are being exam-
ined, including nitrogen, sulfur, ozone, and particulate
matter concentrations.241
ENVIRONMENTAL CHEMICALS
Numerous environmental chemicals have demonstrated
potentially harmful effects on spermatogenesis and infer-
tility (reviewed in depth by Neto et al.29). Lead (paint)
has been demonstrated to disrupt chromatin during
spermatogenesis and damage mannose receptors.242
Cadmium (batteries and pigments) disrupts tight junc-
tions of the BTB and causes failure of spermiation.243,244
Ethylene dibromide (gasoline) has been shown to bind
histones, disrupting the ability of DNA packaging,
resulting in reducing sperm counts, impaired motil-
ity, and abnormal morphology.245,246 1,2-Dibromo-3-
chloropropane (synthesis of fire retardants) results in
increased reactive oxygen species among germ cells
with spermatogonial apoptosis and decreased sperm
counts.247,248 Tetrachlorodibenzo-p-dioxin (herbicide
and pesticide synthesis) impairs BTB tight junctions and
results in impaired semen parameters.249–251 Phthalates
130  Regulation of fertility and infertility in humans
(plastics) result in decreased testosterone production due
to activation of peroxisome proliferator-activated recep-
tors (PPARs) and also impairs the function of both germ
cells and SCs.252,253 Bisphenol A (BPA) (plastics and res-
ins) binds to the androgen receptor (AR), ERβ and ERα,
resulting in impaired androgen and estrogen signaling
and associated poor semen parameters.254,255 It is unclear
what degree of exposure is required in humans to see
negative effects for many of these substances; however,
among men wishing to conceive, the best recommenda-
tion is to avoid potential exposures when possible.
Assisted reproductive techniques
ARTs work to treat infertility in the presence of male or
female factor infertility. Methods for ART include IUI,
IVF, and ICSI. 256 Table 10.2 highlights the sperm counts
required for successful fertilization using assisted repro-
ductive techniques. IUI can be performed in as low as
a 500,000 motile sperm count, but optimal results are
generally reported with at least 5 million sperm. 257,258
IVF typically requires 50,000 to 100,000 sperm per
oocyte.259,260 ICSI on the other hand can be performed
with very few sperm present in the ejaculate or even
a single sperm from the ejaculate or when harvested
using methods such as MESA, PESA, or microTESE. 261
Other methods include cryopreservation of sperm or
oocytes, which are techniques that have been used
successfully. 262,263
CONCLUSION
Male fertility relies first on adequate spermatogenesis
and spermiogenesis producing viable spermatozoa capable
of fertilization. This process involves critical regulatory
steps that are both spatially and temporally important.
Abnormalities during these processes may lead to poor
sperm production and subsequent infertility. Extrinsic
factors such as medications, environmental pollution, and
lifestyle factors may also impact the efficiency of spermato-
genesis or the fertilization potential of the spermatozoa.
Thus, it is important for the clinician to understand the
multitude of factors that are both necessary for optimal
fertility as well as those that may contribute to infertility
to ensure each patient may be optimized for a successful
pregnancy.
Table 10.2  Minimal motile sperm counts required for
respective type of assisted reproductive technique.
Assisted reproductive
techniques (ART) Motile sperm count
IUI (intrauterine 5 million264 (range: 500,000 to
Insemination) 10 million257,258)
IVF (in vitro fertilization) 50,000 to 100,000 motile
sperm/oocyte
ICSI (intracytoplasmic Minimum single sperm261
sperm injection)
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258. Merviel P, Heraud MH, Grenier N, Lourdel E,
Sanguinet P, Copin H. Predictive factors for preg-
nancy after intrauterine insemination (IUI): An
analysis of 1038 cycles and a review of the literature.
Fertil Steril. 2010;93(1):79–88.
259. Michelmann HW. Minimal criteria of sperm qual-
ity for insemination and IVF therapy. Int J Androl.
1995;18 Suppl 2:81–87.
260. Lopez G, Lafuente R, Checa MA, Carreras R,
Brassesco M. Diagnostic value of sperm DNA frag-
mentation and sperm high-magnification for pre-
dicting outcome of assisted reproduction treatment.
Asian J Androl. 2013;15(6):790–794.
261. Merchant R, Gandhi G, Allahbadia GN. In vitro
fertilization/intracytoplasmic sperm injection for
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male infertility. Indian J Urol. 2011;27(1):121–132.
262. Di Santo M, Tarozzi N, Nadalini M, Borini A.
Human sperm cryopreservation: Update on tech-
niques, effect on DNA integrity, and implications for
ART. Adv Urol. 2012;2012:854–837.
263. Argyle CE, Harper JC, Davies MC. Oocyte cryo-
preservation: Where are we now? Hum Rreprod
Update. 2016;22(4):440–449.
264. Dickey RP, Pyrzak R, Lu PY, Taylor SN, Rye PH.
Comparison of the sperm quality necessary for suc-
cessful intrauterine insemination with World Health
Organization threshold values for normal sperm.
Fertil Steril. 1999;71(4):684–689.
Male infertility
Evaluation and treatment
RYAN FLANNIGAN and MARC GOLDSTEIN
INTRODUCTION
Approximately 15% of couples seek evaluation for infer-
tility.1 Male factors contribute to 50% of infertile couples
and are thought to be primarily responsible in 20%.2 Some
forms of infertility can be managed and result in natural
pregnancies; however, more severe forms of infertility may
necessitate assisted reproductive technologies (ARTs), pri-
marily in vitro fertilization (IVF) with intracytoplasmic
sperm injection (ICSI), genetic counseling, donor sperm,
or adoption. In 2014, nearly 170,000 IVF procedures were
utilized in the United States.3 In this chapter, we will iden-
tify appropriate evaluation and management options for
men presenting with infertility.
EVALUATION
The objective of evaluating men presenting with infertility is
threefold: (1) identify modifiable factors to improve fertility
through behavioral change, medical, and/or surgical thera-
pies, (2) identify nonmodifiable factors affecting fertility
potential, and (3) provide appropriate counseling, expecta-
tions, and resources to men and their partners based on their
evaluation. Couples unable to achieve pregnancy within
12 months of regular unprotected intercourse should seek
reproductive evaluation. Earlier evaluation is warranted
after 6 months of regular intercourse among couples where
the female is greater than 35 years of age. Furthermore, men
with medical comorbidities known to impact fertility or
men with concerns regarding future fertility also warrant
evaluation. Nonreproductive specialists may conduct initial
screening on history, physical exam, and semen analysis prior
to referral to a specialist in the case of any abnormalities.
All men undergoing fertility evaluation should initially
undergo a history, physical examination, and semen anal-
ysis. Abnormalities may be further assessed with special-
ized investigations in the following sections.
History
A male reproductive history involves evaluating the couple’s
experience in attempting fertility (i.e., history of present-
ing illness), their social history for behavioral and envi-
ronmental factors potentially affecting fertility potential,
past medical history for conditions predisposing one to
poor reproductive health or abnormal mechanics of inter-
course (i.e., erectile dysfunction, intravaginal ejaculation),
past pelvic, abdominal, inguinal, or scrotal surgical history,
medication history affecting spermatogenesis, emission and
ejaculation or erectile function, family history of infertility
or predisposing conditions, and allergies. Table 11.1 outlines
11
a detailed list of key considerations to elicit on history. Here
it is important to consider that a full cycle of spermatogen-
esis and spermiation takes approximately 72 days; thus,
exposures or illnesses within the past 3 months may impact
present fertility and semen analyses. It is also important to
ensure that the female partner obtains an appropriate eval-
uation, and the male specialist knows the female partner’s
age, past reproductive history, and abnormalities on gyne-
cologic evaluation. Beyond evaluating factors affecting fer-
tility potential, it is essential to determine the couple’s goals
of fertility, including the number of children desired and
the timeline they wish to achieve pregnancies.
Physical exam
All patients should receive a general physical exam.
Particular focus should be made to body habitus, hair dis-
tribution, secondary sex characteristics, and presence of
gynecomastia, stature, and midline defects. An abdominal
examination should be performed to assess for signs of pre-
vious abdominal or inguinal surgical scars. Penile examina-
tion should be performed to assess the location of the meatal
opening. Scrotal examination should be performed in a
warm and comfortable environment to encourage relaxation
of the scrotal dartos muscle, making examination easier and
more accurate. Locally, we use a heating pad on the scrotum
for 2–5 minutes to facilitate scrotal relaxation. The testicles
are palpated for size and consistency; we recommend using
an orchidometer for accurate assessment of testicular size.
The epididymis is palpated for fullness, induration, and ten-
derness. The spermatic cord should be palpated while the
patient is both supine and standing. The vas deferens are
palpated bilaterally and the cords are assessed for varico-
celes with and without Valsalva upright. If the presence of
varicocele is unclear, the patient should be placed supine and
reexamined; if a varicocele is present, the cord will collapse
when supine and become distended when standing.
INVESTIGATIONS
Semen analysis
Beyond history and physical examination, semen analy-
sis is the first investigation performed in evaluating a
man’s fertility status. The present reference values for
semen analyses arise from the World Health Organization
(WHO) 2010 iteration. It is important to recognize how
these values were derived and the limitations they pose.
Here, the study population was defined as men able to con-
ceive a pregnancy within 12 month of attempting regular
unprotected intercourse.6 One thousand nine hundred
139
140  Male infertility

Table 11.1  Key considerations to elicit on history.

Type of history Key considerations
History of presenting illness • Duration of regular unprotected intercourse
• Use of aids or devices for fertility (i.e., ovulation kits)
• Mechanics of intercourse (i.e., intravaginal ejaculation)
• Previous paternal or maternal pregnancies
• Erectile function
• Ejaculatory history
Social • Smoking
• Recreational drug use
• Anabolic steroids
• Exercise (i.e., sedentary or prolonged endurance activity)
• Dietary history
• Heat exposures (i.e., >20-min hot shower, hot tubs, laptop use on thighs)
• Tight underwear
Medical • Abdominal or pelvic radiation therapy4
• Germ cells are significantly more sensitive than somatic cells
• Doses as low as 0.1–1.2 Gy may impair spermatogenesis
• >4 Gy may cause permanent damage
• 20–30 Gy may cause Leydig cell dysfunction
• Diabetes
• Obesity
• Galactorrhea
• Visual field deficits
• Central hypothalamic or pituitary dysfunction
• i.e., Kallman syndrome, pituitary adenoma, prolactinoma
• Klinefelter syndrome
• Sinus and respiratory infections
• Undescended testicle
• Testicular torsion
• Orchitis or epididymitis
• Testicular or pelvic trauma
• Systemic illness or fever
• Age of puberty
• Malignancies
• Reviewed in depth in Wallace et al. 20054
Surgical • Previous abdominal surgery affecting erections or ejaculation
• i.e., retroperitoneal lymph node dissection, rectal surgery, radical
prostatectomy, transurethral resection, or ablation of prostate
• Inguinal surgery or scrotal surgery compromising vas deferens, epididymis,
and/or testicular blood supply
• .i.e., inguinal hernia repair, hydrocelectomy, spermatocelectomy,
orchidopexy, vasectomy
Medications *This topic is reviewed in depth in Samplaski and Nangia 20155
Hormonal medications
• Exogenous testosterone or anabolic steroids
• Suppression of spermatogenesis
• Supplement use
• Various effects
Prostate medications
• Alpha blockers
• Retrograde ejaculation
• 5 alpha-reductase inhibitors
• Reduce sperm counts in 5% of men, sexual dysfunction
(Continued )
 Investigations 141

Table 11.1 (Continued)  Key considerations to elicit on history.

Type of history Key considerations
Psychiatry related
• Selective 5-hydroxytryptamine reuptake inhibitors (i.e., SSRIs, SNRIs, TCAs,
MAOIs)
• Sperm DNA fragmentation, erectile and ejaculatory dysfunction
• Lithium
• Reduced sperm viability
Antihypertensives
• Beta blockers
• Sexual dysfunction, impaired sperm motility
• Diuretics
• Sexual dysfunction, impaired sperm motility and concentration
• Calcium channel blockers
• Decreased sperm concentration, motility and acrosome reaction
• ACE inhibitors
Antibiotics
• Trimethoprim-sulfamethoxazole
• Nitrofurantoin
• Spermatogenic arrest, reduced sperm concentration
• Macrolides
• Impaired sperm motility and viability
• Aminoglycosides
• Impaired spermatogenesis
• Subtilosin
• Impaired sperm motility
• Ketoconazole
• Reduced testosterone production
Oncologic agents**
• Chemotherapy4
• Gonadotoxicity to germ cells, Leydig cells, or Sertoli cells
• High risk: chlorambucil, cyclophosphamide, ifosfamide, busulfan,
melphalan, procarbazine, dacarbazine, and nitrogen-mustard, vincristine,
procarbazine, prednisone (MOPP)
• Intermediate risk: cisplatin, carboplatin, doxorubicin and bleomycine,
etoposide, cisplatin (BEP), and adriamycin, bleomycin, vinblastine,
dacarbazine (ABVD)
• Low risk: vincristine, methotrexate, dactinomycine, bleomycin,
mercaptopurine, vinblastine
• Imatinib
• Leydig cell dysfunction and subsequent impaired testosterone production
Partner history • Female age
• Female evaluation
• Previous pregnancy history
Allergies • Medication allergies
• Latex allergy if considering procedure
Family history • Infertility
• Cystic fibrosis
Goals of fertility • Number of children desired
• Time course couple wishes to conceive
• Restrictions (religious or personal beliefs) of assisted reproductive techniques
* Adapted from Samplaski and Nangia.5
** Adapted from Wallace et al.4
142  Male infertility
Table 11.2  Semen analysis parameters derived from Cooper and colleagues (2010).
Semen analysis parameter
Volume (mL)
Concentration (million sperm/mL)
Total sperm count (million)
Total motility (%)
Progressive motility (%)
Normal morphology (%)
Vitality (%)
Leukocyte count (million/mL)
Source: Splingart, C et al., Int J Androl. 2012;35(3):467–474; Cooper TG et al., Hum Reprod Update.
2010;16(3):​231–245.
Note: Lower 5th percentile values are presented from 1852 semen analyses of men able to conceive
a pregnancy within 12 months of unprotected regular intercourse. Mean values are presented
from a study evaluating 1114 men donating sperm for preservation with a history of fathering
at least 1 child.
fifty-three semen samples were assessed among men from
eight countries on three different continents. The lowest
5th percentile was used as the reference value (Table 11.2).7
It is also important to recognize that values within the
normal range do not guarantee fertility and values below
the 5th percentile do not necessarily prevent men from
obtaining pregnancies as significant overlap exists among
fertile and infertile men with respect to semen analyses.
WHO 5th percentile cutoff values are important to use
to direct further investigation or management targeting
potential sources of impaired fertility; however, mean val-
ues are also of benefit to know (Table 11.2).
Specific semen analysis abnormalities may be defined
as poor sperm concentration (oligozoospermia), reduced
semen volume, poorly motile sperm (asthenozoospermia),
abnormal morphology (teratozoospermia), dead sperm
(necrospermia), or absence of sperm (azoospermia).
Morphology
Kruger and colleagues introduced strict criteria for grad-
ing sperm morphology in 1986 for purposes of prognos-
ticating sperm for success in in vitro fertilization.8 The
criteria were described in additional detail by Menkveld
and colleagues in 1990.9 The normal cutoffs for morphol-
ogy by the WHO has been decreasing to less stringent
values with more recent iterations, thus emphasizing
less importance on the number of sperm required to be
normal for adequate fertilizing potential. However, a
recent study evaluated the receiver operating character-
istics (ROCs) of semen analyses among fertile men and
those requiring IUI. Sperm morphology demonstrated
the greatest area under the curve and thus was most pre-
dictive of fertility.10 Little data exist evaluating the effect
of severely abnormal morphology on natural pregnancy
rate. One study reports natural pregnancy rates of 29.2%
in men with severe 0% normal forms compared to a con-
trol cohort of 55.6%.11 Thus, the fertilizing potential is
likely less; however, natural pregnancy is in fact possible.
The impact of sperm morphology on pregnancy rates
WHO 2010 cutoff
(5th percentile)6 Mean values7
1.5 3.9 ± 1.7
15 93.2 ± 65
39 350.7 ± 279.8
40 –
32 48 ± 16
4 37 ± 24
58 80 ± 12
<1.0 –
with intrauterine insemination (IUI) have demonstrated
heterogeneous results. A meta-analysis from 2001 favored
pregnancy rates among men with >4% normal sperm
morphology12; however, more recent reports have not
found a difference.13 Most importantly, these data suggest
that men with poor morphology <4% are still candidates
for IUI. The literature surrounding IVF and sperm mor-
phology is heterogeneous. A recent meta-analysis from
2011 did not identify normal sperm morphology >4% to
be associated with higher pregnancy rates for either IVF
or IVF-ICSI.14
Motility and progressive motility
The WHO classifies motility as progressive motility
(PM), non-progressive motility (NPM), and immotility
(IM). PM sperm motility refers to active movement of
sperm in a linear or large circular direction. NPM refers
to any other movements with lack of progression and
IM is the complete absence of movement. The impact of
PM on natural pregnancy is heterogeneous. Some stud-
ies suggest PM is important in achieving pregnancies15–17
while recent studies did not find statistically significant
difference once controlled for other semen parameters.18
Sperm motility is likely required for natural pregnan-
cies; however, the proportion of PM or NPM sperm that
are required for a natural pregnancy has not yet been
defined.
ASTHENOSPERMIA
Causes
Asthenozoospermia is present among 25% of men present-
ing for infertility evaluation. The differential diagnosis
includes leukocytospermia, antisperm antibodies, partial
ejaculatory duct obstruction, prolonged abstinence, vari-
coceles, or sperm ultrastructural defects (USDs).19 USDs
may be of the axoneme or periaxonemal structures. These
typically occur among samples with motility less than
7%.20 Kartagener syndrome is a genetic cause of primary
ciliary dyskinesia. It is important to note that immotile

sperm on a semen analysis must be tested with a viability
assay to determine if they are living but immotile sperm or
if they are dead sperm (necrospermia).
Management
Treatment of the underlying cause is always indicated if
identified (i.e., varicocele repair, transurethral resection of
the ejaculatory duct [TURED] for partial ejaculatory duct
obstruction (EDO), and regular intercourse or ejaculation
for prolonged abstinence). For nonmodifiable factors, IVF/
ICSI may be used with live sperm confirmed by the presence
of at least twitching tails after treatment with pentoxifylline.
Semen volume and pH
Semen collection for a semen analysis requires 2–5 days
of abstinence, collection without the use of spermicidal or
toxic lubricants, and processing typically within 1 hour of
collection. Semen should be maintained at room or body
temperature prior to analysis. No upper limit of normal
exists; however, as mentioned earlier, the lower limit of vol-
ume is 1.5 mL. The differential diagnosis for men present-
ing with low semen volume includes incomplete collection,
retrograde ejaculation, testosterone deficiency, obstructed
ejaculatory ducts, absent seminal vesicles, or congenital
bilateral absence of the vas deferens. The latter is diagnosed
by physical examination of the scrotum where no vas def-
erens are palpable. Here, hypoplastic seminal vesicles are
often associated. Obstructed ejaculatory ducts is suspected
with low volume, acidic semen with low fructose, and
absent or few poorly motile sperm. Digital rectal examina-
tion may also be positive for a midline cyst at the level of the
prostate. Transrectal ultrasound (TRUS) is confirmatory
with dilated seminal vesicles >1.5 cm in diameter and often
a midline cystic structure. Management of ejaculatory duct
obstruction includes TURED with the guidance of TRUS.
Low testosterone is diagnosed by serum testing. Men found
to have low testosterone cannot be placed on exogenous tes-
tosterone. The exogenous testosterone will provide negative
feedback to the anterior pituitary and hypothalamus, con-
sequently downregulate the hypothalamic-pituitary-testes
(HPT) axis and inhibit release of luteinizing hormone
(LH) and follicle-stimulating hormone (FSH). This sup-
presses spermatogenesis. Alternatively, many practitioners
use selective estrogen receptor modulators (SERMs) such
as clomiphene to upregulate the HPT axis and endogenous
testosterone production. In central pituitary or hypotha-
lamic abnormalities, human chorionic gonadotropins
(hCG), recombinant FSH, or human menopausal gonado-
tropins (hMGs) may be used.
Postejaculate urinalysis
In men with either low semen volume or a medical con-
dition that predisposes one to retrograde ejaculation,
a postejaculate urinalysis should be obtained to search
for the presence of sperm on microscopic assessment.
Retrograde ejaculation occurs when the bladder neck is
incompetent functionally or anatomically and is the path
of least resistance for semen to travel.
Azoospermia 143
Concentration and sperm count
Eight percent of men presenting with infertility will have a
sperm concentration less than 15 million/mL and be classi-
fied as asthenospermia.22 The minimal number of sperm to
achieve a natural pregnancy is unknown. Several studies have
evaluated the effect of sperm concentration and time to preg-
nancy. Results suggest that an increased trend is observed
among sperm concentrations increasing to 40–55 million/
mL, but the effect levels out at greater concentrations.23,24
Thus, low sperm concentrations below 40 million/mL may
have an impact on time to natural pregnancy among couples.
With respect to IUI, sperm concentration and total motile
count (motility × total sperm count) have been demonstrated
to be the strongest predictors of success. Total motile count of
5 million or greater portends to greater IUI pregnancy rates.25
IVF results are best with 3 million total motile count.26
OLIGOSPERMIA
Causes
Oligozoospermia is directly related to spermatogenesis. The
differential diagnosis includes environmental factors, medi-
cal conditions, and medications. Environmental factors
include heat exposures, pesticides and chemical exposures,
high caffeine intake, diets high in saturated fat, tobacco use,
chronic intense endurance exercise, stress, marijuana use,
opioid abuse, and excessive alcohol use.27 Medical conditions
include varicoceles, systemic illnesses, fever, cryptorchidism,
obesity,28 primary or secondary hypogonadism, radiation
therapy,4 previous testicular trauma or orchitis, malignancy
and genetic causes such as Y-chromosome microdeletions,
and karyotype abnormalities such as Klinefelter syndrome
(progressive testicular failure), and Kallman syndrome (cen-
tral hypogonadism). Medication causes include but are not
exclusive to the following: chemotherapy, nitrofurantoin,
aminoglycosides, diuretic and calcium channel blocker anti-
hypertensives, 5 alpha reductase inhibitors, and exogenous
testosterone or anabolic steroid use.5
Management
Oligozoospermic men with counts less than 10 million/
mL warrant a hormonal evaluation with serum FSH and
testosterone.29,30 Men with Oligozoospermia less than
5 million/mL warrant both hormonal evaluation and
genetic testing including karyotype and Y-chromosome
microdeletion.31 Patients may be counseled to improve
lifestyle or environmental exposures, consider alternative
medications, or treat medical illnesses. Beyond reversible
causes of oligospermia, IUI may be considered in patients
with a total motile count greater than 5 million, IVF for
patients with a total motile count of 3 million, and IVF-
ICSI for severely oligozoospermic patients.
AZOOSPERMIA
Azoospermia is defined as the absence of spermatozoa
after two semen specimens have been examined with
extended search after centrifugation. The etiology may
be due to a blockage within the male reproductive tract,
144  Male infertility

Table 11.3  Investigations to differentiate the etiology of azoospermia.

Post-testicular
Ejaculatory
Investigation Pretesticular Testicular Obstructive ­dysfunction
Testicular size ↓ N/↓ N N
Semen volume N N N/↓ ↓
Post ejaculate N N N N/+ for sperm
urinalysis
Semen pH ≥7.2 N N N/↓ N
FSH ↓ ↑/N N N
T ↓ ↓/N N N
Prolactin N/↑ N N N
TRUS N N N/Abn N
Karyotype N/Abn N/Abn N N
y-Microdeletion N N/Abn N N
CFTR N N N/Abn N
Note: NOA can be classified into pretesticular or testicular abnormalities. Pretesticular, also termed hypogonadal hypogonad-
ism, is due to a central deficiency in the pituitary or hypothalamus. Testicular dysfunction, also termed hypergonadal
hypogonadism, is due to failure of the testicle to produce testosterone and/or spermatogenesis. Post-testicular azoosper-
mia may be due to obstruction in the epididymis, vas deferens, or ejaculatory duct. Ejaculatory dysfunction is best diag-
nosed by history and may comprise the absence of ejaculation failure of emission; here, semen volume is low and a
postejaculate urinalysis must be performed to assess for retrograde ejaculation.

termed obstructive azoospermia (OA), or may be due to
impaired spermatogenesis, classically termed nonobstruc-
tive azoospermia (NOA).
NOA may be due to central hypothalamic or pituitary
abnormalities (pretesticular azoospermia) or due to testic-
ular failure. History, physical exam, serum hormonal pro-
file (testosterone, estrogen, FSH +/− LH and prolactin), and
genetic testing including karyotype and Y-chromosome
microdeletion are part of a standard evaluation to deter-
mine the etiology (Table 11.3). NOA patients often have
small testes on physical exam and the epididymis is flat
and nondilated. Histology of testicles from NOA patients
are classified into four patterns: hypospermatogenesis, late
maturation arrest, early maturation arrest, and Sertoli-
only syndrome.
If the pituitary is intact, pulsatile GnRH 25 to 600 ng/KG
may be delivered subcutaneously, intra­venously, or by pump
every 120 minutes. Otherwise, hCG 1000 to 2500 interna-
tional units (IUs) are injected intramuscularly or subcuta-
neously twice per week and act to stimulate the Leydig and
Sertoli cells, with or without hMG 75 to 150 IU three times
per week; alternatively, recombinant FSH (rFSH) may be
used in place of hMG but is significantly more costly.33
Surgical sperm retrieval
Among patients with NOA, sperm is surgically retrieved
from the testicle. Techniques include testicular sperm aspira-
tion (TESA) (Figure 11.1), testicular sperm extraction (TESE),

Pretesticular
Causes
The differential diagnosis for pretesticular causes of
azoospermia include congenital abnormalities, such as
Kallman syndrome, characterized by midline defects,
anosmia, and tall stature, or acquired abnormalities, such
as pituitary tumor where they may present with headache,
galactorrhea, or hemianopsia, cranial radiotherapy, or
surgical extirpation, or exogenous androgens.32

Management
Treatment of these patients entail two components: medi-
cal correction of their hormonal abnormalities, and surgical
retrieval of testicular sperm if no sperm returns to the ejacu-
late following hormonal modification. GnRH or gonado-
tropins may be used depending on the a­ natomical location
of the defect. Several regimens exist for hormonal therapy.
Figure 11.1  Testicular sperm aspiration (TESA) for sperm
retrieval in men with obstructive azoospermia. This procedure
is often performed in an outpatient setting with local anesthetic
to the scrotal skin and a spermatic cord block is performed. A
23-gauge needle is passed into the testis 20–30 times while
aspirating. The fluid is then placed in sperm transport media.
 Azoospermia 145

Figure 11.2  Microdissection testicular sperm extraction (mTESE) for non-obstructive azoospermia. This procedure is typically
performed under a general anesthetic. The testicle is delivered from the scrotum and bivalved in a transverse equatorial fashion
with a 15-degree microknife. The seminiferous tubules are searched under 15-20x magnification for dilated opalescent tubes. These
tubes are teased out of the surround tissue, placed in a petri dish and minced with sterile scissors, and filtered through a 24-gauge
angiocatheter-tipped syringe to further break down the tissue. The tissue is placed on a microscope slide with a small amount of
sperm transport media and examined for the presence of sperm on a bright field microscope in the surgical theater.

and microdissection testicular sperm extraction (mTESE) Testicular azoospermia

(Figure 11.2). TESA is performed in an outpatient setting
with local anesthesia in the form of a cord block and skin
wheal. A 23-gauge needle is passed into the testis 20–30
times while aspirating. Contents are placed into a sperm
buffer. TESA is successful in 52%–100% of cases.34 TESE
involves a general anesthetic, delivery of the testicle, inci-
sion in the tunica albuginea, and excision of seminifer-
ous tubules. This technique has largely been replaced by
mTESE, which involves the use of an operating micro-
scope and was described in 1999 by Peter Schlegel.35 Here,
seminiferous tubules are visualized under a 10–20 power
operating microscope; dilated tubules are identified and
selectively removed as they are more likely to contain
active spermatogenesis and yield sperm. This technique
results in sperm retrieval rates of 45%–63%, and 70-fold
less tissue is excised compared to conventional TESE.36
Surgical sperm retrieval rates vary by histologic subtype
of NOA: 73%–91% in hypospermatogenesis, 27%–86%
in late maturation arrest, 27%–40% in early maturation
arrest, and 31%–73% in Sertoli-cell-only.37–40 MicroTESE
is considered the gold standard for sperm retrieval of
NOA patients. It is important to note that a semen analysis
should be performed on all men the morning of a sched-
uled mTESE because 5%–10% of NOA patients will have
sperm in their ejaculate despite prior documentation of
azoospermia, thus obviating the need for the surgical
procedure.41
Causes
Testicular azoospermia is the result of the testicles not
responding to appropriate levels of FSH and LH. The dif-
ferential diagnosis for testicular failure can be organized
into congenital and acquired causes. Congenital causes
include Klinefelter syndrome (i.e., XXXY karyotype),
Y-chromosome microdeletion (AZFa, AZFb, AZFc), and
cryptorchidism. Acquired causes include orchitis, vari-
coceles, significant heat exposures, malignancies, chemo-
therapy, or radiation therapy. Successful sperm retrievals
have not been reported in the literature for complete AZFa
and AZFb Y-chromosome microdeletions, but are reported
in 70% of men with AZFc deletions.42
Management
Medical optimization prior to surgical sperm retrieval
in NOA patients with testicular failure has been lightly
studied. One multicenter study determined that use of
clomiphene citrate, hCG, and hMG to elevate presurgical
testosterone levels to 600–800 ng/dL and FSH to 1.5 times
baseline improved sperm retrieval rate from 34% in the
control group to 57% in the medical intervention group.43
Aromatase inhibitors such as anastrozole have also dem-
onstrated benefit in NOA patients with low testosterone
and elevated estrogen levels.44 These medications have lim-
ited evidence and are presently used off-label. Otherwise,
146  Male infertility

surgical sperm retrieval is performed in this patient popu- rectal injury may also be used. Upon relieving the obstruc-
lation as mentioned above with mTESE. tion, cloudy milky fluid is often released.46
Posttesticular Epididymal obstruction and vasal obstruction
Posttesticular Azoospermia is due to a mechanical is diagnosed by history, physical exam demonstrating nor-
obstruction in the male reproductive tract or secondary to mal volume testicles, and normal serum FSH and testos-
ejaculatory or emissary dysfunction. terone. Among patients presenting with a vasectomy and
Obstructive causes previously normal spermatogenesis, then the presence
of normal volume testicles, serum FSH, and testosterone
OA due to mechanical obstruction may be secondary to is acceptable. However, if history identifies risk factors
vasectomy at the level of the vas deferens or at the level of for secondary impaired spermatogenesis or the patient
the epididymis secondary to epididymal blowout in the is presenting with primary epididymal or vasal obstruc-
setting of vasectomy, previous infection causing epididy- tion, then confirmation of spermatogenesis must be per-
mal stricturing, iatrogenic damage to the epididymis or vas formed. The gold standard method for confirmation of
deferens at the time of hydrocelectomy, orchidopexy, sper- normal spermatogenesis is testicular biopsy, demonstrat-
matocelectomy, or inguinal hernia repair. These patients ing active spermatogenesis with at least 20 mature sper-
will present with normal volume azoospermia. Congenital matids per seminiferous tubule cross section.48 However,
bilateral absence of the vas deferens (CBAVD) occurs due use of serum antisperm antibodies is an effective means of
to one of several possible mutations of the cystic fibro- identifying active spermatogenesis in an OA patient; here
sis transmembrane regulatory (CFTR) protein gene and
the area under the curve (AUC) for IgG against sperm tails
results in absence of the vas deferens and an incompletely
has been shown to be 0.92 with a sensitivity of 85% and
formed epididymis on physical exam; these patients will
specificity of 97%, while IgA demonstrated the greatest
often have hypoplastic seminal vesicles and demonstrate
specificity of 99%, a positive predictive value of 99% and
low volume on their semen analysis. They require CFTR
a positive likelihood ratio of 70.49 Patients presenting with
testing for both the patient and partner to provide appro-
epididymal or vasal obstruction can be managed by sperm
priate counseling for the risk of cystic fibrosis in their
retrieval, microsurgical reconstruction, or a combination.
potential offspring. Ejaculatory duct obstruction is another
etiology and often presents with a low-volume, acidic Sperm retrieval in OA
semen analysis with absence of fructose. In the presence
Sperm may be retrieved from the epididymis in OA.
of low semen volume and a pH <7.2, a digital rectal exam
This may be performed percutaneously or microsurgi-
and TRUS ultrasound is indicated to evaluate the patient
cally. Percutaneous epididymal sperm aspiration (PESA)
for cystic dilatation of the ejaculatory duct or enlarged
involves transcutaneous insertion of a 21–26-gauge nee-
seminal vesicles with a diameter greater than 1.5 cm. In the
dle into the epididymis while withdrawing (Figure 11.3).
case of equivocal TRUS findings but strong clinical suspi-
cion, a T2-weighted magnetic resonance imaging with an
endorectal coil may provide enhanced tissue resolution and
enable detection of subtle abnormalities.45 The differential
diagnosis for EDO includes congenital and acquired etiolo-
gies. Congenital causes include ejaculatory duct atresia or
stenosis and cysts of the prostate or utricle. Acquired causes
include seminal vesicle calculi, stricturing postcatheteriza-
tion or instrumentation, prostatitis, procedures for benign
prostatic hyperplasia such as transurethral resection of
prostate, green light laser, or prostate cancer radiotherapy,
or focal therapies such as cryotherapy, or high-intensity
focused ultrasonography (HIFU).46

Management of obstructive azoospermia

Ejaculatory duct obstruction may be managed via laser
incision of the ejaculatory ducts, retrograde balloon
dilatation, retrograde seminal vesiculoscopy using a 9F
scope or transurethral resection of the ejaculatory duct
(TURED).46,47 TURED is both a popular and effective
technique. Here, a resectoscope is introduced into the
urethra and tissue is resected on the inferiolateral veru-
montana. Care is taken to avoid injury to the bladder neck
and external striated sphincter. In addition, intraoperative
use of TRUS to guide depth of resection and avoidance of
Figure 11.3 Percutaneous epididymal sperm aspiration
(PESA) for sperm retrieval in men with obstructive azoosper-
mia. This procedure is often performed in an outpatient set-
ting with local anesthetic to the scrotal skin and a spermatic
cord block is performed. The testicle is secured in the surgeon’s
nondominant hand and a 21-26-gauge needle is inserted into
the epididymis while aspirating for fluid. It is good practice to
examine the fluid under bright field microscopy to confirm the
presence of motile sperm.

Figure 11.4  Microsurgical epididymal aspiration (MESA)
for men with obstructive azoospermia. The testis is deliv-
ered from the scrotum and visualized through an operat-
ing microscope under 15-25x magnification. A 15-degree
microknife is used to incise a dilated tubule and the fluid
is aspirated with a 24-gauge angiocatheter-tipped syringe
or micropipette. The fluid is examined under bright field
microscopy in the surgical theater to confirm presence of
sperm. The fluid collected is then placed in sperm transport
media in Eppendorf tubules.
The fluid is then assessed on a microscope for presence
of motile sperm that can be used for IVF-ICSI. PESA
can be performed with local anesthetic in the office set-
ting. Sperm is successfully retrieved in 61-96% of cases
using PESA.50,51 Microsurgical epididymal sperm aspira-
tion (MESA) is the gold standard method for epididymal
sperm retrieval (Figure 11.4).
MESA is performed under a general or regional anes-
thetic. The testicle is delivered and the epididymis is
examined for dilated tubules. A 15-degree microknife
is then used to incise an “epididymal tubule and the fluid
is aspirated by capillary action into an ordinary 5-µl
capacity laboratory pipette and examined under a bench
microscope to identify motile sperm. MESA is successful
in retrieving sperm in 96%–100% of patients with OA,52,53
typically yielding 15–95 million sperm, which is adequate
for cryopreservation in 98%–100% of patients.34
Table 11.4  Intraoperative vasal fluid is sampled through a hemivasotomy at the time of vasal reconstruction for OA.
Vasal fluid
Copious clear fluid
Cloudy fluid
Creamy yellow fluid
Thick white toothpaste-like fluid
Dry vas with no granuloma
Dry vas with granuloma at vasectomy site Barbotage fluid reveals sperm
Note: The decision to proceed with vasovasostomy compared to vasoepididymostomy is made by examining the appearance of the
vasal fluid and the microscopic findings.
Ejaculatory dysfunction  147
Reconstruction
Epididymal and vasal obstruction are often differenti-
ated intraoperatively depending on characteristics of the
vasal fluid following history of vasectomy (Table 11.4).
These findings direct decision making for the appropri-
ate reconstruction required. Important concepts for both
types of reconstruction include ensuring sperm parts or
copious clear fluid is present on the testicular end of the
anastomosis and an unobstructed abdominal vas. This can
be tested by injecting saline through the vas deferens with
a 24-gauge angiocatheter or placing a 2-0 monofilament
suture into the abdominal vas to ensure it can be passed
freely beyond the inguinal region.
Epididymal obstruction is corrected via a vasoepidid-
ymostomy (VE). Several techniques have been described;
however, the technique with the greatest patency rate is
the longitudinal intussusception vasoepididymostomy
(LIVE) technique (Figure 11.5). This technique has dem-
onstrated a patency rate of 90% in experienced hands.54,55
Vasal obstruction is reconstructed by performing a
vasovasostomy. Several techniques are described in the
literature and are largely differentiated by single and
multilayer techniques. A recent meta-analysis reported
a mean patency rate of 89.4% for VVs and pregnancy
rate of 73.0%. They also determined that no statistically
significant differences existed in the patency of single vs
multilayer techniques.56 However, the highest reported
patency rate in the literature of 99.5% involves a multi-
layer technique involving six microdots to separate plan-
ning from suture placement of corresponding 10-0 nylon
sutures to intricately reapproximate the mucosal layer,
followed by six deep muscularis sutures using 9-0 nylon,
six superficial 9-0  nylon sutures, and a final adventitial
layer (Figure 11.6).57
EJACULATORY DYSFUNCTION
Causes of Ejaculatory Dysfunction Ejaculatory dysfunc-
tion may be due to failure of emission, anejaculation (AE),
or retrograde ejaculation (RE).
Retrograde ejaculation
Retrograde ejaculation occurs when the bladder neck
is incompetent. RE may be implicated in up to 18% of
men presenting with azoospermia.21 RE may be due
Microscopic findings Surgical procedure indicated
No sperm Vasovasostomy
Intact sperm Vasovasostomy
Sperm heads Vasovasostomy
No sperm Vasoepididymostomy
No sperm Vasoepididymostomy
Vasovasostomy
148  Male infertility

(a) (b)

(d)
(c)

(e) (f )

Figure 11.5  Longitudinal intussusception vasoepididymostomy (LIVE) technique is performed in men with obstructive azo-
ospermia. Vasoepididymostomies are performed under a general anesthetic. Once the testicle is delivered and the transition
from dilated to collapsed tubules has been identified, a region of dilated tubules just prior to the transition is selected. The tunica
is incised exposing the dilated tubules (a). A double-armed 10-0 nylon suture is placed longitudinally on one side of the epi-
didymal tubule (b). The needle is only placed halfway through so that the curve of the needle remains in the tubule. The needle
is not driven all the way to prevent decompression of the fluid around the suture, which is smaller diameter than the needle. A
second suture is then placed parallel to the initial suture (b). Four microdots are drawn on the muscularis of the vas deferens. A
microknife is used to incise the tubule longitudinally between the two needles (c). The fluid is sampled for sperm or sperm parts
under a microscope in the surgical theater. If present, the anastomosis is completed; if no sperm or sperm parts are present, then
the surgeon must move proximally. The remaining ends of the double-armed sutures are then placed inside-out through the
microdot and tied (d and e). Then, 9-0 nylon sutures are used to reapproximate the muscularis and adventitia to the tunica of the
epididymis (f).

to congenital abnormalities such as posterior urethral Causes

valves, utricular cysts, or extrophy. RE may also be due
to an anatomic defect following prostatic resection, inci-
sion, or ablation or due to a functional deficit. Functional
deficiencies may be due to use of alpha blockers, psycho-
tropic medications, abdominal or pelvic surgeries such as
retroperitoneal lymph node dissection, aortoiliac vascu-
lar surgery and abdominal perineal resection, or neuro-
logical diseases such as diabetic autonomic neuropathy,
multiple sclerosis, myelodysplasia, multisystem atrophy,
cerebrovascular accident, spinal cord injuries, and lum-
bar sympathectomy.
Medical conditions known to be associated with retro-
grade ejaculation include alpha-blocker medications, pre-
vious transurethral resection of the prostate, diabetes, and
spinal cord lesions.
Management
A trial of a sympathomimetic such as oral pseudoephed-
rine or a tricyclic antidepressant imipramine may be trialed
prior to ejaculation for men with a functionally incompe-
tent bladder neck. Other agents described in the literature
include brompheniramine, midodrine, and ephedrine.
 Ejaculatory dysfunction  149

(a) (b)

(c) (d)

(e)

Figure 11.6  Vasovasostomy microdot multilayer closure performed for men with obstructive azoospermia where the level of
obstruction is in the vas deferens. This procedure is performed under a general anesthetic and the testicle is delivered through
a vertical scrotal incision on the respective side. The site of obstruction is identified and the vas deferens is transected (a).
Patency of the vas deferens is checked on the abdominal side and fluid from the testicular side is evaluated under a microscope
for sperm or sperm parts. Reconstruction is then performed as depicted above. Six microdots are drawn onto the muscularis
layer of the cut surface (b). Three 10-0 double-armed nylon sutures are then placed in an inside-out fashion and tied (c). Three
9-0 sutures are placed in the muscularis between the 10-0 sutures (d). An additional layer of three 9-0 sutures are placed super-
ficially through the muscularis between the previous 9-0s. The vas deferens is then flipped and the second half is completed in
the same sequence. Finally, a fourth layer of 9-0s are placed to reapproximate the adventitia to complete a patent and water-
tight anastomosis (e).

Pseudoephedrine (Sudafed) 60-mg tablets may be taken
four times per day in the 2–3 days leading up to ejacula-
tion.21 If pseudoephedrine is not successful or an anatomi-
cally abnormal bladder neck is present (i.e., transurethral
resection of the prostate), then the postejaculate urine
specimen may be collected, centrifuged, and the pellet of
sperm may be resuspended and used for assisted reproduc-
tive techniques, IUI, or if of poor quality, IVF/ICSI.
Anejaculation
Anejaculation occurs when the patient fails to ejaculate.
This may be due to psychological stress, hyperstimula-
tion of sexual content, chronic medical conditions such
as chronic kidney disease requiring dialysis, selec-
tive serotonin reuptake inhibitor use, low testosterone,
decreased penile sensation, and neurological disorders
such as spinal cord injury (SCI), and some men with
multiple sclerosis.58 Pelvic surgery and retroperitoneal
lymph node dissection may be associated with ejacula-
tory dysfunction or failure of emission where sperm and
semen are transported to the posterior urethra prior to
ejaculation.58
Management of anejaculation and failure
of emission
Management of RE is described above in the manage-
ment of low semen volume. Modifiable factors such as
psychological stressors, hyperstimulation, use of selec-
tive serotonin reuptake inhibitors, and low testoster-
one should be identified and attempts made to address
these factors. Among patients still unable to ejaculate
through intercourse, outercourse, or masturbation, then
vibrostimulation (VS) or electroejaculation (EEJ) should
be considered.
150  Male infertility
Vibrostimulation
Is less invasive than EEJ and should be considered first.
Here, a vibrator device is applied to the frenular surface of
the glans until ejaculation ensues. This technique may be
used for men with psychogenic anejaculation or second-
ary to SCIs above T12. For VS to work successfully, the
patient’s ejaculatory reflex arc must be intact, including
the afferent sensory nerves, S2-4 ejaculatory center, and
the efferent motor nerves. This technique may be per-
formed at home or in the office. The treating physician
should always be aware of autonomic dysreflexia among
patients with a SCI above T6. Sperm may be used for intra-
vaginal insemination, IUI, IVF, or IVF-ICSI.59,60
Electroejaculation
Involves using a rectal probe that serves as an electrode to
deliver electrical stimulation to the prostate and seminal
vesicles. The patient is positioned to right lateral decubitus.
The patient is catheterized prior to the procedure using
mineral oil as lubrication and the urine is drained. Twenty
to 30 mL of sperm transport media (e.g., human tubal fluid
[HTF] buffered with hydroxyethyl-piperazineethane-sul-
fonic acid buffer and plasmanate) is then instilled into the
bladder. Anoscopy is performed to ensure that the rectal
mucosa is healthy. The Seager Electroejaculator© probe
is then inserted into the rectum and either a peaked sine
wave or 1-second block paradigm is used with five stimu-
lations, repeated up to six times. The probe temperature
must be kept below 38 degrees Celsius and below 30-volts
maximum. An assistant holds a sterile specimen cup in
front of the meatus to collect the sample. Anoscopy is
again performed at the end of the procedure. EEJ may be
performed in a surgical theater for sensate patients and
may be performed in either the office or surgical theater
for patients not sensate below the waste. However, the
physician must again monitor for autonomic dysreflexia
(AD) in patients with SCI above T6 and have the ability
to treat respected changes. Many physicians recommend
pretreating >T6 SCI patients with nifedipine; a discus-
sion with the anesthetist preoperatively is recommended.
If AD does ensue, the stimulation should be withdrawn
immediately.21
DNA FRAGMENTATION
Sperm DNA integrity is required for subsequent embryo
development. DNA integrity is thought to be maintained
and protected by chromatin repackaging in the nucleus
by disulfide cross-links among protamines. However, the
DNA can acquire strand breaks and damage. This can
occur intrinsically via dysfunctional DNA repair, prot-
amine deficiencies,61 and incomplete apoptosis.62 Extrinsic
factors are also thought to contribute to DNA fragmen-
tation, such as prolonged abstinence, heat exposures,
radiation, gonadotoxins, and varicoceles.63–65 Increased
DNA fragmentation has been associated with spontane-
ous recurrent miscarriage, impaired fertilization, early
embryo development, implantation, and pregnancy.66 A
meta-analysis determined that elevated DNA fragmenta-
tion was associated with lower clinical pregnancy rates in
both IVF and ICSI techniques. However, the positive and
negative predictive abilities are limited at 36% and 40%,
respectively, for ICSI and are worse for IVF.67 Another
meta-analysis reports improved live birth rates for IVF in
men with low sperm DNA fragmentation.68 Reports have
also determined increased miscarriage rates among men
with elevated sperm DNA fragmentation undergoing ICSI
but not IVF.69
Several assays presently exist: sperm chromatin disper-
sion (SCD) or halo test,70 sperm chromatin structure assay
(SCSA),71 comet assay,72 and terminal deoxynucleotidyl
transferase dUTP nick-end labeling assay (TUNEL).73
SCD, SCSA, and TUNEL measure the proportion of sperm
with DNA strand breaks, while comet assay measures
the degree of DNA fragmentation per sperm. The comet
assay has the ability to detect single- and double strand-
DNA breaks as well as protamine and histone associated
breaks in chromatin, while TUNEL can detect single- and
double-strand DNA breaks, and both SCD and SCSA can
only detect single-strand DNA breaks.
Management
Presently, DNA fragmentation assays are not considered
routine evaluation; however, as more data from clini-
cal studies emerge, the role of DNA fragmentation may
also evolve. However, many practitioners will assess
DNA fragmentation in patients with recurrent preg-
nancy loss via natural conception or ARTs. Strategies to
improve DNA fragmentation are presently limited in
number. Varicocelectomy, use of antioxidants, and use
of testicular sperm have been strategies reported to date.
Varicocelectomies have demonstrated improvements in
DNA fragmentation74; however, a meta-analysis noted lim-
ited improvement of DNA fragmentation.75 Antioxidant
formulations vary widely in content. However, Greco
and colleagues used daily vitamin C and vitamin E in
men with elevated DNA fragmentation and resulted in
76% of men reducing their TUNEL scores to the normal
range for their lab, and also resulted in significantly more
clinical pregnancies using ICSI compared to those not
using antioxidant formulations.76 A meta-analysis assess-
ing the impact of antioxidants on fertility determined a
positive benefit on live birth rates, and the two studies
assessing DNA fragmentation demonstrated improved
rates.77 Studies have demonstrated that among men with
high DNA fragmentation, use of testicular sperm often
has lower DNA fragmentation; two studies have reported
improved clinical pregnancies, live births, and fewer mis-
carriages in couples with high ejaculated DNA fragmen-
tation and subsequent use of testicular sperm with lower
DNA fragmentation.78,79
VARICOCELE
Varicoceles are identified in 12% of men with normal
semen parameters, 40% of men with primary infertility,

and 80% of men with secondary infertility. However,
among all men with varicoceles, approximately 20% will
have difficulties with fertility.80 Varicoceles are most
often associated with oligoasthenoteratospermia (OAT);
however, repair has been shown to improve each of these
semen parameters. Eighty-five to 90 percent of varico-
celes arise on the left side, thought to be secondary to the
90-degree entry of the left spermatic vein into the renal
vein causing higher hydrostatic pressure, compared to the
oblique approach on the right side.81,82 Additional theories
for development of varicoceles include absent or dysfunc-
tional venous valves and compression of the spermatic vein
between the superior mesenteric artery and renal vein.83,84
The negative effect of varicoceles on testicular function
is thought to occur via multiple mechanisms: increased
scrotal temperature,85 hypoxia,86 increased venous pres-
sure,87 oxidative stress,88 reflux of toxic renal and adrenal
metabolites,89 and hormone imbalances.90
Management
Varicoceles are diagnosed by physical exam. In stand-
ing position, grade 1 varicoceles can be palpated with
Valsalva, grade 2 can be palpated even in the absence of
Valsalva, and grade 3 can be visualized through the thin
scrotal skin even in the absence of Valsalva. A varicocele
that is palpable should become not palpable upon chang-
ing positions from standing to supine. In the event of an
isolated right-sided varicocele, abdominal imaging, typi-
cally in the form of an ultrasound, is warranted to assess
for an intra-abdominal mass causing venous compression.
Furthermore, if physical exam is unclear, a scrotal ultra-
sound should be considered. Here, spermatic cord vein
diameters should be measured both supine and standing,
as well as assessment for reversal of blood flow. Vein diam-
eters larger than 3 mm and reversal of flow are considered
clinical varicoceles; veins smaller than 3 mm with reversal
of flow are termed subclinical varicoceles.
Varicoceles may be repaired through various tech-
niques: percutaneous embolization, laparoscopic, ingui-
nal, and microscopic subinguinal (Figure 11.7). The latter
has been the gold standard in the literature with the low-
est recurrence rate of 0.8% to 2.1% and the lowest inci-
dence of postoperative hydrocele formation among the
surgical techniques.91,92 Postoperative hydroceles occur
when lymphatics are indiscriminately ligated during
varicocele repair (VR); however, these can be visually
identified and avoided in the microscopic subingui-
nal technique. VR has been demonstrated to improve
sperm motility, concentration, and morphology, increase
spontaneous pregnancy rates,91 and reduce miscarriage
rates.93 In a meta-analysis, VR has also been shown to
increase live birth rates among oligospermic men; preg-
nancy rates were higher in men with azoospermia as was
surgical sperm retrieval rates among azoospermic men.94
The effects of a VR are typically not observed prior to 3–6
months as one round of spermatogenesis typically takes
at least 64 to 72 days.95
References 151
Genital femoral nerve
Vasal veins (ligated)
Vas deferens
Lymphatic
Testicular artery
Vessel loop
Figure 11.7  Subinguinal varicocelectomy. A 2.5-cm oblique
incision in the lines of Langerhans is made inferior to the exter-
nal inguinal ring and the spermatic cord is delivered. The vas
deferens is identified and excluded. The external and internal
spermatic fascia is opened and the internal spermatic artery is
identified with Doppler ultrasound and marked with a vessel
loop. The veins are then identified, isolated, and ligated with
4-0 silk ties around the artery and titanium clips peripherally.
Deferential and cremasteric arteries are also identified in most
cases (not shown here) and preserved. Lymphatics are also iden-
tified by their scalloped and clear appearance and are preserved.
ACKNOWLEDGMENT
The authors would like to thank Vanessa Dudley for the
figure illustrations.
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Effects of chemical pollutants on
spermatogenesis and implications
in male infertility
CHRIS KC WONG
ENVIRONMENTAL POLLUTION AND REPRODUCTIVE
HEALTH
Introduction
In the past century, the remarkable advancement in indus-
trialization and technology have driven changes to the
environment to a scope that is unprecedented in human
history. An overwhelming number of synthetic chemicals
of highly heterogeneous structures have been produced for
industrial, medical, and domestic purposes. These chemi-
cal substances were initially thought to have negligible
or no biological activity and are extensively used in our
daily lives. The chemical residues are ubiquitous in our
environment and are found to contaminate air, water, and
foods. Public awareness on the potential health impacts
of these man-made chemicals, (i.e., pesticides) was stirred
by the book Silent Spring published in 1962 and written
by American biologist Rachel Carson. In recognition of
toxicities of chemicals in our ecosystem, some man-made
chemicals were excluded for their manufacture and usage.
For example, the insecticide dichlorodiphenyltrichloro-
ethane (DDT) was banned for agricultural use in 1972.
Other chemicals, like polychlorinated biphenyls (PCBs),
the compounds widely used as dielectric and coolant flu-
ids in transformers and capacitors, were phased out in
1979. Although public concern about chemical pollution
in our environment was elevated, the potential impacts
of these chemicals on ecological and animal health were
largely undisclosed up until 1994, when the first World
Wildlife Federation Wingspread Conference took place
and global concerns were raised about the health effects
of chemical pollution.1,2 Some of the more harmful chemi-
cal pollutants were classified as endocrine disrupting
chemicals (EDCs) since they could presumably interfere
with the synthesis, metabolism, and action of endogenous
hormones.3,4 Since then, a considerable number of stud-
ies have been conducted to reveal the ecological distribu-
tion, animal body loading, and health effects of EDCs.
For instance, a study from the U.S. Centers for Disease
Control (CDC) reported that Americans of all ages accu-
mulated over 116 extraneous chemicals into their bodies.5
Over 358 industrial chemicals and pesticides were detected
in cord blood of American infants.6 The data have shown
the ubiquitous contamination of chemical contaminants
in our environment and food-chains, leading to the pres-
ence of these exogenous chemicals in the human body.7
12
A recent review postulated that about 40% of human deaths
(62 million per year) is to a certain degree attributed to
exposure to chemical pollutants via different exposure
routes.8 To safeguard public health, instrumental chemi-
cal analysis has been adopted globally and routinely to
identify and quantify all these contaminants in order to
evaluate the possible risk of human exposure to EDCs
and their metabolites.9 At present, the potentially harmful
effects on human health and ecological well-being caused
by endocrine disruptions are one of the top international
concerns. A 2013 report by United Nations Environment
Programme/World Health Organization (UNEP/WHO)
showed that the prevalence of chronic disease is increas-
ing. An association between EDC exposure and a number
of noncommunicable diseases in humans is advocated.
Plausible causal links between EDC exposures to many
human chronic diseases are widely supported by both lab-
oratory animal and epidemiological studies. The incidence
of reproductive problems is also a prevalent issue globally.
A positive correlation between exposure of chemical pol-
lutants and the increased risk of male subfecundity has
been reported.
Human reproductive health is declining
Data from the World Bank and WHO have revealed a
declining trend (from 4.8 to 2.6) of world total fertility
rates  from 1970–2010, affecting over 80 million people
worldwide.10 Studies indicated that 30% of infertility cases
are related to male factor problems11 such as immunologi­
cal  disorders, structure abnormality, and sperm produc-
tion. Recent data for 2011–2013 from WHO show low
fertility rates in developed countries in North America
(1.6–2.0), Europe (1.4–2.0), and Asia (1.7–3.1) compared
to  developing countries in Africa (4.1–5.7). To unravel
mechanisms attributing to human infertility, a consider-
able amount of resources have been allocated to identify
genetic targets on fertility.12 However, most reported infer-
tility cases remain unexplained. This may be due to our
limited grasp of the empirical mechanisms underlying
fertility12 or that the cases were complicated by nongenetic
factors including diseases and/or exposure to environ-
mental contaminants. In fact, clues regarding the decline
in human reproductive health can be revealed from inves-
tigating numerous historical cases of pollution issues
reported in both wildlife and humans.
155
156  Effects of chemical pollutants on spermatogenesis and implications in male infertility
A great deal of evidence has suggested that the exposure
to EDCs is related to animal reproductive problems world-
wide. Many incidences of pollutant exposure and conse-
quent adverse biological effects on reproductive function
have been described in wildlife. For example, in the 1990s,
adult male alligators in Lake Apopka in Central Florida
exposed to agricultural wastes (dicofol and DDT, its metab-
olites) produced low testosterone levels and presented
micropenis with disorganized testes.13 A coordinated field
and laboratory study reported exposure to the herbicide
atrazine that caused gonadal dysgenesis and testicular
oogenesis in leopard frogs.14,15 A report from CHEM Trust
in 2008 revealed the negative impacts of chemical pollu-
tions on the reproductive health of male vertebrate wild-
life, covering many different species of animals (i.e., fish,
amphibians, reptiles, birds, and mammals).16 Moreover, the
report underlined the mixture effects of different classes of
chemical contaminants on perturbing endocrine systems,
leading to the deformities of sex-linked structures and fer-
tility of animals. In addition to the observations on genital
disruption, effects of chronic, low-dose exposure to meth-
ylmercury on disrupting courtship behaviors and sexual
preference of white ibises were reported.17 This exposure
caused a dose-related increase of male-male pairing behav-
ior and was linked to reproductive deficits. Given the nega-
tive effects of EDC exposure on the genital development
and fertility of wildlife,18 the possible harmful effects of
environmental pollutants/chemicals on human fecundity
do raise public concerns.
In fact, the decreasing trends in human fertility rates
and the manifestation of male reproductive health prob-
lems, cryptorchidism (undescended testis) and poor semen
quality, support the notion.19,20 In highly polluted areas or
chemical plants, human exposure to dioxins, polychlori-
nated biphenyls, polychlorinated dibenzofurans, or mixed
petrochemicals were found to be associated with signifi-
cant reductions in the ratio of male births to total number
of births.21–24 Epidemiological studies have reported an
increased risk of genital malformations and cryptorchi-
dism in the children of workers exposed to pesticides.25,26
In the Study for Future Families in the United States, a
positive correlation between prenatal exposure to several
phthalates and shortening of the anogenital index and
incomplete testicular descent was observed.27,28 Although
we are still far from understanding the mechanisms of the
casual relationship, some data suggest that human hypo-
spadias and cryptorchidism may be related to endocrine
disruption by chemical contaminants.29 Based on pre-
cautionary principles, avoidance of prenatal exposure to
chemical pollutants is recommended.30 In addition to the
possible endocrine-disrupting effects on genital develop-
ment, many studies evaluated the impacts of the exposure
on levels of reproductive hormones and semen quality
(concentration, morphology, mobility, and chromatin
integrity of sperm). Epidemiological cross-sectional stud-
ies reported negative correlations between serum PCBs or
the proxy (2,2’4,4’,5,5’-hexachlorobiphenyl, CB-153) for
total PCB exposure with semen quality (concentration,
motility, and DNA integrity) in Northern Europeans,31–37
Americans,38 and Indians39 and serum testosterone
levels.31,34 In other epidemiological analysis, negative
­correlations of serum levels of DDT and its metabolite
(dichlorodiphenyldichloroethylene p [p’-DDE]),33,36,40–43
perfluorinated compounds (PFCs),44–47 or urinary bisphe-
nol A (BPA)48–51 concentrations with semen quality and
reproductive hormones were observed. The exposure
and accumulation of organophosphate flame retardants
were also linked to decreased semen quality in men.52,53
Although these studies have shown that exposure to
chemical pollutants exerted some negative effects on male
reproductive health, the results across studies are still not
conclusive. Nevertheless, the observed effects were related
to the exposure to certain classes of chemical pollutants
and may be interlinked in the causation.54–56 To elucidate
the possible causations, most prior research delineated
that the actions of EDCs were mediated by estrogenic and/
or (anti)-androgenic pathways. Ubiquitous environmental
contaminants and organic (i.e., DDT, hydroxylated PCBs,
BPA, p-nonylphenol, and dioxins) and inorganic (cad-
mium and mercury) contaminants were found to exhibit
either or both estrogenic and androgenic activity.57–59
Some newly identified emerging contaminants, like PFCs
(perfluorooctanoic acid [PFOA], perfluorooctane sulfo-
nate [PFOS]) and flame retardants were also reported to
possess estrogenic activities.60,61 However, recent studies
have indicated that EDCs can act on different biological
components via diverse mechanisms. Other nuclear hor-
mone receptors are found to be the targets of EDCs.
GENERAL MECHANISTIC ACTIONS OF EDCs
Receptor mediated pathways
Some EDCs or their metabolites perturb estrogenic,
androgenic, and thyroid signaling as their chemical struc-
tures are similar to endogenous hormones.62 A common
molecular mechanism is their direct interactions with
nuclear receptors (NRs) to modulate downstream signal-
ing pathways and gene expressions. Numerous studies
have demonstrated the stimulatory or inhibitory effects
of some EDCs on estrogen receptors (ERs), androgen
receptors (ARs), retinoid X receptors (RXRs), peroxi-
some proliferator-activated receptors (PPARs), constitu-
tive androstane receptors (CARs), pregnane X receptors
(PXRs), glucocorticoid receptors (GRs), and thyroid hor-
mone receptors (TRs).63 Moreover, different EDCs may
share common receptor targets and interfere with vari-
ous NRs expressed in several organs with different affini-
ties and intensities. Therefore, EDC-elicited effects may
result from perturbations at multiple tissue levels through
cross-talking among different hormonal signaling path-
ways. Although investigations on the multiple interac-
tions would benefit our understanding of the fundamental
mechanism of EDCs, there are still criticisms about inter-
nal doses of EDCs and their potency in mimicking or
inhibiting the biological actions of endogenous hormones.
It is true that some EDCs (dioxins, diethylstilbestrol [DES]
 Reproductive toxicity of EDCs from laboratory animal and cell studies  157
and 17α-ethinylestradiol [EE2]) show high or compara-
tive affinity to the endogenous ligands in receptor binding
(i.e., aryl hydrocarbon receptor [AhR] or ERs), but most
EDCs have low (by several orders of magnitude) bind-
ing affinity to the receptors.64 The concentrations of most
EDCs in human body fluid (i.e., blood) are 100–1000-folds
lower than the experimental conditions that were used to
demonstrate receptor binding or activation. Although the
additive/synergistic effects of EDC mixtures cannot be
neglected, it seems unlikely that EDCs can compete with
the endogenous ligands for receptor binding. Therefore,
with regard to the impact of EDCs (i.e., BPA, phthal-
ates, PFCs, and flame retardants) with weak or unidenti-
fied hormonal activity, their mechanistic action leading
to animal infertility cannot be fully explained by direct
endocrine activities. Hence, the key question that needs to
be addressed is whether the experimental observation of
receptor activation/inhibition by EDCs could happen in
real life. If this is not the case, could EDC-elicited action
be due to some other spectrum of effects? Other than the
receptor-mediated actions, EDC-elicited oxidative stress
has been recognized as the most common mechanism in
affecting cellular structures and functions.65 Interestingly,
an elevated level of reactive oxygen species (ROS) has
been found in the semen plasma of infertile men.66,67
This inflammatory/oxidative effect may ultimately affect
gonadal functions for the production of healthy gametes
and lead to reduced fertility and fecundity in animals.
Epigenetic changes on animal early development
Fetal growth and development are influenced by both
genomic and environmental factors.68,69 Presumably the
exposure to EDCs of high potency in early life can pro-
foundly influence the development and long-term health
of infants via alteration of signaling, gene expression, and
epigenetic modifications.70–72 We have learned these lessons
from human exposure to synthetic estrogen, DES, and the
sedative drug thalidomide, which induced changes in the
development of reproductive tracts in offspring, increased
incidence of vaginal cancer, and caused severe develop-
mental malformations.73,74 Converging evidence supports
the notion that the modulatory effects of different EDCs
on epigenetic programming, cell signaling, and tissue
functions would lead to chronic and/or transgenerational
effects on the exposed animals or increase disease suscep-
tibility in their offsprin.75,76 Numerous reports have dem-
onstrated the effects of EDCs on DNA methylation in early
development of mice,77–80 abnormal embryonic karyotype
and chromosome synapsis defects in humans,81,82 spine
synapses formation in nonhuman primates,83 fetal mouse
mammary gland development and stromal-epithelial
signaling,84,85 and Hox gene expression and genitouri-
nary development.86,87 Using in utero exposure studies in
rodent models, transient exposure of gestating female rats
to a fungicide (vinclozolin) or a pesticide (methoxychlor)
decreased male infertility of their offspring.77 The effects
were found to be manifested via an alternation of DNA
methylation patterns in germ line. Dietary exposure to
BPA in maternal mice showed a shift of coat color distri-
bution in their offspring via reducing CpG methylation in
the Agouti gene.80 These examples illustrate that the leak-
ing of EDCs via placenta barriers to developmental fetus
is possible. Fetal life is vulnerable as the structural and
functional events are rapidly developed. Therefore, the
perturbing effects of placenta-derived EDCs may lead to
considerable changes in the developmental paths in the
fetus, leading to a disturbance on the inherited genetic
program, the commitment of cells to specific lineages, and
the structural/functional differentiation in organogenesis,
leading to chronic and/or transgenerational effects on the
exposed animals or increase disease susceptibility or dys-
function in their offspring.88
REPRODUCTIVE TOXICITY OF EDCs FROM
LABORATORY ANIMAL AND CELL STUDIES
One of the current hypotheses proposes that EDCs cause dis-
turbances on the regulatory pathways at the hypothalamic-
pituitary-gonadal (HPG) axis to perturb the process of
steroidogenesis in affecting the feedback circuit and circu-
lating levels of gonadotropin and sex hormones. EDCs may
affect the growth and maturation of gonads and the forma-
tion of functional gametes. Indeed, the inability to produce
sufficient numbers of healthy sperm or nonobstructive
azoospermia is one of the major reasons for male subfecun-
dity and infertility.11 Numerous studies have revealed that
the male reproductive system is the major target of chemi-
cal toxicants. Almost 60 years ago, Parizek and coworkers
identified that the testis was sensitive to cadmium toxic-
ity, which caused testicular injury and male sterility.89 In
recent years, experimental and epidemiological studies
demonstrated the effects of EDC exposure on the manifes-
tation of testicular dysgenesis syndrome or the reduction of
male fertility through formulations of the biological rela-
tionship of EDC exposure (i.e., dioxin, PCBs, pesticides,
BPA, PFCs, and flame retardants) to the reduction of tes-
tosterone and sperm production.30 Therefore, understand-
ing the actions and effects of EDCs on the feedback circuit,
steroidogenesis, and spermatogenesis can provide clues to
test the hypothesis and to elucidate the roles of EDCs on
perturbing reproductive functions.
Hypothalamic-pituitary gonadal axis
The hypothalamic kisspeptin (KiSS1)/G-protein-coupled
receptor (GPR) 54 system is essential for puberty matu-
ration and gonadal function.90 The activation of KiSS1/
GPR54 stimulates the release of GnRH, which controls the
synthesis of gonadotrophin luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) by the pituitary. LH
and FSH act on testicular Leydig and Sertoli cells to regu-
late the processes of steroidogenesis and spermatogenesis,
respectively.91,92 In rat models, neonatal BPA exposure (sub-
cutaneous injection) were found to reduce hypothalamic
kisspeptin fiber density and KiSS-1 mRNA expression at the
prepubertal stages.93,94 A similar BPA exposure to neonatal
rats affected GnRH signaling at the hypothalamic-pituitary
axis.95 Oral gavage male rats with BPA at early puberty
158  Effects of chemical pollutants on spermatogenesis and implications in male infertility
showed significantly greater numbers of ERα expression
in neurons in the arcuate nucleus and medial preoptic area
of the hypothalamus,96 indicating a perturbation of neural
circuit of estrogen signaling. Perinatal exposure to BPA
and di(2-ethylhexyl) phthalate (DEHP) caused a reduction
in the male-to-female sex ratio and sizes of testes in male
pups.97 The expression levels of some genes (i.e., antimul-
lerian hormone, androgen receptor, cyclin A, and steroido-
genic acute regulatory protein [StAR]) in relation to the
growth and maturation of testes were significantly reduced.
In a pregnant mouse model, oral gavage of BPA to mothers’
perturbed mRNA expressions of KiSS-1, GnRH, and FSH at
the hypothalamic-pituitary axis and reduced the expression
of steroidogenic enzymes, leading to a decrease of testoster-
one levels in male pups.98 Retrospectively, a recent hypoth-
esis has underlined that gonadal steroidogenesis is one of
the possible targets for EDCs.99,100 The modulatory effects of
EDCs on steroidogenesis were reported in both in vivo and
in vitro studies.101,102 Using a testicular Leydig R2C cell-line
model, a treatment with BPA increased the expression of
aromatase.103 Moreover, a recent study revealed that EDCs
might target on ATP-binding cassette transporters in the
blood-testis barrier (BTB) to alter the process of testoster-
one secretion.104 Therefore, EDCs could interfere with the
process of steroidogenesis and modulate the release of the
endogenous steroid hormones and also the hormonal feed-
back circuity. Collectively, altered serum levels of the ste-
roid hormones may interfere with the feedback regulatory
mechanisms at the HPG axis, thereby perturbing normal
reproductive function.
Spermatogenesis
The process is undertaken in the seminiferous epithelium
where an interaction of Sertoli cell–spermatogonium/
spermatid mediate cell cycle progression of germ cells,
spermiogenesis, and spermiation.105 Therefore, disrup-
tion of the epithelial homeostasis at the Sertoli-Sertoli and
Sertoli-germ cell interface in the seminiferous epithelium
would result in the loss of germ cell adhesion, sperma-
tid orientation, and dysregulation of spermatogenesis.106
Emerging evidence has revealed that EDC, like cadmium
or BPA elicited oxidative stress may disrupt the integrity
of the BTB in seminiferous tubules, leading to an impair-
ment of sperm production.107,108 This observation has
revealed the specific but nonhormonal-effect of EDCs on
testicular function, in particular the empirical action of
EDCs on the regulatory roles of “polarity proteins” and
BTB-associated proteins in the seminiferous epithelium.
Current evidence suggests that BTB-associated junctional
proteins and protein kinases are the potential targets of
some EDCs, since those affect seminiferous epithelial
function and sperm production. In addition to sperm
number, the integrity of sperm chromatin is known to
be a clinical marker of male fertility. The high rate of cell
division of spermatogonium would provide a vulnerable
time window for EDC’s actions. This scenario is depicted
in a recent whole genome sequencing study in which
fathers passed nearly four times more new mutations to
the developing child’s genome than did mothers.109 Using
a Caenorhabditis elegans (C. elegans) model, an impair-
ment effect of BPA on the double-strand break repair
machinery in the germ line was demonstrated.111 In rhe-
sus monkeys, disturbing effects of BPA on the prophase
events at the first meiotic division during early oogenesis
has been reported.110 Even though the corresponding data
in males of mammals are lacking, it is sensible to assume
that a similar effect may happen during the meiotic divi-
sion of male germ cells. In the latter stage of spermato-
genesis, the shaping and nuclear condensation of sperm
head are tightly regulated processes involving extensive
chromatin remodeling. Dysregulation of the BTB junc-
tion proteins in seminiferous epithelial homeostasis may
cause an improper change in histone retention and epi-
genetic modification of the developing sperm.112 Previous
studies using the chemotherapeutic drug cyclophospha-
mide113 or vinclozolin77 have demonstrated their effects
on DNA methylation in male germ cells. Other studies
have verified the epigenetic action of BPA or phthalate
exposure on DNA methylation,80,114–120 histone modifica-
tion,121 and microRNA expression122 in the germ/somatic
cells of offspring or the subsequent passages in the cell-
line models. Collectively EDCs interfere with seminifer-
ous epithelial function by disrupting the role of junctional
proteins, which in turn impede spermatogenesis and alter
genetic and epigenetic changes in the developing sperm.
Remarkably, anomalous methylation profiles in human
spermatozoa were reported in men with low sperm counts
and infertility.123,124 Alteration in the normal epigenetic
state of sperms from infertile patients undergoing in vitro
fertilization (IVF) has also been proposed as the under-
lying cause of increased anomalies in IVF offspring.125,126
Further studies are recommended to delineate the effects
of EDCS on seminiferous epithelial function and the
acquired epigenetic modification during spermatogenesis.
Taken together, emerging evidence supports the notion
that exposure to EDCs may cause adverse effects on ani-
mal reproductive functions via different mechanisms.
However, it is still challenging to establish the causal rela-
tionship between the exposure to the observed functional
disability. In fact, humans are exposed to a complex mix-
ture of environmental contaminants with heterogeneous
chemical structures. The effects of their interactions with
macromolecules at molecular levels may result from their
agonized or antagonized actions. Moreover, the outcomes
of the mixture effect are complicated by different routes,
durations, and doses of the exposure at different devel-
opmental windows of animals. In the following section,
the historical background and reproductive toxicity of the
emerging persistent organic contaminant, PFCs (synthetic
fluorinated hydrocarbons), is the focus of the discussion.
PFCs, HUMAN EXPOSURE RISK, AND REPRODUCTIVE
HEALTH
PFCs are a family of synthetic fluorinated hydrocarbons
(C 4-C14) originally produced by 3M as fluorosurfactants in
1949. PFCs have been used in fabric protectors and stain

repellents for textiles and leather and oil-resistant coatings
for food wrappers, waxes, and polishes.127 Due to their
numerous applications in commercial products, exposure
of the general population to PFCs is widespread. Since
the DuPont Washington Works fluorochemical manu-
facturing plant in Parkersburg, West Virginia, began to
release PFCs into the environment, a C8 Health Project
has been launched to measure serum concentrations of
PFCs in exposed residents. In a recent report from the C8
Health Project, high levels of PFCs (i.e., PFOA [32.9  ng/
mL], PFOS [19.2 ng/mL], and perfluorohexane sulfonate
[PFHS] [3.3  ng/mL]) were detected in blood samples
from 69,030 participants enrolled between 2003–2006.128
Additional studies have reported the widespread con-
tamination of human populations with PFCs (1–20 ng/
mL).129,130 More importantly, placental transfer of PFCs
was demonstrated in humans by analyzing paired mater-
nal and umbilical cord blood samples from patients in
various countries, including Japan, Norway, Germany,
Korea, and Canada.131–135 The evidence of intrauterine
exposure was further strengthened by comparing PFC
(i.e., PFOA, PFOS) levels in blood samples from pregnant
and nonpregnant women.136 These observations imply a
widespread and high risk of exposure to PFCs for fetuses
during early development. Moreover, breastfeeding is rec-
ognized as a route of PFC exposure in infants.137 Because
of the stability of carbon-fluoride bonds, PFCs are highly
resistant to biodegradation/metabolism. Indeed, lengthy
serum elimination half-lives were reported in humans
(5 years)138,139 and mice (20 days).140 As a result, bioaccu-
mulation of these chemicals can lead to greater adverse
health effects in humans.
Epidemiological studies demonstrated that the pres-
ence of PFCs was implicated in male reproductive dys-
function, including reduced sperm count and semen
quality, in humans. High serum PFC levels correlated
with low semen quality in a population of 105 Danish
men44 and in 588 men from Artic and European popu-
lations.47 A study of 247 Danish men also demonstrated
a negative association between serum levels of PFCs and
testosterone production.45 Additionally, PFOA exposure
was positively correlated with a reduction in sperm DNA
integrity in 199 subjects from Greenland.46 In the studies
of 1240 pregnant women from the Danish National Birth
Cohort141 and 910 women enrolled in the Norwegian
Mother and Child Cohort,142 high maternal levels of PFCs
(i.e., PFOA and PFOS) were associated with a longer time-
to-pregnancy. Furthermore, a follow-up study by a preg-
nancy cohort in Denmark demonstrated a link between
in utero exposure to PFCs and reduced semen quality in a
group of 169 adult men.143
The toxicity of PFCs to male reproductive health has
primarily been evaluated using animal and cell culture
models. Early studies using 14C-labeled perfluorodeca-
noic acid (PFDA) and PFOA demonstrated covalent
binding of these compounds to cellular proteins in the
plasma, liver, and testes of rats.144 Interestingly, renal
excretion of PFOA was significantly slower in male rats
PFCs, human exposure risk, and reproductive health  159
(T1/2 = 15 days) than in females (T1/2 = 1 day). Although
the underlying mechanism has yet to be elucidated,
testosterone was a key factor in the sex-specific differ-
ence in PFOA elimination.145 Adult rats treated with
0.05%–0.5% PFOA exhibited reduced activities of
testicular-metabolic enzymes.146,147 Meanwhile, chronic
exposure of adult rats to PFDA (0.2, 0.5 mg·kg−1·day−1)
resulted in disruption of testicular steroidogenesis.148
Using microsomal preparations of human and rat tes-
tes, PFCs were found to target the steroidogenic enzyme
3β-hydroxysteroid dehydrogenase, resulting in a reduc-
tion in testosterone production.149 To elucidate the mech-
anisms of PFC-induced male reproductive disorder, a
proteomic analysis of testes from adult mice exposed to
PFOA (0.21–20  mg·kg−1·day−1) for 4 weeks was utilized
to screen for molecular targets of PFOA.150 In addition
to a reduction in testosterone synthesis and lower sperm
quality, this study demonstrated that PFOA treatment led
to reduced expression of the Leydig cell marker (insulin-
like factor 3) and of steroidogenic enzymes, including the
cytochrome P450 cholesterol side-chain cleavage enzyme
and 17β-hydroxysteroid dehydrogenase. The modulating
effects of PFCs on testicular steroidogenesis were then
supported by studies using human adrenocortical carci-
noma (H295R) cells. In this cell line, exposure to PFOA,
PFOS, or perfluorononanoic acid (PFNA), at concentra-
tions of 6 nM–600 μM, resulted in a reduction in the
mRNA expression levels of CYP11A.151 A similar study
using H295R cells illustrated the inhibitory effects of six
PFCs on the expression levels of steroidogenic enzymes
and on testosterone synthesis.152 These results are con-
sistent with observations made in zebrafish embryos
exposed to PFOS (500 μg/L) in interfering with functions
of NRs and steroidogenesis.153
In addition to its effects on steroidogenesis and sex hor-
mone production, studies have investigated the effects of
PFC exposure on sperm production. Qiu and coworkers
reported that exposure of CD-1 mice to PFOS (0.25–50
mg·kg−1·day−1) was accompanied by disorganization of
the BTB and reduced sperm count.154 These data were
supported by a follow-up experiment using cultured pri-
mary Sertoli cells, which revealed that PFOS (5–60 μM)
disrupted BTB integrity in vitro by affecting the localiza-
tion of junctional proteins. PFOS (50 μM) treatment also
resulted in an oxidative stress-mediated reduction in the
expression of cytoskeletal proteins and a defect in cyto-
skeletal organization in a coculture of neonatal gonocytes
and Sertoli cells.155 Correspondingly, in utero exposure
of female rats to PFOS (5 or 20 mg·kg−1·day−1 maternal
dose) from gestational day 11–19 reduced the number
of fetal Leydig cells and impaired cell functions in com-
parison to the untreated control group, as evidenced by
a decrease in steroidogenic activity and in testosterone
levels.156 Oral exposure to PFOS (0.5–6 mg·kg−1·day−1) for
4 weeks caused a disruption in the reproductive axis, in
particular a loss of spermatozoids, in adult male rats.157
PFOS-elicited degeneration of spermatogonium was also
reported in the testes of Xenopus laevis.158 Furthermore,
160  Effects of chemical pollutants on spermatogenesis and implications in male infertility
PFOA exposure (1, 3, 5, 10, 20, or 40 mg·kg-1·day-1) in
timed-pregnant CD-1 mice (GD 1-17) enhanced sexual
maturation in male offspring.159 Exposure of adult male
CD-1 mice to PFOS (1–10 mg·kg−1·day−1) caused pro-
gressive deterioration of testicular signaling manifested
by significant reductions in testosterone synthesis and
epididymal sperm counts.160 Collectively, these findings
support the human epidemiological studies to indicate
the negative association between high serum levels of
PFCs and testicular steroidogenesis, testosterone pro-
duction, and semen quality.
Perfluoroalkyl chemicals and their mechanistic
activities
PFCs are classified as peroxisome proliferators. This class
of molecules activate ligand-dependent transcription
factors of the PPAR family to alter lipid metabolism and
cell differentiation.161–164 PPARs belong to the steroid hor-
mone receptor superfamily and are able to interact with
steroid receptors to modify the activities of other steroid
hormones.165 The identification of PPARs in human testes
and developing rat testes suggests PPAR-signaling may
play a role in testicular cell differentiation during early
development.166 Animal and cell culture studies revealed
that an activation of PPARs in the testes was associated
with gonadal steroidogenesis,167 spermatogenesis,168 and
Sertoli cell metabolism.169 Conversely, inactivation of
PPAR-elicited peroxisome β-oxidation in mouse testes
was found to negatively affect spermatogenesis and cause
male infertility.170 Although various studies had illus-
trated the involvement of the PPAR-pathways in PFC-
elicited intoxication, studies of wild-type and PPAR-α
null mice treated with PFOS (3–10 mg·kg−1·day−1) dem-
onstrated PPAR-independent effects on reproductive
health.171,172
In previous studies, exposure to PFOS resulted in
increased expression of the arachidonic acid (AA)-
metabolizing cytochrome P450 (CYP) isoforms, CYP2, and
CYP4.173,174 These enzymes convert AA into hydroxyeicosa-
tetraenoic acids (HETEs) or epoxyeicosatrienoic acids
(EETs). It was proposed that the PFOS-mediated perturba-
tion of fatty acid metabolism and the resulting physiologi-
cal outcomes were due to the modulation of lipid signaling
via dysregulation of the AA cascade.175 Interestingly, a
study reported that certain endocrine-disrupting chemi-
cals (i.e., BPA and phthalates) inhibited the prostaglandin
(PG) signaling pathway in a mouse Sertoli cell line and
in ex vivo rat testes.176 PGs are a group of lipid signaling
mediators generated by the oxidation of AA. AA and the
lipoxygenase-catalyzed products of the AA cascade play
critical roles in steroidogenesis, early male sexual devel-
opment, and masculinization.177–187 Interestingly, most
lipid-mediators (i.e., PGs and HETEs) produced by this
cascade are also agonists of PPARs.188,189 Using primary
mouse Sertoli cell cultures, PFOS treatment (10–20
μM) disrupted actin polymerization and bundling via
affecting the levels and/or localization of cell adhesion
proteins and activity of focal adhesion kinase at Sertoli
cell BTB. Significant reductions of actin crossing linking
proteins (filamin A, palladin), tight junctional proteins
(zonula occludens-1, occludin), adherens junction pro-
teins (β-catenin, N-cadherin), and the gap junction (GJ)
protein connexin-43 (Cx43) were observed.190 The over-
expression of Cx43 rescued PFOs-disrupted TJ barrier.191
Intriguingly, CX43 is known to be a downstream target
of the lipid mediator PG. Therefore, further investigation
is necessary to determine whether PFC exposure disrupts
AA metabolism, and the PG signaling pathway in particu-
lar, resulting in male sexual dysfunction.
CONCLUSION
EDC-related health issues have been widely reported in
scientific journals, appearing extensively in the media,
and have sparked considerable discussion in society.
Many reports have suggested that EDCs could modulate
the masculinization process during human fetal develop-
ment and result in the manifestation of reproductive dis-
order similar to testicular dysgenesis syndrome. Given the
adverse effects of EDC exposure on reproductive health,
one can surmise that the global increase in the prevalence
of subfertility in humans is likely due to an increased
exposure to EDCs. While the increasing incidence of
reproductive problems is a global issue, the mechanism
by which EDCs predispose humans to reproductive dys-
function is still unclear. Identification of the effects and
mechanistic actions of EDCs on reproductive functions in
humans would help to understand their influence on ani-
mal health. Changes in government policies, manufactur-
ing best practices, and even in the lifestyles of people can
help to eliminate the excessive use of man-made chemicals
and thus lessen the immense financial burden of EDC-
elicited diseases on the global health care system.
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Advances in our understanding
of human spermatogenesis
QING WEN, ELIZABETH I. TANG, TITO JESUS, BRUNO SILVESTRINI,
and C. YAN CHENG
INTRODUCTION
In the mammalian testis, spermatogenesis takes place in
the testis. Spermatogenesis is divided into three distinctive
cellular events. First, during the premeiotic steps, sper-
matogonial stem cells, known as Asingle (As) in rodents,1,2
which are capable of undergoing self-renewal, enter mitosis
and transform to Apaired (Apr) and then Aaligned (Aal) undif-
ferentiated spermatogonia (Figure 13.1). Apr and Aal in
rodents that are not synchronized with the seminiferous
epithelial cycle are further expanded to spermatogonial
clones of differentiated spermatogonial A1, A2, A3, and A4,
which are now synchronized with the epithelial cycle.3,4
Finally, these type A differentiated spermatogonia divide
and transform to intermediate spermatogonia and type B
spermatogonia, which are the interconnected cohorts of
spermatogonia that are then transformed to spermato-
cytes (Figure 13.1). In humans, Adark spermatogonia in
men, considered to be human testicular stem cells and the
regenerative reserve5 capable of undergoing self-renewal,
transform to Apale undifferentiated spermatogonia, which
are the male germline progenitor and the functional
reserve.5 An undifferentiated Apale spermatogonium in
humans undergoes one mitotic division and transforms
to two differentiated type B spermatogonia, which in turn
undergo another mitotic division to form four spermato-
cytes.1 It is noted that all the spermatogonia are residing
outside the blood-testis barrier (BTB) in the basal com-
partment or at the stem cell niche (for spermatogonial
stem cells).2 Type B spermatogonia transform to prelep-
totene spermatocytes and which the germ cells that are
transported across the BTB to become leptotene, zygotene,
and pachytene spermatocytes in the adluminal compart-
ment. Second, the most prominent series of cellular events
is meiosis, during which one spermatocyte undergoes
meiosis I to form two secondary spermatocytes, and each
of these undergoes meiosis II to form two haploid sperma-
tids. Third, once haploid spermatids arise, they undergo
rapid morphological changes via spermiogenesis so that
elongated spermatids via 16 and 19 steps in mice and
rats6–8 versus six steps in humans9 eventually transform to
spermatozoa, which will be released into the tubule lumen
during spermiation to complete spermatogenesis, entering
the epididymis1,10–13 for further development. In theory, a
single As in mice or rats can become 1024 spermatocytes
and thus 4096 haploid spermatids versus 16 haploid sper-
matids from one Apale spermatogonium (Figure 13.1).
These series of events take place nonstop in rodents and
168
13
men after puberty at ~35–45 days of age in mice and rats
versus ~12 years of age in humans to produce 30–50 mil-
lion versus 200 million functional sperm daily in rodents
versus humans in order to fertilize the egg. In humans,
rats, and mice, spermatogenesis takes 64–68, 48–58, and
34–35 days, respectively, to complete.9,12,14,15 During the
past two decades, advances have been made in under-
standing the molecular mechanisms that regulate these
events. In this chapter, we focus our discussion on stud-
ies in humans except for specific topics where few reports
focused on human studies are found in the literature, we
turn our discussion to rodents. The goal is to provide a
brief overview of a few specific areas of research that might
interest investigators so that more emphasis can be placed
on studies that bridge the gap between studies in rodents
and humans.
GERM CELL DEVELOPMENT
From undifferentiated spermatogonia to haploid
spermatids
In humans, a small group of cells with a unique morphol-
ogy designated human primordial germ cells (hPGCs)
(also known as prespermatogonia, which are the gamete
founder cells in the male) is derived from extraembryonic
tissues surrounding the yolk sac in week 4 embryos.16,17
Male gonadal development begins at ~6 weeks postcon-
ception when Sertoli cells make their first appearance,18
and these changes are initiated and governed by the testis-
determining genes Sry and Sox9.19,20 hPGCs are known
to express key pluripotency markers such as POU5F1,
NANGO, and SSEA1 and to proliferate until the first tri-
mester (weeks 10–12).21 It is noted that hPGCs migrate to
the gonadal ridge and differentiate into gonocytes, and
together with the Sertoli cells they begin to form the semi-
niferous or testis cords, the precursors of the seminifer-
ous tubules. In short, during the second trimester (weeks
13–28), most of hPGCs progressively become mitotically
inactive (or mitotic arrest) and differentiate into prosper-
matogonia, and some of these prospermatogonia trans-
locate to the basal region of the tubules and differentiate
into spermatogonia, forming type A spermatogonia dur-
ing the third trimester (weeks 28–40), and some type B
spermatogonia are found by birth.21 During embryonic
development, peritubular myoid cells originating from
the mesonephros also migrate to the site under the influ-
ence of Sry, which is necessary to assemble functional
 (a) Rodent Testis
in vivo BTB

+ =

PGC Gonocyte As Apr Aal A1 A2 A3 A4 In B Spc sS rS ES Oocyte

(1) (2) (4-8-16) (16) (32) (64) (128) (256) (512) (1024) (2048) (4096) (4096)

Migration Quiescence Mitosis Differentiation Mitosis Meiosis I Meiosis II Spermiogenesis Fertilization

piRNAs

tsRNAs
miRNAs
(Epididymis maturation)

(b) Human Testis (in vivo)

in vivo BTB

+ =

PGC Gonocyte Adark Apale B Spc sS rS ES Oocyte

(1) (1) (2) (4) (8) (16) (32)
Spermatogonial stem cell
Migration Quiescence Differentiation Mitosis Meiosis I Meiosis II Spermiogenesis Fertilization Undifferentiated
spermatogonium
piRNAs level? miRNAs level? tsRNAs level?

Figure 13.1  A schematic illustration of spermatogenesis in rodents (a) and humans (b). The numeral bracketed below each germ cell type (see below) denotes the number of daughter cells
derived from an earlier progenitor cell via either mitosis or meiosis. It is noted that meiosis takes place behind the blood-testis barrier (BTB) in both rodents and humans. Theoretically, 4096 elon-
gated spermatids (ES) (i.e., spermatozoa) are formed from a single Asingle spermatogonium in rodents (versus 32 ES and sperm are formed from a single Adark or Apale spermatogonium in humans).
However, more than 75% of germ cells, most notably spermatocytes and spermatids, undergo apoptosis and/or degeneration to avoid overwhelming the population of Sertoli cells in the testis
since, unlike germ cells, Sertoli cells in adult testes by day ~17-19 in the rat testis (or after puberty in humans after age ~12-year-old) are differentiated cells [44,146]. Thus, the number of spermatozoa
derived from a single spermatogonium can be considerably less than the theoretical number. The BTB divides the seminiferous epithelium into the basal and apical (adluminal) compartment in
rodent and human testes. Spermatogonial mitotic proliferation and differentiation take place in the basal compartment. Type B spermatogonia differentiate into preleptotene spermatocytes (at
stage VIII and III of the epithelial cycle in rat and human testes, respectively) which are the germ cells connected in clones via intercellular bridges to be transported across the BTB at stage VIII or
stage III of the epithelial in rat and human testes, respectively) so that meiosis I/II and spermiogenesis take place behind the BTB in the apical compartment. Post-meiotic spermatid development
through spermiogenesis also takes place behind the BTB until the release of fully developed spermatids (i.e., spermatozoa) at spermiation to empty sperm into the tubule lumen. Subsequent
sperm maturation takes place in the epididymis so that fertilization can take place in the fallopian tube in the female to give rise of new offspring. Studies have also shown the involvement of
small no-coding regulatory RNAs (ncRNAs) such as piRNA (piwi-interacting RNA), miRNA (microRNA) and tsRNA (tRNA-derived small RNA) that regulate different stages of the spermatogenesis in
rodents [144], however, their involvement in regulatory human spermatogenesis remains largely unknown. Abbreviations: In rodents: A, type A spermatogonium; In, intermediate spermatogo-
nium; B, type B spermatogonium; Spc, spermatocyte; sS, secondary spermatocyte; rS, round spermatid (step 1-7); ES, elongating/elongated spermatid; As, Asingle spermatogonium, Apr, Apaired sper-
Germ cell development  169

matogonium, Aal, Aaligned spermatogonium in rodents are undifferentiated spermatogonia; A1-A4 are differentiated spermatogonia. In humans: Adark and Apale are undifferentiated spermatogonia.
170  Advances in our understanding of human spermatogenesis
testis cords.22 However, factors and/or genes that provide
the necessary signaling functions to regulate these events
remain largely unknown. For instance, recent studies in
the mouse have shown that this is regulated, at least in
part, by the Sertoli cell Wt1 gene,23 yet the involvement of
Wt1 in human testis assembly remains unknown. At the
same time, outside these testis cords, Leydig cells differ-
entiate12 along with rapid proliferation of germ cells and
Leydig cells 24,25 to develop into the testis.
The somatic cells of the newly formed testis begin to
secrete anti-Mullerian hormone26 and testosterone,27
which are necessary to confer male differentiation of the
secondary sex organs. Also, primordial germ cells and
Sertoli cells within the testis cords and Leydig cells found
outside the cords rapidly proliferate via mitotic divisions
without embarking on meiosis as seen in the fetal ovary.18
For instance, the number of germ cells per human testis
at week 6 postconception is ~3000 which rises to ~30,000
by week 9, with a germ:Sertoli cell ratio of ~1:11 versus a
germ:somatic cell (including Sertoli, Leydig, peritubular
myoid, and mesenchymal cell) ratio of ~1:44 at 6- versus
9-week postconception, respectively.24 Studies in rodents
have shown that a number of paracrine factors are crucial
to confer migration, differentiation, and proliferation of
primordial germ cells and Sertoli cells during testis devel-
opment. These include members of the TGFß family (e.g.,
TGF-ßs, activins, and inhibins),28,29 nerve growth factor
(NGF) family (e.g., NGF, brain-derived neurotrophic fac-
tor [BDNF], neurotrophin [NT] 3, NT4 [also known as
NT5], and NT6, and pituitary adenylate cyclase-activating
polypeptide [PACAP]).30–32 However, the roles of these
factors in human testis development also remain to be
investigated.
In this context, it is of interest to note that epigen-
etic reprogramming, in particular genomic-wide DNA
demethylation in hPGCs, is somewhat similar to mouse
primordial germ cells.33 For instance, hPGCs undergo
extensive demethylation at week 5 at the time of their colo-
nization of embryonic gonads in humans, which continue
thereafter.33 The mechanism for epigenetic reprogram-
ming in human and mouse germ cells appear to be well
conserved between species.34,35
In human testes, spermatogonia undergo self-renewal
via mitosis, and they are classified into three subtypes based
on their heterochromatin content known as Adark, Apale, and
B spermatogonia.36,37 At present, Adark spermatogonia are
considered to be the true testicular stem cells (also known
as quiescent stem cells) or the regenerative reserve, which
are characterized by their low mitotic activity and possibly
involved in preserving the genome integrity in the germ
line in humans.1,5 However, Apale spermatogonia are con-
sidered to be the male germline progenitor (or the active
stem cells), the functional reserve, which are capable of
undergoing mitosis through cyclic proliferation to main-
tain its population. But some Apale cells undergo mitosis so
that each Apale spermatogonial cell divides and transforms
to two type B spermatogonia to meet the need of produc-
ing 200 million sperm daily from an adult human male.1,5
Studies in rodents have shown that the decision for undif-
ferentiated spermatogonia to take the route of self-renewal
via mitosis, differentiate to type B, or become apoptotic is
regulated by Sertoli cell- and peritubular myoid-­produced
biological factors, cytokines, paracrine factors, and/or
hormones as well as Leydig cell-produced testosterone and
possibly microRNAs (miRNAs).38–41 For instance, GDNF
is involved in spermatogonial self-renewal, whereas stem
cell factor (SCF), colony stimulating factor 1 (CSF-1),
bone morphogenetic protein 4 (BMP4), retinoic acid (RA),
and the Notch1/Jagged2 signaling system are involved
in differentiation, and miRNAs are involved in deter-
mining spermatogonia cell fate.40 The number of germ
cells in the testis is also determined by the population of
Sertoli cells10,42,43 that become differentiated in humans by
puberty at ~10–13 years of age.44 Thus, many of the devel-
oping germ cells, including differentiated spermatogonia,
spermatocytes, and spermatids, during spermatogenesis
elect, being programmed or mandated (or instructed)
by Sertoli cells, to go through the apoptotic pathway to
keep the proper ratio of Sertoli:germ cells at ~1:30–1:50 in
rodents.45 As such, as much as ~75% of germ cells undergo
apoptosis46 to avoid overwhelming the fixed population of
differentiated Sertoli cells in the testis following puberty in
humans at ~12–13 years of age versus ~17 days postpartum
(dpp) in rodents. Since Sertoli cells are the only somatic
cells in the seminiferous epithelium to support germ cell
development, germ cells, in particular the highly differen-
tiated postmeiotic spermatids, rely almost exclusively on
Sertoli cells for the provision of nutrients, cytokines, and
paracrine factors and also structural support, in particu-
lar their transport across the seminiferous epithelium to
coordinate with the seminiferous epithelial cycle. Thus,
fully developed spermatids (i.e., spermatozoa) line up at
the adluminal edge of the apical compartment near the
tubule lumen in early stage VIII tubules in rodents versus
stage II in humans to prepare for their release at spermia-
tion in the corresponding late stage VIII versus II. Since
it is known that developing spermatids are metabolically
quiescent cells lacking the ultrastructures of lamellipodia
and filopodia to initiate cell movement, they rely solely on
the Sertoli cell to support their transport across the semi-
niferous epithelium during spermiogenesis.7,47
It is noted that all spermatogonia reside in the basal
compartment of the seminiferous epithelium. Type B
spermatogonia transform into preleptotene spermato-
cytes, marking the end of the mitotic proliferation phase
of spermatogenesis. Furthermore, preleptotene sper-
matocytes are the only primary spermatocytes to be
transported across the BTB, which physically divides the
seminiferous epithelium into the basal and the adluminal
(apical) compartment.7 According to stages of prophase 1,
primary spermatocytes in humans and rodents are classi-
fied into preleptotene, leptotene, zyogtene, and pachytene
spermatocytes, which eventually transform into diplotene
spermatocytes to begin meiosis I.12 In men and rodents,
DNA recombination that is being used to exchange genetic
material between homologous chromosomes is mediated
 Development of round spermatids to functional spermatozoa through spermiogenesis  171
by the synaptonemal complex, which takes place at the
pachytene stage of spermatocytes with strict checkpoints
from metaphase 1 through anaphase 1 during meiosis.
In men with nonobstructive azoospermia (NOA), a type
of idiophatic infertility, about 10% of these infertile men
display a higher recombination failure rate versus normal
fertile men, and they are also at risk of producing chromo-
somally compromised offspring.48,49
The rationale for technological advance and a better
understanding of obtaining haploid spermatids from sper-
matogonia is to assist men with NOA to father their own
children. Recent studies in rodents using 3-dimensional
(3-D) in vitro organ cultures with fragments of neonatal
mouse testes embedded in soft agarose gel in a gas-liquid
interphase method have generated some excitement in the
field. For instance, it was shown that using this in vitro cul-
ture system, functional haploid spermatids were obtained
from undifferentiated mouse spermatogonia. However,
the efficacy of this approach was exceedingly low, at about
<2%,50,51 thereby making this approach unsuitable for
widespread adoption for human studies. Interestingly, a
recent report has provided important advances wherein
functional spermatids were efficiently produced from
undifferentiated spermatogonia when cultured in vitro.52
This approach must now be tested in studies in humans.
In fact, a recent report using human spermatogonial
stem cells (SSCs) obtained from cryptorchid patients and
cultured in vitro were found to develop into functional
haploid spermatids,53 illustrating the feasibility of this
approach. As such, it is likely that functional spermatids
could be developed from undifferentiated spermatogonia
among infertile men, perhaps within the next decade, so
that men with nonobstructive azoospermia could father
their own children.
Blood-testis barrier (BTB)
In rodents and humans, the BTB is constituted by coex-
isting actin-based tight junction (TJ), basal ectoplasmic
specialization (ES, a testis-­specific actin-rich adherens
junction [AJ]), and gap junction, as well as intermediate
filament-based desmosome between adjacent Sertoli cells
near the basement membrane.14,54-56 However, in rodents,
the BTB is also considerably contributed by the peritubular
myoid cells in the tunica propria since administration of
electron-dense substances (e.g., colloidal carbon, thorium,
lanthanum) failed to penetrate the peritubular myoid cell
layer in ~85% of the tubules examined in rodent testes.57,58
However, in primate testes, the peritubular myoid cells
were shown to be less effective in blocking the penetration
of electron-dense markers across the tubules,59 implicat-
ing that the human BTB may share similar characteristics.
More important, BTB integral membrane proteins such
as occludin, which is the best-studied TJ protein in the
rodent testis, is not found in humans.60,61 Also, claudin-3,
an important BTB integral membrane protein in the
mouse testis62 and shown to be delicately involved in the
transport of preleptotene spermatocytes across the BTB,63
is absent in the rat testis.64 Interestingly, testosterone,
which is known to play an important role in maintain-
ing the BTB integrity in rodents,65 may not be essential to
the BTB homeostasis in humans since BTB integrity was
not compromised in healthy men following suppression
of spermatogenesis treated with testosterone or a combi-
nation of testosterone and levonorgestrel (LNG), and the
claudin-3 level was not down-regulated.66 A recent study
used testis biopsies from healthy men treated with a testos-
terone plus LNG,67 similar to the earlier study,66 but also
with a GnRH antagonist (acyline) or 5α-reductase inhibi-
tor (dutasteride) that provided a ~25%–40% added sup-
pression of spermatogenesis versus testosterone and LNG
alone,68,69 for BTB integrity assessment. It was found that
the BTB in these men with spermatogenesis suppression
was compromised (i.e., the BTB was “leaky”), with exten-
sive downregulation and mislocalzation of claudin-11,
connexin 43, and vinculin at the BTB, but the distribu-
tion of ZO-1 and ß-catenin was unaffected.67 Also, in men
with primary seminiferous tubule failure, such as meiotic
arrest that led to, Sertoli-cell-only syndrome, the spatial
localization of claudin-11 and connexin 43 at the BTB
was grossly disorganized, failing to localize at the BTB to
confer barrier integrity, thereby causing spermatogenesis
dysfunction.70 Collectively, these findings illustrate that
the human BTB is likely regulated analogous to rodents,
at least in part, but there are some substantial differences.
Thus, studies using rodent testes as study models should
be cautiously interpreted before applying the pertinent
information to humans. Therefore, the use of an in vitro
culture system involving human Sertoli cells may be an
alternative approach to study human BTB function and
its role in spermatogenesis before human biopsy samples
are used for additional analysis.71 The BTB is important
to spermatogenesis since its disruption, at least in rodents,
is known to cause infertility. For instance, environmen-
tal toxicants such as cadmium and glycerol that cause
irreversible BTB damage in rodents are known to cause
sterility.72–74 However, it remains to be established if some
infertile men with idiopathic infertility are associated
with irreversible BTB disruption.
DEVELOPMENT OF ROUND SPERMATIDS
TO FUNCTIONAL SPERMATOZOA THROUGH
SPERMIOGENESIS
Spermiogenesis
In rodents and humans, primary spermatocytes at the
beginning of meiosis I are diploid but have 4n amount of
DNA, and secondary spermatocytes derived from meio-
sis I contain the haploid number of chromosomes but 2n
DNA content. There is a brief interphase between meio-
sis I and II without DNA synthesis. Almost immediately,
secondary spermatocytes begin meiosis II, moving from
prophase through metaphase, anaphase, and telophase
to form round spermatids that are haploid and contain-
ing 1n DNA content. In humans, studies have shown that
maturation arrest at this stage is rare.75,76 Once formed,
round spermatids undergo a series of transformation and
172  Advances in our understanding of human spermatogenesis
development, forming elongating and elongated sperma-
tids via spermiogenesis. Spermatids are also divided into
different stages known as steps, with 16, 19, and 6 steps in
mice, rats, and humans, respectively.9,77 Due to the unique
association of developing spermatids with Sertoli cells in
cross sections of the seminiferous epithelium through-
out spermatogenesis, Leblond and Clermont78 were able
to divide the epithelium into different stages. This stag-
ing was aided in part by using the periodic acid-Schiff
(PAS) reaction to visualize changes in the Golgi region of
spermatids (to identify the steps of spermatids) with the
development of the acrosome and known as the epithelial
cycle.6–8,12,78,79 In mouse, rat, and human testes, there are
12 (I–XII), 14 (I–XIV), and 6 (I–VI) stages of the cycle, and
the duration of a complete seminiferous epithelial cycle is
8.6, 12.8, and 16 days, respectively.6,12,14 Since an undiffer-
entiated spermatogonium (Asingle in rodents versus Adark in
humans) takes ~35, 53, and 64 days to develop into step
16, 19, and 12 spermatids in the testis of the mouse, rat,
and human, respectively, it thus takes a spermatogonium
to go through the cycle ~4.5 times to develop into func-
tional sperm. The development of round spermatids into
functional sperm is known as spermiogenesis, referring to
the transformation of a spherical spermatid to a sperm-
like elongated spermatid known as spermatozoon. During
this process, no DNA synthesis or cell division takes place
except for a series of morphological changes in the cyto-
sol and the nucleus, such as condensation of the genetic
material and their transport to the spermatid head overly-
ing with the acrosome, and coupled with the elongation
of the tail.77,80,81 As such, the genetic material of sperma-
tids is considered to be transcriptionally inactive except
that spermatids, similar to other germ cells, contain con-
siderable amounts of small miRNAs and siRNA82,83 that
are capable of regulating Sertoli cell function, possibly
through gap junctions or intercellular bridges. This pos-
sibility, however, has not been investigated. During sper-
miogenesis, defects in acrosome assembly in human testes
leads to globozoospermia, a rare (incidence <0.1% among
human males) but severe disorder of male infertility84 in
which spermatozoa lack acrosomes, which is difficult to
manage therapeutically except with the use of intracyto-
plasmic sperm injection (ICSI) to ensure successful preg-
nancy.85 Table 13.1 lists some defects in fertility associated
with mutations of specific genes that regulate spermato-
genesis in men.
Studies using genetic models in mice have identi-
fied a number of genes associated with different cellular
events during spermiogenesis.86 These include acrosome
biogenesis (e.g., Gopc [Golgi-associated PDZ and coiled-
coil motif containing protein] and Gba2 [ß glucosidase
2]), flagella assembly (e.g., Tekt2 [Tektin 2], Tek4 [Tektin
4], Vdac3 [voltage-dependent anion channel 3]), energy
metabolism for motility (e.g., Catsper1-4 [cation chan-
nel, sperm associated 1, 2, 3, and 4], Gapdhs [glyceral-
dehyde-3-phospahte dehydrogenase sperm-specific]),
nuclear condensation (e.g., Tnp1, [transition protein 1],
Tnp2, Prm1 [protamine 1] Prm2), and cytoplasm removal
(e.g., Spem 1 [sperm maturation 1]). In this context, it is
of interest to note that early stages of spermiogenesis are
monitored with various checkpoints, perhaps up to step
8 spermatids in rodents. For instance, round sperma-
tids with defects are usually removed from the epithe-
lium, such as through the formation of multinucleated
spermatids—a common feature of abnormal spermio-
genesis87 that leads to infertility. It appears that these
multinucleated spermatid cells are formed, at least in
part, as a result of defects in intercellular bridges, such
as observed after colchicine treatment,88 which is known
to induce microtubule disassembly.89 Also, deletion of
genes important to early spermiogenesis, such as genes
pertinent to acrosome biogenesis (e.g., Gopc), leads to
infertility.90 However, a similar quality control mecha-
nism is absent in late spermiogenesis because deletion of
late spermatid-associated genes (e.g., Catsper1-4, Spem
1) in mice fail to affect testis weights, and sperm counts
are normal and development of defective late spermatids
continue to proceed to spermiation even though abnor-
mal sperms are found in these mice.86 At least spermatids
from these mice could be used for ICSI to obtain success-
ful pregnancy;however, the biology of spermiogenesis in
humans remains largely unexplored.
Spermiation
At the end of spermiogenesis, fully developed spermatids,
namely spermatozoa, appear and they line up at the edge
of the tubule lumen to prepare for their release at sper-
miation, which takes place at stage VIII of the epithelial
cycle in rodent testes versus stage II in human testes.6,7,9,47
Interestingly, the biology of spermiation in humans, in
particular the underlying regulatory mechanism(s), is vir-
tually unknown and much of the findings in the literature
are based on studies using the rodent testis.87,91,92 As such,
the following discussion is based on studies in rodents
unless otherwise specified. Spermiation is a highly com-
plicated cellular process, consisting of at least three sepa-
rate series of cellular events.
1. Active spermatid transport and endocytic vesicle-­mediated
protein trafficking event. First, spermiation begins with
the active transport of spermatids across the adluminal
compartment toward the luminal edge of the tubule
lumen starting at stage VII in rodents (versus stage I
in humans), and by about mid-stage VIII (versus early
stage II in humans), elongated spermatids have lined
up at the luminal edge of the tubule lumen. During
this series of events, apical ES, the only anchoring
device that anchors spermatids onto the Sertoli cell as
it makes its initial appearance at stage VIII of the epi-
thelial cycle by replacing desmosome and gap junction,
undergo some remarkable changes. These changes are
first detected at the concave (ventral) side of the sper-
matid head, where apical ES transforms into an ultra-
structure known as the apical tubulobulbar complex
(apical TBC) as named by some investigators.93–95 In
essence, the TBC represents endocytic vesicle-mediated
 Development of round spermatids to functional spermatozoa through spermiogenesis  173

Table 13.1  Genetic anomalies or mutation associated with male infertility and associated phenotypes in men.
Genetic anomalies Phenotype
Chromosomic alterations, such as Klinefelter’s syndrome, Azoospermia, oligozoospermia, or
Robertsonian translocations, reciprocal translocations147–149 normozoospermia
Y chromosome microdeletions, such as deletion of AZFa, AZFb, Azoospermia, oligozoospermia, SCOS,
or AZFc 150,151 spermatogenic arrest, NOA or normozoospermia
Genetic Mutation
Nuclear receptor 5A1 (NR5A1), also known as steroidogenic Azoospermia or oligozoospermia
factor 1 (SF-1)152
Kallmann syndrome 1 sequence (KAL-1)153,154 Kallmann syndrome-hypogonadotropic
hypogonadism
Androgen receptor (AR)155 Azoospermia or oligozoospermia
CFTR (cystic fibrosis transmembrane conductance regulator)156 Obstructive azoospermia
INSL3- RXFP2 (insulin-like factor 3-its receptor, relaxin/insulin-like Cryptorchidism
family peptide receptor 2)157,158
CATSPER1 (Cation channel sperm-associated protein 1)159 Oligo-astheno-theratozoospermia
AURKC (aurora kinase C)160 Macrozoospermia
SPATA16 (spermatogenesis-associated 16)161 Globozoospermia
DPY19L2 [Dpy-19 like 2 (C. elegans)] 162,163 Globozoospermia
DNAI1 (dynein axonemal intermediate chain 1), DNAH5 (dynein Asthenozoospermia
axonemal heavy chain 5) and DNAH11 (dynein axonemal heavy
chain 11)164
SNRPA1* (Small nuclear ribonucleoprotein polypeptide A’)165 NOA
RHOX* (reproductive homeobox)166 Poor semen quality
DAZL* (deleted in azoospermia-like)167 Spermatogenic failure
MTHFR* (methylenetetrahydrofolate reductase) 168,169 Spermatogenic defects
KATNB1* (katanin regulatory subunit B1)129 Oligoasthenotheratozoospermia
RABL2A* (RAB, member of RAS oncogene family-like 2A)170 Oligoasthenozoospermia
SHBG* (sex hormone–binding globulin)171 Spermatogenic defects and poor semen quality
TAF7L* (TATA-box binding protein associated factor 7 like)172,173 Spermatogenic failure
USP26* (ubiquitin specific peptidase 26)174–177 Azoospermia
FSHR* (follicle stimulating hormone receptor) 178,179 Spermatogenic defects
ESR1* (estrogen receptor 1) and ESR2* (estrogen receptor 2)180,181 Oligozoospermia
ETV5* (ETS variant 5)182 NOA and SCOS
*Candidate genes correlated with male infertility; AZF: azoospermia factor; NOA: nonobstructive azoospermia; SCOS: Sertoli
cell–only syndrome.

intracellular trafficking organelles, similar to giant
endosomes, to elicit endosome-mediated recycling and
also protein degradation87,96 (Figure 13.2). In fact, TBC
is constituted by proteins found in endocytic vesicles,
including clathrin, cortactin, and other endosome
markers.97–100 TBC is also found at the basal ES, is
associated with the BTB, and is known as basal TBC101
clearly visible under electron microscopy.102 In short,
proteins at the apical ES are rapidly endocytosed and
likely recycled to assemble the newly formed apical ES
when step 8 spermatids appear at stage VIII of the epi-
thelial cycle in the rat testis.
2. Remodeling/degeneration of the apical ES and residual
body disposal. Second, elongated spermatids undergo
extensive remodeling near or at the luminal edge,
including the formation of spermatozoa from step 19
spermatids in the rat testis (versus step 16 and step 6
spermatids in the mouse and human testis) and grad-
ual degeneration of the apical ES. This event is tightly
associated with the condensation of the spermatid
remaining cell cytosol and eventually shed as the resid-
ual body to be engulfed by the Sertoli cells and actively
transported to the base of the tubule. Phagosomes are
then fused with lysosomes to form phagolysosomes,
which are to be degraded and eliminated in the Sertoli
cell.12,103 As such, the Sertoli cell is also known as a
scavenger in the epithelium.
3. Sperm release. At late stage VIII in rodents (versus
stage II in humans), spermatozoa transformed from
elongated spermatids are released into the tubule
lumen due to a complete degeneration of the apical ES.
Studies have shown that apical ES degeneration that
facilitates the release of sperm in the rat testis is medi-
ated, at least in part, through the action of MMP2,104
174  Advances in our understanding of human spermatogenesis

which likely induces proteolysis of the laminin frag-
ments at the apical ES, such as laminin-γ3 chain.105–107
This, in turn, generates biologically active fragments
from laminin-γ3 and laminin-β3 chains.106 One of
these peptides, known as F5-peptide, was shown to be
capable of inducing BTB restructuring106,108 so that the
events of sperm release and BTB remodeling that take
place simultaneously across the epithelium at stage
VIII of the cycle can be coordinated.109,110 Studies have
shown that the events pertinent to spermiation are
regulated by p-FAK-Tyr407 and p-FAK-Tyr397 through
their effects on the actin-based cytoskeleton at the api-
cal ES.108,111
Regulation of spermatogenesis
Studies have shown that during spermatogenesis, in
particular spermiogenesis and spermiation, besides the
timely expression of specific genes necessary to regu-
late spermatid transformation, packaging of the genetic
material into the spermatid head, DNA condensation,
elongating of the tail, and others, 86,112,113 the actin- and
microtubule (MT)-based cytoskeletons are also nota-
bly involved. 8,87,92,114 (Figure 13.2). This notion was first
noted in studies using toxicant models in which rodents
were treated with toxicants, such as 2,5-hexanedione
and carbendazim. It was shown that the Sertoli cell
cytoskeletons, in particular MT-based cytoskeleton,
were grossly disrupted.114–116 This in turn affects sper-
matid and luminal fluid transport, causing retention of
spermatids in the seminiferous epithelium and/or fluid
accumulation in the tubule lumen. For instance, sper-
matids at or near the luminal edge are emptied into the
tubule lumen, whereas spermatids inside the epithelium
fail to be transported to the luminal edge.117,118 This fail-
ure of spermatid transport, such as premature release of
spermatids in nonstage VIII tubules and/or retention of
late spermatids in the epithelium, such as step 19 sper-
matids found in stages IX-XI tubules, is also found in
rat testes following in vivo knockdown of genes known
to be involved in conferring cytoskeletal function, such

Spermiation

Late stage VII - early stage VIII Late stage VIII

Residual body (outside)

Spermatid cytoplasm Phagosome (inside)

Tubulobulbar complex (TBC) Fluid-phase endocytosis

Microtubule-based cytoskeleton Phagolysosome

Actin-based cytoskeleton Dissolution of phagosome

Figure 13.2  A schematic illustration of the developing spermatids and residual bodies that are transported across the seminifer-
ous epithelium during the epithelial cycle of spermatogenesis, and are supported by the actin- and microtubule (MT)-based cyto-
skeletons in adult rat testes. The left panel illustrates the cross section of a seminiferous tubule at stage VII-early VIII of the epithelial
cycle vs. a tubule at late stage VIII on the right panel when the release of sperm takes place. The transport of elongated spermatids
across the epithelium in a stage VII-VIII tubule is supported by the track-like structures of MTs and actin filaments so that sperm can
be lined up near the tubule lumen for their release at spermiation in late stage VIII of the cycle (right panel). Also, at stage VIII, phago-
somes derived from residual bodies engulfed by the Sertoli cell are being transported to the base of the tubule for their degradation
via the lysosomal degradation pathway.
 Development of round spermatids to functional spermatozoa through spermiogenesis  175
as actin nucleation protein formin 1119 and actin bun-
dling protein plastin 3.120
Role of actin- and MT-based cytoskeletons
Rats treated with adjudin, 1-(2,4-dichlorobenzyl)-1H-in-
dazole-3-carbohydrazide were shown to induce extensive
germ cell exfoliation, but in some tubules, spermatids
remained embedded deep inside the seminiferous epithe-
lium even though apical ES was compromised and degener-
ated.121 A recent report using the adjudin model has shown
that the tracklike structures conferred by both actin- or
MT- (visualized by α-tubulin or ß-tubulin staining, which
are the building blocks of MTs) based cytoskeletons are
truncated and grossly downregulated within 6 hours fol-
lowing adjudin treatment.122 In short, no distinctive track-
like structures of actin- or MT-based cytoskeletons are
found in the testis of adjudin-treated rats (50 mg/kg b.w. by
oral gavage) from 6 through 24 hours,122 and these changes
are associated with extensive spermatid exfoliation, lead-
ing to transient infertility in these rats. More detailed
analysis of these rat testes revealed that the apical ES at
the spermatid-Sertoli cell interface in spermatids located
near the tubule lumen and in spermatids embedded deep
inside the epithelium were equally disrupted. However,
spermatids embedded inside the epithelium failed to be
transported across the epithelium to be emptied into the
tubule lumen. The plausible explanation is due to the lack
of “tracks” conferred by cytoskeletons, analogous to the
absence of subway tracks for transporting cargo via sub-
way cars. It was also shown that these unwanted changes in
the actin- and MT-based cytoskeletons are due to changes
in the spatiotemporal expression of Arp3 and Eps8122 such
that actin microfilaments at the apical ES fail to organize
into bundles to support apical ES function. Studies have
shown that the dynamic conversion of actin microfila-
ments between their branched/unbundled and bundled
configuration at the apical ES are conferred by Arp3, a
branched actin polymerization protein that causes nucle-
ation of an actin microfilament at its barbed ends, creat-
ing a branched actin network. On the other hand, Eps8,
an actin barbed end capping and bundling protein that
causes microfilaments to organize into bundles.123 Thus,
the tracklike structures conferred by F-actin were grossly
disrupted following adjudin treatment due to disruptive
spatial expression of these actin regulatory proteins. For
MT conferred tracklike structures, it was shown that the
spatiotemporal expression of EB1 (end-binding protein 1, a
plus (+) -end microtubule tracking protein [+TIP]) was also
disrupted following adjudin treatment, so that the tracklike
structures conferred by MTs were grossly disrupted follow-
ing adjudin treatment.122 In this context, it is important to
note that cytoskeletons, in particular F-actin, also provide
the attachment site for adhesion protein complexes, such
as α6ß1-integrin, JAM-C, JAM-A, nectin-2, nectin-3, and
others at the apical ES.54,124 In this context, it is of interest to
note that apical ES is a hybrid adherens junction (AJ) since
it is constituted by proteins that are usually found at the TJ
(e.g., JAM-A, JAM-C), AJ (e.g., N-cadherin), gap junction
(e.g., connexin-43, connexin-33), and focal adhesion com-
plex (also known as focal contact) (e.g., ß1-­integrin).125,126
A disruption of the actin-based cytoskeleton thus com-
promises adhesion protein complexes at the apical ES,121
thereby causing germ cell loss from the epithelium, leading
to infertility.
Cytoskeletal reorganization
As noted above, cytoskeletons that provide the tracklike
structures are crucial to the transport of spermatids, resid-
ual bodies, and phagasomes across the epithelium during
the epithelial cycle. However, the actin- and MT-based
cytoskeletons also require dynamic changes to confer
plasticity to Sertoli cells so that the Sertoli cell can mod-
ify its cell shape to accommodate changes in spermatid
morphology during spermiogenesis. As such, cleavage
proteins specific to the actin- and MT-cytoskeletons are
necessary to confer cytoskeletal dynamics. Recent studies
have shown that mutation or inactivation/deletion of a MT
cleavage protein, katanin, causes defects in spermiogen-
esis, leading to infertility.127,128 In fact, genetic mutation of
katanin gene in humans lead to oligoasthenotetratozoo-
spermia in humans, an infertile condition manifested by
low spermatozoa number, abnormal sperm morphology,
and poor motility.129 On the other hand, Sertoli-cell-
specific deletion of N-WASP, an upstream activator of the
Arp2/3 complex, perturbs the ability of actin cytoskeleton
to reorganize from its bundled to unbundled/branched
configuration also shown to cause infertility in male
mice.130 A detailed analysis using these Sertoli cell-­specific
N-WASP deleted mice has shown that the loss of the
branched actin polymerization capability in Sertoli cells
leads to spermiogenesis arrest, since meiosis can be seen in
some tubules, but none of the round spermatids undergo
spermiogenesis to advance into elongating spermatids.131
Collectively, these findings illustrate the importance of
cytoskeletal reorganization to support spermiogenesis.
Hormonal regulation
Studies have shown that intratesticular testosterone (T)
level is maintained at ~100-fold higher than the systemic
circulation in both rodents and humans to support sper-
matogenesis.132–134 Thus, T implants are used to perturb
the hypothalamic-pituitary-testicular axis in rodents by
increasing the serum T level, which in turn shuts down
the release of GnRH from the hypothalamus. This thus
reduces LH secretion from the pituitary gland, lowering
the intratesticular T level due to reduced T production
by Leydig cells. These changes thus contribute to exfolia-
tion of germ cells from the epithelium.135,136 An elevated
intratesticular T level is also crucial to m
­ aintain spermio-
genesis.137 Studies have also illustrated the significance of
estrogen in male reproductive function since earlier stud-
ies demonstrated the role of estrogen in fluid reabsorption
of luminal fluid in efferent ducts in the rodent testes.138,139
Interestingly, by deleting the Cyp19 gene encoding P450arom
was found to perturb estrogen synthesis capability in
these mice without perturbing the levels of gonadotropins
176  Advances in our understanding of human spermatogenesis
or androgens in the systemic circulation and in the tes-
tis. Also, Cyp19 KO mice were found subfertile, having a
reduced number of round and elongated spermatids by
the time they reached ~1-year of age.140 Thus, it is likely
that the relative ratio of androgen:estrogen in the micro-
environment of the testis is crucial to support spermio-
genesis and/or spermiation. These findings will need to be
better examined in humans to explore the role of T and
estradiol-17ß on human spermiogenesis and spermiation.
Genetic regulation
Studies using genetic models have shown that more
than 400 genes are essential for male fertility, involving
­k nockout/knockin/gene-trapped, transgenic, and chem-
ical-mediated point mutant mice.112 In humans, genetic
factors also play a crucial role in maintaining fertility in
men since a number of reproductive disorders in men are
now known to be the result of genetic anomalies and muta-
tions (or genetic variations) of specific genes113,141; some of
these findings are listed in Table 13.1. Furthermore, recent
studies have shown that abnormal expression of miRNAs
in human testes also leads to NOA. These include upreg-
ulation of miR-302a, miR-491-3p, miR-141, miR-429, and
miR-7-1-3p, and also downregulation of miR-520d-3p and
miR-383 in men with NOA.142,143 These findings thus illus-
trate the complexity of regulation pertinent to spermato-
genesis. Future studies should focus on identifying the
common signaling and/or regulatory pathway(s) utilized
by multiple sets of genes to regulate specific cellular events
during spermatogenesis. In this context, it is of interest to
note that certain paternally acquired traits are known to
be memorized or impregnated in germ cells as epigenetic
information through RNAs and/or sperm RNA modifica-
tions.144 In short, epigenetic inheritance can be acquired
through sperm, and studies have shown that this is medi-
ated mainly through sperm RNAs and RNA modifica-
tions.144 These findings also illustrate that much research
is needed to better understand the genetic material of hap-
loid spermatids.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
We briefly review advances in the field regarding molecu-
lar mechanisms that regulate some aspects of spermato-
genesis in selected areas that have implications to human
males. Nonetheless, studies in understanding the biol-
ogy of spermatogenesis in humans are far behind stud-
ies in rodents. One of the major obstacles is the lack
of human samples for analysis and functional studies.
However, some recent advances in the field, such as the
use of human Sertoli cells that are capable of undergoing
mitotic proliferation when cultured in medium contain-
ing ~5%–10% fetal calf serum61,145 and the development
of simple in vitro testis-like cultures in rodents,50,52 sug-
gest that these human Sertoli cells can be used in func-
tional studies. In fact, human Sertoli cells have been used
to probe the molecular mechanism(s) by which environ-
mental toxicants induce male reproductive dysfunction.61
Furthermore, advances in genetic analysis have identified
mutations of many genes that are crucial to maintain sper-
matogenesis in humans (Table 13.1). As noted in various
sections discussed above, many questions remain to be
answered. However, the next decade is likely to be very
promising in terms of providing new tools and techniques
to manage some male infertility, such as helping men with
unobstructed azoospermia to father their own children.
ACKNOWLEDGMENTS
The authors have no conflicts of interest to declare. This
work was supported by grants from the National Institutes
of Health, NICHD R01 HD056034 to C.Y.C., and U54
HD029990 Project 5 to C.Y.C.
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A look into the testis as a reservoir for
HIV and ZIKV—A reproductive biologist’s
perspective
ELIZABETH I. TANG, CHRISTOPHER L. ROBINSON, CHI NOK CHONG,
SHUIBING CHEN, and C. YAN CHENG
INTRODUCTION
Immune privilege is a concept which arose from a group
of work that revealed that specific sites in the body, such
as the eye, brain, testis, and others, can protect grafted
tissue from rejection.1,2 There are two distinct aspects of
immune privilege: privileged sites and privileged tissues,
which should be noted.3 A privileged site describes a site
in which foreign tissue grafts can survive longer, or indefi-
nitely, than in a nonprivileged site. A privileged tissue
refers to tissue that resists rejection when grafted into a
nonprivileged site. The testis is a unique organ because it
encompasses both aspects of immune privilege.4 The first
evidence that the testis is immune-privileged dates back to
1786, when John Hunter transplanted a cock testis into the
abdomen of a hen.5 This was the first recorded indication
that the testis can induce tolerance when transplanted into
an allogeneic recipient, as the testis transplant appeared to
have no noticeable effects on the hen. During the 1970s,
almost 200 years after Hunter’s experiment, evidence that
the testis can receive foreign cells and suppress rejection
arose. In these studies, tissue allografts or parathyroid
glands were implanted in the interstitial space of rat testis
and survived.6 These discoveries paved the way for later
cotransplantation studies in which pancreatic islet cells
were transplanted with testicular Sertoli cells (SCs), which
have been found to suppress immune responses, helping
the islet cells to survive.7–12 Sertoli cells have been deemed
the “nurse” cells of the testis and serve many roles, one of
which is conferring immune privilege.
Immune privilege of the testis is necessary for safe-
guarding the propagation and proliferation of genetic
material, such as during meiosis and postmeiotic sperma-
tid development through spermiogenesis and the release
of sperm at spermiation (Figure 14.1). The testis is an
essential organ of the male reproductive system and is
the site of mammalian spermatogenesis. Spermatogenesis
is a complex process in which undifferentiated hap-
loid germ cells (GCs) develop into differentiated hap-
loid spermatozoa, which takes place in the seminiferous
tubules of the testis. The testis protects developing GCs
from systemic circulation via local immunoregulatory
mechanisms, peritubular myoid cells, Leydig cells, as
well as by the blood-testis barrier (BTB) formed by Sertoli
cells. This cumulative defense system of the testis con-
fers its immune-privileged nature, which is largely why
14
this organ has few major diseases. Although the testis is
immune-privileged, it does not mean that it is entirely
immune from infections. Studies have shown viruses,
such as mumps, human immunodeficiency v­ irus-(HIV) 1,
human papillomavirus (HPV), and Zika virus (ZIKV)
can afflict the testes.13–15 Some of these viruses are sexu-
ally transmitted, as studies detected the presence of the
viruses in semen.13,16–18 There is increasing evidence sug-
gesting that the testis may serve as a sanctuary site for
viruses, which take advantage of the immune-privileged
nature of the testis, making the testis a safe haven.19,20 This
chapter explores what is currently known in the literature
about the presence of select viruses in the testis and how
they may evade or exploit the protective mechanisms of
the testis. Further understanding on how the viruses may
harbor themselves in the testis will be helpful in develop-
ing treatments for these viruses such as their eradication.
CONTRIBUTING FACTORS OF TESTIS IMMUNE
PRIVILEGE
Systemic immune tolerance, local immunoregulatory
mechanisms, as well as the physical structure of the tes-
tis are all contributing factors of testis immune privilege.
In a typical cross section of a testis, one can see that the
testis is primarily composed of the seminiferous tubules
with interstitial tissue (space) between them (Figure 14.1).
The interstitial tissue contains blood vessels, immune
cells, such as macrophages, dendritic cells, lymphocytes,
and mast cells, which confer the local immunoregulatory
mechanisms of the immune-privileged testis (Figure 14.1).
These immunoregulatory mechanisms have been reviewed
recently4,21,22 and also appear elsewhere in this book, and
thus will not be discussed further herein.
The structure of the seminiferous tubules is also impor-
tant for immune privilege of the testis. The seminiferous
tubules are surrounded by peritubular myoid cells, which
together with SCs help to form the basal lamina (also
known as the basement membrane) of the seminiferous
epithelium through their secretory proteins and other
constituent products (e.g., laminins), providing structural
integrity to the tubules.23,24 The seminiferous epithelium is
a specialized microenvironment for spermatogenesis and
is composed of only two cell types: SC and GC at various
stages of their development. As previously mentioned, SCs
have innate immunosuppressive properties, which is why
183
184  A look into the testis as a reservoir for HIV and ZIKV—A reproductive biologist’s perspective

Seminiferous
tubule

Interstitial
space

Seminferous tubules:
Pachytene Spermatogonium Myroid cell
Elongated spermatocyte
spermatid Blood-testis Spermatogonial Lymph Sertoli
barrier stem cell niche cell
Round Preleptotene Basement membrane Lymphatic
spermatid spermatocyte and type I collagen layer endothelium

Interstitial space:

Leydig Mast Dendritic Blood

Macrophage
cell cell cell vessel

Figure 14.1  Morphological features of the seminiferous epithelium in mammalian testes. The micrograph in the left panel is a
cross section of an adult rat testis. The schematic drawing in the right panel illustrates the basic morphological features of the semi-
niferous epithelium. The blood-testis barrier, constituted by coexisting actin-based tight junctions, basal ectoplasmic specialization,
and gap junction, as well as intermediate filament-based desmosome, divides the epithelium into the basal and adluminal (apical)
compartment. Both undifferentiated and differentiated type A spermatogonia and preleptotene spermatocytes derived from type
B spermatogonia are found in the basal compartment, whereas late spermatocytes (including pachytene/zygotene/diplotene sper-
matocytes, secondary spermatocytes, and spermatids [steps 1–19 in the rat testis]) are found in the adluminal compartment. Blood
vessels, Leydig cells, dendritic cells, and macrophages, however, are all found in the interstitial space, whereas only Sertoli cells and
germ cells at different stages of their development constitute the seminiferous epithelium. Seminiferous epithelium lays next to the
basement membrane (a modified form of basal lamina in the testis), underneath of which is the type 1 collagen, to be followed by
the myoid cell layer and then the lymphatic vessel, constituting the tunica propria.

they are used to cotransplant pancreatic islet cells. In addi-
tion, SC, or “nurse” cells, have numerous essential roles
in the testis, such as providing nutritional and structural
support for differentiating GCs and formation of the BTB.
The BTB is one of the tightest blood-tissue barriers and
divides the seminiferous epithelium into two compart-
ments, adluminal (also known as apical) and basal.25–28
It is comprised of specialized junctions between adjacent
SCs near the base of the epithelium, serving as an immu-
nological barrier.29–33
During GC development, autoantigens are transiently
expressed by GCs undergoing meiosis as well as by devel-
oping spermatids.22,34 The BTB protects GCs, postmeiotic
spermatids in particular, from immunological response
to their autoantigens. Interestingly, premature GCs such
as spermatogonia and preleptotene spermatocytes that lie
outside the BTB also express autoantigens35 as well as many
cancer/testis (CT) antigens and oncogenes,36 yet male
rodents and humans do not develop antibodies against
them except in pathological conditions.36–39 However,
when these autoantigens are injected in other parts of the
body, a strong autoimmune response is directed against
them, which is compelling evidence to support the unique
immunosuppressive properties of the testis.21,40 Thus,
suppression of immunological response to expression of
autoantigens by either GCs outside the BTB in the basal
compartment or by GCs in the adluminal compartment is
an indication of the highly regulated immunoregulatory
mechanisms of the testis.
VIRUSES AND THE TESTIS
The mammalian testis is known to have a sophisticated
system to defend against viral infections. It has been
hypothesized that viral entry into the testis via blood is
subject to three lines of defense: (1) Leydig cells and mac-
rophages in the interstitial space, (2) peritubular myoid
cells that lay at the periphery of the seminiferous tubules,
and (3) Sertoli cells.41 How viruses can evade the first two

lines of defense is still unclear. Overcoming the third line
of defense to invade the seminiferous epithelium proves to
be a daunting task. Part of the SC antiviral defense system
consists of interferon response; SCs constitutively express
interferons (IFN), and after exposure to virus the levels of
multiple interferons increase considerably.41 As previously
discussed, SCs protect developing late spermatocytes
and postmeiotic germ cells through the formation of the
BTB. In short, the BTB segregates the events of meiosis I/
II and postmeiotic spermatid development from systemic
circulation and the host immune system. Interestingly,
it was found that GCs per se, unlike SCs, express lower
levels of IFNs and following exposure to virus, GC IFN
levels change minimally.42 This possibly suggests that GCs
are more susceptible to viral infection if a virus can pass
through the BTB in the first place. Behind the BTB, GCs
express autoantigens during differentiation and are pro-
tected from immune response due to their presence. Thus,
this also suggests the possibility of a suppressed immune
response if viruses penetrate the BTB and stay behind in
the adluminal compartment. On the other hand, SCs and
GCs secrete defensins, which are a family of antimicrobial
peptides that may also target viruses.43,44 It is known that
some viruses can evade the testicular antiviral system,
infecting GC/sperm, and remain in considerable quanti-
ties in semen; however, how they do so is still unknown.
Sexual transmission of viruses is a public health con-
cern. HIV/acquired immunodeficiency syndrome (AIDS)
is a global health pandemic and ZIKV is emerging as a
novel sexually transmitted virus. These two unrelated
viruses do share something in common: both are found in
immune-privileged sites such as the testis and brain when
their viral RNA levels are virtually undetectable in serum.
This chapter explores what is currently known about HIV
and ZIKV in the testis.
HIV
HIV is a retrovirus that causes HIV infection, and if left
untreated can lead to the potentially life-threatening con-
dition, AIDS. It is a virus that attacks the immune sys-
tem, specifically CD4 cells (T cells), which help the body’s
immune system fight off infections. The loss or a declining
population of T cells makes the body more prone to infec-
tion and other diseases such as pneumonia. HIV is most
commonly transmitted through contact with bodily flu-
ids, such as blood, semen, vaginal fluids, and breast milk.
Even with treatment, HIV cannot be fully eliminated
from the body and remains throughout an infected indi-
vidual’s lifetime. However, with the use of HIV medicines
or antiretroviral therapy (ART), people with HIV can lead
longer, healthier lives while reducing the risk of transmis-
sion to other individuals. The goal of ART is to achieve and
maintain viral suppression. Monitoring plasma HIV RNA
(viral load) is an important indicator of positive response
to ART. However, measuring viral load does not measure
the presence of HIV in sites protected from systemic cir-
culation, such as the central nervous system (CNS) and
genitourinary tracts.
Viruses and the testis  185
Both the CNS and genitourinary tracts have been
proposed to be reservoirs for latent HIV infection.19,45
Sanctuary sites in the body are anatomical sites that are
nearly inaccessible by drugs. The CNS is a well-established
sanctuary site due to the blood-brain barrier (BBB), a
selective semipermeable membrane that segregates the
brain from the circulatory system. The brain is an ideal
reservoir for HIV as it is an immune-privileged site and
because the BBB protects ART drugs from effectively
entering the brain. The testis is also an immune-privileged
site and has its own blood-tissue barrier namely the BTB,
which also effectively prevents ART drugs from passing
into the testis. Both these sites are efficiently protected
from leukocyte trafficking, making these ideal sanctuary
sites for HIV, where they can hide and eventually evolve
and emerge as more virulent.46
As mentioned above, the testis is an ideal site for HIV to
hide because it is immune-privileged. Features of immune
privilege allow the testis to facilitate fertility, such as by
tolerating GC autoantigens that are transiently expressed
during differentiation. HIV has been detected in semen
but its origins in the genitourinary tract are still not clearly
defined. HIV was found in semen as free viral particles,
which proved that infected lymphocytes and macrophages
from the blood were not the only sources of the virus.47
Subsequently, it was shown that free viral particles in
semen can be different from those isolated from infected
leukocytes in semen, suggesting that HIV can be compart-
mentalized in genital tissues.48
Studies have shown that men receiving ART have reduced
viral load. However, some of these men can still shed HIV-1
in their semen, suggesting that the testis is an antiretroviral
drug sanctuary.49–51 Similar results were found in simian
immunodeficiency virus (SIV)-infected macaques.52 ART
reduced viral load in blood but did not affect viral DNA
levels in genital tissues compared to untreated macaques.
In another study, human testes samples from virally sup-
pressed patients were examined and confirmed primate
study findings.14 In this study, expression of C-C chemokine
receptor 5 (CCR5), the main coreceptor of HIV, increased as
compared to expression of the same coreceptor in peripheral
blood in these virally suppressed patients, suggesting that
the human testis is a sanctuary site for HIV and provides
an immune-tolerant environment for the virus. However,
one limitation of this study is that whole testis lysates were
used for examination, thereby preventing investigators
from identifying which testicular cell type(s) was infected
or harboring the virus.
Since HIV viruses are detected in the testis and semen,
it is quite possible that spermatozoa/sperm may be carri-
ers or infected by the virus. However, whether or not HIV
viruses are detected in sperm remains controversial.53–55
Depending on the technique used, HIV can be found with
sperm.56 There are reports in the literature suggesting that
HIV is present on sperm. One such study detected CD4
receptor, which has a high affinity for HIV, on the surface
of sperm, indicating that it may be a vehicle for HIV.56
Another study, using transmission electron microscopy
186  A look into the testis as a reservoir for HIV and ZIKV—A reproductive biologist’s perspective
to examine sperm obtained from AIDS patients, which
illustrated the presence of virus-like particles in sperm
cytoplasm, supporting the notion that viruses could infect
sperm.53,57 These and other studies in which GC/sperm
were cultured in vitro also showed that they can become
infected or be hosts for HIV.53,58 In contrast, other reports
challenged those findings as very low levels of CD4 recep-
tor were detected on sperm surface57 and that virus-like
particles were in fact acrosomal reaction vesicles.54
Other possible routes of HIV association or infec-
tion of sperm may be through early infection of GCs.
Spermatogenesis is the process by which spermatogo-
nial stem cells or premature GCs develop into sperm.
Spermatogonial stem cells reside outside the BTB as they
develop into preleptotene spermatocytes, which are the
only GC type able to pass through the BTB and enter the
adluminal compartment for subsequent development.
Infection of GCs may be a path by which HIV may harbor
in the testis, a sanctuary site. In one study in which tes-
tes of AIDS patients were examined, investigators found
evidence of decreased spermatogenesis and atrophy in the
testes. Investigators also detected considerable loss of GCs
as well as HIV positivity in residual GCs, supporting the
notion that HIV-infected GCs may be partially responsible
for sexual transmission of HIV.59 Additionally, other find-
ings suggest the possibility of direct infection of GC by cell-
free virus as HIV-1 proviral DNA was detected in the nuclei
of GCs at all stages of differentiation.60,61 Collectively,
these findings suggest that GCs could be infected by HIV.
ZIKV
ZIKV is a flavivirus that can cause microcephaly and
Guillain-Barré syndrome as well as other neurological
complications.62–64 Typical symptoms of infected individu-
als are usually mild, characterized by fever, headache, rash,
joint pain, conjunctivitis, and muscle pain (www.cdc.gov)
lasting for a few days to a week. Since symptoms are mild,
most infected individuals do not realize they are infected.
In 2015, a large-scale outbreak of ZIKV in Brazil was cause
for alarm. By 2016, ZIKV was declared a global health
emergency by the World Health Organization (WHO).65
Prior to this, small outbreaks were detected in other conti-
nents, ranging from Africa to Southeast Asia and Oceania.
Conventionally, ZIKV is considered an arborvirus since it
is transmitted by mosquitoes. However, recent outbreaks
have revealed that ZIKV can also be spread via sexual
(both homo- and heterosexual) and vertical transmission
routes unlike most flaviviruses.17,18,66–70 Zika viruses can
pass through the placenta during pregnancy and infect the
fetus to cause microcephaly as well as cause Guillain-Barré
syndrome in adults.71 Clinical observations in male ZIKV
patients detected the presence of the virus in semen for up
to 6 months, suggesting the possibility of transmission long
after the appearance of symptoms.72 These findings among
others, in addition to the declaration of a global health
emergency, spurred scientists worldwide to better under-
stand the pathogenesis of ZIKV. ZIKV and its impact on
male reproductive health is the subject of this section.
Finding a suitable model to study ZIKV has proven
challenging. To date, a small number of studies investi-
gating the role of ZIKV and its consequences in the male
reproductive tract have emerged. In humans, ZIKV targets
transcription factor STAT2 and initiates its degradation,
disrupting type I IFN signaling.73,74 However, when wild-
type (WT) mice, a typical study model, were infected with
ZIKV they were found to have little or no infectious virus/
viral RNA in their tissues.71 Interestingly, ZIKV does not
target STAT2 in WT mice, which may be one of the rea-
sons why WT mice are resistant to ZIKV infection.71,73,74
Due to the general resistance of immunocompetent mice
to ZIKV, scientists have used other approaches to establish
a working ZIKV mouse model.
Currently, scientists use immunocompromised mice to
study the pathogenesis of ZIKV and persistence in the male
reproductive tract. In one study, researchers inoculated
INF α/ß receptor-deficient mice (Ifnar1-/-) or WT mice
treated with anti-Ifnar1 MAb (IFNα receptor 1 monoclo-
nal antibody) to block IFN signaling. Findings from this
study were primarily from the Ifnar1-/- mice data.75 They
examined the genital tract of these mice and reported that
ZIKV was present in the testis and epididymis but not in
the prostate and seminal vesicles. Progressive testicular
damage was observed in these mice. At 8 dpi (days postin-
fection), mouse testes already began to display signs of
orchitis, degeneration of GC and Leydig cells, and lym-
phocyte and leukocyte infiltration inside the seminiferous
tubules. By 30 dpi and longer, seminiferous tubules were
structurally damaged and testes atrophic. Additionally,
these investigators also detected the presence of ZIKV in
specific testicular cell types and found it in spermatogo-
nia, spermatocytes, sperm, and SCs.
A subsequent study published by a different group used
WT mice treated with anti-Ifnar1 antibody and infected
them with a mouse-adapted ZIKV strain. They discov-
ered that ZIKV caused significant testicular damage as
well, in addition to decreased sperm count and ­fertility.76
Cell types infected in this study were SC, GC/sperm, and
possibly Leydig cells in the testes but also in the epididymis.
Since then a number of studies have used similar approaches
to study the pathogenesis of ZIKV. What these studies have
in common is that they all utilize ZIKV-infected immuno-
compromised mice. As seen in Table 14.1, defining which
cell types are targeted by ZIKV is inconclusive. Results
vary depending on the ZIKV strain in combination with
the type of immunocompromised mice. Understanding
which cell types are susceptible to ZIKV infection can help
to further uncover the biology of the virus, especially how
the virus is able to evade the highly immune-privileged
nature of the testis and male r­ eproductive tract.
CONCLUDING REMARKS
The clinical implications of HIV and ZIKV are drasti-
cally different; however, both are global health concerns.
Further understanding of how both viruses evade the
immunoregulatory mechanisms of the testis, as well as
other sanctuary sites like the brain, will be helpful for
 Concluding remarks  187

Table 14.1  Results of selected study models investigating Zika virus infection in the male genitourinary tract.
Infected cell
Zika virus strain Study model type(s) Cell location Technique d.p.i. Ref.
From patient returning ZIKV positive semen Sperm Semen IF 56a 81

from trip to French from human patient

Guyana, 2016

ZIKA-SMGC-1 (isolated ZIKV infected Spermatogonia/GC & Testes & epididymis IF 8, 16 & 30 75

from Chinese patient Ifnar1-/- mice peritubular myoid

returning from Fiji cells; epididymal
and Samoa, 2016) epithelial cells (EEC)
Testicular & epididymal Testes & epididymis qRT-PCR 8, 16 & 30
cells

Mouse-adapted ZIKV infected C57BL6 Testicular, epididymal Testes, epididymis, & qRT-PCR 7, 14 & 42 76

Dakar 41519 mice + anti-Ifnar1 cells & sperm epididymal fluid

(Senegal, 1984) or H/ antibody Sperm Epididymis Plaque assay 7
PF/2013 (French Spermatogonia, Testes ISH 7 & 14
Polynesia, 2013)b primary
spermatocytes, & SC

Mouse-adapted Dakar ZIKV infected Axl-/- Indiscriminate All reproductive tract qRT-PCR 7
41519 (Senegal, mice + anti-Ifnar1 tissues
1984) antibody SC & GC Testes & epididymis ISH 7
ZIKV infected Rag-/- Indiscriminate All reproductive tract qRT-PCR 7
mice + anti-Ifnar1 tissues
antibody SC & GC Testes & epididymis ISH 13

American-derived ZIKV infected Leydig cells (LC) Interstitial space IHC 5 82

MEX2-81 (Mexico, Ifnar1-/- mice Sperm & EEC Epididymis IHC 5

2016) Isolated LC from WT or Interstitial space Plaque assay 1-5
Ifnar1-/- mice testesc
Testicular & epididymal Testes & epididymis qRT-PCR 5 & 21
cells

Paraiba_01/2015 ZIKV infected Rag1-/- GC (likely Seminiferous IHC & ISH 17 or 21 83

(Brazil, 2015) mice + anti Ifnar1 spermatogonia/ epithelium

antibody primary
spermatocytes)

ZIKV/Homo sapiens/ ZIKV infected Sperm Caudal epididymis qRT-PCR 7, 14 & 21 84

PRI/ Ifnar1-/- mice Sperm Caudal epididymis IF 28

PRVABC59/2015 SC & GC Seminiferous IHC 7, 14 & 21
(Puerto Rico, 2015) epithelium
EEC & cells in Epididymis IHC 7, 14 & 21
epididymal lumend

CAS-ZK01 (Asian ZIKV infected AG6 SC Seminiferous IF (in vivo & in 2, 5 & 8 85

strain) mice epithelium vitro)

Macrophages Interstitial space IF
Abbreviations: d.p.i., days post-infection; EEC, epididymal epithelial cells; SC, Sertoli cells; GC, germ cells, LC, Leydig cells; IF, immunofluorescence
analysis; qRP-PCR, quantitative reverse transcription-polymerase chain reaction; ISH, in situ hybridization; IHC,
immunohistochemistry.
a Days postappearance of symptoms.

b Dakar 41519 replicated to higher levels than H/PF/2013 strain and was used for majority of this study.

c Leydig cells (LCs) were infected after isolation.

d Cells in lumen contained sperm and degenerating/sloughed necrotic epithelial cells.

188  A look into the testis as a reservoir for HIV and ZIKV—A reproductive biologist’s perspective
developing drugs and treatments. It also remains to be
determined whether viruses that penetrate the SC BTB to
gain entry into the adluminal compartment of the seminif-
erous epithelium is mediated through transfection of SC
or is the result of paracellular transport of viral particles
at the BTB. Reports in the literature suggest that HIV is
utilizing one of its exterior envelope glycoproteins, gp120,
to induce breakdown of tight junctions (TJs) between
endothelial cells at the BBB.77 HIV gp120 also downregu-
lated the expression of ZO-1 and ZO-2, two key adaptor
proteins that support TJ integral membrane proteins such
as occludin, claudins, and junctional adhesion molecules,
thereby causing the TJ barrier to become “leaky,” facilitat-
ing their entry into the brain.77 Furthermore, HIV gp120
was also found to induce production of matrix metallo-
proteinases (MMPs) in vaginal epithelial cells, leading to
degradation of TJ proteins in vaginal epithelium, thereby
facilitating the passage of HIV across the vaginal epithelial
barrier.78 Additionally, human T-cell leukemia virus type
1 (HTLV-1) was found to penetrate through a tight human
epithelial barrier via a transcytosis mechanism without
involving any disruption or infection of the epithelium.79
This information is crucial since it presents evidence that
viruses have the ability to pass through tight tissue barri-
ers. These findings provide the foundation for developing
future treatments to prevent viral entry through the BTB
and other tissue barriers. Perhaps the BTB can be tight-
ened by manipulating signaling pathway(s) that regulate
it80 to deny entry of viral particles to the testis, eliminating
a haven for these viruses.
CONFLICTS OF INTEREST AND ACKNOWLEDGEMENTS
Conflicts of interest: Nothing to declare.
This work was supported by grants from the National
Institutes of Health (NICHD R01 HD056034 to C.Y.C.;
U54 HD029990 Project 5 to C.Y.C.).
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Cytoskeletons (F-actin)
and spermatogenesis
LIZA O’DONNELL and PETER G. STANTON
INTRODUCTION
Spermatozoa are produced during the complex and
elegant process of spermatogenesis. These specialized
cells arise from immature diploid spermatogonia, which
undergo meiosis to produce haploid gametes. The sperm
structure develops during spermiogenesis, where a round
haploid germ cell (round spermatid) is transformed into
a spermatozoon by the development of a motile cilium, a
streamlined nucleus, and an acrosomal cap required for
fertilization. All aspects of male germ cell development are
supported by the somatic Sertoli cell; this large and struc-
tural complex cell simultaneously supports multiple germ
cells at different phases of development, providing both
the structural and nutritional support required to execute
the spermatogenic program. Spermatogenesis is highly
dynamic and precisely controlled in space and time and is
accompanied by extraordinary changes in each cell’s cyto-
skeleton. This chapter will address the role of a particular
cytoskeletal component, actin, in spermatogenesis.
Actin is one of the most abundant proteins in cells and
indeed on Earth.1 The amino acid sequence of actin mono-
mers (also known as globular actin or G-actin) is highly
conserved between species. There are three main isoforms
of actin monomers, alpha, beta, and gamma, encoded by
different genes. Actin monomers are polymerized into
filamentous actin (F-actin), which in turn is arranged into
higher-order structures via interactions with numerous
actin binding and regulatory proteins. Actin participates
in many cellular functions, including division, migration,
polarity, shape, intercellular adhesion (including cell-cell
and cell-substrate adhesion), and intracellular transport.
Emerging evidence has revealed actin can also func-
tion within the nucleus, regulating gene expression and
responding to extracellular cues.2,3
F-ACTIN BIOGENESIS, DYNAMICS, AND FUNCTION
Actin monomers are relatively flat proteins with a cleft
that  binds tightly to ATP, which is hydrolyzed during
polymerization. Monomers spontaneously dimerize then
polymerize into a helical single-stranded filament (Figure
15.1a). Due to the orientation of actin monomers at each
end, F-actin has a “pointy end,” which is slow to polymer-
ize, and a “barbed end,” which rapidly polymerizes and
grows in length (Figure 15.1). Profilin is a small protein
that plays a major role in actin polymerization via binding
to monomers (Figure 15.1a) and to the polymerized fila-
ment and cytoskeletal proteins.4 Once polymerized, actin
filaments can be cross-linked into higher-order structures
15
via association with various microtubule-associated proteins
(Figure 15.1b), (reviewed in Pollard 2016).1 The complex-
ity of actin structures is further increased by branching,
facilitated by the Arp2/3 complex. The Arp2/3 complex,
in association with nucleation promoting factors such
as WASH and WASP, binds to the side of a filament and
nucleates a daughter branch,5 whereas cortactin stabilizes
the branches (Figure 15.1b).
Actin filaments and higher-order structures are innately
dynamic, possessing elastic and mechanical properties
that allow them to fulfill multiple roles within the cell;
such properties are influenced by actin’s association with
a wide range of binding proteins.1,6 Along with profilin,
F-actin elongation can be regulated by formins, homodi-
meric proteins that bind to the barbed ends of F-actin
and promote further polymerization by stabilizing actin
dimers. The growth of F-actin barbed ends can be inhib-
ited by actin capping proteins, whereas tetramers of Ena/
VASP proteins facilitate elongation by inhibiting actin
capping. F-actin dynamics are further regulated by sever­
ing of the filaments (Figure 15.1b) via the small protein
cofilin and the larger protein complex gelsolin, whereas
the actin-associated protein tropomyosin can protect fila-
ments from severing. Cofilin can sever filaments or inhibit
their elongation but can also promote filament assembly
at higher concentrations.1,7 A multitude of actin cross-
linking proteins are also essential for the organization of
F-actin into higher-order structures (Figure 15.1b). These
linking proteins are rapidly exchanged, giving rise to the
unique properties of F-actin networks; these networks are
relatively stiff when rapidly deformed but can be deformed
over a longer time scale (tens of seconds) due to exchange
of linking proteins. This explains why cells are stiff and
elastic when subject to a rapid force but can be deformed
by longer periods of applied pressure.1
Actin generates forces that underlie many cellular
functions and provides a signal-responsive cytoskeletal
system that responds to many cues. While polymeriza-
tion of actin itself can generate force, many cellular move-
ments and force generation involve F-actin binding to
myosins, a family of ATPase motor proteins. Myosins are
large heterodimeric proteins consisting of heavy and light
chain subunits. They possess a globular head that binds
to F-actin and has ATPase activity and a tail region that
confers specificity for different binding proteins. Upon
ATP hydrolysis, the head region undergoes a conforma-
tional change, allowing it to “walk” along the actin fila-
ment while moving vesicles or other proteins associated
191
192  Cytoskeletons (F-actin) and spermatogenesis

ATP binding
cleft

Pointed end Barbed end

(slow (rapid Profilin
Barbed end polymerization) polymerization)
groove
Actin Actin
(a) monomer filament

Cortactin
Cofilin

N-WASP

Arp2/3 Actin-
Formins capping
proteins
(b) Branching Elongation Severing Cross-linking and bundling

Figure 15.1  Actin filament biogenesis and dynamics. (a) Actin monomer and filament structure and profilin’s role in regulat-
ing actin polymerization by binding to the monomer pool. Actin monomers contain an ATP-binding cleft and a so-called barbed
end that binds to target proteins. The polymerization of the actin monomers, facilitated by profilin, creates a helical filament with
a slowly polymerizing end and a rapidly polymerizing end that can grow rapidly in length. (b) Overview of the regulation of actin
dynamics by interacting proteins. Branched filaments are created by the Arp2/3 complex in association with other nucleation pro-
moting factors such as N-WASP, and the branches are stabilized by interactions with cortactin. Actin filament elongation is promoted
by formins, but can be inhibited by actin capping proteins. Filaments can be severed (e.g., by cofilin) and arranged into various
higher-order structures by a multitude of actin-binding and bundling proteins.

with the tail region. Myosin activity is regulated by the
phosphorylation of the regulatory light chain subunit.8,9
The forces generated by actin filaments and networks,
as well as those generated during myosin-mediated move-
ments, create a mechanically-sensitive dynamic state,
reviewed in De La Cruz and Gardel 2015.10 This mechani-
cally sensitive state allows the cell to respond to external
stimuli, for example to shear stress, but also to respond
to internal cues such as forces generated within the cyto-
skeleton itself. Depending on the type of filament cross-
linking and the abundance of myosin motors, the actin
network can be arranged into various higher-order struc-
tures with different physical properties. For example, cell
motility is achieved by the polymerization of branched
actin filaments in lamellipodia effectively pushing the
leading edge of a migrating cell forward. Actin networks
can be contractile; actomyosin (actin + myosin complex)-
mediated contractility is involved in many cellular func-
tions such as exo- and endocytosis and cell shape changes.
Actin networks can also be organized into ring structures;
constricting actomyosin rings create the cleavage fur-
row between dividing cells and facilitate cytokinesis, and
stabilizing actin rings are components of cell adhesion
junctions.11
Finally, it should also be noted that there is extensive
cross talk between the actin and microtubule cytoskel-
etons.12 For example, while cell motility requires actin
polymerization in membrane protrusions, directional cell
movement involves growing microtubules being captured
by the cortical actin foci at the leading edge of the cell.12
Actin-binding formins appear to be a key player in the
cross talk between the actin and microtubule cytoskel-
eton, with an ability to regulate both actin polymerization
and microtubule stabilization.13
From the above it is clear that the actin cytoskeleton
plays multiple roles in all cells and thus the actin cytoskel-
eton will drive many aspects of spermatogenesis, such as
the extraordinary changes in Sertoli and germ cell shape
as they proceed through the spermatogenic cycle. The fol-
lowing section will provide an overview of the actin cyto-
skeleton in spermatogenesis, highlighting the role of actin
dynamics in particular processes in the seminiferous epi-
thelium. Other reviews14–18 also address this topic.
F-ACTIN IN SPERMATOGENIC CELLS
F-actin in spermatogonia and meiotic cells
Spermatogenesis originates from the spermatogonial stem
cell (SSC) niche. SSCs both renew their own population to
provide a lifelong supply of stem cells and differentiate to
become spermatogonia that ultimately develop into sper-
matozoa; the establishment and maintenance of this niche
is the foundation of successful spermatogenesis. Various
lines of evidence suggest that the actin cytoskeleton is
involved in maintaining the SSC niche. Spermatogonial
transplantation experiments show that the ability of SSCs
to colonize the niche is dependent on their ability to express
β1-integrin, which links the actin cytoskeleton with the
extracellular matrix.19 Glial cell line-derived neurotrophic
factor (GDNF), secreted by Sertoli cells, is a key regulator

of the SSC niche, regulating the migration, differentiation,
and self-renewal of SSCs. GDNF induces the expression
of VASP in undifferentiated spermatogonia, suggesting
that VASP-dependent changes in the actin cytoskeleton
influences the ability of SSC to migrate into the niche.20
BMP4, another factor that regulates SSC differentiation,21
stimulates SSCs to transcribe genes involved in the actin
cytoskeleton and actin-dependent adhesion junctions.22
AIP1, an actin disassembly factor, is required in both
spermatogonia and Sertoli cells for spermatogonial migra-
tion and establishment of the SSC niche in mice,23 and
profilin-dependent stabilization of F-actin is required for
the maintenance of SSCs in Drosophila.24 Therefore, main-
tenance of the SSC niche involves the regulation of actin-
dependent adhesion and cytoskeletal structures.
Differentiating spermatogonia ultimately enter meiosis,
the process that generates haploid, genetically diverse gam-
etes. Early in meiosis, preleptotene spermatocytes must
traverse the blood-testis-barrier to complete their develop-
ment within a specialized microenvironment; their trans-
port across this barrier is dependent on F-actin dynamics
within Sertoli cells (see section “Dynamics of Sertoli cell
F-actin-containing structures during spermatogenesis”).
During the first meiotic prophase, homologous chromo-
somes pair, form synaptonemal complexes, and undergo
recombination to ensure genetic diversity of the male
germline. Actin and myosin-dependent processes are likely
involved in synaptonemal complex formation. Ablation
of ALKBH4, a protein that influences F-actin’s ability to
associate with nonmuscular myosin II, caused failure of
synaptonemal complexes to form, resulting in spermato-
cytes entering apoptosis and failing to complete meiosis.25
Interestingly, nonmuscular myosin II is also required for
the completion of cytokinesis at the end of meiosis.26 The
movement of chromosomes during prophase is thought
to be reliant on microtubule-based processes;27 however,
there is some evidence that actin is involved in chromo-
some positioning in late diakinesis just prior to anaphase.28
F-actin during spermiogenesis
After the last meiotic division, haploid spermatids enter
spermiogenesis as small, round, very transcriptionally active
cells, yet they finish the process as long, transcriptionally
inert spermatozoa with dense, characteristically shaped
nuclei and long flagella capable of motility. This transfor-
mation is accompanied by extraordinary cytoskeletal and
nuclear changes, in which actin dynamics play key roles.
There is abundant evidence that spermatids specifically
express novel transcripts of components of the F-actin
cytoskeleton. Spermatids specifically express novel actin
monomers29–31 that only share 40%–50% identity with
conventional actin genes but retain conserved functional
motifs; these intron-less genes are conserved in rodents
and humans, have unique regulatory elements, and may
have arisen from retrotransposons during evolution.29
Sperm uniquely express novel actin-related proteins that
form a major constituent of the sperm head32 as well as
a unique actin-capping protein,33 novel testis-specific
F-actin in spermatogenic cells  193
profilins,34 and a novel isoform of αT-catenin35 that can
regulate actin cytoskeleton dynamics. Therefore, it is pos-
sible that spermatids have evolved a novel F-actin machin-
ery that may be functionally different to other actin-based
cytoskeletal structures.
Male germ cell division is incomplete and germ cells
at various stages of development remain attached to one
another by intercellular bridges that allow the movement
of cytoplasmic contents between cells. These bridges are
required for germ cell development36 and facilitate shar-
ing of gene products between adjacent cells;37 this is likely
important for synchronous development of cohorts of hap-
loid spermatids. Intercellular bridges contain actin,18,36,38
which is essential for their maintenance, as disruption of
actin filaments dissolves the bridges38 ultimately resulting
in the formation of multinuclear symplasts followed by
germ cell death.
Round spermatids employ sophisticated mechanisms
to control gene transcription and translation during sper-
miogenesis.39,40 Such mechanisms are required to execute
the spermiogenic program as active gene transcription
ceases midway through spermiogenesis when the sperma-
tid nucleus compacts. Interestingly, an actin-related pro-
tein, ARPC5, a member of the Arp2/3 complex proteins,
has been shown to participate in translational repression
during spermiogenesis,41 and the F-actin cytoskeleton is
associated with translational repression mechanisms in
other systems.42 The chromatoid body in round spermatids
is an organizing center for RNA and facilitates noncod-
ing RNA-dependent translational silencing pathways.39
Although the chromatoid body contains actin,43 it remains
intact after cytochalasin D-induced actin disassembly,37
suggesting actin dynamics might not be required for the
maintenance of chromatoid body structure. Spermatids
also express novel actin proteins in the nucleus during
elongation.31,44 Given the abundant evidence for actin and
its associated proteins operating within the nucleus to reg-
ulate transcription, translation, and chromatin dynam-
ics,3,42,45,46 the role of nuclear F-actin in gene transcription
and chromatin remodeling during spermiogenesis war-
rants further study.
During early spermiogenesis, round spermatids develop
the acrosome, a vesicle containing proteolytic enzymes
required to penetrate and fertilize the oocyte. Acrosome
development involves F-actin dynamics, as the acrosome
fails to spread across the nucleus after treatment with
cytochalasin D.18 Acrosome development is accomplished
by F-actin–rich structures present in the subacrosomal
space; these structures have been termed the acroplax-
ome (an actin-based scaffold that anchors the acrosome to
the nuclear membrane) and the marginal ring (an actin-
rich structure at the leading edge of the spreading acro-
some).47,48 The acrosome arises from vesicles in the Golgi;
these vesicles coalesce and spread across the nuclear sur-
face in a process facilitated by the actin motor myosin Va
together with its receptor RAB27A/B.49 Proteins involved
in the regulation of F-actin dynamics are present in the
subacrosomal space and acroplaxome and may regulate
194  Cytoskeletons (F-actin) and spermatogenesis
acrosome development, including FerT, profilin III and
IV, and phosphorylated cofilin, reviewed in Kierszenbaum
et al. 2007.47
As spermiogenesis progresses, the nucleus polarizes to
one side and the spermatid begins to elongate, with the
nucleus undergoing extensive condensation and remod-
eling. The perinuclear theca is a narrow, condensed layer
of cytoskeleton surrounding the spermatid nucleus as it
elongates,50 extending beyond the acrosome as the post-
acrosomal sheath or calyx.51 This calyx contains short
F-actin filaments bound to the protein calicin, thought to
impart a structural rigidity,52 and other actin-associated
proteins, reviewed in Toshimori 2003.53 The spermatid
nucleus is remodeled into its species-specific shape via the
microtubule-based manchette. 54,55 However, F-actin-
based mechanisms operating within the manchette and
acroplaxome, accompanied by “sculpting forces” from the
actin-based Sertoli cell ectoplasmic specialization (see
section “F-actin in ectoplasmic specializations”), are
also proposed to participate in nuclear shaping during
spermiogenesis.17,47 Toward the end of spermatid develop-
ment and in epididymal sperm, F-actin is found in specific
regions of the perinuclear theca in the sperm head56 along
with a testis-specific isoform of an actin-­bundling protein,
FSCN3.57 As spermatids continue through spermiogen-
esis, however, F-actin may depolymerize to G-actin and
become redistributed.18 Interestingly, F-actin in the sperm
head increases during capacitation and the acrosome reac-
tion, suggesting that polymerization of monomeric actin
is involved in the activation of mature spermatozoa.58
Therefore, F-actin is present in the sperm head and may
play multiple roles in sperm head development and fertil-
izing ability.
Finally, F-actin may play a role in the development of
the spermatid flagella. Flagella biogenesis begins in round
spermatids, with the formation and extension of the
microtubule-based axoneme. Sperm-specific accessory
structures such as outer dense fibers and the mitochon-
drial sheath are then assembled around the axoneme in
elongating spermatids. This latter stage of development
is proposed to involve the transport of proteins along the
manchette to the developing flagellum.59 While the man-
chette is largely microtubule-based, small tracks of F-actin
are visible within the microtubule bundles and contain
myosin-based vesicles,49 testis-specific profilins34 and the
actin regulatory protein palladin.60 Current theories sug-
gest that the transport of vesicles along the manchette
microtubule and F-actin tracks delivers at least some of
the proteins required for flagella assembly.59
F-ACTIN IN SERTOLI CELLS
The cytoskeleton in Sertoli cells is comprised of actin fila-
ments, intermediate filaments, and microtubules. F-actin
tends to be concentrated at intercellular junctions between
adjacent Sertoli cells where they contribute to the blood-
testis barrier (BTB), and at junctions between Sertoli cells
and elongating spermatids (step 8 and onward in rats
and mice) prior to their release from the epithelium at
spermiation. These actin-based structures within Sertoli
cells perform various essential functions required for suc-
cessful sperm production.
F-actin in ectoplasmic specializations
The Sertoli cell ectoplasmic specialization (ES) consists
of a layer of hexagonally packed actin filaments recog-
nizable by electron microscopy61,62 located between the
plasma membrane and a cistern of endoplasmic reticu-
lum. Opposing ES structures are found at intercellular
junctions between Sertoli cells near their basal aspect and
are referred to as basal ES (Figure 15.2); these comprise
an important part of the BTB (see section “Dynamics of
Sertoli cell F-actin-containing structures during sper-
matogenesis”). In contrast, single ES structures are seen in
Sertoli cells opposite elongating spermatids (Figure 15.2);
these are referred to as apical ES.
The ES has adhesive properties because the intercellular
gap is narrow and contains adhesion proteins such as α6β1
integrin,63 nectin-2,64 and N-cadherin.65 The basal ES forms
part of the inter-Sertoli–cell basal junctional complex; var-
ious other junction types (tight-, gap-, ­desmosome-like-)
are also present at this site and have links to the actin cyto-
skeleton66,67 (Figure 15.2). The adhesive function of this
junctional complex is spatiotemporally regulated to allow
restructuring when basal spermatocytes translocate into
the adluminal c­ ompartment (Figure 15.2).
Various actin-binding proteins are found at the basal ES,
with functions including actin bundling and cross-linking
(espin, ezrin, fascin 1, palladin, plastin family), branched
actin polymerization (Arp2/3 complex, N-WASP), end-
capping (Eps8), and branch stabilization (cortactin).68,69
Selective RNAi-mediated knockdown of individual actin-
binding protein expression tends to disturb Sertoli cell
junction function in vitro as well as F-actin and associ-
ated junction protein organization (e.g., targeting fascin
170). However, under these circumstances ES function is
not completely ablated, suggesting that multiple proteins
contribute to actin dynamics at the basal ES. In contrast,
targeted disruption of the branched actin polymerization
pathway by knockout of N-Wasp (which acts upstream
of Arp2/3) in mouse Sertoli cells caused spermatogenic
arrest, loss of expression of connexin-43 and occludin
(markers for gap and tight junctions, respectively), and
loss of BTB function.71 These studies clearly show con-
trol of branched actin polymerization to be an essential
component of the maintenance of seminiferous epithelial
structure and function.
In contrast to basal inter-Sertoli cell junctions, apical
ES junctions between Sertoli cells and advanced sperma-
tids are heterotypic and consist of ES located only on the
Sertoli cell side; no junctional structures are discernible
in the spermatid. The apical ES has much the same struc-
ture as the basal ES, comprised of hexagonally packed
actin filaments located between the Sertoli cell plasma
membrane and an underlying endoplasmic reticulum.
In the rat, the apical ES first appears in Sertoli cells adja-
cent to round spermatids as they polarize at step 8, and
 F-actin in Sertoli cells  195

5
6

3
Adluminal
4 4
4 7
2 Sertoli cell
8
9

1 Basal

10

Figure 15.2  Summary of the major roles of F-actin structures (green) in the seminiferous epithelium. In certain Sertoli cell struc-
tures, F-actin is located near cisternae of endoplasmic reticulum (denoted in red). 1. Actin dynamics are involved in the migration of
spermatogonia and the establishment of the spermatogonial stem cell niche. 2. Actin dynamics are proposed to be involved in the
formation of synaptonemal complexes between meiotic chromosomes. 3. In early spermiogenesis, actin is involved in formation of
the acrosome and its attachment to the nucleus, as well as in maintaining intercellular bridges. 4. During the elongation phase of
spermiogenesis, F-actin gradually disappears from the perinuclear region of the spermatid head, but abundant F-actin is present
in large bundles in the Sertoli cell cytoplasm adjacent to the spermatid head in an adhesion structure known as the ectoplasmic
specialization (ES). 5. At the end of spermiogenesis, the actin bundles and the ES are disassembled, coincident with the formation
of tubulobulbar complexes (TBCs). TBCs are surrounded by dendritic actin and are involved in the removal of adhesion junction
components and remodeling of the spermatid. 6. Just prior to sperm release, spermatids remain attached to the Sertoli cell by an
integrin-based focal adhesion type junction that may associate with cytoplasmic actin filaments. 7. Transport and phagocytosis of
the residual body (the remainder of the spermatid cytoplasm) is thought to rely on actin dynamics. 8. Inter-Sertoli cell junctions
include ES junctions with opposing bundles of actin, TBCs, and other adhesion junctions (gray bars) including desmosome-like
and occluding junctions. 9. As early spermatocytes translocate into the adluminal compartment, the inter-Sertoli cell junctions are
remodeled, disappearing above and reappearing below the spermatocytes as they migrate. 10. Peritubular myoid cells surrounding
the seminiferous tubules contain abundant actin filaments; the actin-based contractility of these cells is important for the contrac-
tion of the tubules as they propel released sperm toward the epididymis. The tight inter-Sertoli cell junctions (8) effectively divide
the seminiferous epithelium into a basal and adluminal compartment (denoted by the blue lines); germ cells in the basal compart-
ment have free access to factors from the interstitium whereas germ cells in the adluminal compartment develop in a specialized
microenvironment determined by the Sertoli cell.

remains associated with elongating spermatids until it is
removed during the process of spermiation.72 The apical
ES functions to firmly attach the elongating spermatid
to Sertoli cells, which in turn draws them deep into the
epithelium as part of the spermatid maturation process.62
The orientation and polarity of adluminal spermatids is
likely maintained by F-actin dynamics at the apical ES,
as knockdown of various proteins involved in F-actin
dynamics leads to the loss of correct positioning sper-
matid in the epithelium and ultimately in their failure to
complete spermatogenesis.73,74
F-actin in tubulobulbar complexes
The tubulobulbar complex (TBC) is a novel structure
comprised of a tubular extension of plasma membranes
surrounded by a cuff of cross-linked actin (Figure 15.2)
to which various actin-binding proteins are localized. At
the end of this F-actin-associated tubular extension is a
so-called bulbous structure, surrounded by endoplasmic
reticulum but not actin, which extends into a clathrin-
coated pit. Many studies have shown that TBCs are a
novel endocytic structure similar to podosomes, and their
major function is the removal and recycling of intercel-
lular junctions.69 TBCs are readily found at apical junc-
tions between Sertoli cells and advanced spermatids and
are well described at this site.69 These structures form on
the ventral side of the spermatid nucleus at the begin-
ning of spermiation (stage VII in rodents) and they persist
throughout spermiation, where they are involved in the
removal of ES and other junction-associated proteins as
well as remodeling the spermatid plasma membrane prior
to its release into the lumen.69
TBCs are also present in the basal junctional complex
between Sertoli cells 69; see Figure 15.2. Here, the long
tubular extension from one Sertoli cell projects into an
invagination in the neighboring cell, and the distal region
196  Cytoskeletons (F-actin) and spermatogenesis
including the clathrin-coated pit enlarges to form a bul-
bous structure before it buds off from the complex and is
internalized via endocytic mechanisms.75 Various junc-
tion proteins, including claudin-11, connexin-43, and
nectin-2 are found within basal TBCs, and indeed whole
tight and gap junction fragments have been observed
there by electron microscopy,75 highlighting the role of
basal TBCs in remodeling of inter-Sertoli-cell junctions.
TBC formation and function depends on F-actin
dynamics. Various actin-binding proteins are present in
the proximal cuff region of the TBC, including espin, cor-
tactin, cofilin, paxillin, vinculin, zyxin, Eps8, N-WASP,
and Arp2/3.69 These proteins are likely important for the
formation, elongation, and maintenance of the tubu-
lar portion of the TBC76,77; however, relatively little is
known about their regulation. Knockdown of Arpc1b,
an e­strogen-responsive subunit of the Arp2/3 complex
involved in actin nucleation and branching, compro-
mised BTB integrity in vivo.78 Both FSH and testosterone
regulate the expression of a panel of Sertoli cell miRNAs
in vitro that target proteins in the actin cytoskeleton and
focal adhesions.79 Importantly, disruption of F-actin net-
work using cytochalasin D caused the tubular regions of
basal TBCs to become swollen with disorganized actin
foci, confirming the importance of actin networks in nor-
mal TBC function.76
Dynamics of Sertoli cell F-actin-containing
structures during spermatogenesis
The BTB is a functional barrier that sequesters meiotic
and postmeiotic germ cells into a controlled adlumi-
nal environment separate from the immune system that
would otherwise see them as foreign. In broad terms the
BTB has three components: the inter-Sertoli-cell junctions
(mentioned above) that provide a physical barrier, plus
Sertoli cell transporters that control substance movement
and create a physiological barrier, and an immunologi-
cal barrier contributed by immunoregulatory factors and
tolerance mechanisms.80,81 This barrier effectively divides
the seminiferous epithelium into two compartments: the
basal compartment, where germ cells “below” the inter-
Sertoli-cell junctions have free access to factors from the
interstitium and circulation, and the adluminal compart-
ment, where germ cells reside “above” the barrier and
develop within a restricted environment (Figure 15.2). The
barrier also enables maturing germ cells to develop within
a unique specialized environment that is strictly regulated
by the Sertoli cell.
It is well known that the Sertoli cell responds to the hor-
mones FSH and testosterone with multiple structural and
functional changes.40 The formation of the BTB and basal
ES occurs concomitant with the pubertal rise in FSH and
LH.82 Androgen action on Sertoli cells is required for BTB
formation, whereas FSH action appears to be permissive
but not mandatory.82 However, FSH stimulation of F-actin
organization into ES structures in Sertoli cells has consis-
tently been observed in vitro83,84 and in vivo,85 highlight-
ing the importance of FSH in promoting proper Sertoli
cell F-actin organization. Interestingly, palladin, which
functions as a scaffold to bundle actin fibers, is found at
the basal ES in vivo86 but FSH-mediated intercellular sig-
naling directs its localization to the Sertoli cell nucleus
where it may regulate transcription, potentially providing
a direct link between nuclear function and the status of
the F-actin cytoskeleton.87
Remodeling of the BTB and inter-Sertoli-cell junctions
is required for early spermatocytes to enter the adlumi-
nal compartment and complete their development (Figure
15.2). This remodeling is accompanied by marked changes
in F-actin-based structures, and the disturbance of vari-
ous proteins involved in F-actin organization can impair
both the structure and function of inter-Sertoli-cell junc-
tions.68,88 TBCs are most prevalent between Sertoli cells
prior to inter-Sertoli-cell junctions being remodeled to
allow germ cell translocation.89 The fact that these TBCs
contain morphologically discernible fragments of gap
and tight junctions, as well as internalized junction-
associated molecules, supports the hypothesis that TBC
formation between Sertoli cells is important for the
remodeling of inter-Sertoli-cell junctions prior to germ
cell translocation.69
TBCs are also involved in junction removal between
Sertoli cells and spermatids during spermiation. Sper­
miation is the process by which mature spermatids
undergo a final remodeling, including removal of their
extensive cytoplasm and remodeling of the nucleus, prior
to their disengagement or release from the Sertoli cell.72
In order for spermiation to occur, the extensive ES junc-
tion present around the spermatid (Figure 15.2) needs to
be removed. The removal of ES likely involves the disas-
sembly of the extensive actin bundles by proteins involved
in F-actin dynamics (e.g., formin 190). In addition, TBCs
likely remove ES structures. TBCs form and internal-
ize both Sertoli cell and spermatid plasma membranes
together with the junction-associated proteins.69,91 The
bulbar portion of the TBC then buds off and becomes
internalized into the Sertoli cell as an endocytic vesicle; in
this way TBCs are able to remove ES and associated adhe-
sion junctions between the Sertoli cell and the spermatid
in preparation for its final disengagement.69,91 There are
now many lines of evidence to show that F-actin dynamics
control TBC formation and their ability to remove ES and
other junction-associated proteins to ready the sperma-
tid for disengagement. For example, disruption of F-actin
with cytochalasin D prevents TBC formation and normal
spermiation.92 The F-actin “cuffs” around the tubular por-
tion of TBCs contains various proteins involved in actin
branching, including the Arp2/3 complex, N-WASP and
paxillin.69 Estradiol treatment reduces F-actin-associated
proteins within TBCs, causing defective TBC formation
and spermiation failure.78 Importantly, knockdown of
cortactin, the protein that stabilizes actin branches, pre-
vents normal TBC formation and impairs spermiation.93
The release of mature sperm into the lumen is mediated
by a focal adhesion type junction containing integrins and
focal adhesion kinase (FAK).73 Focal adhesions in other

systems link to, and are regulated by, the actin cytoskel-
eton, mediating instantaneous loss of adhesion events.
Thus, the actin cytoskeleton within the Sertoli cell at the
time of disengagement likely participates in spermatid
disengagement, perhaps explaining why many inhibitors
of actin dynamics can impair sperm release.72,94 Finally,
when spermatids disengage, the remnants of their cyto-
plasm are left behind as the residual body (Figure 15.2).
The transport and phagocytosis of residual bodies may
also rely on F-actin dynamics.95
ROLE OF F-ACTIN IN SEMINIFEROUS TUBULE
CONTRACTION
The seminiferous tubules are surrounded by one or more
continuous layers of peritubular myoid cells.96 These cells
facilitate the contraction of seminiferous tubules to pro-
pel sperm and fluid along the tubules toward the rete tes-
tis.96 Consistent with their contractile nature, peritubular
myoid cells contain layers of actin filaments (along with
myosin motors) oriented in different directions (longi-
tudinal and circular) in rodent97,98 and human testis.99
Contraction of the peritubular myoid cells is accompanied
by marked rearrangements in the F-actin cytoskeleton,100
highlighting the fact that F-actin dynamics are important
for seminiferous tubule contractility.
CONCLUSIONS
This chapter has highlighted the major roles for the F-actin
cytoskeleton during spermatogenesis. F-actin dynamics
are involved in many processes within germ cells, par-
ticularly in the dramatic remodeling of spermatids as they
develop into spermatozoa. Sertoli cells contain numerous
actin-based structures, some of which are unique to the
testis, that maintain the integrity of the seminiferous epi-
thelium and support male germ cells through their dra-
matic transformation into motile spermatozoa.
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Roles of mTOR signaling
in spermatogenesis
16
LAN YE and KE ZHENG

INTRODUCTION transplantation. Substantial evidence suggests that the

mTOR complexes and its inhibitor rapamycin dysregulation of the mTOR pathway is associated with
cancer pathologies, and hyperactivation of mTORC1 is
The mechanistic target of rapamycin (mTOR) (TOR1/
highly prevalent in human cancers. In support of this view,
TOR2 in yeast) was originally identified in budding yeast
the mutations of genes encoding for many tumor suppres-
through a genetic screen in 1991,1,2 and it is the target of
sors promote mTORC1 activation.25,26 For example, P53,
a small molecule called rapamycin. mTOR is a large con-
a well-known tumor suppressor and a major checkpoint
served serine/threonine protein kinase that belongs to the
protein, regulates mTOR activity through AMP-activated
phosphoinositide 3-kinase (PI3K)-related kinase family,
kinase (AMPK) and the tuberous sclerosis (TSC) 1/TSC2
and it forms two complexes in mammals called mTORC1
complex.27 PTEN, a putative protein tyrosine phospha-
and mTORC2.3–6 mTORC1 consists of mTOR, Raptor,
tase gene mutated in many types of human cancers, lies
mLST8/GβL, PRAS40, and DEPTOR, and mTORC2 con-
upstream of the mTOR complexes and negatively regulates
sists of mTOR, Rictor, mLST8/GβL, mSIN1, Protor1/2, and
the PI3K pathway.25,28 Additionally, mTORC2 is required
DEPTOR. Raptor and PRAS40 are specific components
for the development of tumors from human prostate epi-
of mTORC1 complex that regulates ribosomal biogenesis,
thelial cells caused by Pten deletion.29 LKB1 is a critical
translation of mRNAs containing pyrimidine-rich 5’TOP
upstream kinase for AMPK activation and is also involved
or TOP-like motifs, and autophagy through downstream
in cancer development. Mutations in the Lkb1 gene are
targets including ribosomal protein S6 kinase (S6K),
associated with a cancer-prone disease, Peutz-Jeghers
eukaryotic initiation factor 4E binding protein-1 (4E-BP1),
syndrome (PJS).30,31 Previous studies demonstrated LKB1
and ULK1/Atg13/FIP200 complex (unc-51-like kinase-1/
phosphorylates and activates TSC2, which further regu-
mammalian autophagy-related gene 13/focal adhesion
lates the small GTPase Rheb and subsequently inhibits
kinase-interacting protein of 200 kDα).7–11 In skeletal mus-
mTORC1.26,32 This evidence suggests that modulating of
cle tissues and cells, Raptor is required for maintaining
the mTOR pathway by rapamycin and its analogs (rapa-
mitochondrial respiration.3,12,13 In contrast, Rictor, mSIN1,
logs) may have great potential to treat cancer pathogenesis.
and Protor1/2 are specific components of mTORC2 com-
So far, there are two mTOR inhibitors approved by the U.S.
plex, which is involved in metabolism through AKT and
Food and Drug Administration (FDA) for treating specific
serum/glucocorticoid induced kinase (SGK).14,15 It also sus-
cancer types. Temsirolimus, the first approved rapalog, is
tains cytoskeleton through protein kinase C (PKC-α).16
an intravenous drug for advanced stage of renal cell car-
Rapamycin (clinically referred to as sirolimus) was dis-
cinoma. The second approved rapalog is everolimus for
covered in the soil of Easter Island as a macrolide compound
treatment of renal cell cancer and also for a genetic disease
produced by Streptomyces bygroscopicus.17 Mechanistically,
caused by Tsc1/Tsc2 mutation.26
rapamycin forms an intracellular complex with FKBP12 to
In addition to the critical role of the deregulated mTOR
inhibit mTOR kinase through binding its FRB domain.18,19
pathway implicated in cancer progression, the dysregu-
Rapamycin is known as a potent immunosuppressant
lation of this pathway is also involved in many patho-
for the prevention of transplant rejection20,21 and it also
logical conditions, including obesity, type 2 diabetes,
inhibits the growth and proliferation of mammalian cells
neurodegenerative disorders, as well as aging processes.
with the therapeutic potential in treating human can-
For example, the dysfunction of autophagy is impli-
cers.22 mTORC1 complex is sensitive to rapamycin and
cated in neurodegenerative diseases, such as Parkinson’s
nutrient signals, whereas mTORC2 was originally consid-
disease, Alzheimer’s disease, and Huntington’s dis-
ered a rapamycin-resistant complex. However, prolonged
ease.26 mTORC1 regulates autophagy and inhibition of
rapamycin treatment also disrupts mTORC2 assembly and
mTORC1 by rapamycin reduces the accumulation of
inhibits phosphorylation of mTORC2 downstream effec-
protein aggregates and subsequently has a beneficial role
tors in cultured cell lines and tissues.23,24
in alleviating neurodegenerative-associated symptoms.33
Importantly, reduced TOR signaling either by genetic
PHARMACOLOGICAL APPLICATIONS OF RAPAMYCIN manipulation or small inhibitors extends the lifespan
AND RAPALOGS IN HUMAN DISEASE of model organisms including yeast, worms, and flies. 34
In 1999, rapamycin received FDA approval as an immu- In mammals, rapamycin is the small molecule that
nosuppressant agent to prevent rejection in organ robustly extends lifespans in mice across three sites by

201
202  Roles of mTOR signaling in spermatogenesis
the National Institute on Aging Interventions Testing
Program (ITP).35,36 Supportingly, genetic attenuation
of mTORC1 signaling or deletion of the mTORC1 sub-
strate S6 kinase 1 (S6K1) is sufficient to extend lifespan
in mice.23,37 In contrast, depletion of Rictor, an essential
protein component mTORC2, decreases the lifespan in
male mice.38 These studies indicates that rapalogs of spe-
cifically targeting mTORC1 may promote longevity and
have beneficial effects in treating aging-associated dis-
eases in humans.
CONNECTING mTOR SIGNALING TO MALE FERTILITY
As mentioned above, LKB1 is an essential tumor sup-
pressor and mutation of Lkb1 is associated with human
cancers. LKB1 is a serine/threonine kinase that directly
phosphorylates AMPK, which further phosphory-
lates and activates the negative upstream regulator of
mTORC1, TSC2. 39 Mutation of Lkb1 was identified in
patients with PJS and is believed to have a causal role in
this disease. Importantly, clinical evidence shows that
PJS patients have defects in spermatogenesis, includ-
ing substantial loss of germ cells, and are at high risk
of developing Sertoli cell tumors.40,41 Moreover, male
patients who are prescribed with sirolimus in organ
transplantation and cancer therapies have high preva-
lence of infertility.42 This evidence indicates that the
mTOR pathway regulates multiple physiological pro-
cesses, and dysregulation of the mTOR pathway is asso-
ciated with pathological conditions such as cancer and
infertility in humans.
A close connection between the mTOR pathway and
male fertility has also demonstrated in lower model
organisms. In Caenorhabditis elegans (C. elegans), Vellai
and colleagues found that depletion of mTOR homo-
log let-363/CeTor by RNA interference reduces fertility.43
The reproductive defects are also observed in mutants of
daf-2 gene, which encodes the C. elegans insulin/IGF-like
receptor.44–47 Insights into the regulation of the mTOR
pathway on fertility have also been provided by studies in
Drosophila. In Drosophila, the release of insulin-like pep-
tides (ILPs) responds to changes in nutritional availability
and couples metabolism, longevity, and fertility through
a TOR/Raptor-dependent mechanism.48 Loss of function
of PRAS40, an inhibitory subunit of TORC1, completely
rescues the sterility of insulin receptor substrate (chico)
mutant flies.49,50 In contrast, Drosophila mutants of Rictor
or Sin1, two members of TORC2, grows normally and
are fertile.51 Therefore, the mTORC1 pathway integrates
nutrient status in animals to regulate fertility in a man-
ner that is highly conserved from flies to mammals (Table
16.1). More recently, an accumulating body of evidence
suggests an essential role of the mTOR pathway in mouse
spermatogenesis. Genetic manipulation for inhibition/­
activation of mTOR signaling in mice is complicated,
and both mTORC1 and mTORC2 play important roles in
multiple aspects of testicular function as shown in greater
detail in Table 16.1.
ROLES OF mTOR SIGNALING IN MAINTAINING
SPERMATOGONIAL FUNCTION
The lifelong spermatogenesis in mammals is founded by
the self-renewal potential of the spermatogonial popu-
lation, which resides on the basement of seminiferous
tubules. In mouse testes, spermatogonial population con-
tains A, intermediate, and B spermatogonia, and type A
spermatogonia are further divided into undifferentiated
(As, Apr, and Aal) and differentiated spermatogonia (A1-4)
with a distinct gene expression pattern that predicts dif-
ferential self-renewal potential.78 One of the questions in
stem cell biology is, What is the fundamental mechanism
that coordinates various stimuli to regulate the balance
between spermatogonial stem cell maintenance and dif-
ferentiation? Recently, genetic studies in mouse models
demonstrated that the signaling network anchored by the
mTOR kinase is one of the mechanisms.
The glial cell line-derived neurotrophic factor (GDNF)
is a crucial growth factor required for self-renewal of sper-
matogonial stem cells (SSCs).79 Mechanistically, GDNF
binds the cell surface receptor GFRA1/RET, which are
expressed in a subset of undifferentiated spermatogo-
nia (spermatogonial progenitor cells, SPCs).80 Feng and
colleagues first indicated that P70 S6 kinase, one of the
downstream targets of mTORC1, mediates the role of stem
cell factor (SCF) on the proliferation of cultured spermato-
gonial cells.81 PLZF is an essential transcriptional factor
for SPCs maintenance, evidenced by compromised self-
renewal potential in Plzf-/- testes. Hobbs and colleagues
demonstrated that mTORC1 signaling is enhanced in
cultured SPCs isolated from prepubertal Plzf knockout
testes, and the aberrant mTORC1 activation inhibits the
response of SPCs to GDNF through suppressing expres-
sion of GDNF receptor components GFRα and c-Ret.82 The
RNA-binding protein Nanos2, another essential factor
for spermatogonial self-renewal, marks the undifferenti-
ated spermatogonial populations in testes. Similar to the
observations in Plzf-/- testes, hyperactivation of mTORC1
was also apparent in germline stem cells from Nanos2 con-
ditional knockout testes. Indeed, Nanos2 interacts with
other mRNP components to promote SPCs self-renewal
through repressing mTORC1 by sequestrating mTOR in
mRNPs.83 Together, these findings indicate that mTORC1
signaling mediates the function of several essential
growth factors or transcriptional factors in maintaining
SPCs homeostasis, but they did not show genetic evidence
to support a direct role of mTOR signaling in spermato-
gonial self-renewal and differentiation. Hobbs and col-
leagues further define the mTORC1 signaling network in
controlling SPCs function by conditionally deleting TSC2,
a key upstream regulator of mTORC1.63 TSC2 negatively
regulates mTORC1 by inactivating Rheb, which stimu-
lates mTORC1 activity.3 Intriguingly, different subpopula-
tions of undifferentiated spermatogonia exhibits distinct
staining intensity of mTORC1 activity characterized by its
downstream targets phospho-S6 and 4EBP1.63,65 mTORC1
activity is lower in SSCs with high self-renewal potential
 Roles of mTOR signaling in maintaining spermatogonial function  203

Table 16.1  Genetic evidence of TOR pathway in reproduction.

Species Genetic strategies Phenotypes References
Drosophia dTorA948V mutant Abnormal testes with enlarged apical tip 52

dTORp (P insertions in the dTOR) Infertile 53

dTorW1251R, dTorP2293L mutants GSCs maintenance defects 54

Tsc1Q87X mutant GSCs loss 54

chico (insulin receptor substrate) mutants Infertile 50,55

PRAS40 null Fertile, but rescues the infertility of chico 49

mutants
InR (insulin-like receptor) mutants Sterile 52

Tsc1 mutant Precocious differentiation of GSCs and 56

loss
Tsc2 mutant GSCs loss 56

Rictor mutant Fertile 51

Sin1 mutant Fertile 51

C. elegans CeTor RNAi Reduced fertility 43

Daf-15 (Raptor) mutant Sterile 57

CeTor mutant Gonadal developmental arrest 58

rict-1 (Rictor) mutant Reproductive defects 59

rsks-1(S6K) mutant Reduced fertility 60

rsks-1 mutant Germline progenitors loss 61

CeTor RNAi Reduced germline progenitors 61

Daf-15 (Raptor) RNAi Reduced germline progenitors 61

ife-1 (eIF4E) deletion Reduced germ cells 61

Daf-18 (Pten) mutant Reproductive defects 62

Daf-2 (insulin receptor family) Reproductive defects 44

M. musculus Tsc2fl/-Stra8-Cre Normal testicular phenotype 63

Tsc2fl/-Vasa-Cre SPCs maintenance defects 63

Tsc2fl/-Amh-Cre Defective spermatogenesis in juvenile 63

but recovered in adulthood

Rheb fl/-Vasa-Cre Oligoasthenoteratozoospermia 64

Tsc1fl/-Vasa-Cre Precocious spermatogonial 65

differentiation
mTOR fl/-Vasa-Cre Arrested at differentiated spermatogonia 66

Constitutive activation of AKT Promotes proliferation of SSCs 67

Foxo1fl/-Vasa-Cre SSC defects, sterile 68

Foxo1/3/4 triple knockout Severe spermatogenic defects 68

Pten fl/-Vasa-Cre Depletion of germ cells 68

Pdk1 fl/-Vasa-Cre Normal spermatogonia 68

Raptor fl/-Vasa-Cre Spermatogonial defects 69

Raptor fl/-Ngn3-Cre Meiotic arrest 70

Rictor fl/-Ngn3-Cre Spermatogonial and BTB integrity 71

defects
Rictor RNAi BTB integrity defects 72

LKB1S mutant Abnormal spermiogenesis and 73,74

spermiation
Lkb1 fl/-Amh-Cre Sertoli-cell-only tubule 75

Tsc1fl/-Amh-Cre Sertoli-cell-only tubule 75

Tsc2fl/-Amh-Cre Sertoli-cell-only tubule 75

Ptenfl/-Amh-Cre Normal testicular phenotype 75

Rictorfl/-Amh-Cre BTB integrity defects 76

Raptor fl/-Ksp-Cre Disrupted spermatozoa 77

204  Roles of mTOR signaling in spermatogenesis

but induced in differentiation-committed progenitor cells.
In their study, three mouse models were made with con-
ditional deletion of Tsc2 in germ cells and somatic cells
within testes by using Vasa-Cre, Stra8-Cre, and Amh-
Cre strategies, respectively. Through genetic and cell bio-
logical analyses, they demonstrate that the self-renewal
potential of undifferentiated spermatogonia is impaired
in Vasa-Cre-mediated Tsc2-deleted testes (Figure 16.1),
while the spermatogonial function appears unaffected
in Tsc2-deficient testes using Stra8-Cre and Amh-Cre.63
TSC1, another upstream regulator of mTORC1, inter-
acts with TSC2 to form a complex to negatively regulate
mTORC1 activity. Wang and colleagues observed preco-
cious spermatogonial differentiation at the expense of
germline maintenance and subsequently testes atrophy
were observed in Tsc1 knockout testes (Figure 16.1).65
These findings suggest mTORC1 acts cell-autonomously
in the maintenance of the germline stem cells, and this
regulation takes place in a distinct subset of SPCs with
a differential dependency on mTORC1 activity. Shortly
after this work, Busada and colleagues showed the role of
mTORC1 in SSCs by treating neonatal mice with rapamy-
cin, a well-known mTOR inhibitor.84 They showed that
activation of mTORC1 is required for the spermatogonial
differentiation. Specially, inhibition of mTOR signaling
by rapamycin promotes the self-renewal potential of sper-
matogonia, since rapamycin treatment causes an accu-
mulation of undifferentiated spermatogonia, and blocks
spermatogonial differentiation partially through transla-
tional control of a subset mRNAs including Kit, Sohlh1,
and Sohlh2 (Figure 16.1).84 Recently, they provided direct
genetic evidence of mTOR signaling in spermatogenesis by
specifically inactivating mTOR using Vasa-Cre. In agree-
ment with their previous studies, mTOR deficiency sub-
stantially blocks spermatogonial differentiation; hence,
spermatocytes are absent at different ages (Figure 16.1).66

PI3K
mTORC2
Deptor
mSin1 PIP3 PTEN Depletes germ cells
mTOR mLST8
BTB integrity defects
Impaired spermatogonial Rictor PDK1 No defects in SSCs maintenance
differentiation Protor

Constitutive
activation
Promotes SSCs proliferation Akt Foxo1 SSCs maintenance defects

Precocious spermatogonial TSC1 TSC2 Impaired undifferentiated spermatogonia

differentiation

GTP-Rheb GDP-Rheb

mTORC1
mTOR Blocked spermatogonial differentiation
Deptor mLST8
Raptor
Meiotic arrest, MSCI defects
PRAS40
Rapamycin Inhibits spermatogonial differentiation
Activate Inhibit

Figure 16.1  mTOR signaling and deletion of its components influences spermatogenesis. mTOR forms two distinct protein
complexes, mTORC1 and mTORC2. mTORC2 phosphorylates Akt kinase at its serine 473 site. Conditional deletion of Rictor in Sertoli
cells disrupts cytoskeletal organization and cell polarity. Conditional deletion of Rictor in germ cells impairs spermatogonial dif-
ferentiation and disrupts intercellular adhesion and BTB integrity. PTEN, a negative regulator of the PI3K/AKT signaling, inactivation
of Pten results in AKT hyperphosphorylation and severe defects in SSCs self-renewal, while inactivation of Pdk1 has no obvious
effects on SSCs maintenance. AKT phosphorylates and inactivates FOXO1 while conditional deletion of Foxo1 causes defects in SSCs
maintenance. TSC1 interacts with TSC2 and forms a TSC complex, which inactivates Rheb by converting GTP-Rheb into its inactive
GDP-Rheb. Deletion of either Tsc1 or Tsc2 activates mTORC1 signaling and results in precocious spermatogonial differentiation and
impaired SSCs maintenance. In contrast, specifically knocking out Rheb caused defects in meiotic progression and spermatogenesis.
Inhibition of mTORC1 by acute rapamycin treatment blocks spermatogonial differentiation, and conditional knockout of mTOR using
Vasa-cre also results in defects in spermatogonial differentiation. Specifically inactivating Raptor using Ngn3-Cre leads to derepres-
sion of sex chromosomes-linked genes and meiotic arrest.
 Roles of mTOR signaling in maintaining spermatogonial function  205
Interestingly, a subset of undifferentiated spermatogonia,
PLZF and GFRα1 positive, remained in mTOR-deficient
testes, indicating spermatogonial self-renewal potential
is not compromised. Consistently, Xiong et al. revealed
that enhanced mTORC1 activity, induced by P53 loss,
also promotes spermatogonial differentiation.85 Taken
together, mTORC1 is involved in the GDNF response of
SSCs through crosstalk with transcriptional factors, and it
regulates the balance of spermatogonial self-renewal and
differentiation.
Compared to mTORC1, mTORC2 complex is less
sensitive to rapamycin. However, prolonged rapamy-
cin inhibits mTORC2 signaling in vitro and in vivo.23,24
mTORC2 directly activates AKT at its hydrophobic motif
(Ser473) and with phosphorylation at other sites further
phosphorylates substrates including forkhead box O1/3a
(Foxo1/3a), TSC1/2, and glycogen synthase kinase alpha/
beta (GSK3α/β). Through a germline stem cell culture
system, Lee and colleagues have reported that activa-
tion of AKT kinase is observed in GDNF-treated SSCs.67
Consistent with our recent study, phosphorylated AKT
is one of the dramatically increased phosphorylated
proteins upon GDNF stimuli identified by quantitative
mass-spectrometry-based proteomic and phosphopro-
teomic analyses.69 Constitutive activation of AKT by
transfecting with myr-Akt-Mer plasmid substantially
promotes proliferation of SSCs even in the absence of
GDNF (Figure 16.1).67 Goertz and colleagues generated
germline conditional Foxo1 knockout and Foxo1/3/4 tri-
ple knockout mice to investigate the function of Foxos in
the mouse testes, particularly in SSCs self-renewal and
differentiation.68 Conditional Foxo1-deficient mice are
sterile, with distinct defects in SSCs maintenance and
initiation of spermatogenesis (Figure 16.1). Although
Foxo3 and Foxo4 are dispensable for germ cell devel-
opment, Foxo1/3/4 triple knockout mice exhibit more
severe spermatogenic defects, indicating the members of
Foxo family compensate their functions. These findings
indicate the transcriptional factor FOXO1, one down-
stream target of AKT and AKT kinase itself are required
for maintaining SSCs. Goertz and colleagues further
conditionally inactivated Pten or 3-phosphoinositide-
dependent protein kinase 1 (Pdk1) to determine whether
Foxos regulate SSCs function through PI3K-AKT signal-
ing. Indeed, Pten mutant gradually depletes germ cells in
an age-dependent manner, which indicates Pten shares
similar roles in maintaining SSCs function with FOXOs
(Figure 16.1).68 Pdk1 loss attenuates AKT phosphoryla-
tion and subsequently constitutive Foxo1 activation, and
no obvious defects in SSCs self-renewal and differentia-
tion were observed in Pdk1 mutant testes (Figure 16.1).68
These results strongly suggest Foxo1 acts through PI3K-
AKT to regulate SSCs self-renewal and PI3K-AKT sig-
naling is crucial for SSCs maintenance. Moreover, Rictor
has a germline-specific role in maintaining normal sper-
matogonial differentiation, suggesting mTORC1 and
mTORC2 functions may overlap during spermatogonial
differentiation.71
mTOR signaling in meiosis
As discussed above, mTOR signaling is an essential
pathway in the organism to coordinate the balance
of spermatogonial self-renewal and differentiation.
Spermatogenesis begins with spermatogonial mitotic
proliferation, followed by two meiotic divisions, and
mature into haploid spermatozoa through spermiogen-
esis. Meiosis is a specialized division process in germ
cells that contains several key stages including chro-
mosomal pairing, chromosomal recombination, and
accurate segregation. Recently, we showed that Raptor,
an essential component of mTORC1, expresses in iso-
lated spermatogonia, pachytene spermatocytes, and
round spermatids, especially with high abundance in
pachytene spermatocytes, suggesting the potential of
mTOR signaling functions beyond the spermatogonial
development. To characterize the role of mTORC1 dur-
ing meiosis, we generated Raptor conditional knock-
out mice using Neurogenin Ngn3-Cre, which has been
shown to fully disrupt protein expression in testes at
postnatal day 9. Indeed, Raptor-deficient testes arrest
at pachytene stage with a particularly high frequency
of X-Y asynapsis and defects in recruiting silencing fac-
tors and epigenetic modifiers into the entire sex chro-
matin, which is illustrated in Figure 16.2.70 However,
conditional ablation of mTORC2 component Rictor by
employing the same knockout strategy shows Rictor
is dispensable for meiotic progression and recombina-
tion, suggesting the nonoverlapping functionality of
mTORC1 and mTORC2 during meiosis.71
mTOR signaling in Sertoli cells
Spermatogenesis relies largely on an exclusive type of
nongerm cells called Sertoli cells that provide nutritional
and physical support for germ cells at different stages of
their development within seminiferous tubules.86 These
somatic cells, extending from the basement membrane
almost throughout the seminiferous epithelium, keep
in frequent contact with differentiating germ cells and
direct their migration toward the epithelia lumen, for
which Sertoli-Sertoli and Sertoli-germ cell interactions
are critical.87,88 The blood-testis barrier (BTB) is formed
between adjacent Sertoli cells with the structures of tight
junction (TJ), basal ectoplasmic specialization (basal
ES), gap junction (GJ), and desmosome (DS).89–91 As an
immunological barrier, the BTB protects germ cells at
the basal compartment against immune responses from
those at the apical compartment. An extensive network
of actin filament bundles that lie perpendicular to the
Sertoli cell plasma membrane at above adhesion junc-
tions except for DS is recognized as the major force by
which BTB is formed as one of the tightest BTBs.72,86,92 In
order to facilitate the translocation of preleptotene sper-
matocytes to initiate meiosis, BTB undergoes remodel-
ing via cyclic junctional events during stage VIII–XI of
the epithelial cycle.92 Spermatocytes continuously travel
toward the luminal edge of the seminiferous tubule,
206  Roles of mTOR signaling in spermatogenesis
(a) WT (b) Raptorcko
SYCP3 SYCP3
rH2AX rH2AX
(c) WT (d) Raptorcko
SYCP3 SYCP3
H3K9me3 H3K9me3
(e) WT pachynema (f ) Raptorcko pachynema
Silencing of Impaired silencing of
sex chromosomes sex chromosomes
X
X of Peutz-Jeghers syndrome, relevant to gene mutations
Y
Y tumors impairing spermatogenesis. 30,31,40,41 These studies
Figure 16.2  Mechanisms responsible for meiotic arrest
in conditional Raptor knockout testes. (a) In wild-type
pachynema, γH2AX disappeared from synapsed auto-
somes and accumulated on sex chromatin. (b) In Raptorcko
pachynema, γH2AX was diminished on sex chromatin and par-
tially remained on autosomes. (c) In wild-type pachynema, the
repressive epigenetic maker H3K9m3 is enriched on sex chro-
matin and centromeric regions of autosomes. (d) In Raptorcko
pachynema, sex chromosomes were largely exposed except
for the centromeric region of X chromosome. (e) In wild-type
pachynema, autosomes are synapsed except for XY chromo-
somes, which only pair at a short region and are silenced.
(f) In Raptorcko pachynema, extensive asynapsis including
autosomes and sex chromosomes takes place, and sex chro-
mosomes are not efficiently silenced.
synchronizing with the dynamic Sertoli-germ cell inter-
action. Compelling evidence has implicated the cen-
tral involvement of the mTOR pathway in Sertoli cell
physiology and function during spermatogenesis.93,94
Follicle-stimulating hormone (FSH) is the major Sertoli
cell mitogen and regulates Sertoli cell proliferation via
a PI3K/Akt/mTORC1 pathway.95 17b-estradiol pro-
motes Sertoli cell proliferation via mTOR activation.96
Human Sertoli cells, when stimulated with rapamycin,
present accelerative glucose consumption, increased
sensitivity to oxidative stress, and altered mitochon-
drial bioenergetics.97,98 In prepubertal mice, short-term
in vivo rapamycin treatment does not affect the state of
Sertoli cells.84 Recent studies have reported that mTOR
signaling participates in actin cytoskeleton reorganiza-
tion and BTB remodeling.92 mTOR is also involved in the
extensive reorganization of F-actin network to modulate
the BTB dynamics. Disrupting mTOR in Sertoli cells
affects meiotic progression probably due to improper gap
junction alpha-1 protein distribution.99 A component of
the basement membrane laminin α2 is essential for the
Sertoli cell TJ barrier through mTORC1 signaling.100
Phospho-S6 is a downstream signaling molecule  of
mTORC1 that colocalizes with zonula occludens-1 (ZO-1),
a BTB adaptor protein that connects TJ to actin cytoskel-
eton and TJ-barrier function.101 A master upstream pro-
tein kinase LKB1 has a critical role in spermiation due
in part to defects in the breakdown of the junctions of
ESs.73,74 Specific loss of Lkb1 in Sertoli cells causes dra-
matic defects in Sertoli cell polarity and testicular junc-
tional complexes by ectopic activation of mTOR, leading
to Sertoli-only phenotype in spermatogenesis.75 As LKB1
positively regulates mTOR signaling that is mediated via
TSC1/2,102 conditional deletion of Tsc1 and Tsc2, but not
Pten, phenocopys the Lkb1 mutant.75 Clinically, patients
in the LKB1-mTOR signaling, often develop Sertoli cell
together reinforce the importance of the mTOR signal-
ing for ensuring normal Sertoli cell function and male
fertility.
Raptor and Rictor localize in the seminiferous epithe-
lium near the basement membrane. Rictor is a crucial BTB
regulator, protecting the Sertoli cell TJ-barrier function
in vitro and the BTB integrity in vivo.72 RNAi-mediated
knockdown of Rictor in cultured Sertoli cells led to a
reduced PKC-α phosphorylation and actin reorganization,
with reduced F-actin to support the BTB TJ structure.72 In
mouse testis, Sertoli cell-specific ablation of Rictor led to
disregulation of cytoskeleton and disruption of BTB integ-
rity resulting in azoospermia.76 Our recent study demon-
strates an equivalent essentiality of the germline-specific
Rictor in protection of BTB structure and function71
implicating an interconnection of  mTORC2-mediated
BTB regulatory pathway between somatic and germinal
cells. A “yin” and “yang” model has been proposed to
explain the stage-specific expression of the components of
mTOR complexes Raptor and Rictor during the epithelial
cycle and how mTORC1 and mTORC2 exert antagonistic
regulation on BTB.92 BTB dynamics is regulated by both
mTORC1 and mTORC2 signaling. Coinciding with the
timing of BTB restructuring at stage VIII–IX of the epi-
thelial cycle, ribosomal protein S6 (S6) is phosphorylated
and specifically activated at the BTB site to facilitate the
opening of BTB, possibly coupled with transient downreg-
ulation of the TJ protein claudin-11.101 The mTORC2 sig-
naling might be involved in maintaining the BTB integrity
during all the stages of the epithelial cycle of spermato-
genesis except at stage VIII–IX when Rictor is downreg-
ulated and the BTB is under restructuring.72 Therefore,
the “assembly” and “disassembly” of the BTB is precisely
coordinated by mTORC2 and mTORC1, respectively.

CONCLUSION
The signaling anchored by mTOR kinase is one of the
fundamental pathways responding to the nutritional
state of the organisms to regulate cell growth, metabo-
lism, and reproduction. In this review, we have described
­reproduction-associated observations in low model organ-
isms with mutation/deletion in mTOR pathway genes and
further detailed the current advances on the function of
the mTOR pathway during mouse spermatogenesis, espe-
cially in the process of spermtogonial self-renewal and
differentiation, meiosis, and Sertoli cell physiology. These
findings suggest mTOR signaling is a highly evolutionally
conserved pathway for germ cell development.
Notably, inhibition of mTOR by rapamycin treatment
significantly promotes spermatogonial self-renewal poten-
tial and restored the functional capacity in SPCs from Plzf
knockout mice. On the other hand, hyperactivation of
mTORC1 signaling due to deletion of Tsc1, Tsc2, or Pten
impairs the functional capacity of SPCs. This phenomenon
is also observed in somatic cells. For example, reduced
mTORC1 signaling in Paneth cells mediates the beneficial
effects of calorie restriction on intestinal stem-cell (ISC)
function, and rapamycin increases the proliferation of
ISC.103 These results suggest inhibition of mTOR signal-
ing has a protective effect on stem cells, and hyperactive
mTORC1 promotes excessive proliferation or precocious
differentiation, which subsequently depletes stem cell pop-
ulation. Helsel and colleagues recently reported that SSCs
are prone to utilize lactate through glycolysis as their pri-
mary bioenergetics process, and glycolysis-optimized cul-
ture media boost the regenerative capacity of the SSCs.104
Interestingly, it was reported that mTOR inhibition by
rapamycin reduced mitochondrial oxidative phosphory-
lation and enhanced glycolysis and hence produced more
lactate.105 This suggests the metabolic shift from oxidative
phosphorylation to glycolysis by inhibition of mTOR may
mediate its effects on maintaining SSCs. However, the
downstream effectors of mTORC1 or the molecular link
that mediates this effect remains to be identified. It is impor-
tant to note that rapamycin robustly extends life spans in
genetically heterogeneous mice,36 and reduced mTORC1
signaling through genetic manipulation in mouse model
is sufficient to increase longevity without any side effects
on glucose metabolism.23 Future studies will be needed to
explore the mechanism of rapamycin used in the longevity
study on spermatogenesis, and ultimately provide implica-
tions for designing new molecules for targeting mTOR to
confer their beneficial effects on human health.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
No potential conflicts of interest were disclosed.
ACKNOWLEDGMENTS
We would like to thank C. Yan Cheng at the Center for
Biomedical Research (Population Council, New York) for
critical reading of this manuscript. We thank all members
of the Zheng and Ye labs, particularly Mengneng Xiong,
Shun Bai, Ruping Zhu, Suwen Tian, Shuya Zhang, Xin Li, and
References 207
Mengrou Liu for helpful discussions and providing refer-
ences. The Zheng lab is supported by National Key R&D
Program of China (2016YFA0500902), National Natural
Science Foundation of China (31471228, 31771653), Jiangsu
Science Foundation for Distinguished Young Scholars
(BK20150047), and Natural Science Foundation of Jiangsu
Province (BK20140897, 14KJA180005, 14KJB310004).
The Ye lab is supported by National Natural Science
Foundation of China Grant (81471502), Innovative and
Entrepreneurial Program of Jiangsu Province, and Natural
Science Foundation of Jiangsu Province (15KJA180006).
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Does planar cell polarity matter during
spermatogenesis?
LINXI LI, HAIQI CHEN, QINGQUAN LIAN, REN-SHAN GE, and C. YAN CHENG
INTRODUCTION
Cell polarity is important to spermatogenesis since it con-
fers apico-basal (A/B) polarity in Sertoli cells and devel-
oping spermatids in the seminiferous epithelium during
the epithelial cycle of spermatogenesis1–3 (Figure 17.1).
This is important and necessary so that the maximal
number of developing germ cells can be packed within
the limited space of the seminiferous epithelium in semi-
niferous tubules, such that millions of sperm can be pro-
duced in the testes of an adult male in rodents or humans.
Furthermore, cell polarity also confers A/B polarity to
developing cells and tissues during embryogenesis and
morphogenesis.4 However, besides cell polarity, studies
have shown that planar cell polarity (PCP) is essential to
many cellular processes, in particular convergent exten-
sion during embryogenesis such as gastrulation when tis-
sue narrows (i.e., converges) along one axis alongside with
elongation (i.e., extension) along a perpendicular axis due
to polarized cell movement to generate antero-posterial
(A/P) axis.5–7 In short, PCP proteins are important to
establish cell polarity within an epithelial plane and reg-
ulates tissue morphogenesis during embryonic and neo-
natal development.8 Earlier studies have mostly focused
on the role of PCP proteins in regulating directional cell
movement during embryogenesis, such as neutral tube
formation, and asymmetric orientation of cell projections,
such as cilia in cochlear hair cells.8 PCP proteins are also
important to the assembly of cilia in multiciliated cells of
the skin and on neuroepithelial cells of the neural tube
during development.9 In adult animals, PCP is most nota-
bly detected in cells and/or tissues wherein PCP refers to
the alignment of a field of polarized cells (e.g., elongating/
elongated spermatids) within the plane of a cell epithe-
lium such as the Sertoli cell epithelium in the seminiferous
tubule, mostly notably in stage VII-VIII of the epithelial
cycle (Figure 17.1)10 versus hair cell alignment on skin or
cochlea. Without the coordinated efforts of polarity pro-
teins and PCP proteins in the seminiferous epithelium
to confer the proper alignments of polarized spermatids
across the plane of Sertoli cells, the tremendous cellular
output of generating millions of spermatozoa per testis
on a daily basis is not sustainable. In this context, it is of
interest to note that spermatid PCP is highly sensitive to
toxicants such as environmental toxicants (e.g., cadmium
chloride) or male contraceptives (e.g., adjudin) since a
brief exposure of the testis to these toxicants can induce
rapid loss of PCP (Figure 17.1). Studies in other mam-
malian epithelia have shown that PCP proteins can be
17
classified into (1) PCP core proteins (e.g., Van Gogh-like
[Vangl] protein such as Vangl2, dishevelled [Dvl] such as
Dvl2, and frizzled [Fzd] class receptor such as Fzd 3 and
Fzd5), (2) PCP ligands (e.g., Wnt5a), (3) PCP effectors (e.g.,
fuzzy, multiple wing hair [Mwh], Fritz, inturned), and
PCP signaling proteins (e.g., dachsous cadherin-related
protein [Dchs] such as Dchs1).7,11,12 Due to the indispens-
able role of PCP proteins to confer axial versus vectorial
asymmetry during embryonic development (Figure 17.2),
PCP proteins are very well conserved and are found from
worms, flies, and mammals, and their sequences are well
conserved. While PCP proteins are important to confer
PCP alignment of developing spermatids in the Sertoli cell
epithelium, PCP proteins have not been studied in the tes-
tis regarding their role in spermatogenesis until recently.
Interestingly, virtually all major PCP proteins in each of
the four classes of PCP have been detected in the testis.13
Herein, we sought to carefully evaluate the currently avail-
able data in the field, which will provide important guides
and insightful information for future studies so that the
role of PCP proteins in spermatogenesis can be carefully
delineated.
PCP PROTEINS AND SPERMATOGENESIS
Background
Much of the information regarding PCP proteins is
based on studies in cuticular hair in Drosophila. Table
17.1 summarizes the best-studied core PCP proteins/
genes found in vertebrates, the corresponding homologs
in Drosophilia. Table 17.1 also summarizes their func-
tion in rodents including their role in the testis regard-
ing spermatogenesis/fertility based on studies using gene
knockdown approach by RNAi in rats. Also listed are
diseases caused by their mutations and/or deletions in
humans (Table 17.1). As shown in Figure 17.2, alignment
of PCP proteins can be either axial (or bipolar) asymme-
try or vectorial (or unipolar) asymmetry.7,11 It is generally
accepted that axial asymmetry is conferred by integral
membrane proteins Celsr (or flamingo [Fmi] in inverte-
brates) and furrowed (Fw) (no equivalent homolog has
been found in vertebrates thus far) in which they local-
ize to both poles along one planar axis (Figure 17.2).7,11
Vectorial asymmetry is conferred by integral membrane
proteins frizzled (Fzd) and Van Gogh (Vang, or Vangl,
such as Vangl1, Vangl2) in which they localize prefer-
entially to one pole of each cell (Figure 17.2).7,11 Also,
Diego (Dgo) and dishevelled (Dsh) are the cytoplasmic
211
212  Does planar cell polarity matter during spermatogenesis?

Toxicants

CdCl2
(3 mg/kg b.w., i.p.)

Control adult rat testis CdCl2 (16 hr)

Adjudin
(50 mg/kg b.w., oral gavage)

Control adult rat testis Adjudin (12 hr)

Figure 17.1  A schematic drawing that illustrates features of PCP regarding the alignment of a field of polarized developing
spermatids within the plane of the Sertoli cell epithelium in adult rat testes. In the top left panel, PCP is noted in this schematic
drawing in which a field of polarized spermatid heads are aligned across the plane of a Sertoli cell epithelium. This is also supported
by fluorescence microscopy and histological analysis using cross sections of adult rat testes on the middle left and lower left panel,
respectively. In the corresponding right panel, spermatids had lost their PCP in which spermatid heads displayed polarity defects
following treatment of rats with either environmental toxicants (e.g., cadmium chloride, 3 mg/kg b.w. via i.p.) or a male contraceptive
(e.g., adjudin, 50 mg/kg b.w. by oral gavage). Besides defects in spermatid polarity (spermatids with polarity defects are annotated
by yellow arrowheads), many elongated spermatids remained trapped deep inside the epithelium (such as following adjudin treat-
ment) due to a disorganization of actin microfilaments and also microtubules (MTs) across the epithelium, and failure to support
proper transport of spermatids to the adluminal edge of the apical compartment to prepare these spermatids for their release at
spermiation in late stage VIII tubules. Scale bar, 40 µm in fluorescence images; 50 µm in hematoxylin and eosin stained images.
 PCP proteins and spermatogenesis  213

Mwh Actin

Rho GTPase

BTB
Sertoli cell

Vangl2 Prickle (pk)

Frizzled (Fzd) Dishevelled (dvl)

Celsr Inversion (invs)

Axial asymmetry Vectorial asymmetry
Actin filament bundles

Figure 17.2  Asymmetric distribution of core PCP proteins. Asymmetrical distribution of core PCP proteins confers either axial
asymmetry or vectorial asymmetry in cells, such as in cuticular hair cells in Drosophila or cochlea hair cells in mammals. It is noted that
Celsr (cadherin EGF LAG seven-pass G-type receptor including Celsr 1, Celsr 2, and Celsr 3) is the mammalian homolog of Flamingo
(Fmi, also known as starry night [stan]) found in Drosophila, which displays axial asymmetry in which Celsr can be localized to inter-
cellular junctions along one tissue axis but excluded from orthogonal junctions. However, Van Gogh (Vang, such as Van Gogh-like
2, Vangl2)-Prickle (Pk) and Frizzled (Fzd or Fz in Drosophila)-Dishevelled (Dsh in Drosophila or Dvl in vertebrates)-Diego (Dgo, its
homolog is Inversin [Invs] in mammals) protein complexes display vectorial asymmetry in which each protein complex localizes to
the opposite end of the cell. The enlarged image depicts the Fzd/PCP core component interactions across a two-cell border in mam-
malian cells such as in Sertoli cells. Based on studies in Drosophila and also in Sertoli cells (see text for details), it is now known that
the Vangl2/Pk complex exerts its regulatory effects to promote actin filament disorganization via an inhibition of actin polymeriza-
tion through multiple wing hairs (Mwh). However, the homolog of Mwh remains unknown in the testis. In contrast, the Fzd/Dvl/Invs
protein complex promotes actin organization through its action on Rho GTPase by inducing actin polymerization. It is known that
Vangl2 and Celsr or Vangl2 and Fzd can antagonize each other as depicted herein.

components of Fz, whereas prickle (Pk) is the cytoplas-
mic component of Vang. It is of interest to note that Dgo/
Dsh and Pk antagonize each other and are capable of acti-
vating separate intracellular signaling pathways, playing
an important role in maintaining the vectorial asymme-
try (Figure 17.2). The Vang/Pk complex and the Fz/Dgo/
Dsh complex are working in concert to regulate vectorial
asymmetry through their corresponding action on actin-
based cytoskeletal function (Figure 17.2). It is now gener-
ally accepted that the Vang/Pk complex recruits Mwh (a
PCP effector molecule12)45 to exert its regulatory effects
by inhibiting polymerization of actin microfilaments,
whereas the Fz/Dgo/Dsh complex promotes polymeriza-
tion of actin microfilaments through activation and/or
recruitment of Rho-family GTPases and the downstream
effectors (e.g., Rho-associated kinase [ROK])7,11,46,47
(Figure 17.2). These findings thus suggest that the Vang/
Pk complex and the Fz/Dgo/Dsh complex have antago-
nistic effects on cellular F-actin polymerization.
Study in vitro
In this context, it is of interest to note that in a recent
report, Vangl2 (a homolog of Vang) was shown to inhibit
actin-based cytoskeletal function in the testis13 similar to
the function of Vang/Pk complex in Drosophila, by pro-
moting F-actin disorganization in Sertoli cells, making
the Sertoli cell tight junction (TJ) barrier “leaky.”13 For
instance, following overexpression of Vangl2 in Sertoli
cell epithelium with an established functional TJ perme-
ability barrier that mimicked the Sertoli cell blood-testis
214  Does planar cell polarity matter during spermatogenesis?

Table 17.1  Core PCP proteins/genes in Drosophilia and vertebrates.

Drosophila Vertebrate
genes genes Function in rodents Disease found in humans via mutation
Disheveled Dvl1 Regulates gut microbiota composition to Autosomal-dominant Robinow syndrome15
(dsh) confer intestinal homeostasis14 Inflammatory bowel disease16
Dvl2 Involves in depression using a mouse social Inflammatory bowel disease18
model17
Dvl3 Involves in heart development19 Autosomal-dominant Robinow syndrome20
Frizzled (fz) Fzd1 Oocyte maturation and cumulus cell function Inflammatory bowel disease,18 diabetic kidney
in mice21 disease22
Fzd2 Diabetic kidney disease22
Fzd3 Axonal development23 Inflammatory bowel disease18
Fzd4 Inflammatory bowel disease18
Fzd5 Inflammatory bowel disease,18 diabetic kidney
disease22
Fzd6 Diabetic kidney disease22
Fzd7 Serves as Clostridium difficile toxin B (TcdB) Diabetic kidney disease22
receptor in mice24
Fzd8 Involved in arthritis pathogenesis based on an Involved in drug resistance in breast cancer
adjuvant-induced arthritis (AIA) model in pathogenesis26
rats25
Fzd9 Hippocampal development,27 osteoblast
function28
Fzd10 Breast cancer metastasis29
Van Gogh-like Vangl1 Neural tube development 30 Neural-tube defects31
(Vangl) Vangl2 Neural tube development, lung 30 Neural-tube defects33
morphogenesis, and a regulator of
32

ectoplasmic specialization dynamics in adult

rat testes by promoting blood-testis barrier
remodeling13
Flamingo (fmi) Celsr1 Lung morphogenesis during embryogenesis32 Induces ischemic stroke,34 and involved in
Huntington’s disease pathogenesis35
Celsr2 Facilitates circulation of the cerebrospinal An increase in blood low-density lipoprotein
fluid36 cholesterol37 or an increase in serum
cholesterol,38 involved in Huntington’s disease
pathogenesis35
Celsr3 Facilitates circulation of the cerebrospinal Involved in Huntington’s disease
fluid36 and axonal development23 pathogenesis35
Prickle (pk) Pk1 Neuronal morphogenesis, its deletion leads Involved in progressive myoclonus epilepsy40
39

to seizure in mice40
Pk2 Susceptible to seizures after knockout in mice40 Involved in progressive myoclonus epilepsy40
Diego (dgo) Diversin Deletion causes PCP defects in inner ear Neural tube defects42
(ankrd6) sensory organs 41

inversin (invs) Deletion leads to defects in kidney and urinary Involved in nephronophthisis pathogenesis44
tract development43

barrier (BTB) in vivo, long stretches of actin microfila-
ments across the Sertoli cell cytosol were grossly disrupted
since they were defragmented.13 The truncation of actin
microfilaments, in turn, perturbed Sertoli cell adhesive
function. This is because BTB-associated adhesion protein
complexes such as claudin 11-ZO-1 and N-cadherin-ß-
catenin all utilized F-actin for attachment. The disrup-
tion of F-actin thereby impeded the function of these cell
adhesion proteins. Thus, claudin 11, ZO-1, N-cadherin,
and ß-catenin all became dislocalized, and this thus
contributed to the TJ permeability barrier disruption.13
Most importantly, the overexpression of Vangl2-induced
F-actin disorganization was shown to be mediated by
two actin regulatory proteins wherein (1) Eps8 (an actin
barbed end capping and bundling protein) was found to
redistribute from the cell-cell interface to the Sertoli cell
cytosol, and (2) Arp3 (an actin barbed end nucleation pro-
tein that effectively converts bundled actin filaments to an

unbundled/branched configuration) was shown to become
more intensely localized to the cell cortical zone.13 These
changes thus contribute to extensive truncation of actin
microfilaments at the Sertoli cell cortical zone following
overexpression of Vangl2, making the TJ-barrier “leaky.”
In contrast, a knockdown of Vangl2 in the Sertoli cell epi-
thelium by RNAi to transiently silence Vangl2 expression
led to (1) a redistribution of Eps8, which became intensely
localized to the Sertoli cell-cell interface, and (2) a reduced
expression of Arp3 at the Sertoli cell cortical zone.13 These
changes contribute to a strengthening of the actin micro-
filaments at the Sertoli cell basal ES/BTB, “tightening”
the TJ barrier. As such, a knockdown of Vangl2 in Sertoli
cell epithelium by RNAi was indeed found to promote the
Sertoli cell TJ permeability barrier, making the TJ bar-
rier tighter.13 Studies by immunofluorescence microscopy
also support the concept that Vangl2 per se in the testis
promotes actin microfilament disorganization since its
knockdown by RNAi indeed was found to promote the
recruitment of TJ protein complex (e.g., claudin 11/ZO-1)
and basal ES protein complex (e.g., N-cadherin/ß-catenin)
at the Sertoli cell-cell interface due to better maintenance
of actin filament organization at the cell cortical zone.13 In
short, these findings thus suggest that Vangl2 in the nor-
mal testis likely promotes actin filament disorganization
during the epithelial cycle of spermatogenesis, such as in
stage VIII tubules to facilitate the transport of prelepto-
tene spermatocytes across the immunological barrier.
Study in vivo
Studies in vitro as briefly discussed above have illustrated
the significance of Vangl2, and also other PCP proteins, to
the organization of actin microfilaments in the seminifer-
ous epithelium during the epithelial cycle. However, it is
envisioned that a delicate balance of Vangl2 across the sem-
iniferous epithelium is needed to support spermatogenesis,
in particular through its changes in spatiotemporal expres-
sion, but also with the involvement of other PCP proteins
that contribute to the proper organization of actin micro-
filaments at different stages of the epithelial cycle. When
Vangl2 was knocked down by ~50% using Vangl2-specific
siRNA duplexes for transfection in the adult rat testis in
vivo, considerable changes in the status spermatogenesis
were noted13 (Figure 17.3). For instance, following Vangl2
knockdown in the testis in vivo, defects in spermatid and
phagosome transport were found in ~40% of the stages
IX-X tubules.13 For instance, many step 19 spermatids were
found embedded deep inside the epithelium in both stage
IX and X tubules when spermiation had occurred.13 This is
due to the persistent presence of residual F-actin network at
the apical ES to support spermatid adhesion when it should
have been degenerated completely to facilitate the release
of sperm at spermiation.13 This observation is also con-
sistent with the findings in vitro that Vangl2 knockdown
supported the actin filament organization at the basal ES/
BTB to make the BTB barrier “tighter”13 (Figure 17.3). This
unwanted persistence of the F-actin network at the ES in the
epithelium also perturbed proper organelle transport across
Concluding remarks and future perspectives  215
the epithelium. For instance, phagosomes derived from the
engulfment of residual bodies by Sertoli cells that should
have been transported into the base of the seminiferous
epithelium for their eventual degradation through the lyso-
somal degradation pathway  48,49 were persistently found near
the luminal edge of the adluminal compartment in stages
IX and X tubules in testes following Vangl2 knockdown.13
Additionally, the defects in organizing the F-actin network
at the Sertoli-spermatid interface in response to changes in
the epithelial cycle following Vangl2 knockdown also per-
turbed apical ES plasticity, perturbing spermatid adhesion
and spermatid PCP so that considerable spermatids with
defects in polarity were noted13 (Figure 17.3). In this context,
it is of interest to note that environmental toxicants (e.g.,
cadmium chloride) or contraceptives (e.g., adjudin) are also
shown to perturb spermatid PCP (Figure 17.1) since these
toxicants, similar to Vangl2, also exert their disruptive
effects by altering the organization of actin microfilaments
at the ES, thereby perturbing PCP function.
In short, Vangl2 promotes BTB disruption since a
knockdown of Vangl2 in Sertoli cells in vitro makes the
Sertoli cell TJ barrier “tighter,” whereas its overexpres-
sion causes the TJ barrier to be “leaky” through changes
in the organization of actin microfilaments in Sertoli
cells13 (Figure 17.3). In brief, Vangl2, the integral mem-
brane protein of the Vangl2/Pk complex, is a core PCP
protein in the seminiferous epithelium, which exerts
its regulatory effects through the actin-based cytoskel-
eton. F-actin is not only crucial to spermatid and Sertoli
cell adhesion in the seminiferous epithelium, but also
intracellular trafficking events, such as the transport of
residual bodies and phagosomes during the epithelial
cycle of spermatogenesis.50,51 Therefore, it is not entirely
unexpected that a knockdown of Vangl2 in the testis in
vivo by RNAi also perturbed the transport of residual
bodies/phagosomes across the epithelium at stage VIII
of the cycle13 so that they can be degraded via lysosomes
near the base of the stage IX tubules. In this context, it is
of interest to note that the frequency of meiosis I/II was
also grossly reduced at stage XIV of the epithelial cycle.13
Since meiosis is also an F-actin-dependent cellular event
by supporting the segregation of the genetic material
across the cell cytosol at anaphase, thus a Vangl2 knock-
down that impedes F-actin organization would perturb
meiosis. Based on the available data and information
in the literature, we propose that Vangl2 is working in
concert with other core PCP proteins, such as Fzd/Dvl/
Invs and Celsr, to induce BTB restructuring and changes
in apical ES function to support the transport of prelep-
totene spermatocytes across the immunological barrier
and the release of sperm at spermiation, respectively, as
noted in Figure 17.3.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
In brief, Vangl2 regulates PCP function in the testis
through its effects on the actin-based cytoskeleton. It is
noted that Sertoli cell adhesion that contributes to the
BTB integrity is mediated predominantly by the actin
216  Does planar cell polarity matter during spermatogenesis?

Spermiation

Degenerating
apical ES

Sertoli cells

Germ cell
transport
Intact
apical ES

Intact Remodeling
BTB BTB

Sertoli cells
Preleptotene
Preleptotene spermatocytes
spermatocytes

Vangl2 Prickle (Pk) Actin filament bundles

Frizzled (Fzd) Dishevelled (Dvl) Actin filaments

Celsr Inversin (Invs) Branched actin network

Figure 17.3  A schematic drawing that illustrates the likely mechanism by which core PCP proteins regulate ectoplasmic special-
ization (ES) dynamics to support germ cell transport in the testis. Actin microfilament organization at the apical and the basal ES
integrity is maintained by Vangl2, since its overexpression promotes Sertoli cell TJ-barrier disruption. In contrast, Vangl2 silencing
by RNAi in the testis in vivo also impedes spermatid transport due to changes in actin microfilament organization at the ES (see text
for details). In short, an upregulation of Vangl2 promotes ES disruption, which in turn facilitates the transport of preleptotene sper-
matocytes across the immunological barrier and the release of spermatids at spermiation.

microfilament bundles at the basal ES (which together
with TJ create the BTB).52–57 Additionally, spermatid
(steps 8–19 or 8–16 spermatids in the rat or mouse testis,
respectively) adhesion to the Sertoli cell in the seminif-
erous epithelium is also supported by the microtubule
(MT)-based cytoskeleton at the apical ES.58,59 Thus, it
remains to be determined regarding the role of Vangl2
in MT dynamics in the testis. Furthermore, a number of
issues remain to be investigated: (1) if Pk is a functional
partner of Vangl2 in the testis, (2) if Mwh is involved in
the Vangl2/Pk complex functionally downstream, and
(3) if other protein partners and/or signaling proteins are
involved in the Vangl2-induced ES remodeling. Based
on studies of the other PCP core proteins, such as the
Fz/Dsh complex, which is known to play an important
role to promote actin polymerization,7,11 it remains to be
determined if the Fz/Dvl (note that dishevelled is sym-
bolized as Dvl in vertebrates versus Dsh in Drosophila)
complex is working in concert with the Vangl2/Pk com-
plex to confer vectorial asymmetry in the mammalian
testis as they are in Drosophila. It is likely that the Fz/Dvl
complex is found in the testis to promote F-actin polym-
erization to maintain the Sertoli TJ barrier integrity by
making the barrier “tighter” and to maintain spermatid
adhesion during spermiogenesis (Figure 17.3). As such,
the Vangl2/Pk and the Fz/Dsh complexes are working
partners to regulate apical and basal ES dynamics (i.e.,
disassembly, reassembly, and stabilization), support-
ing not just adhesion of Sertoli cells and spermatids,
but also intracellular trafficking events through their
effects on F-actin organization. This speculation is not
entirely unexpected since Dsh has been shown to work
with clathrin AP-2 to modulate Fz endocytosis60 and is
involved in the endocytosis of the Wnt/ß-catenin signal-
ing complex,61 illustrating its role in endocytic protein
trafficking. In fact, studies have shown that the PCP
pathway plays a crucial role in endocytic protein traffick-
ing,62 which is crucial to support spermatogenesis so that
proteins, in particular apical and basal ES proteins, can
be recycled without overwhelming the limited number

of Sertoli cells in the testis.59,63 In short, protein traffick-
ing events must be tightly regulated since it is estimated
that a single Sertoli cell provides nutritional, hormonal,
paracrine, and structural support to ~30 to 50 develop-
ing germ cells during spermatogenesis.64 These possi-
bilities must be carefully evaluated in future studies. In
short, better understanding of the role of PCP proteins in
the seminiferous epithelium during the epithelial cycle
is crucial to unravel the biology of spermatogenesis, to
develop new approaches for nonhormonal male contra-
ception, and to restore spermatogenesis in infertile men.
ACKNOWLEDGMENTS
This work was supported by grants from the National
Institutes of Health, NICHD R01 HD056034 to C.Y.C.,
and U54 HD029990 Project 5 to C.Y.C.; National
Natural Science Foundation of China (NSFC 81601264
to L.L.), Zhejiang Provincial Natural Science Foundation
(LQ16H040004 to L.L.), Department of Education of
Zhejiang Province (Y201534170 to L.L.).
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Computational characterization and
integrative analysis of proteins involved
in spermatogenesis
PRANITHA JENARDHANAN, MANIVEL PANNEERSELVAM,
and PREMENDU P. MATHUR
INTRODUCTION
Increasing population and declining male fertility rates
are two different but corelated serious issues being faced
among humans. Lack of sex education and reproduction
control measures remain causes for increasing population,
while diverse genetic, environmental, and unidentified fac-
tors also affect fertility rates. While reports forecast that
the world will have a human population of about 10.1 bil-
lion in 2050,1 other reports also state that both developed
and developing nations will face a decline in fertility rates
far below 2.1, the average number considered to withstand
population size.2 Approximately 15% of couples worldwide
are observed to face infertility problems, with about half
of the total count falling under male infertility factors.3–6
Either for understanding the causes for male infertility
or for design of male contraceptives to achieve population
control, it becomes necessary to understand the funda-
mental process, spermatogenesis. Spermatogenesis is the
complex, precisely coordinated, and regulated event that
results in the production of sperms. Success of spermato-
genesis lies in the proper regulation and coordination of
events that dictates (1) proper renewal of stem cells, pro-
liferation by mitosis, and differentiation, (2) generation
of preleptotene spermatocytes from type B spermatogo-
nia, (3) transit of preleptotene spermatocytes across the
blood-testis barrier (BTB), (4) division of preleptotene to
zygotene and diplotene spermatocytes that enters meiotic
divisions I and II to form haploid spermatids, (5) contin-
ued association of spermatids with Sertoli cells and their
maturation by spermiogenesis, and (6) precise removal of
mature spermatozoa from seminiferous epithelium into
the lumen. Deregulation at any of these stages either by
genetic or environmental factors affects the successful
production of mature fertile sperms.7–11
Besides understanding the molecular mechanism of
spermatogenesis through basic research, it is also of ben-
efit to translate the findings for medical applications such
as identification of biomarkers for designing effective
noninvasive methods for diagnosis of male infertility as
well as to identify drug targets for designing therapeu-
tic strategies to create nonhormonal contraceptives. The
combined use of genomics, transcriptomics, metabolo-
mics, and bioinformatics methods has been significantly
beneficial for understanding the basic mechanisms as well
220
18
in identification of suitable biomarkers and drug targets.
In this chapter, we discuss selected applications of these
techniques in identification of gene, miRNA, and protein
biomarkers for diagnosis and treatment of male infertility.
Later we discuss the application of reproductive toxicants
and contraceptive-based models in identification of drug
targets that can be used for diagnosis and design of potent
nonhormonal male contraceptives.
ROLE OF OMICS AND BIOINFORMATICS METHODS IN
TRANSLATIONAL RESEARCH OF MALE REPRODUCTION
Translational research in reproduction involves a com-
bination of multiple areas beginning with understand-
ing reproductive developmental biology, reproductive
tract biology, reproductive genetics, reproductive endo-
crinology, and reproductive medicine. The output of all
these disciplines is used for understanding the molecular
mechanisms associated with successful reproduction and
understanding the causes for its failure and for identifica-
tion of novel biomarkers for developing proper diagnosis
and treatment methods. Genomics, transcriptomics, pro-
teomics, and bioinformatics methods are proven to pro-
vide successful understanding of reproductive disorders
specifically for male infertility.12 Profiling of gene/miRNA/
protein expression profiling associated targeted inhibition
of proteins and understanding their structure and interac-
tion mechanism helps in high throughput screening, iden-
tification, and characterization of potential biomarkers.13
To understand the effect of mutations, copy number
variation, single nucleotide polymorphisms, and altera-
tion pattern of genes related to spermatogenesis, various
sequence-related methods like DNA microarray, next gen-
eration sequencing methods, miRNA-based microarray,
real-time polymerase chain reaction (RT-PCR), and small
RNA sequencing methods are employed. Proteomics
methods use two-dimensional electrophoresis (2-DE),
­differential-in-gel electrophoresis (DIGE), matrix-assisted
laser desorption mass spectrometry (MALDI-MS),
surface-­enhanced desorption/ionization mass spectrom-
etry (SELDI-MS), multidimensional protein identifica-
tion technology (MUDPIT), and liquid chromatography
and mass spectrometry (LC–MS).14 The net result of these
techniques provides the list of genes and proteins that are
differentially regulated in the given condition.

With the identification of differentially expressed genes/
proteins, it becomes necessary to understand their bio-
logical significance. Application of bioinformatics finds its
place in this step, where several integrated bioinformatics
tools are employed. The basic understanding of biologi-
cal significance aims at understanding different aspects
of gene products such as their cellular location, func-
tional classification, structural classification, and analy-
sis of their domain, structures, relationship to biological
pathways, and diseases. Several integrated ­bioinformatics
tools like the Database for Annotation, Visualization and
Integrated Discovery (DAVID), gene ontology, and Kyoto
Encyclopedia of Genes and Genomes (KEGG) are being
used for this purpose. DAVID tool performs gene-list-
based annotations15,16 that takes gene list as the input
and identifies data related to gene ontology17 and meta-
bolic pathway analysis from KEGG.18 With identifica-
tion of protein targets it becomes essential to understand
their interaction partners, which can be achieved using
web-based protein-protein interaction databases such
as String.19 Later, structural analysis of predicted targets
is achieved by three-dimensional structure prediction of
proteins by the MODELLER suite20 and the Rosetta suite.21
With analysis of individual protein structures it becomes
essential to understand how selected protein interacts
with its targets using protein-protein docking software
such as HADDOCK 22 and protein-ligand docking such as
Schrodinger Glide.23 Finally, the conformational changes
induced by complex formation are evaluated using molec-
ular dynamics simulation studies that use software such as
the Gromacs suite.24
With the application of diverged protein sequence
and structure-based analysis tools, the overall function
of the identified targets can be postulated. Finally, based
on the significance of the role played by the identified
targets, the  potent biomarkers and drug targets can be
identified.
APPLICATION OF GENOMIC, TRANSCRIPTOMIC,
AND PROTEOMIC APPROACHES IN IDENTIFICATION
OF POTENT BIOMARKERS
The conventional techniques of diagnosing male infertil-
ity depends on semen analysis, which can be segregated
into macroscopic and microscopic factors that include
coagulation, color, viscosity, and agglutination, sperm
count, and concentration, respectively. Indeed the analy-
sis of semen suffers from serious challenges. These factors
potentiate the need for identification of novel biomarkers
that can pave the way for easy identification and diag-
nosis of diseases. Genes, miRNAs, and proteins are such
potential biomarkers, the presence of which can be easily
identified, further promoting their selection as potent bio-
markers. In addition, with an understanding of their func-
tion and structure, potent therapeutic strategies can also
be designed. The following sections discuss the application
of genomics, transcriptomics, and proteomics in identifi-
cation of potent biomarkers and a list of a few proteins that
Genomic and transcriptomic biomarkers  221
can be used as biomarkers for diagnosis and treatment of
male infertility.
GENOMIC AND TRANSCRIPTOMIC BIOMARKERS
Lin et al. first characterized testis-specific genes in humans
and identified Septin 4, zinc metallopeptidase, Ca+/
calmodulin protein kinase IV (CaMKIV) and calspermin
(CAS) as some of the important genes found to be specific
to the testis and play an important role in spermatogen-
esis.25 Later, Feig et al. obtained testis from normal and
azoospermic patients and identified testis-specific genes
in humans. Results identified about 348 genes in premei-
otic, 81 genes in postmeiotic, and 38 genes in termination
phase in testis.26 Liu et al. identified testis-specific genes
in humans, mouse, and rat and with their findings cre-
ated a precise testis-specific gene database. In their study,
authors employed digital differential analysis where they
screened Unigene libraries, retrieved testis-specifc genes,
and used RT-PCR studies to validate their findings. They
found about 317, 449, and 147 testis-specific genes in
human, mouse, and rat, respectively.27
In addition to approaches to identify testis- and sperm-
specific genes from databases, several other studies have
been involved in analyzing genes/miRNA that are dif-
ferentially expressed in different conditions pertaining
to male infertility. Malcher et al.28 attempted to charac-
terize the genetic causes of male infertility and in their
study compared the gene expression profiles between
normal and oligozoospermic patients. Results identi-
fied differential expression of about 4946 genes, wherein
genes A-kinase anchoring protein (AKAP4), ubiquilin 3
(UBQLN3), calpain 11 (CAPN11), gametogenetin (GGN),
sperm acrosome associated 4 (SPACA4), apermatogen-
esis associated 3 (SPATA3), and family with sequence
similarity 71, member F1 (FAM71F1) were the most sig-
nificantly downregulated in infertile patients. Expression
analysis revealed that AKAP4 is expressed in spermato-
zoa29 UBQLN3, CAPN11, and SPATA3 were expressed in
spermatocyte,30,31 and GGN was expressed in early round
spermatids.32 Molecular characterization of all these pro-
teins shows that their knockout impairs spermatogenesis,
thus favoring their selection as potential biomarkers for
diagnosing male infertility.
In addition to the genomic studies, miRNA profiling
has also helped in identifying the potential biomarkers
for diagnosis of male infertility. Abu-Halima et al.33 com-
pared microRNA expression profiles in normozoosper-
mic, asthenozoospermic, and oligoasthenozoospermic
donors. Nearly 50 miRNAs were upregualted in astheno-
zoospermic males and 42 miRNAs were upregulated in
oligoasthenozoospermic men in comparison with normal
men. In a similar study,34 the same authors compared the
miRNA expression profile in normal men with infer-
tile men characterized with Sertoli-cell-only (SCO) syn-
drome, mixed atrophy (MA), and germ cell arrest (GA)
phenotypes and 197, 68, and 46 miRNAs were observed to
have differential expression in these groups, respectively.
222  Computational characterization and integrative analysis of proteins involved in spermatogenesis
Both these studies identified miR-141, miR-200a, miR-
122, miR-34b, miR-34c-5p, and miR-16, and hsa-mir-34b*,
hsa-mir-34b, hsa-mir-34c-5p, hsa-mir-449a, and hsa-mir-
449b as potential miRNA-based biomarkers for diagnosis
of male infertility. miR-122 is involved in posttranscrip-
tional downregulation of transition protein 2 (TNP2).35
miR-34b and miR-34c regulate Notch gene homologue 1
(NOTCH1) that is found to regulate survival and differen-
tiation of germ cells.36 miR-449a in turn regulates BCL-2
and miR-26a regulates the function of estrogen receptors.
These miRNAs and their target mRNAs were validated
by RT-PCR studies and are shown to be the effective bio-
markers for the diagnosis of male infertility.
The combined expression profiling of both miRNA and
their target mRNAs in normal versus impaired sperm
donors was performed by Zhuang et al. In their study,37
they collected the testes from normal, obstructive azo-
ospermia, and nonobstructive azoospermic patients.
The differential expression of the miRNAs and mRNAs
obtained was studied and results found that about 93
miRNAs and 4172 mRNAs were differentially expressed
in obstructive azoospermia and nonobstructive azoosper-
mic patients. Functional classification of these miRNA/
mRNA pairs revealed that they are highly associated with
spermatogenesis, cell meiosis, cell cycle, and development
of secondary sexual characteristics. Interestingly, identi-
fied miRNAs were found to target multiple genes such as
hsa-miR-199a-5p that targets spermatid-associated pro-
tein (SPERT), SPACA4, actin-like protein 7A (ACTL7A),
testis-specific serine kinase 6 (TSSK6), and UBQLN3. Of
these, the functional role of TSSK6 and UBQLN3 has been
well studied. TSSK6 belongs to the testis-specific serine/
threonine kinase family, and the downregulation asso-
ciated with TSSK6 is observed to result in production of
abnormal sperm morphology and is also associated with
affecting sperm-egg fusion.38,39 UBQLN3, on the other
hand, belongs to the family of ubiquitin-like proteins, and
is a testis-specific gene essential for sperm function.40
PROTEOMIC MARKERS AND DRUG TARGETS
The systematic analysis of protein expression and charac-
terization in a given tissue/cell under a particular condition
or at a given time, called proteomics, plays a crucial role in
understanding the expression of the pattern of vital pro-
teins that function as cohort to ensure progression of spe-
cific functions. Proteomic approaches are highly beneficial
in characterizing the protein signature of germ cells specific
to each stage of spermatogenesis and in comparison to their
expression between fertile and infertile patients. They also
prove vital in identifying a specific set of proteins during
the exposure of testis/Sertoli cells/sperm to a wide range of
environmental factors. The results of all these approaches
are crucial to understanding the underlying mechanisms
of fertile sperm production and to understanding the
molecular basis for development of male infertility.
The testis is a unique somatic counterpart that pos-
sesses endocrine and exocrine factors that regulate sper-
matogenesis and proteomics has played a crucial role in
characterizing the proteomic fingerprint of testes. The tes-
tis is also unique in that it has a high level of alternative
splicing at the transcriptional level, resulting in heteroge-
neous proteome. This was analyzed by Guo et al., where
the authors employed two-dimensional gel electrophore-
sis to analyze testis-specific proteins, alternative isoforms
of testis-enriched proteins, and those that exhibit unique
expression between normozoospermic and azoospermic
patients.41 They identified 462 unique proteins and charac-
terized 52 proteins that showed heterogeneous forms. They
also found 27 proteins that expressed differential expres-
sion between normal and infertile donors. As well, these
results were embedded and developed into a testis-specific
proteome. Following this study, numerous authors have
been involved in characterizing the testis proteome under
different conditions. Recently, Kathrein et al. character-
ized the expression of proteins in testis in the presence of
spermatogonia and compared them to testis with an SCO
data set.42 SCO appearance is the characteristic form of
male factor infertility and the results of this study identi-
fied 239 specific genes that showed differential expression.
The results postulated that the proteins fibroblast growth
factor receptor 3 (FGFR3) and desmoglein 2 (DSG2) are
specific biomarkers of spermatogonia.
In another study, Djureinovic et al. characterized the
testis-specific proteome based on transcriptomics and
antibody-based profiling and identified more than 1000
gene products that were testis-specific and further charac-
terized them based on their location, such as in spermato-
gonia, spermatocytes, spermatids, sperm, Sertoli cells, and
Leydig cells.43 The results reveal that doublesex- and mab-
3-related transcription factor 1 (DMRT1) and P antigen
family member 1 (PAGE1) are specific to spermatogonia.
DMRT1 protein is involved in male sex determination and
differentiation,44 while the function of PAGE1 is unknown.
Similarly, the deleted-in-azoospermia-like (DAZL) gene
product was reported to show high enrichment in sper-
matocytes, while the transition protein 1 (TNP1) gene
and sperm mitochondria-associated cysteine-rich protein
(SMCP) and actin-like protein 7B (ACTL7B) were specific
to spermatid. Meanwhile, defensin, beta 119 (DEFB119)
was found to be specific to Sertoli cell, the A kinase (PRKA)
anchor protein 4 (AKAP4) was specific to Sertoli cell and
glyceraldehyde-3-phosphate dehydrogenase (GAPDHS)
gene products are specific to sperm. These findings provide
useful insights into understanding the proteomic signature
of testis.
Like Djureinovic et al., several other authors have also
characterized the proteomic signature in sperm-milieu,45,46
seminal plasma,47 and sperm tail.48 However, over time,
applications of proteomics has drifted toward identifica-
tion of biomarkers that exhibit differential expression
between normal and infertile patients. Hashemitabar et al.
compared the expression of proteins in sperm tail between
normozoospermic and asthenozoospermic donors.48
Asthenozoospermia, a common form of male infertility,
is characterized by reduced sperm motility, and the pro-
teomic study conducted by Hashemitabar et al. identified
 Models based on exogenous compounds to identify potential biomarkers in spermatogenesis  223
14 unique proteins that exhibited adistinct expression
­pattern.48 Of the total 14 proteins, 11 proteins [A-kinase
anchor protein 4 (AKAP4), Tubulin beta 2B (TUBB2B);
outer dense fiber protein 2 (ODF2), cytochrome c oxidase
subunit 6B (COX6B) phospholipid hydroperoxide gluta-
thione peroxidasemitochondrial (PHGPx), glutathione
S-transferase Mu 3 (GSTMu3);glyceraldehyde-3-phosphate
dehydrogenase, testis-specific (GAPDS), voltage-depen-
dent anionselective channel protein 2 (VDAC2), heat
shock-related 70 kDa protein 2 (HSPA2); stress-70 protein,
mitochondrial (HSPA9), and sperm protein associated
with the nucleus on the X chromosome B (SPANXB)] are
specific to normal sperm tails.
On the other hand, proteins such as keratin, type II
cytoskeletal 1 (KRT1), isoaspartyl peptidase/lasparaginase
(ASRGL1), and clusterin (CLU) were enriched in astheno-
zoospermia data set. Besides the ASRGL1 and SPANXB
proteins, the remaining 12 proteins were assigned to five
functional classes: (1) sperm movement and structural
organization (TUBB2B, ODF2, AKAP4, KRT1, and CLU);
(2) energy and metabolism (COX6B, GAPDS, PHGPx),
(3) stress response and turnover (HSPA2, HSPA9), (4) sig-
naling and transportation (VDAC2), and (5) antioxidant
activity (GSTMu3).
Among the proteins enriched in sperm tails from nor-
mozoospermic donors, TUBB2B and ODF2, which showed
decreased expression in asthenozoospermic sperm tails,
were found to be crucial for sperm tail motility. Tubulin
beta 2B is a component of falagellar microtubule49 and
outer dense fiber protein 2 forms the component of outer
dense fibers, which are the accessory structures found sur-
rounding the axenome of sperm tail and are involved in
regulating elastic rigidity of sperm flagellum.50,51 Reduced
expression of these proteins is obvious to impart nega-
tive effects in sperm tail movement in asthenozoospermic
patients. Similarly, A- AKAP4 is the abundant struc-
tural protein in fibrous sheath that anchors cyclic-AMP-
(cAMP)-dependent protein kinase A (PKA) to the fibrous
sheath, therein increasing phosphorylation activities lead-
ing to activation of fibrous sheath. Deletion of AKAP4 is
shown to inactivate fibrous sheath and successively impair
sperm tail motility.52,53 GAPDS is another important pro-
tein that is downregulated in asthenozoospermic patients.
GAPDS is located along the principal piece of sperm tail
and plays a role in ATP production in the distant site of
flagellum. Gene knockout studies proved that a GAPDS-
deficient mouse is deficient in sperm tail movement.54
Similarly, the proteins COX6B, PHGPx, VDAC2, and
HSPA2 and HSPA9 also can be used as biomarkers for
understanding male infertility.
In another study, 55 Liu et al. analyzed the proteome
expression pattern in patients affected with obesity. The
authors found that 105 proteins shown less abundance
while 22 were enriched in obesity-associated asthenozoo­
spermic patients. They further analyzed the functionality
of these proteins and found them involved in actin orga-
nization, flagellar assembly, vesicule traffic, signaling,
metabolism, protein degradation, and stress resistance.
They observed decreased expression of tektin, myosin,
and septin, which are involved in ­acrosome formation
and sperm motility, and therefore, their low abundance in
obesity-associated asthenozoospermic patients renders the
sperm immotile.55 Low ­expression of proteins involved in
glucose metabolism, such as ­fructose-bisphosphatealdolase,
glyceraldehyde-3-­phosphate dehydrogenase, aldose reduc-
tase, glyoxylatereductase­, cytochrome c oxidase, and pyru-
vate dehydrogenase, were also observed, which directly
implies that their low expression affects energy metabo-
lism in sperms. As well, for the first time they reported
the association with the decreased expression of two novel
proteins ERp57 and actin-related protein ACTRT2 with
the progression of obesity-associated asthenozoospermia,
which they proposed as the potential biomarkers for diag-
nosis and therapeutic intervention.
Proteomics study has also been involved in analyzing
the impact of environmental factors in impairing repro-
ductive abilities in humans. In a study conducted by Li,56
the authors analyzed the proteomic pattern in patients
subjected to ionizing radiation, such as cosmic radiation,
diagnostic X-rays, and radiotherapy for cancer. More pre-
cisely, cancer patients receiving radiotherapy are often
reported to have infertility problems,57 implying the need
for a detailed investigation on the effect of radiation on
male fertility. With the help of proteomic approaches,
Li  et  al.56 have identified profound diminution in the
expression pattern of Hsp70-2, phospholipases C, PHGPx,
β-tubulin, and GAPDHS. All these proteins are also
observed to have decreased expression in the sperm tails
of asthenozoospermic patients, suggesting that these pro-
teins might act as potential biomarkers for diagnosis and
therapeutic strategies. Huang et al. have also characterized
the proteomic signature in response to exposure of arsenic
in humans.58 In their study they identified that the pro-
teins, voltage-dependent anion-selective channel protein
3, heat shock 70 kDa protein 4L, microtubule-associated
protein 1B, glutathione peroxidase 4, and Y-box-binding
protein 3 are significantly downregulated, and they
reported the involvement of ERK/AKT/NF-κB signaling
pathway in arsenic-induced male reprotoxicity.58
In general, a diverse set of proteomic studies have been
carried out recently to assess the protemic signatures of
infertile and fertile men under diverse conditions. These
studies are continuously updating several proteins as
biomarkers for diagnosis and therapeutic strategies,
and detailed experimental validations are anticipated to
unravel the potential of these biomarkers.
MODELS BASED ON EXOGENOUS COMPOUNDS
TO IDENTIFY POTENTIAL BIOMARKERS
IN SPERMATOGENESIS
Although genomics, transcriptomics, and proteomics
methods are used to profile an expression pattern of genes,
miRNA, and proteins in different conditions pertaining to
male infertility, the resulting number of targets still needs
experimental validation, thus delaying their selection as
biomarkers. On other hand, the study of spermatogenesis
224  Computational characterization and integrative analysis of proteins involved in spermatogenesis
events in the presence of foreign substances such as toxi-
cants and chemical substances such as contraceptives has
proven to be a valid approach for identifying potential tar-
gets. Spermatogenesis, the coordinated series of precisely
regulated events that takes place in the microenviron-
ment of the testis, is organized into four phases involving
(1) spermatogoniogenesis, (2) spermatocyte differentiation,
(3) spermiogenesis, and (4) spermiation. These processes
take place in the seminiferous epithelium of the testis,
where the basal and adluminal compartments are por-
tioned by the presence of the testis-specific BTB, which
serves as an immunological barrier and is involved in the
transport of preleptotene spermatocytes into the adlumi-
nal compartment where it further differentiates to form
zygotene and diplotene spermatocytes that follow two
meiotic divisions to form haploid spermatids. Haploid
spermatids then enter into a spermiogenesis event where
they mature into round and elongated spermatids, which
throughout their maturation process remain attached with
somatic nurse cells called Sertoli cells. The cytoskeleton
network of Sertoli cells constituted by actin and micro-
tubules remains crucial for attachment and transport of
spermatids across the adluminal compartment toward the
lumen where the matured elongated spermatids get trans-
ferred into lumen as sperms.
The precise restructuring of a BTB, apical ES, and
regulation of Sertoli cells, actin- and microtubule-based
network is essential for successful termination of sper-
matogenesis. Various studies have established that BTB,59
apical ES,60 actin- and microtubule-based network,61,62 and
Sertoli cells63 serve as the potent targets of diverse repro-
ductive toxicants and contraceptives. Extensive studies
using these exogenous compounds as models has identi-
fied various potent biomarkers that can be used for diag-
nosis of male infertility as well as in the design of potent
nonhormonal male contraceptives.
BPA-BASED MODEL FOR IDENTIFICATION OF CRUCIAL
DRUG TARGETS
In a study model based on BPA, researchers have identi-
fied several crucial proteins that can be used as potential
biomarkers. BPA, bisphenol A, [2,2-bis(4-hydroxyphenol)
propane] (BPA), is a ubiquitously occurring endocrine dis-
ruptor, and it is characterized as a Category 3 reproductive
toxicant by the European Chemicals Bureau. (A  Class 3
reproductive toxicant causes irregularities affecting human
fertility or results in developmental defects as evidenced by
animal studies.)64
The studies of Li et al. show that BPA disrupts BTB by
declining the steady state level of N-cadherin, occludin,
and connexin 43 (Cx43).65 N-Cadherin forms the ecto­
plasmic specialization protein found at apical and basal
ES, while occludin is a part of the occluding-ZO-1 adhe-
sion complex found at tight junctions and Cx43 is a part
of the gap junction proteins. A decrease in the steady state
level of these proteins substantially affects the BTB integ-
rity that in turn affects the normal progression of sper-
matogenesis. In a similar study, Li et al. discussed the effect
of BPA on the steady state level of Cx43, and using BPA as a
model, they show that Cx43 is crucial for maintaining the
integrity of BPA.66 In their studies, treatment of BPA has
decreased the steady state level of Cx43 and other BTB-
associated proteins such as occludin, ZO-1,N-cadherin,
β-catenin, and α-catenin. However, after 24 hours of BPA
removal, these junction proteins, except for Cx43, relocal-
ized toward the cell-cell surface, but the level of Cx43 was
maintained at a low level, while knockdown of Cx43 was
shown to affect the relocalization of these proteins. Studies
show that ERK1/2 signaling induced by BPA could play a
crucial role in BTB disruption but in the junction reas-
sembly. Other studies also show that BPA can effectively
decrease the level of Cx43 and therefore it can affect the
localization of N-cadherin and ZO1 in BTB and reduce
the sperm count.67 Together, these studies indicate that
Cx43 is the potential cellular target of BPA although it can
downregulate the expression of other BTB proteins.
In another study, Xio et al. elucidated that BPA can affect
F-actin organizations and can impair Sertoli cell adhesive
functions.68 In this study, BPA exposure altered the local-
ization of actin regulatory proteins such as Arp3, Eps8,
c-SRC, and annexin-II. Actin-related protein 3 (Arp3)
and epidermal growth factor receptor substrate 8 (Eps8)
are two critical proteins that are expressed differentially
at BTB near the basal ES and are shown to regulate the
organization of actin microfilaments. Studies show that
Eps8 is essential in bundling of actin microfilaments while
Arp3 induces the nucleation of branched actin filaments.69
Changes from bundled to branched architecture is pivotal
for restructuring the BTB. At the given concentrations
BPA has altered the localization of these proteins and has
resulted in the conversion of bundled actin filaments to
branched structures. Further, this structural change is
associated with impairing the association of Jam-ZO1
and N-cadherin-β-catenin complexes with actin bundles,
which results in BTB disruption at undesirable stages of
the epithelial cycle.
BPA is also shown to induce apoptosis in Sertoli cells
through varied mechanisms. Qian et al. has shown the
involvement of CaM-CaMKII-ERK1/2 in mediating the
Sertoli cell apoptosis.70 Here, the authors show that BPA
increased the expression of CaM and subsequently acti-
vated the phosphorylation of CamKII and on the other
hand BPA affected the Ca2+–ATPase activity and increased
the Ca2+ concentration in Sertoli cells. In general, CaM
is shown to mediate the Ca2+ mediated mitochondrial
damage, where its activation leads to its association with
CaMKII, which drives the opening of the mitochondrial
permeability transition pores.71 The opening of the mito-
chondrial permeability transition pores substantially
increases the permeability in the inner mitochondrial
membrane leading to mitochondrial depolarization, ATP
depletion, mitochondrial swelling, and subsequent release
of cytochrome C to cytosol. Together with an increase in
cytosolic cytochrome C, BPA also increased the expres-
sion of caspases and downregulated expression of Bcl-2
antiapoptotic proteins leading to Sertoli cell apoptosis.

BPA is also shown to decrease the serum testosterone
level in rats exposed to BPA from postnatal days 21 to 35,
where a decrease in the testosterone level, accompanied
with upregulation of Fas and FasL, activated Fas/FasL sig-
naling and upregualted the cytochrome C and Bax pro-
teins leading to Sertoli cells apoptosis.72
In another study, D’Cruz et al. showed that BPA
decreased the activity of antioxidant enzymes such as
superoxide dismutase, catalase, as well as it affected the
activities of glycolytic enzymes such as hexokinase, phos-
phofructokinase, insulin receptor subtype-2 (IRS-2), and
glucose transporter 8 (GLUT-8) and increased the level
of insulin. In doing so, BPA effectively induced oxidative
stress, impaired glucose homeostasis and insulin signal-
ing, and affected steriodogenesis in rat testis.73 In a simi-
lar study, D’Cruz et al. reported that exposure of BPA also
decreased the level of IRS-1 and GLUT-2 and impaired
the activities of enzymes involved in steroidogenesis such
as 17β-hydroxysteroid dehydrogenase (17β-HSD) and
3-β-hydroxysteroid dehydrogenase (3β-HSD), and down-
regulated steroidogenic acute regulatory protein (StAR)
protein and affected testosterone production.74
Together these studies identify connexin Cx43, occludin,
ZO-1,N-cadherin, β-catenin, Arp3, Eps8, CaM-CaMKII-
ERK1/2 signaling proteins, Fas and FasL, superoxide
dismuatase, catalase, hexokinase, phosphofructokinase,
IRS-2, GLUT-8, 17β-HSD, 3β-HSD, and StAR as the key
targets for diagnosis of male infertility and for develop-
ment of male contraceptives.
CADMIUM-BASED MODEL SYSTEMS
Cadmium is a well-studied heavy metal that is shown
to impart reproductive toxicity. Studies elucidate that
cadmium can effectively affect BTB functions in differ-
ent ways and can act on the Sertoli cell cytoskeleton.68,75
Cadmium is shown to perturb tight junction permeability
by selectively targeting occludin and it is shown to acti-
vate JNK and p38 MAPK pathways.75–77 It is also shown to
downregulate the expression of p-FAK-Tyr576 and p-FAK-
Tyr397, and therefore cadmium destabilizes apical ES
integrity as well as the functions of BTB.78
MALE CONTRACEPTIVE-BASED MODEL
Adjudin 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide
is a well-studied nonhormonal male contraceptive designed
and patented by the C. Yan Cheng lab.79 In their experi-
ments, scientists from this lab used this compound to
identify various protein targets that can be exploited for
the use of potent nonhormonal male contraceptives.80,81
Their studies highlight that adjudin more prominently
exerts its effects in the proteins localized in the api-
cal ectoplasmic specialization (ES).82–84 Apical ES is the
testis-­specific cell junction constituted by adherens junc-
tions, focal contact, and tight junction proteins.85 Adjudin
is observed to disrupt F-actin organization at apical ES,
downregulate the expression of actin bundling/barbed
end capping protein Eps8, palladin, and barbed  end
Male contraceptive-based model  225
nucleation complex Arp2/3. 86 By downregulating the
expression of proteins adjudin typically affects the apical
ES restructuring, leading to the release of immature sper-
matids into the lumen. Adjudin also affects the dynamics
of the Sertoli cell microtubule network by downregulating
the expression of microtubule affinity regulating kinase 4
(MARK4).87 MARK4 is a typical member of the MARK
family that shows direct binding with microtubules.88,89
Adjudin treatment is shown to delocalize MARK4 from
the concave side of the spermatid head, affecting sperm
polarity and resulting in premature release of immature
spermatid into the lumen.87 The adjudin model has also
highlighted the function of the Fascin 1 protein.90 The
Fascin protein (Fascin 1-3) is involved in binding with
and bundling actin filaments by cross-linking acting fila-
ments to form tight bundles.91 Fascin 1 is highly expressed
in Sertoli cells and in germ cells. Its localization has been
observed up to stage VIII near the basal ES at the BTB, in
stages VII to late-stage VIII in apical ES, and is restricted
to stage 19 spermatids of the seminiferous epithelium.
Adjudin-based models highlight that Fascin 1 is crucial
for conferring polarity to the sperm head of spermatids
adhered to apical ES and its targeted disruption affects
sperm polarity and results in premature sperm release into
the lumen.90
Although adjudin treatment provides useful insights
into the underlying mechanisms of BTB and apical ES
assembly/disassembly, the efficacy of adjudin remains very
low due its limited availability.59,92 Studies using an adju-
din model were therefore designed to identify factors that
affect bioavailability and results show that P-glycoprotein
(a member of the multidrug resistance gene MDR1),93
multidrug resistance-related protein 1(MRP1), a member
of the multidrug resistance-related protein family,93–95
and breast cancer related protein ([BCRP], ATP-binding
cassette, subfamily G, member 2)96 are three crucial drug
transporters located in the BTB that are involved in drug
efflux across the BTB. Adjudin-based studies show that a
steady state level of P-glycoprotein was increased after the
exposure of adjudin, which returns to their normal level
following the elimination of adjudin.93 Results highlight
that P-glycoprotein is actively involved in elimination of
adjudin, and that coimmunoprecipitation revealed that
P-glycoprotein associates with occludin, Claudin-11, and
JAM-A of the BTB and aids in further regulation of BTB
integrity therein to prevent entry of adjudin into the adlu-
minal compartment to prevent the contraceptive actions
of adjudin.93 In another study by Qian et al., the authors
showed that BCRP is expressed by both Sertoli cells and
germ cells although it is not located in BTB but is observed
in apical ES. Expression of BCRP was observed in Sertoli
step 18–19 spermatid interface in apical ES and is limited
to stages VI to early VIII tubules, implying that matur-
ing spermatids utilize the potential of BCRP to safeguard
themselves from the harmful effects of drugs.96
Adjudin models also highlight the function of three
members of the Src family kinases (c-Src, c-yes, and FAK)
in spermatogenesis.97–99 The protein c-Src is expressed in
226  Computational characterization and integrative analysis of proteins involved in spermatogenesis
both Sertoli cells and germ cells, and is upregualted at api-
cal ES, conferring its role in restructuring apical ES for the
release of mature spermatozoa into the lumen.100 Adjudin
models have confirmed this function of c-Src, particularly
of its phosphorylated form p-c-Src-Tyr419.101–103 Protein
c-Src is shown to physically associate with N-cadherin/β-
catenin, β1-integrin, which is a component of the α6β1-
integrin-lamnin-α3β3γ3 complex.104 Adjudin treatment
is shown to enhance the phsophoryaltion activity of c-Src
that is in turn shown to phosphorylate the cellular targets
of c-Src, leading to dismantling of apical ES components
favoring release of immature spermatids into the lumen.104
Activity of c-Src is also observed at the BTB, where it colo-
calizes with N-cadherin/β-catenin, and also binds and
phosphorylates occludin, hampering the binding of occlu-
din with Zoa-1 that is shown to affect the tight junction
(TJ) barrier in the BTB.105
As well, c-Yes, another member of the SRC family of
kinase, is also a crucial target of adjudin that shows abun-
dant expression in the BTB.106 It is shown to associate with
N-cadherin and occludin and is also shown to be involved
in restructuring of the BTB.106–108 Similarly, FAK is also
involved in phosphorylation, a component of the BTB, and
apical ES. Specifically, studies show that p-FAK-Tyr407,
-Tyr397, and -Tyr576 are highly expressed in the BTB and
also in apical ES,109 but are predominantly expressed in the
BTB. In the BTB they are involved in disrupting the Sertoli
cell TJ permeability by regulating the phosphorylation
status of occludin, while in apical ES it is involved in the
restructuring of F-actin filaments, thus implying that FAK is
also involved in regulating the disassembly of both the BTB
and apical ES.98 Results indicate that the phosphorylation
function of these proteins are also involved in deciding the
bioavailablity of adjudin, and therefore understanding their
function is crucial for developing better contraceptives.
Together, the results of these models highlight the poten-
tial of several proteins in the BTB, apical ES, and Sertoli
cells as crucial drug targets whose deregulation can help
impaired spermatogenesis and can also be used to design
drugs for developing nonhormonal male contraceptives.
ROLE OF BIOINFORMATICS IN UNDERSTANDING
THE STRUCTURE AND FUNCTION OF BIOMARKERS
IDENTIFIED USING OMICS APPROACHES
With the identification of potential protein biomarkers, it
becomes essential to understand their structure and inter-
action mechanism with their cellular partners in order to
design strategies to inhibit its biological function. Besides
targeted inhibition, understanding the structural mecha-
nism underlying the functional activities carried out by
the selected proteins also becomes essential to explain
the observed biological phenotypes that result upon their
inhibition in in vitro and in vivo studies.
In the study conducted by Xiao et al, the authors have
attempted to predict the structural basis of the molecu-
lar association of occludin with c-Src, c-Yes, and FAK.110
Since these Src kinases are observed to bind with and
phosphorylate occludin and are shown to regulate the bio-
availability of adjudin, understanding how these kinases
interact with both occludin and with adjudin gains func-
tional importance. Results of their study highlighted
the presence of important interactions that mediate the
association of c-Src, c-Yes, and FAK with occludin and
also of adjudin with c-Src, c-Yes, and FAK. These results
also showed that adjudin can bind with the hinge region
between the two lobes of kinase in a similar way as with
reported drugs and can alter the activity of these pro-
teins, thus explaining how this association can regulate its
bioavailability.
In another study, Su et al. detailed how adjudin binds
with P-glycoprotein.111 P-glycoprotein is potentially
involved in altering the bioavailability and hence in this
study, by utilizing homology modeling and docking stud-
ies, the authors predicted the structure for inward facing
conformation of P-glycoprotein and analyzed the binding
association of adjudin. The results suggest designing com-
pounds with better structural components that can help
them traverse across the BTB for targeting proteins in the
adluminal compartment.
Similar to P-glycoprotein, BCRP has also been shown
to affect bioavailability of adjudin, such that under-
standing how adjudin binds with BCRP can also help in
designing potent contraceptives. In view of this, Qian et
al. and Cheng et al., in independent studies, predicted the
inward and outward facing conformations of BCRP as
well as tetrameric arrangement of BCRP.112,113 BCRP, like
P-glycoprotein, has N-terminal DNA binding domain fol-
lowed by a transmembrane region at its C-terminal NH2-
NBD-TM-COOH. BCRP adopts a characteristic V-shaped
conformation at its inward-facing conformation and
adopts an X-shaped conformation at its outward confor-
mation. The switch between the conformations helps in
traversing or repelling out the molecules away from the
BTB barrier. The results show that adjudin forms strong
hydrogen bonds, pi interactions, electrostatic interactions,
and van der Waals interactions with residues of BCRP.
Interestingly, it switches between the interaction with cat-
alytic R465 and R482 in accordance with inward and out-
ward conformations, and in both cases, 3-carbohydrazide
moiety of adjudin helps in anchoring adjudin with BCRP,
and thus a suitable replacement of this moiety can help
adjudin to escape these interactions for traversing into the
adluminal compartment.
In addition to the studies aimed at improving efficacy of
adjudin, other studies were also conducted to identify new
promising leads that can be used as male contraceptives.
In a study conducted by Jenardhanan et al., the authors
analyzed the structure of MARK4,114 which is shown to be
important in conferring polarity to maturing spermatids
that are attached with apical ES and targeted inhibition of
MARK4 is shown to impair spermatogenesis, resulting in
premature release of immature spermatid into the lumen.
In this study, the authors predicted the three-dimensional
conformation for the kinase-UBA construct of MARK4
 Conclusion and future perspectives  227

and identified MARK4-specific inhibitors.114 MARK4
belongs to the serine/threonine kinases and is the mam-
malian homolog of partition-defective 1 protein. MARK4
has four important regions: N-terminal kinase domain,
autoregulatory UBA domain, disordered spacer region,
and a terminal kinase-associated domain. As a means of
identifying novel inhibitors for MARK4, the authors have
predicted the structure for the kinase–UBA construct
in an inactive catalytic conformation and revealed that
MARK4 has unique structural features such as DFG-in-
αC-helix-out inactive conformation that differentiates
it from other ser/thr kinases. The presence of a unique
DFG-in-αC-helix-out mode necessitates the design of
MARK-specific inhibitors. In doing this, the authors uti-
lized the pharmocophoric feature of 9-oxo derivatives that
are shown to be MARK-specific and used them to screen
a library of kinase inhibitors. The results identified 10
potent leads that on molecular dynamics simulation study
maintained their binding association and showed steady
pulling of N- and C-terminal lobes of kinase domain,
thereby arresting the catalytic conformation from phos-
phorylating its substrates. The identified leads function
as potent ATP competitors such that with unique DFG-in
conformation they can be used to inhibit both active and
inactive states of MARK4, thus favoring their selection for
the design of next generation nonhormonal contracep-
tives. There are few studies that illustrate how structural
bioinformatics can be used to understand the underlying
structural mechanism behind the biological function of
selected biomarkers.
See Table 18.1 for more details.
CONCLUSION AND FUTURE PERSPECTIVES
With an increasing population and declining male infertil-
ity rate, the need for designing potent techniques to achieve
efficient diagnosis and treatment strategies also increases.
Although the severity of the problems increase the poten-
tial of diagnosis and treatment, strategies still suffer
from various unknown factors. The potency of advanced
genomic, proteomic, and computational techniques should
be integrated for translating the understanding of funda-
mental mechanisms of spermatogenesis toward identify-
ing targets that mark the presence of unusual conditions
such as male infertility. Further, these identified targets
should be evaluated for their potency to act as drug targets
for designing effective treatment strategies. This chapter
discussed the application of genomics, proteomics, and
bioinformatics methods in translating the basic under-
standing of spermatogenesis toward biomarker and drug
target identification. Various studies in these areas should
be designed in the future by integrating these techniques,
and several targets should be identified in the near future
so that diagnosis and treatment strategies can be rein-
vented for overcoming the problems of male infertility and
increasing population growth.

Table 18.1  Details of biomarkers and drug targets discussed.

Protein
GO NCBI ID
S.no Target identified Localization class GeneID uniprot PDB Id
Genomics Study
1. Septin 4 (SEPT4) Sperm Sperm Mitochondria 5414 O43236 Not available
organization
2. Zinc metallopeptidase Testis Metalloendopeptidase 10269 O75844 2YPT, 4AW6
(ZMPSTE24)
3. Ca+/calmodulin protein Testis Calmodulin-dependent 814 Q16566 2W4O
kinase IV (CaMKIV) protein kinase activity
4. Calspermin (Camk4) Testis Calmodulin-dependent 25050 P13234 Not available
protein kinase activity
5. A-kinase-anchoring protein Sperm flagellum Flagellated sperm motility 8852 Q5JQC9 Not available
(AKAP4) Sperm principal
Piece
6. Ubiquilin 3 (UBQLN3) Testis Cell cycle progression 50613 Q9H347 1YQB
in spermatogenesis
7. Calpain 11 (C APN11) Acrosome Cytoskeletal remodeling 11131 Q9UMQ6 Not available
and signal transduction
8. Gametogenetin (GGN) Sperm Spermatogenesis 199720 Q86UU5 Not available
9. Sperm acrosome-associated 4 Acrosome Not defined 171169 Q8TDM5 Not available
(SPACA4)
10. Spermatogenesis-associated 3 Spermatocyte Not defined 130560 Q8NHX4 Not available
(SPATA3)
(Continued)
228  Computational characterization and integrative analysis of proteins involved in spermatogenesis

Table 18.1 (Continued)  Details of biomarkers and drug targets discussed.

Protein
GO NCBI ID
S.no Target identified Localization class GeneID uniprot PDB Id
11. Family with sequence similarity Testis Not defined 84691 Q96KD3 Not available
71, member F1 (FAM71F1)

Proteomics Study
12. Doublesex- and mab-3-related Spermatagonia Male sex determination 1761 Q9Y5R6 4YJ0
transcription factor 1 (DMRT1) and differentiation
13. P antigen family member 1 Testis Uncharacterized 8712 O75459 Not available
(PAGE1)
14. Transition protein 1 (TNP1) Spermatid Sperm motility, spermatid 7141 P09430 Not available
developemt
15. Sperm mitochondria- Sperm motility 4184 P49901 Not available
associated cysteine-rich
protein (SMCP)
16. Actin-like protein 7B (ACTL7B) Testis-cytoskeleton 10880 Q9Y614 Not available
17. Defensin, beta 119 (DEFB119) Sertoli cell, testis Uncharacterized 245932 Q8N690 Not available
18. A kinase anchor protein 4 Sertoli cell, sperm Sperm motility 8852 Q5JQC9 Not available
(AKAP4)
19. Glyceraldehyde-3-phosphate Sperm Energy production in sperm 26330 O14556 3H9E
dehydrogenase (GAPDHS)
20. Tubulin beta 2B (TUBB2B) Sperm tail Constituent of microtubules 347733 Q9BVA1 Not available
21. Outer dense fiber protein Sperm tail Sperm tail outer dense fibers 4957 Q5BJF6 Not available
2,ODF2 constituent
22. Cytochrome c oxidase subunit Sperm tail Cytochrome C oxidase activity 1340 P14854 Not available
6B (COX6B)
23. Phospholipid hydroperoxide Sperm tail Glutathione peroxidase activity 2879 P36969 2GS3
glutathione
peroxidasemitochondrial
(PHGPx)
24. Voltage-dependent Sperm tail Mitochondrial membrane 7417 P45880 Not available
anionselective channel activity
protein 2 (VDAC2)
25. Heat shock-related 70-kDa Sperm tail Spermatogenesis, meiosis 3306 P54652 5FPN
protein 2 (HSPA2)
26. Sperm protein associated with Sperm tail Spermatid development 728695 Q9NS25 Not available
the nucleus on the X
chromosome B (SPANXB)
27. Keratin, type II cytoskeletal 1 Sperm tail Regulation of kinase activity 3848 P04264 4ZRY
(KRT1) of its partners like protein
kinase A
28. Isoaspartyl peptidase/ Sperm tail L-asparaginase and beta- 80150 Q7L266 4O0C
Lasparaginase (ASRGL1) aspartyl peptidase activity
29. Clusterin (CLU) Sperm tail Chaperone binding 1191 P10909 Not available

Exogenous Compound Based Models

30. N-cadherin Testis Assembly and disassembly 1000 P19022 Not available
of BTB and apical ES
31. β-catenin Testis 1499 P35222 4DJS
32. Occludin Testis 100506658 Q16625 1WPA,
1XAW,3G7C
33. Connexin (Cx43) Testis 30236 P17302 2LL2
(Continued)
 References 229

Table 18.1 (Continued)  Details of biomarkers and drug targets discussed.

Protein
GO NCBI ID
S.no Target identified Localization class GeneID uniprot PDB Id
34. Zona occludens protein 1 Testis 7082 Q07157 4Q29
(ZO-1)
35. Actin-related protein 3 (Arp)3 Testis 10096 P61158 Not available
36. Epidermal growth factor Testis 2059 Q12929 2E8M
receptor substrate 8 (Eps8)
37. P-glycoprotein Testis Drug Transporters at BTB for 5243 P08183 Not available
38. Breast cancer-related protein Testis effecting protection to 9429 Q9UNQ0 Not available
(BCRP) developing spermatids form
drugs and other exogenous
compounds
39. Fascin 1 Testis Regulates ectoplasmic 6624 Q16658 1DFC
specialization
40. c-SRC Testis Kinases located at cell 6714 P12931 2HSH
41. c-Yes Testis junctions for modulating 7525 P07947 2HDA
42. Focal adhesion kinase (FAK) Testis assembly and disassembly 5747 Q05397 1MP8, 1K04
of BTB and apical ES
43. Microtubule affinity regulating Testis Conferring polarity to 57787 Q96L34 5ES1
kinase (MARK4) elongating spermatids
at apical ES
Maintains dynamics of
microtubules

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Effects of chemical exposures on testis
cell-cell interactions and endocrine
function
RACHEL C. KNIGHT, JENNIFER R. PANIZZI, and BENSON T. AKINGBEMI
INTRODUCTION
Increased exposure to environmental endocrine dis-
rupting chemicals (EDCs) and possible association with
negative reproductive effects in the population continue
to raise public health concerns. An apparent decline in
sperm production capacity in men, a higher incidence of
urogenital anomalies such as cryptorchidism and hypo-
spadias in newborns, testicular cancer in young adults, as
well as the obesity epidemic in the population1 apparently
correspond to a marked increase in the use of industrial
chemicals in the last 40 years.2,3 The general population is
exposed to EDCs present in food, air, water, and soil while
those with occupations in the agricultural, petrochemi-
cal, and construction industry are subject to even greater
exposures.4 Therefore, it is imperative that further stud-
ies be conducted to determine the precise effects chemical
toxicants have on human reproductive and overall health.
CLASSES OF ENDOCRINE DISRUPTING CHEMICALS
EDCs are classified based on their applications, chem-
istry, and/or hormonal activity (Table 19.1). Chemicals
are considered estrogenic if they bind to estrogen recep-
tors (ESRs) and mimic activity of the natural estrogen
17β-estradiol (E2). Compounds that prevent E2 binding
of the ESR and/or suppress E2 biosynthesis are consid-
ered antiestrogenic. Chemicals with ESR agonist activ-
ity (called xenoestrogens) include pesticides (e.g., 1, 1,
1-trichloro-2, 2-bis [p-chlorophenyl] ethane [DDT],
methoxychlor, dimethoate, and chlordecone) and indus-
trial pollutants [e.g., polychlorinated biphenols (PCBs)],
alkylphenols, and bisphenol A (BPA). While PCBs are
present in industrial applications,5 BPA is used in the
manufacture of food packaging products and dental seal-
ants while alkylphenols are used in the manufacture of
industrial detergents. Also contributing to EDC levels in
the environment are pharmaceuticals in human and ani-
mal waste (e.g., the oral contraceptive ethinyl estradiol)
and dietary estrogens (e.g., soy isoflavones).5 Additionally,
other compounds interfere with and/or suppress andro-
gen receptor- (AR-) mediated activity acting as antian-
drogens, such as the fungicide vinclozolin, the persistent
DDT metabolite p, p-chlorophenyl, ethylene (p-p’-DDE),
and phthalate esters used in the manufacture of plastics.6
234
19
EXPRESSION OF STEROID HORMONE RECEPTORS
IN TESTICULAR CELLS
The testis consists of a tubular compartment of seminif-
erous tubules containing Sertoli cells and germ cells at
different stages of development.7 An interstitial compart-
ment between seminiferous tubules includes connective
tissue (i.e., collagen fibers, fibroblasts, mesenchymal cells,
and immune cells), blood vessels, lymphatics, nerves, and
Leydig cells (Figure 19.1). Leydig cells are the major source
of androgens, including testosterone (T) and dihydrotes-
tosterone (DHT).7 Cloning of the ESR1 (formerly ERα) and
impairment of spermatogenesis in ESR1 knockout mice
indicated a physiological role for estrogen in the testis.8 A
subsequently cloned estrogen receptor subtype was desig-
nated ESR2.9 In rats, ESR1 was expressed in Sertoli cells,
peritubular myoid cells, as well as in rat fetal and adult
Leydig cells, while ESR2 is found in rat germ cells10–12 and
was localized to adult mouse Leydig cells.13,14 In human
testes, ESR2 expression began at 16 weeks of gestation,15
and ESR1 and ESR2 were both expressed in Sertoli cells,
spermatocytes, and elongating spermatids in the adult.16,17
Only ESR2 was present in human Leydig cells.18 The aro-
matase enzyme, which catalyzes conversion of androgens
into estrogens, was found predominantly in prepubertal
rat Sertoli cells and germ cells while expression appeared
in Leydig cells after 21 days of age.19 Notably, estrogen bio-
synthesis and aromatase activity occur mostly in adipose
tissue in male subjects because the testis synthesizes only
about 25% of E2 in circulation.20
GENERAL MECHANISMS OF ENDOCRINE DISRUPTION
Estrogenic chemicals
Steroid hormones regulate target cell activity by activation
of their cognate receptors (ESR1, ESR2, ARs) and bind-
ing of estrogen and androgen response elements (EREs
and AREs) in promoters of target genes.21,22 Estrogens
may also induce rapid nongenomic responses through
plasma membrane receptors (mESR1 and mESR2) and
cause Ca++ release and changes in intra- and extracellular
signaling.23,24 A seven-transmembrane estrogen receptor,
GPR30, is present in the brain, placenta, ovary, testis, and
prostate.25 ESR and GPR30 agonists induced activation of
 General mechanisms of endocrine disruption  235

Table 19.1  Classes of endocrine disrupting chemicals.

Substances Category Mechanism of action
Bisphenol A (BPA) Plasticizer Estrogen receptor agonist
Phthalates Plasticizer Androgen receptor antagonist
Dichlorodiphenyl-trichloroethane (DDT) Pesticide Estrogen receptor agonist
Polychlorinated biphenyls (PCBs) Industrial by-product Thyroid hormone receptor antagonist
Diethylstilbestrol Synthetic, non-steroidal estrogen Estrogen receptor antagonist
Alkylphenol Surfactant Estrogen receptor agonist
Vinclozolin Fungicide Androgen receptor antagonist
Soy isoflavones Dietary estrogens Estrogen receptor agonists
Dioxins Environmental toxins Estrogen receptor agonists

Elongated spermatids
Round spermatids

Secondary spermatocytes
Sertoli
cell
Sertoli Primary spermatocyte
cell

Basal lamina Spermatogonium

Fibroblast
Peritubular myoid
cells

Capillary Capillary

Leydig cells

Figure 19.1  Cellular composition of seminiferous tubules in testis: Supporting Sertoli cells and germ cells at different stages of
development (spermatogonia, primary and secondary spermatocytes, and round and elongated spermatids); and, interstitial con-
nective tissue containing fibroblasts and Leydig cells (not drawn to scale).

secondary messengers and signaling cascades mediated by
tyrosine kinase receptors and protein kinases (e.g., insulin
growth factor- [IGF-] insulin receptor [IR], protein kinase
B [Akt], mitogen-activated protein kinases [MAPK], pro-
tein kinase A [PKA], and protein kinase C [PKC]).26,27
Interestingly, BPA exhibited high binding affinities for
GPR30 in ESR-negative cells,28 implying that EDC action
may be mediated by multiple transcription factors.
Antiandrogenic chemicals
Chemicals may block AR binding and activation by endog-
enous androgen. For example, the fungicide vinclozolin
has been shown to bind DNA and inhibit dihydrotestos-
terone- (DHT-) induced transcriptional activation of the
AR.29 Similarly, the phthalate esters dibutyl phthalate
(DBP) and di(2-ethylhexyl) phthalate (DEHP) inhibited
AR transcriptional activity and disrupted sexual differen-
tiation in male rats.30,31 DDT-related compounds inhibited
hepatic mono-oxygenase activity, thereby promoting
androgen degradation and disruption of AR-mediated
activity.32 Interestingly, BPA decreased E2 secretion while
DEHP increased aromatase expression and E2 biosynthe-
sis in rodent testis, which increased serum E2 concentra-
tions (Figure 19.2).33,34 Therefore, the estrogenic and/or
antiandrogenic activity of EDCs possibly varies with the
tissue and stage of development, and dosage and duration
of chemical exposure.
Antithyroid agents
The thyroid hormone receptor (TR) belongs to the nuclear
family of transcription factors.35 Thus, EDC-induced
disruption of thyroid function may result from altered
TR expression, changes in triiodothyronine (T3) and
tetraiodothyronine (T4) concentrations, disruption of
coactivator recruitment, and/or interference with bind-
ing of TR-ligand complexes to thyroid hormone response
236  Effects of chemical exposures on testis cell-cell interactions and endocrine function

0.12

Basal leydig cell E2 production

* 1.2 *
*
Serum E2 levels (ng/mL) 1.1 1.0

(ng/106 cells • 3h)

Pituitary LHβ subunit protein/ACTB

Pituitary LHβ subunit protein/ACTB
0.08 0.8 1.0
0.9 0.8
0.8
0.7 0.6
0.04 0.4 0.6
0.5 *
0.4
0.4
0.00 0.0 0.3
(a) (b) 0.2
0.2
2.4 0.8 0.1
*
production (ng/106 cells • 3 h)

0.0
LH-stimulated leydig cell E2

* 0.0 25 0 50
Aromatase mRNA levels 0
1.8 (aromatase/S16) 0.6 (a) Dose of BPA (µg/kg bw) (b) Dose of DEHP (µg/kg bw)

*
1.2 0.4

Pituitary FSHβ subunit protein/ACTB

1.0

Pituitary FSHβ subunit protein/ACTB

0.6
0.0
0 mg/kg/day DEHP Aromatase
290 bp
10 mg/kg/day DEHP
S16
100 mg/kg/day DEHP 150 bp
Dose of DEHP (mg/kg/day)
(c) (d)
Figure 19.2  Serum E2 concentrations (a) were associated
with increased Leydig cell E2 production (b and c) and increased
aromatase gene expression (d) after DEHP treatment of rats
from 21 to 48 days of age (n = 10). Leydig cells were incubated
in culture media for 3 h, and E2 was measured in aliquots of the
spent media by RIA and normalized to nanograms per 106 cells.
S16, rat ribosomal protein S16; *, p < 0.01 compared to control.
Data are presented as mean +/– SEM. (See further Proc. Natl.
Acad. Sci. U S A. 2004; 103:775-780.)
elements (TREs).21,36–38 For example, organohalogen
chemicals decreased thyroid hormone biosynthesis,39,55
increased biliary excretion of T4 by induction of uridine
5’-diphospho (UDP)-glucuronyl transferase activity,40
and prevented T4 binding to the TR.41,42 BPA phthalates,
and DDT induced T3 binding to the retinoid X receptor/
thyroid receptor complex and regulated SF-1 and steroido-
genic acute regulatory protein (StAR) gene expression in
mouse Leydig cells.43 As well, serum phthalate, BPA, and
cadmium concentrations were inversely related to thyroid
hormone levels in male subjects.44
Endocrine disrupting chemical (EDC) affection
of neuroendocrine control of testicular function
Hypothalamic pathways regulate gonadotropin-releasing
hormone (GnRH) secretion and control pituitary luteiniz-
ing hormone (LH) and follicle-stimulating hormone (FSH)
secretion and release while gonadal steroids and other fac-
tors acting through feedback mechanisms regulate GnRH
and gonadotropin secretion. Estrogenic activity was asso-
ciated with regulation of hypothalamic GnRH pulses
and GnRH receptor gene expression in gonadotropes.45,46
0.8
0.2
0.8
0.6
0 0.6
0.4
0.4 *
*
0.2
0 10 100 0.2
0.0 0.0
0 25 0 50
(c) Dose of BPA (µg/kg bw) (d) Dose of DEHP (µg/kg bw)
Figure 19.3  Male rats were exposed to BPA and DEHP by
maternal gavage from gestational day 12 to 21. Gonadotropin
subunit protein was analyzed in pituitary glands from 90-day-old
male rats (n = 6 animals/group): LHβ (a and b) and FSHβ (c and d).
Protein measurements were done by Western blotting proce-
dures using antisera specific to rat LHβ and FSHβ and the appro-
priate secondary antibodies. Assays of samples were repeated
at least four times and protein levels normalized to beta-actin
(ACTB). *, p < 0.05 vs control. (See further Endocrinology. 2015;156
(12):4672–4685.)
Men with inactivating mutations of the aromatase and
ESR1 gene had elevated serum FSH and LH concentra-
tions.47,48 Exposure of newborn male rats to environmen-
tal estrogens results in delayed and sustained high levels
of prolactin,49 a potent inhibitor of hypothalamic GnRH
production50 Additionally, gestational exposures of male
rats to BPA and DEHP decreased pituitary LH protein
expression in adulthood51 (Figure 19.3). Therefore, neuro-
endocrine pathways regulating the reproductive axis are
targets for chemical agents.
EDC TARGETING OF TESTICULAR CELLS
Chemical exposure effects are presumably exerted in mul-
tiple testicular cells with implications for exocrine and
endocrine function. The primary androgen T stimulates
sexual differentiation in the prenatal period, promotes
development of male secondary sex characteristics, sup-
ports hormonal imprinting of the liver, prostate, and hypo-
thalamus, and maintains the male phenotype and fertility.6
 EDC targeting of testicular cells  237

Sertoli cells phosphorylation of steroid hormone receptor coactiva-

Somatic Sertoli cells produce several factors, including
androgen-binding protein (ABP), sulfated glycoproteins
(SGP) 1/2, transferrin, and inhibin B, which support germ
cell development. Exposures to perfluorooctanesulfonic
acid (PFOS) decreased inhibin B expression in testes of adult
mice and disrupted feedback regulation of pituitary FSH
secretion.52 Exposure to bisphenol AF (an analog of BPA)
increased serum gonadotropin concentrations associated
with decreased testicular inhibin B mRNA in male rats, and
serum BPA concentrations were inversely related to inhibin
B concentrations in infertile male subjects.53,54 As well, peri-
natal exposures to dioxin decreased serum inhibin B and
elevated FSH levels in male subjects.55 Prenatal exposures
to BPA activated Raf1 and extracellular regulated kinase
(ERK)1/2 in rat Sertoli cells56 and disrupted junctional bar-
riers in vivo and in vitro.57,58 Furthermore, BPA exposures
were associated with redistribution of junctional proteins
such as occludin, zonula occludens 1, N-cadherin, β-catenin,
or connexin 43 in the rat and human testes, which dis-
rupted intercellular communication.57,59–62 Chemical dis-
ruption of the blood-testis barrier (BTB) and ectoplasmic
specializations (ES) in Sertoli cells likely renders the testes
more vulnerable to chemical exposure effects.
Germ cells
Germ cells begin differentiation as primordial germ cells
(PGCs) in the embryo. E2 and ESR agonists induced
tors, ERK1/2, Akt, and promoted survival and prolifera-
tion of PGCs.63 Other agents such as cadmium and BPA
caused germ cell depletion in the seminiferous epithelium
acting via mitogen-activated protein kinases (MAPKs).64
Recent studies have demonstrated that exposures to the
EDC vinclozolin disrupted epigenetic pathways leading to
transgenerational effects by modifying germ cell differen-
tiation through dysregulation of microRNAs (mmu-miR-
23b and mmu-miR-21) and disruption of the Lin28/let-7/
Blimp1 pathway in germ cells.65 Interestingly, inactivating
mutations of the ESR1 gene impaired sperm viability and
fertility66 and ESR1 gene deficiency was associated with
decreased fertility in transgenic mice.8
Leydig cells
Fetal Leydig cells arise from interstitial mesenchymal stem
cells (MSCs) and disappear soon after birth with a small
remnant of cells carried over into the postnatal period.
Thus, it is conceivable that chemical exposure effects
imprinted on MSCs early in development persist in adult
tissues and/or are passed in the germ line to subsequent
generations.67,68 The LH signaling pathway is the primary
regulator of Leydig cells, which enzymatically convert
the cholesterol substrate into androgen metabolites and
androgen (Figure 19.4). Chemical-induced inhibition of
T secretion may be receptor-mediated or occur by tran-
scriptional regulation of steroidogenic enzyme activity.

LH
Chol Gs Gi
esters Adenylate
cyclase

Cholesterol cAMP

Steroidogenic
PKA enzyme gene
transcription

StAR
DNA
PBR
mRNA

SER Nucleus

Mitochondrion

Testosterone

Figure 19.4  The LH-stimulated signaling pathway is the primary regulator of Leydig cells. LH binding of its cognate receptors causes
cyclic AMP production and protein kinase A (PKA) activation and recruitment of cholesterol as substrate for androgen biosynthesis.
Cholesterol (CHOL) is moved into mitochondria by transport proteins (PBR and StAR) for enzymatic cleavage into pregnenolone
which then crosses into the SER for further enzymatic action by other enzymes (3β-hydroxysteroid hydrogenase, cytochrome
P45017α-hydroxylase, and 17β-hydroxysteroid dehydrogenase). Finally, T diffuses into the seminiferous tubules or enter into blood
capillaries for circulation to peripheral tissues. LH, luteinizing hormone; PBR, peripheral benzodiazepine receptor; StAR, steroido-
genic acute regulatory protein; SER, smooth endoplasmic reticulum.
238  Effects of chemical exposures on testis cell-cell interactions and endocrine function

LH
BPA
Octylphenol
LH Dimethoate
receptor
N
T
StAR SER
17β-HSD
AR
ER P45017α Octylphenol
CHO 3β-HSD

PREG
M P450scc
HPTE
Dimethoate
Ligand

Figure 19.5  Endocrine disruptor (ED)-induced inhibition of Leydig cell testosterone biosynthesis. Both ER (ESRs) and ARs are
present in Leydig cells. Several steps in the androgen biosynthetic pathway are inhibited by EDs, which decrease steroidogenic
enzyme activity. HPTE (2,2-bis-[p-hydroxyphenyl]-trichloroethane) and dimethoate both reduced cholesterol side-chain cleavage
(P450scc) activity while octyphenol decreases P45017α activity. Gene transcription for the protein that causes movement of choles-
terol into mitochondria for side-chain cleavage, steroidogenic acute regulatory protein (StAR), was inhibited by dimethoate. Some
agents such as octylphenol interfered with luteinizing hormone (LH) binding of its receptor. AR, androgen receptor; CHO, choles-
terol; ER, estrogen receptors; M, mitochondria; N, nucleus; PREG, pregnenolone; SER, smooth endoplasmic reticulum; T, testosterone.
(See further Ann Med. 2001;33(6):391–403.)

For example,  2, 2-bis-(p-hydroxyphenyl)-trichloroethane act as autocrine or paracrine regulators (Table 19.2). It is

(HPTE) and dimethoate both decreased cholesterol side-
chain cleavage (P450scc) enzyme activity while octylphenol
decreased P45017α activity. Other chemicals interfered with
LH binding to receptors in Leydig cells (e.g., octylphenol)
(Figure 19.5). Inactivation of the ESR gene in Leydig cells
affected androgen biosynthesis,8 and males with complete
androgen insensitivity syndrome due to a mutation in the
AR gene exhibited female external and internal genita-
lia.69 Thus, decreased Leydig cell androgen secretion and
disruption of AR-mediated activity both adversely affect
reproductive development and function.
EDC DISRUPTION OF PARACRINE RELATIONSHIPS
BETWEEN TESTICULAR CELLS
Testicular cells express receptors for pituitary gonadotro-
pins FSH, LH, and steroid hormones and are therefore reg-
ulated by gonadotropins and steroid hormones. Testicular
cells also secrete growth factors and other cytokines that
likely that EDC effects on individual testicular cells alter
the patterns of cell-cell interactions necessary to optimize
testicular function.
FSH
Receptors for the gonadotropin FSH are present only in
Sertoli cells70,71 while inhibin B, produced by Sertoli cells,
provide feedback to the hypothalamus and pituitary gland
to regulate FSH secretion. FSH and androgen appear to
be the primary factors controlling spermatogenesis in
the rodent.72–74 Inhibin B secretion appears to vary with
changing roles of Sertoli cells in the immature and adult
testes,75 and its levels are negatively correlated with those
of FSH.76 Serum FSH concentrations were increased after
treatment of male rats with aromatase inhibitors and in
ESR1- and aromatase-deficient male subjects.77 Prenatal
exposures of male rats to BPA and DEHP were associ-
ated with increased LHβ and FSHβ protein expression in

Table 19.2  Paracrine regulators of testicular cells.

Hormone/Growth factor Source Target
AMH Sertoli cells Leydig cells, germ cells
Androgen Leydig cells Sertoli cells, peritubular myoid cells
Estrogen Sertoli cells, Leydig cells Germ cells
IGF-1 Sertoli cells Multiple tissues
Stem cell factor Sertoli cells Leydig cells, germ cells
Transforming growth factors (α and β) Sertoli cells Leydig cells, germ cells

the prepubertal period.51 Thus, EDC disruption of steroid
hormone feedback regulation of the hypothalamus and
pituitary gland likely affects FSH secretion and germ cell
development.
Anti-Müllerian hormone (AMH)
AMH, a product of Sertoli cells, which is required to induce
mesonephric cell migration during mammalian testis for-
mation in the embryo, has receptors (AMHR2) in germ
cells and Leydig cells.78,79 In the postnatal period, AMH
concentrations were negatively correlated with testicular
and epididymal weight.80 Transgenic mice with deletion
of AMH or AMHR2 showed decreased testicular ste-
roidogenic capacity while AMH overexpression increased
serum T concentrations.81,82 In immature animals and
in tfm mice, FSH administration increased AMH levels,
suggesting FSH regulates AMH secretion.83 Interestingly,
prenatal exposure of male rats to BPA and DEHP was asso-
ciated with increased serum AMH levels in adulthood51
(Figure 19.6). As well, AMH expression was downregu-
lated by development of meiotic germ cells while serum
AMH concentrations correlated with increased FSH and
T levels and decreased inhibin B concentrations in male
subjects after administration of chemical toxicants (cis-
platin and busulfan).80,84 AMH may be a good candidate
for development as a biomarker for impaired testicular
development and/or damage as reflected in serum levels
and due to lack of diurnal variations and relative ease of
measurement.
LH and androgen
Pituitary LH is the primary regulator of Leydig cells,
and circulating androgens act on ARs in hypothalamic
AMH
ACTB
* 2.4
1.4
Serum AMH protein/ACTB
Serum AMH protein/ACTB
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 25
(a) Dose of BPA (µg/kg bw) (b)
Figure 19.6  Male rats were exposed to BPA and DEHP by
maternal gavage from gestational day 12 to 21. Adult male
rats at 90 days of age were processed to obtain serum (n = 10
animals per group) to measure AMH protein (a and b). Protein
analysis was achieved using Western blotting procedures and
antisera specific to rat AMH. Assays per sample were repeated
at least four times and protein levels were normalized to ACTB.
*, p < 0.05 vs control. (See further Endocrinology. 2015;156(12):​
4672–4685.)
Conclusion 239
GnRH neurons to control GnRH secretion.85 Deletion
of ARs from Sertoli cells caused meiotic arrest in germ
cells,86,87 indicating that proliferation and maturation
of Sertoli cells is androgen-dependent. In the testis, T  is
bound to Sertoli cell-specific ABP, which provides high
androgen concentrations necessary for spermatogenesis.88
Thus, EDCs that target Sertoli cells, ABP expression, and/
or androgen secretion likely disrupt spermatogenesis.89
Moreover, maintenance of cell adhesions at the BTB
(Sertoli-Sertoli junctions) and ES (Sertoli-spermatid junc-
tions) is androgen-dependent.61,90–94 Exposure of male rats
to BPA, CdCl2, and PFOS95 downregulated Cx43 expres-
sion in Sertoli cells and was associated with oxidative
stress-induced damage to junctional complexes and germ
cell apoptosis.52,93,96–98 Thus, chemical-induced changes in
androgen secretion may compromise BTB and ES integ-
rity and thereby disrupt germ cell development.
Estrogen
Similar to androgen, estrogen is critical to maintenance of
junctional complexes between testicular cells and orderly
progression of spermatogenesis.99,100 Aro­ matization of
androgens to estrogens occurs in testicular cells that
express the cytochrome P450 aromatase enzyme.55,101
However, overexpression of aromatase and excessive E2
exposure increased germ cell apoptosis and impaired
spermatogenesis.102,103 Importantly, exposure of rodents
to several estrogenic compounds (e.g., BPA, dioxin, cad-
mium, and phthalates) interfered with estrogen homeo-
stasis in the testicular microenvironment and disrupted
germ cell development.104
Growth factors
Somatotropin or the growth hormone (GH), acting in
concert with IGF-1, serves as a paracrine regulator of
testicular cells. While GH is produced by the pituitary
* gland, IGF-1 is secreted by Sertoli cells and Leydig cells
in response to FSH and LH stimulation in order to sup-
2.0
port both Sertoli cell maturation and testicular steroido-
1.6 genesis in an autocrine and paracrine manner.105 Exposure
of rats to dioxin decreased serum IGF-1 concentrations106
1.2
while PFOS inhibited IGF-1 secretion and downregulated
0.8 IGF-1R expression in the liver and testis.107 These observa-
tions imply that growth factor receptors (e.g., epidermal
0.4 growth factor receptors and IGF1-Rs) may act in concert
0.0
with other transcription factors to mediate EDC activity
0 50 in the testis.
Dose of DEHP (µg/kg bw)
CONCLUSION
Chemical exposures occurring in the fetal period are
linked to altered sexual differentiation that impact
gonadotropin subunit expression in the pituitary gland
and gonadal secretion of AMH and steroid hormones
(Figure 19.7). Because developing organisms are more
sensitive to EDCs, children and adolescents are especially
vulnerable segments of the population. EDCs are widely
distributed in the environment but only a few studies
of mixed chemical exposure effects have been reported.
240  Effects of chemical exposures on testis cell-cell interactions and endocrine function
Germ cells
AMH
IGF-1
Stem Cell Factor
Transforming Growth Factors
(α & β)
Sertoli cells
Figure 19.7  Paracrine regulation of testicular cells. Sertoli cells and Leydig cells produce factors which self-regulate (autocrine
regulation) or pass to other cells as paracrine regulators.
For example, exposures of male rats to a phthalate mix-
ture caused greater effects than those due to individual
chemicals.108 Thus, studies of single individual chemicals
may mask biological effects that are present in the body.
Nevertheless, identification of molecular mechanisms
related to EDC effects in the male reproductive tract
will identify signaling molecules and pathways mediat-
ing chemical exposure effects.109,110 Studies addressing
identified research gaps will generate data to support risk
assessment of the population.
ACKNOWLEDGMENTS
This work was supported in part by funding from the
Animal Health and Disease Research grant award from
the College of Veterinary Medicine at Auburn University,
and NIH grant ES 15886 (to BTA).
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Environmental toxicants on Leydig
cell function
LEPING YE, XIAOHENG LI, XIAOMIN CHEN, QINGQUAN LIAN, and REN-SHAN GE
INTRODUCTION
Leydig cells reside in the interstitium of the testis and make
androgens and peptide hormones for the male reproduc-
tive system. In rodents, there are two different populations
of Leydig cells: fetal Leydig cells and adult Leydig cells.
The primary function of Leydig cells is to make androgens
(mainly testosterone and dihydrotestosterone). During the
fetal period, testosterone and its more potent 5α-reduced
metabolite dihydrotestosterone are very important for ini-
tiating the development of the male reproductive tract and
for inducing testis descent.1 The disruption of the andro-
gen biosynthesis in fetal Leydig cells causes various types
of malformity, including male-to-female sex reversal,
cryptorchidism, and hypospadias. During the postnatal
period, androgens are critical for initiating pubertal devel-
opment in male adolescents, maintaining the accessory
sex organs,2 stimulating spermatogenesis,3 and promot-
ing sexual behavior in adults.4 Although adult Leydig cells
only account for about 1%–5% of all testicular cell types,
they make up about 95% of the total amount of testoster-
one in the body.5
Besides the production of androgens, Leydig cells also
secrete insulin-like 3, a peptide hormone. Insulin-like 3,
secreted by rodent fetal Leydig cells, is critical for the ini-
tial testis descent.6,7 Insulin-like 3 (encoded by Insl3) pro-
duced by adult Leydig cells plays a role in spermatogenesis.8
Interestingly, primates, including humans, have a third
generation of Leydig cells, called neonatal Leydig cells,
which exist in the testis for a very short time from birth
to 6 months.9 However, their functions are still not clear,
and therefore, we only discuss fetal and adult Leydig cells
in this chapter.
Many environmental chemicals can affect the develop-
ment of both generations of Leydig cells and their func-
tions. They can disrupt the production of androgens and
insulin-like 3. Because there are increased uses of indus-
trial compounds (some of which are potential Leydig cell
toxicants) in modern society, increasing incidences of
cryptorchidism, hypospadias, testicular cancers, and male
infertility over the past 40 years has been noted.10,11 The
association of many environmental toxicants with the mal-
formity of male reproductive tracts, testicular cancer, and
male infertility has been reported.12 These environmental
chemicals can disrupt the normal male reproductive sys-
tem in the experimental animal (mainly rat) models.
Several classes of compounds, including perfluoroalkyl
substances, phthalates, bisphenols, benzophenones, organo-
chlorines, carbamates, polycyclic aromatic hydrocarbon,
20
organophosphates, azole compounds, 1, 2-dibromo-3-​
chloropropane, heavy metals, and plant toxicants have
been observed to disrupt Leydig cell functions. These
compounds may target Leydig cells, acting on multiple
levels to disrupt the normal physiological functions of
Leydig cells via blocking their development, downregulat-
ing the expression levels of the genes for the biosynthesis
of hormones (androgen and insulin-like 3), and/or directly
inhibiting androgen biosynthetic and metabolizing
enzyme activities, and/or inducing Leydig cell apoptosis.
In this chapter, we discuss some of these environmental
toxicants on Leydig cells.
ANDROGEN BIOSYNTHESIS
In adult Leydig cells, at least four enzymes are involved
in testosterone synthesis. Biosynthesis starts with the
substrate cholesterol. The first enzyme is P450 cyto­chrome
cholesterol side chain cleavage enzyme (CYP11A1). This
enzyme resides in the inner membrane of the mitochon-
dria of the Leydig cells.13 The enzyme catalyzes choles-
terol into pregnenolone. Pregnenolone diffuses from the
mitochondria into outside cellular milieu, which are full
of smooth endoplasmic reticulum. Three steroidogenic
enzymes reside in the smooth endoplasmic reticulum: 3β-​
hydroxysteroid dehydrogenase (3β-HSD), cytochrome
P450 17α-hydroxylase/17,20-lyase (CYP17A1), and 17β-​
hydroxysteroid dehydrogenase 3 (17β-HSD3).14 Preg­
nenolone is finally converted into testosterone by the
sequential reactions of these enzymes. Because there are
differences in the affinity of the steroidogenic enzymes
between human and rodents, the catalytic reactions go
down different pathways (Δ4 or Δ5 pathway) with dif-
ferent steroid intermediates (Figure 20.1). Rodents
predominantly take the Δ4 pathway: pregnenolone →
progesterone → 17α-hydroxyprogesterone → andro-
stenedione → testosterone.14 Humans mainly take the Δ5
pathway: pregnenolone → 17α-hydroxypregnenolone  →
dehydroepiandrosterone → androstenedione → testoster-
one.14 However, fetal Leydig cells in mice lack 17β -HSD3,
which is expressed in Sertoli cells. Therefore, fetal Leydig
cells make androstenedione, which is transferred to
Sertoli cells where testosterone is synthesized by 17β-
HSD3.15 The Leydig cell precursors in the adult Leydig cell
population also contain a significantly high level of type
1 5α-reductase, and thus 5α-reduced androgen is made in
these Leydig cells.16
When testosterone is produced, it is metabolized
to dihydrotestosterone, a more potent androgen than
245
246  Environmental toxicants on Leydig cell function

testosterone, in Leydig cells or peripheral tissues by sev-
eral types of 5α-reductase (SRD5A1, 2 and 3). Of three
isoforms of 5α-reductase, SRD5A2 has a high affinity for
testosterone and is particularly important for the genera-
tion of dihydrotestosterone.17 Dihydrotestosterone is criti-
cal for the development of male reproductive tracts during
the fetal period.18,19
ENZYMES FOR TESTOSTERONE BIOSYNTHESIS
AND METABOLISM
CYP11A1
CYP11A1 is encoded by human CYP11A1 or rat Cyp11a1.
20
Thus far, no additional isoforms have been found for this
enzyme.
CYP11A1 is a complex enzyme, requiring NADPH as
the cofactor to provide the electrons (Figure 20.2). The
electrons are transferred from NADPH to CYP11A1 via
two electron transfer proteins: adrenodoxin reductase
and adrenodoxin.22,23 All three proteins constitute the
cholesterol side-chain cleavage complex.23 The complex
catalyzes three sequential reactions using three mol-
ecules of cofactor NADPH for electron transfers via the
mitochondrial electron transfer system.14 The reaction
catalyzed by this enzyme occurs in the inner membrane
of the mitochondria. Two hydroxyl groups are added
to cholesterol (at C20 and C22) first. Then, the cleavage
between the added hydroxyl groups is followed, leading
to the formation of pregnenolone.14 CYP11A1 is the first
enzyme for the formation of all steroids and it expresses
in adrenals, gonads (including Leydig cells), and placen-
tas. Thus, the disruption of CYP11A1 not only impairs
Leydig cell functions but also the functions of other
endocrine systems.
3β-HSD
21 There are several isoforms of 3β-HSD, each encoded by
a different gene. In humans, there are two isoforms of
3β-HSD (3β-HSD1 and 3β-HSD2). Human 3β-HSD1 and
3β-HSD2 are encoded by human HSD3B1 and HSD3B2
(review in Simard et al.24), respectively. Human HSD3B1
and HSD3B2 genes have 81.9% identity. Human HSD3B1
is primarily expressed in the placenta. Human HSD3B2 is
predominantly expressed in adrenals and gonads (includ-
ing Leydig cells). Therefore, in this chapter, we discuss
human HSD3B2 and its encoded protein 3β-HSD2. In
rats, there are four isoforms of 3β-HSD.14 Each isoform
is encoded by a distinct gene.14 In rat Leydig cells, the

LH/hCG
HDL
SCARB1 LHCGR

Cholesterol
Triclosan
cAMP cAMP
StAR Chlorpyrifos

Cholesterol MXC
CYP11A1 Lindane
Etomidate
Pregnenolone Gossypol

Pregnenolone

3β-HSD

Progesterone Androstenedione Testosterone

Tributyltin
CYP17A1 17β-HSD3 SRD5A
Triphenyltin
Genistein
Dihydrotestosterone

PFASs Bisphenol A PFASs

Phthalates Prochloraz Phthalates
Bisphenol A Triphenyltin Bisphenol A
MXC Tributyltin Etomidate
Etomidate DBCP Triphenyltin
Prochloraz Gossypol Gossypol
Tributyltin MXC
Gossypol Genistein

Figure 20.1  The steroidogenic pathway in rodent Leydig cells and the possible inhibitory sites of the environmental toxicants.
Hormones: LH, luteinizing hormone; hCG, human choriogonadotropin. Receptor: LHCHR, luteinizing hormone/choriogonado-
tropin receptor. Signaling: cAMP, cyclic adenosine monophosphate. Cholesterol transporters: SCARB1, scavenger receptor class B
member 1; StAR, steroidogenic acute regulatory protein; Enzymes: CYP11A1, P450 cholesterol side chain cleavage enzyme; 3β-HSD,
3β-hydroxysteroid dehydrogenase; CYP17A1, P450 17α-hydroxylase/17.20-lyase; 17β-HSD3, 17β-hydroxysteroid dehydrogenase 3;
SRD5A1, 5α-reductase.

predominant isoform is 3β-HSD1, which is encoded by
Hsd3b1.25
Human 3β-HSD2 primarily catalyzes the conversion
of dehydroepiandrosterone to androstenedione, while
rat 3β-HSD1 predominantly catalyzes pregnenolone to
progesterone in the Leydig cells. Both require the cofac-
tor NAD+. The catalysis by 3β-HSD undergoes two steps
(Figure 20.1). The first step of the enzyme is to catalyze
dehydrogenation and is the rate-limiting. The second step
is to isomerize steroid molecule. 3β-HSD is critical for the
biosynthesis of all biologically active steroids in adrenals,
gonads (including Leydig cells), and placentas.26 Thus, the
disruption of 3β-HSD not only impairs Leydig cell func-
tions but also the functions of other endocrine systems.
Patients having the HSD3B2 mutation27,28 cannot make
testosterone, thus leading to male-to-female sex reversal
or a partial feminization.
CYP17A1
CYP17A1 is encoded by CYP17A1 (human)29 or Cyp17a1
(rat).30 Thus far, no additional CYP17A1 isoforms have
been identified.
CYP17A1 is a P450 cytochrome enzyme. It is located
in the smooth endoplasmic reticulum. CYP17A1 cataly-
sis depends on cytochrome P450 oxidoreductase, cyto-
chrome b5, and phosphorylation.31 Cytochrome b5 acts
to facilitate CYP17A1 17, 20-lyase. Human CYP17A1 is
phosphorylated on serine and threonine residues by a
cAMP-dependent protein kinase and the phosphorylated
CYP17A1 has increased 17,20-lyase activity.31 Human
CYP17A1 requires cytochrome P450 oxidoreductase for
the transfer of electrons. Both human and rat CYP17A1
enzymes catalyze two steps of reaction: 17α-hydroxylation
and 20-lyation. Each reaction requires cofactor NADPH.14
Human CYP17A1 primarily catalyzes pregnenolone to
17-hydroxyprogenolone and then to dehydroepiandros-
terone (Figure 20.1).32 However, rat CYP17A1 primarily
catalyzes progesterone into 17-hydroxyprogesterone and
then to androstenedione.33
A mutation of human CYP17A1 alters the steroidogen-
esis in the males, leading to defective masculinization that
can range from partial to complete pseudohermaphrodit-
ism and breast enlargement.34,35
17β-HSD3
There are 14 different 17β-hydroxysteroid dehydrogenase
isoforms.25 Different isoforms of 17β-HSD are encoded by
different genes. The only isoform involved in testosterone
production in Leydig cells is 17β-hydroxysteroid dehydro-
genase isoform 3 (17β-HSD3), which is encoded by human
HSD17B336 or rat Hsd17b3.16
17β-HSD3 is located in the smooth endoplasmic reticu-
lum in Leydig cells. It catalyzes the final step of androgen
biosynthesis, converting androstenedione into testos-
terone (Figure 20.1).16 17β-HSD3 requires NADPH as its
cofactor for the electron transfer.
The production of testosterone is considered as the
end-product in adult Leydig cells. The mutation of human
Cell signaling for testosterone biosynthesis in Leydig cells  247
H
H
H H
(a) HO
O
H
H
H H
HO
(b)
Figure 20.2  (a) Chemical structures of cholesterol and
(b) pregnenolone.
HSD17B3 causes various phenotypes, including pseudo-
hermaphroditism, with very low circulating testosterone
in males.37,38
SRD5A
Three distinct isoforms of SRD5A in both humans and
rats have been characterized. Human genes encode type
1 (SRD5A1),39 2 (SRD5A2),40 and 3 (SRD5A3).41 Rat genes
encode the isoforms of SRD5A with a similar designation
(Srd5a1, Srd5a2, and Srd5a3).
SRD5A catalyzes the activation of testosterone into a
more potent androgen, dihydrotestosterone (Figure 20.1).42–44
These enzymes use NADPH as a cofactor to transfer elec-
trons.16,45 The reaction by SRD5A involves an irreversible
breakage of the double bond between Δ4, 5  carbons with
the facilitation of NADPH.
No clear mutation of human SRD5A1 has been found to
be associated with human diseases. The human SRD5A2
mutation is associated with male pseudohermaphrodit-
ism.46 Human SRD5A3 encodes a protein that not only
catalyzes testosterone into dihydrotestosterone but also
converts polyprenol to dolichol.41 The human SRD5A3
mutation causes a congenital glycosylation disorder but
does not affect reproduction.44
CELL SIGNALING FOR TESTOSTERONE BIOSYNTHESIS
IN LEYDIG CELLS
Steroidogenesis in Leydig cells is regulated by lutein-
izing hormone (LH), which is secreted by the pituitary.
LH reaches Leydig cells via circulation and it binds to
the LH receptor (LHCGR, encoded by human LHCGR
or rat Lhcgr) on the surface of Leydig cells. This receptor
is mainly coupled to adenylate cyclase pathways.47 After
the LHCGR is bound by LH, the receptor causes a rapid
increase of intracellular cAMP level. The increased cAMP
level induces a series of downstream signaling to increase
248  Environmental toxicants on Leydig cell function
the expression levels of cholesterol-transporting proteins
and steroidogenic enzymes (Figure 20.1).
One cholesterol-transporting protein is scavenger recep-
tor class B member 1 (SCARB1, also known as SR-BI)
(Figure 20.1). SCARB1 is encoded by the human SCARB1
gene48 or rat Scarb1 gene.49 In rat testis, SCARB1 is primarily
expressed by Leydig cells.50 SCARB1 is a receptor for high-
density lipoprotein, and is a 509-amino acid protein with
two cytoplasmic C- and N-terminal domains separated by
a large extracellular domain. SCARB1 activity depends on
lipoprotein binding to its extracellular domain and subse-
quent lipid exchange at the plasma membrane to transport
cholesterol from the extracellular location into the cytosol.51
The second cholesterol-transporting protein is the ste-
roidogenic acute regulatory protein (StAR) (Figure 20.1).
StAR is encoded by human STAR and rat Star.52 StAR
mobilizes cytosolic cholesterol into the inner membrane
of the mitochondria. The mechanism by which StAR helps
cholesterol movement remains unclear but possible mech-
anisms are as a cholesterol-transporting shuttle53 or by
binding to cholesterol.54 Star is the rate-limiting step for
androgen production in Leydig cells, and thus the protein
is critical for androgen production.
Adrenals and Leydig cells share many common
enzymes, including CYP11A1 and 3β-HSD. These two
compartments may interact with each other. The exces-
sive glucocorticoids secreted by adrenal cortex can inhibit
testosterone production in Leydig cells by downregulat-
ing Lhcgr, Scarb1, Star, Cyp11a1, and other steroidogenic
enzymes55–57 or by inducing the apoptosis of Leydig cells.58
Interestingly, Leydig cells express two glucocorticoid
metabolizing enzymes,59–61 11β-hydroxysteroid dehy-
drogenase isoform 1 (11β-HSD1) and 2 (11β-HSD2). 11β-
HSD1 is an oxidoreductase and uses NADP+/NADPH as
cofactors and primarily acts as an oxidase in adult Leydig
cells, and 11β-HSD2 is a unidirectional NAD+-dependent
oxidase. Both protect Leydig cell function by inactivating
excessive glucocorticoids in Leydig cells.59–61 Thus, inhibi-
tion of both these enzymes in Leydig cells could disrupt
the Leydig cell function.
LEYDIG CELL DEVELOPMENT
In both humans and rats, Leydig cells develop from
stem Leydig cells.5 The first cells that have biomarkers in
the Leydig cell lineage are the progenitor Leydig cells.62
Progenitor Leydig cells are spindle-shaped cells and express
steroidogenic enzymes, including CYP11A1, CYP17A1, and
3β-HSD.63 Stem and progenitor Leydig cells have high pro-
liferative capacity and the increased numbers of these cells
are critical for the population of fetal Leydig cells and adult
Leydig cells. They eventually develop into mature types of
Leydig cells (fetal Leydig cells and adult Leydig cells) with
full capacity to produce androgens and insulin-like 3.5
ENVIRONMENTAL TOXICANTS TARGETING
LEYDIG CELLS
Environmental toxicants can block Leydig cell develop-
ment, downregulate the expression levels of testosterone
biosynthesis-related genes and insulin-like 3, or directly
inhibit testosterone biosynthetic and metabolizing enzyme
activities (Figure 20.1). Disrupting one or more steps in
Leydig cell development and steroidogenesis can poten-
tially cause lower testosterone and/or insulin-like 3 levels.64
Table 20.1 lists environmental toxicants that block Leydig
cell development and/or downregulate the expression lev-
els of testosterone biosynthesis- and metabolism-related
genes and insulin-3, and/or induce Leydig cell apopto-
sis. Table 20.2 lists environmental toxicants that directly
inhibit testosterone-biosynthetic enzyme activities. Table
20.3 lists the toxicants that directly inhibit steroid metabo-
lizing enzyme activities. The spectrum of environmental
toxicants on Leydig cells has been expanded to many cat-
egories of compounds, which include industrial materials
(perfluoroalkyl substances [PFASs], phthalates, bisphe-
nols, benzophenones, organochlorines, carbamates, poly-
cyclic aromatic hydrocarbon, organophosphates, azole
compounds, 1,2-dibromo-3-chloropropane [DBCP]), heavy
metals, and plant toxicants.
PFASs
PFASs are widely used for processing industrial and con-
sumer products as coatings of textiles, paper, and uphol-
stery and as reaction additives in various industrial
processes.65–67 The molecules of PFASs contain carbon and
fluorine atoms. Some PFASs also contain oxygen, hydro-
gen, or sulfur atoms. PFASs differ from one another in
the chain length (the number of carbon atoms) and other
elements besides carbon and fluorine atoms. For example,
perfluorododecanoic acid (PFDoA, C12) contains 12 car-
bon atoms, perfluorononanoic acid (PFNA, C9) contains
nine carbon atoms, perfluorooctanoic acid (PFOA, C8)
contains eight carbon atoms, perfluorooctane sulfonate
(PFOS, C8+S1) contains eight carbon atoms and a sulfur
atom, perfluorohexane sulfonate (PFHxS, C6+S1) contains
6 carbon atoms and a sulfur atom, and perfluorobutane
sulfonate (PFBS, C4+S1) contains 4 carbon atoms and a
sulfur atom. PFASs, especially PFOA, PFOS, and PFHxS,
are widespread around the world in part because of their
unique properties in the environment.68
When absorbed into the human body, they are
extremely persistent because their elimination half-lives
are over 4 years in humans.69 PFAS levels in the blood of
humans are related to their exposure levels and duration.
The blood levels of PFOS, PFOA, and PFHxS in the general
population in the United States in 2006 were 14.7, 3.4, and
1.5 ng/mL, respectively.70 Because of their high levels and
potential toxicities, PFOS and PFOA were no longer man-
ufactured in the United States after 2015. However, their
production in other countries, such as in China, is signifi-
cantly increased. As a result, the levels of PFOS and PFOA
in the cord blood were 2.48 and 6.96 ng/mL, respectively,
in residents of Shanghai in 2012.71
Evidence shows that PFASs interfere with the Leydig cell
functions. A survey of professional workers in a branch of
the 3M Company in Cottage Groove, Minnesota, which
manufactures PFOA, was conducted, and the study found
 Environmental toxicants targeting Leydig cells  249

that workers with higher serum PFOA levels had lower tes-
tosterone levels.72,73 A study of 225 Taiwanese 13–15-year-
old adolescents from 2009 to 2010 demonstrated that
higher levels of PFASs were associated with lower testos-
terone levels in males.74 A cross-sectional study of 247 men
in Denmark from 2008 to 2009 showed that there was a
negative association between serum PFOS levels and tes-
tosterone concentrations.75 Animal studies also showed
that rats and mice had lower testosterone secretions after
exposure to PFOS or PFOA.76–78
PFASs may have multiple mechanisms to lower the bio-
synthesis of testosterone and insulin-like 3 by (1) reducing
Leydig cell numbers, (2) downregulating the expression
levels of Leydig cell steroidogenesis-related genes, and
(3) inhibiting androgen-biosynthesizing enzyme activities.
Exposure to 5- and 10-mg/kg body weight (BW)/day
PFDoA to adult rats for 14 days lowered serum testos-
terone levels and downregulated Scarb1, Star, Cyp11a1,
Hsd3b1, and Cyp17a1.77 Low doses of PFDoA (0.2 and
0.5 mg/kg BW/day) for long-term (110 days) treatment also

Table 20.1  Toxicants that inhibit Leydig cell development, downregulating the expressions of testosterone
biosynthesis-related genes and insulin-like 3, and inducing cell apoptosis.
Action Sites Compounds Use
Leydig cell development Perfluorooctane sulfonate Surfactant
Dipropyl phthalate Plasticizer
Dibutyl phthalate Plasticizer
Dipentyl phthalate Plasticizer
Diisoheptyl phthalate Plasticizer
Bis(2-butoxyethyl) phthalate Plasticizer
Dicyclohexyl phthalate Plasticizer
Diethylhexyl phthalate Plasticizer
Di-n-octyl phthalate Plasticizer
Diisononyl phthalate Plasticizer
Methoxychlor/hydroxychlor Insecticide
Dimethoate Insecticide
Dichlorvos Insecticide
Downregulating the expressions of Perfluorooctane sulfonate Surfactant
testosterone biosynthesis-related genes Perfluorooctane acid Surfactant
and insulin-like 3 Perfluorododecanoic acid Surfactant
Dipropyl phthalate Plasticizer
Dibutyl phthalate Plasticizer
Dipentyl phthalate Plasticizer
Diisoheptyl phthalate Plasticizer
Bis(2-butoxyethyl) phthalate Plasticizer
Dicyclohexyl phthalate Plasticizer
Diethylhexyl phthalate Plasticizer
Di-n-octyl phthalate Plasticizer
Di-isononyl phthalate Plasticizer
Bisphenol A (bisphenol AF) Plasticizer
TCDD Industrial waste
Aroclor 1254 Industrial waste
PCB126 Industrial waste
Methoxychlor/hydroxychlor Insecticide
Zearalenone (zearalenol) Mycotoxin
Aflatoxin B1 Mycotoxin
T2-toxin Mycotoxin
Citrinin Mycotoxin
Causing Leydig cell apoptosis Heavy metals Industrial waste
Zearalenone (zearalenol) Mycotoxin
Deoxynivalenol Mycotoxin
Aflatoxin B1 Mycotoxin
cAMP signaling Triclosan Fungicide
Chlorpyrifos Insecticide
250  Environmental toxicants on Leydig cell function

Table 20.2  Toxicants that directly inhibit testosterone biosynthetic enzyme activities.
Enzyme Compounds Use Mode of action
CYP11A1 Methoxychlor/hydroxychlor Insecticide Noncompetitive
Lindane Insecticide Unknown
Etomidate Drug Competitive
Gossypol Plant toxin Unknown
3β-HSD Perfluorooctane sulfonate Surfactant Competitive
Perfluorooctane acid Surfactant Competitive
Dipropyl phthalate Plasticizer Competitive
Dibutyl phthalate Plasticizer Competitive
Dipentyl phthalate Plasticizer Competitive
Bis(2-butoxyethyl) phthalate Plasticizer Competitive
Dicyclohexyl phthalate Plasticizer Competitive
Bisphenol A Plasticize Competitive
Methoxychlor/hydroxychlor Insecticide Noncompetitive
Etomidate Drug Competitive
Triphenyltin Biocide Unknown
Tributyltin Biocide Unknown
Gossypol Plant constituent Competitive
Genistein Plant ingredient Competitive
CYP17A1 Bisphenol A Plasticizer Competitive
Prochloraz Fungicide Unknown
1,2-Dibromo-3-chloropropane Insecticide Unknown
Triphenyltin Biocide Unknown
Tributyltin Biocide Unknown
Gossypol Plant constituent Unknown
17β-HSD3 Perfluorooctane sulfonate Surfactant Noncompetitive
Perfluorooctane acid Surfactant Noncompetitive
Bis(2-butoxyethyl) phthalate Plasticizer Competitive
Dicyclohexyl phthalate Plasticizer Competitive
Bisphenol A Plasticizer Competitive
Benzophenones UV blocker Unknown
Methoxychlor/hydroxychlor Insecticide Noncompetitive
Triphenyltin Biocide Unknown
Tributyltin Biocide Unknown
Gossypol Plant constituent Competitive

reduced serum testosterone levels and downregulated Star
and Cyp11a1 expressions.79 PFDoA in vitro downregulated
Star expression at its promoter region in mouse mLTC-1
Leydig cells and primary rat Leydig cells.80 Exposure to
3- and 5-mg/kg BW/day PFNA to adult rats for 14 days
significantly lowered serum testosterone levels.81 Exposure
to 5- or 20-mg/kg/day PFOS to prenatal rats significantly
lowered fetal Leydig cell numbers and downregulated the
expression levels of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1,
and Cyp17a1.82 Exposure to 1-, 5-, or 10-mg/kg PFOS to
adult mice for 7, 14, or 21 days dose-dependently down-
regulated the expression levels of Star, Cyp11a1, Cyp17a1,
and Hsd3b1.78
PFOS and PFOA competitively inhibited rat testicu-
lar 3β-HSD activity with IC50 values of 1.3 and 53.2 μM,
respectively.68 Surprisingly, PFASs did not inhibit human
testicular 3β-HSD activity at ≤250 μM.68 In contrast,
PFOS and PFOA competitively inhibited human testicular
17β-HSD3 with IC50 values of 6.0 and 127.6 μM, respec-
tively.68 PFOA inhibited rat Leydig cell 17β-HSD3 with an
IC50 value of 17 μM.83 The inhibition of 3β-HSD and 17β-
HSD3 activities clearly caused the reduction of testoster-
one secretion by rat Leydig cells.83 PFOS and PFOA also
competitively inhibited human 11β-HSD1 with IC50 values
of 7.56 and 37.61 μM and rat 11β-HSD1 with IC50 values
of 3.45 and 45.31 μM, respectively.84 PFOS competitively
inhibited human and rat 11β-HSD2 with IC50 values of 48
and 293 nM, respectively.85 The potencies to inhibit human
and rat 11β-HSD1 and 11β-HSD2 are PFOS > PFOA >
PFHxS > PFBS.84,85 The inhibition of both 11β-HSD1 and
11β-HSD2 in Leydig cells could cause the inhibition of
Leydig cell steroidogenesis by excessive glucocorticoids.
 Environmental toxicants targeting Leydig cells  251

Table 20.3  Toxicants that directly inhibit steroid metabolizing enzyme activities.
Enzyme Compounds Use Mode of action
SRD5A1 Tributyltin Biocide Competitive
Genistein Food ingredient Unknown
SRD5A2 Triphenyltin Biocide Noncompetitive
Tributyltin Biocide Noncompetitive
Genistein Food ingredient Unknown
11β-HSD1 Perfluorooctane sulfonate Surfactant Competitive
Perfluorooctane acid Surfactant Competitive
Dipropyl phthalate Plasticizer Competitive
Dibutyl phthalate Plasticizer Competitive
Dipentyl phthalate Plasticizer Competitive
Bis(2-butoxyethyl) phthalate Plasticizer Competitive
Dicyclohexyl phthalate Plasticizer Competitive
Bisphenol A Plasticize Competitive
Methoxychlor/hydroxychlor Insecticide Competitive
Ziram Fungicide Competitive
11β-HSD2 Perfluorooctane sulfonate Surfactant Competitive
Perfluorooctane acid Surfactant Competitive
Dipropyl phthalate Plasticizer Competitive
Dibutyl phthalate Plasticizer Competitive
Dicyclohexyl phthalate Plasticizer Competitive
Mono-(2-ethylhexyl) phthalate Plasticizer Competitive
Bisphenol A Plasticizer Unknown
Methoxychlor/Hydroxychlor Insecticide Competitive
Ziram Fungicide Noncompetitive
Thiram Fungicide Noncompetitive

Phthalates Worldwide, approximately 8.4 million tons of plasticiz-

Phthalates are synthetic compounds that are esters cre-
ated by joining a phthalic anhydride with two alcohols
that range from methanol (C1) up to tridecyl alcohol
(C13). Phthalates can have either a straight chain or a
branching chain. They are widely used as plasticizers
and solvents in a variety of consumer products.86 Low-
molecular-weight phthalates (those with 1–6 carbons
in the alcohol moiety), including dimethyl (DMP, C1),
diethyl (DEP, C2), dipropyl (DPrP, C3), dibutyl (DBP, C4),
diisobutyl (DIBP, C4), dipentyl (DPeP, C5), butyl benzyl
(BBzP, C4/C6), dihexyl (DHP, C6), bis(2-butoxyethyl)
(BBOP, C6+O), and dicyclohexyl (DCHP, C6) phthalate,
are dominantly used in personal care products (such as
perfumes, lotions, and other cosmetics) or as solvents for
cellulose acetate, and in making lacquers and varnishes.87
High-molecular-weight phthalates (those with 7–13 car-
bons in the alcohol moiety), including diisoheptyl (DIHP,
C7), di(2-ethylhexyl) (DEHP, C8), di-n-octyl (DNOP, C8),
diisononyl (DINP, C9), diisodecyl (DIDP, C10), diundecyl
(DIUP, C11), and ditridecyl (DTDP, C13), are primarily
used as plasticizers in the manufacture of flexible vinyl
that is used in consumer products such as flooring and
wall coverings, food containers and covers, and medical
devices.87
ers are consumed every year, of which 88% are phthal-
ates (data from 2005).88 Phthalates are added to polymers
without chemically binding to the polymers and easily
leak out into the environment. The leached products in
the environment are significant because phthalates usu-
ally make up to 40% of the volume of the plastics.89 The
most abundantly used phthalates are DEHP, DBP, DEP,
and DINP.90
When absorbed into the human body, phthalate dies-
ters are rapidly converted into monoester metabolites; for
example, DEHP into mono-ethylhexyl phthalate (MEHP)
and DBP into mono-butyl phthalate (MBP).91 Some mono-
ester metabolites are believed to be more potent than their
parent compounds in their toxicity. It was found that
MEHP was 10 times more potent than DEHP to disturb
rat testis function92 and that MBP was 1000 times more
potent than DBP to inhibit androgen production and
downregulate steroidogenic gene (Cyp11a1 and Hsd3b1)
expression.93
A human can be exposed to phthalates via various
sources such as indoor air, dust ingestion, and food.94 For
example, DEHP is absorbed via food contamination.94
Low-molecular-weight phthalates such as DEP, DBP, and
BBzP may be dermally absorbed.95 Human blood and fat
tissue samples have significant levels of phthalates. DEHP,
252  Environmental toxicants on Leydig cell function
DEP, and DBP were detected at levels of 5.71 mg/L in
serum, of 0.30 mg/L in semen, and of 0.72 mg/kg in fat
samples.96
Evidence shows that phthalates interfere with Leydig
cell functions. Epidemiological studies claim that expo-
sure to phthalates may be associated with male reproduc-
tive malformity in male babies.97–99 A survey of 483 men
showed that MEHP was inversely associated with serum
testosterone levels.100 A study for serum levels of insulin-
like 3 (the Leydig cell biomarker) and testosterone and 14
phthalate metabolites in 1066 men showed that insulin-
like 3 levels were negatively associated with MEHP and
that MBP was negatively associated with total testosterone
and free testosterone levels.101 Animal studies showed that
rats and mice had lower testosterone secretions after expo-
sure to phthalates.102
Phthalates may have multiple mechanisms to lower
the biosynthesis of testosterone and insulin-like 3 by
(1)  disrupting Leydig cell development, (2) downregu-
lating Leydig cell steroidogenesis-related gene and Insl3
expressions, and (3) inhibiting the androgen-biosynthetic
enzyme activities.
Phthalates have been classified as antiandrogens.
However, phthalates do not block androgen receptors.90
Many in vivo studies using animal models indeed showed
that phthalates were antiandrogens, inducing androgen-
deficient reproductive malformations, lowering testoster-
one levels, and causing infertility.103–108
Phthalates induced abnormal Leydig cell aggrega-
tion in the fetal rat testis. Studies on DBP,109–111 DCHP,112
DEHP,113,114 and DINP115 showed that all these phthalates
induced abnormal Leydig cell aggregation in the fetal rat
testes. The induction of fetal Leydig cell aggregation by
phthalates was independent of their inhibition of andro-
gen.110 Phthalate-induced fetal Leydig cell aggregation
may contribute to the focal dysgenesis in the testis, which
causes reduced fertility.116
Phthalates downregulate the expression levels of
steroidogenesis-related genes and Insl3 in fetal Leydig
cells. Studies on DBP,117 BBzP,118 DPeP,119,120 DCHP,112
DEHP,113,114,118,121 and DINP115 showed that some of
the expression levels of these genes, including Insl3,
Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, and Cyp17a1, were
downregulated.
Studies showed that DEHP and DBP inhibited Leydig
cell differentiation in the rat Leydig cell regeneration
model,113,122,123 although low doses of these phthalates may
increase the proliferation of Leydig cell precursors124–126 and
the commitment of stem cells into the Leydig cell lineage.123
Studies showed that some phthalates directly inhibited
testosterone biosynthetic enzyme activities. Some low-
molecular-weight phthalates, including DPrP, DBP, DPP,
BBOP, and DCHP significantly inhibited 3β-HSD activi-
ties with IC50 values of 123.0, 24.1, 25.5, 50.3, and 25.5 μM
for human enzyme, and 62.7, 30.3, 33.8, 82.6, and 24.7 μM
for rat enzyme, respectively.127 BBOP and DCHP potently
inhibited human (IC50 values of 23.3 and 8.2  μM) and
rat (IC50 values of 30.24 and 9.1 μM) 17β-HSD3 activity,
respectively.127 High-molecular-weight phthalates or some
low-molecular-weight phthalates (DMP and DEP) did
not inhibit these enzyme activities at ≤1mM.127 Modes
of action of these phthalates to inhibit 3β-HSD and 17β-
HSD3 are competitive against steroid substrates.127 Some
phthalates also directly inhibited human 11β-HSD2 activ-
ities. DPrP, DBP, and DCHP competitively inhibited rat
11β-HSD2 with IC50 values of 85.59, 13.69, and 32.64 μM,
respectively. Diphthalates with 1–2 and 7–10 carbons in
the alcohol moiety did not inhibit 11β-HSD2 at 1 mM.128
MEHP competitively inhibited human 11β-HSD2 with
IC50 values of 110.8 and 121.8 μM, while DEHP did not.128
Bisphenols
Bisphenols are a group of compounds with two hydroxy-
phenyl groups. The most abundantly produced bisphenol
is bisphenol A (BPA). BPA is used to make certain plas-
tics and epoxy resins. A human can be exposed to BPA
via various sources such as indoor air, dust ingestion, and
food.129 Human urine samples had significant levels of
BPA with 95% detection.130–132 Men had higher BPA levels
(1.49 ng/mL) than women (0.64 ng/mL), possibly because
of androgen-related metabolism of BPA.133
Concerns about BPA have arisen because BPA binds to
estrogen receptors.134,135 Evidence shows that bisphenols
(specifically BPA) interfere with the Leydig cell functions.
A study with 560 adult men in China showed the inverse
association of serum BPA levels and testosterone levels.136
A National Health and Nutrition Examination Survey
(NHANES) 2011–2012 survey of serum testosterone levels
and urinary BPA levels in adolescents showed the inverse
association between these two indicators in boys.137
BPA may have multiple mechanisms to lower the testos-
terone biosynthesis by (1) blocking Leydig cell development
via as an androgen receptor antagonist, (2)  downregu-
lating Leydig cell steroidogenesis-related gene expres-
sions, and (3) inhibiting androgen-biosynthetic enzyme
activities.
BPA is androgen receptor antagonist because it binds to
the human androgen receptor, blocking dihydrotestosterone-
mediated androgen receptor transcription activity134,135
with a similar potency as the androgen receptor antago-
nist flutamide.134,135 Androgens have been shown to criti-
cal for Leydig cell development, as shown by the evidence
of lower expression levels of CYP17A1 and 17β-HSD3 in
the androgen receptor insensitive or knockout rats and
mice.138–140
Exposure to BPA caused a lower testosterone produc-
tion in rodents.141,142 Male rats exposed to BPA (20, 100,
and 200 mg/kg BW/day, subcutaneously) from the pre-
pubertal to the adult period for 6 weeks had a reduced
plasma and testicular testosterone levels and lower expres-
sions of steroidogenic enzymes.143 Male rats exposed to
BPA (2.4  μg/kg/day, gavage) from the prepubertal to the
pubertal period for 14 days had a reduced serum testos-
terone level and the treatment of BPA to rat adult Leydig
cells significantly inhibited testosterone production and
Cyp17a1 expression.141 Adult male rats exposed to BPA

(400 or 800 μmol/kg BW/day) for 14 days had significantly
lower testosterone production and Cyp17a1 expression.144
Adult mice intraperitoneally exposed to BPA (0.5, 50, and
100 μg/kg BW/day) for 60 days had increased oxidative
stress and lower testosterone production and downregu-
lated Star levels.145
BPA directly inhibits testosterone biosynthetic enzyme
activities. Although BPA did not directly inhibit CYP11A1
activity,146 it inhibited three other testosterone biosyn-
thetic enzyme activities.147 BPA inhibited human and rat
testicular 3β-HSD with IC50 values of 7.9 and 26.5 μM, and
human and rat CYP17A1 activities with IC50 values of 18.9
and 64.6 μM, as well as human and rat 17β-HSD3 with IC50
values of about 100 μM, respectively.147 BPA is a competi-
tive inhibitor for both 3β-HSD147 and CYP17A1148 against
steroid substrates, possibly because of its similar chemi-
cal structure to that of steroid substrates. BPA also inhib-
ited human and rat 11β-HSD1 activities with IC50 values
of 14.81 and 19.39 μM, respectively.149 BPA weakly inhib-
ited human and rat 11β-HSD2 activity with IC50 values of
about 100 μM.149
Besides BPA, its analogues (bisphenol S, bisphenol AF,
bisphenol F, and tetrabromobisphenol) that were recently
used as alternatives had similar actions to BPA. Human
fetal testes exposed to 10-nM BPA, bisphenol S, or bisphe-
nol AF had lower basal testosterone secretion.150 Adult
male rats exposed to different doses of bisphenol S (1–50
μg/kg BW/day) had significantly higher testicular reactive
oxygen species (ROS) and lower testosterone levels.151 Male
rats exposed to bisphenol AF (50 and 200 mg/kg BW/day)
for 14 days had lower testosterone production and Star
expression levels.152 BPA, bisphenol F, and tetrabromobi-
sphenol antagonized human androgen receptors with IC50
values of 39, 20, and 982 nM, respectively.153
Benzophenones
Benzophenones are a family of compounds to protect
from ultraviolet (UV) light. They are used in personal care
products such as lip balm and nail polish as well as in inks,
imaging, and clear coatings in the printing industry. These
chemicals structurally differ in the positions and num-
bers of hydroxy groups and methoxyl groups in phenol.
Benzophenone-2 and benzophenone-3 are common ingre-
dients in sunscreen. Benzophenones are widely exposed
because they migrate into food from packaging.154
Although there are no human epidemiological data,
in vitro testing showed that many benzophenones have
antiandrogenic activities. An in vitro study using human
17β-HSD3 to test the inhibitory potencies of different ben-
zophenones (benzophenones 1–8 and 12) showed that ben-
zophenone 1 was the most potent inhibitor with an IC50
value of 1 μM while others had IC50 values of 47–111 μM.155
Benzophenone 1 inhibited human 17β-HSD3 more spe-
cifically than other 17β-hydroxysteroid dehydrogenases
(17β-HSD1 and 17β-HSD2) since the IC50 values for 17β-
HSD1 and 17β-HSD2 are over 20 μM and it did not inhibit
17β-HSD5.155 Benzophenone 1 also inhibited testosterone
production in mouse and rat testes.155
Environmental toxicants targeting Leydig cells  253
Organochlorines
Polychlorinated biphenyls (PCBs), pentachlorophenol
(PCP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
dichlorodiphenyltrichloroethane (DTT), and methoxy-
chlor are organochlorines. PCBs are widely used as dielec-
tric and coolant fluids in electrical apparatus, carbonless
copy paper, and in heat transfer fluids. There are over 200
PCB congeners. PCP is an organochlorine compound used
as a pesticide and a disinfectant. TCDD is formed as a side
product in organic synthesis and burning of organic mate-
rials. These chemicals may have a common mechanism of
actions via aryl hydrocarbon receptor (AHR).
Exposure to TCDD (1–25 μg/kg BW/day) decreased
serum testosterone levels and 3β-HSD and 17β-HSD
activities in rats.156 Further study showed that TCDD dis-
rupted cholesterol-transporting protein (possibly StAR)
and CYP11A1.157 Rats exposured to TCDD (100 ng/kg BW/
day) for 15 days downregulated StAR and steroidogenic
enzyme expressions.158 Exposure to TCDD (0.3–1 μg/kg
BW/day) to pregnant dams also inhibited testicular tes-
tosterone production.159 In vitro treatment of TCDD sig-
nificantly downregulated Cyp11a1 transcript and reduced
testosterone output in rat adult Leydig cells.160 Using Ahr
knockout mice, it was demonstrated that TCDD down-
regulated Lhcgr and Cyp11a1 expressions via the AHR
pathway.161
Exposure to Aroclor 1254 (a mixture of PCB, 1, 2, and
5 mg/kg BW/day) to lactating rats for 21 days downregu-
lated the expressions of Srd5a1 and androgen receptor.162
Aroclor 1254 (10–100 nM) in vitro significantly inhibited
basal and LH-stimulated testosterone production and
CYP11A1 and 3β-HSD activities163 and downregulated
Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3 mRNA lev-
els in isolated rat adult Leydig cells.164
Rats exposed to PCB126 or PCB169 (3 or 30 μg/kg BW/
day) from fetal days 7 to 21 reduced testosterone levels
in male pups at 21 days postpartum.165 PCB126 inhib-
ited the conversion of progesterone and androstenedione
into testosterone, suggesting that 3β-HSD and 17β-HSD3
were inhibited.166 PCB126 also inhibited Cyp11a1 expres-
sion in Leydig cells with a mechanism similar to TCDD.167
Continuous exposure to Aroclor 1242 (8 and 80 μg/kg
BW/day) to lactating dams reduced androgen production
of adult male offspring.168
DDT is an organochlorine used as an insecticide and
was banned due to its persistent pollution. It has several
metabolites that also have toxicities to the Leydig cells.
3-Methylsulfonyl-DDE is a potent Leydig cell toxicant
formed from DDT and is widely present in human blood,
milk, and fat tissue and can inhibit LH-stimulated testoster-
one production. Studies showed that 3-methylsulfonyl-DDE
disrupted the mitochondrial proteins of porcine  Leydig
cells.169,170
Methoxychlor is another synthetic organochlorine
pesticide used as an alternative to DDT. Methoxychlor
has applications for protecting crops, o ­rnamentals,
livestock, and pets against fleas, mosquitoes, and
254  Environmental toxicants on Leydig cell function
cockroaches. Methoxychlor was banned in the
European Union in 2002 and in the United States in
2003. However, it is still widely used in other countries
(see reviews171,172). After being absorbed to mammalian
bodies, methoxychlor can be metabolized into the toxic
metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroeth-
ane (hydroxychlor).173 Methoxychlor may have multiple
mechanisms to lower the testosterone biosynthesis by
(1) blocking Leydig cell development, (2) downregulat-
ing Leydig cell steroidogenesis-related gene expressions,
and (3) directly inhibiting the androgen-­biosynthetic
enzyme activities.
Exposure to methoxychlor (100 and 200 mg/kg BW/
day) to male rats from postnatal day 21 through puberty
significantly reduced testosterone production without
affecting LH secretion.174 Adult male rats exposed to 100
or 500 mg/kg BW/day methoxychlor for 28 days had lower
testosterone levels.175 Administration of methoxychlor
(40 and 200 mg/kg BW/day) to adult male rats for 14 days
also reduced serum testosterone levels.176 Purified rat
Leydig cells after treatment with hydroxychlor had a lower
androgen output because Cyp11a1 was downregulated.173
Exposure to methoxychlor (10 and 100 mg/kg BW/day) to
adult male rats for 25 days delayed Leydig cell regenera-
tion.177 Interestingly, prenatal exposure to methoxychlor
(10, 50, or 100 mg/kg/day) to rats increased fetal Leydig
cell numbers; however, at a high dose methoxychlor
directly inhibited testosterone production.178
Hydroxychlor in vitro directly inhibited testoster-
one secretion by rat Leydig cells via inhibiting CYP11A1
activity at ≥100 nM.179 Methoxychlor is an irreversible pig
CYP11A1 inhibitor.180 Methoxychlor noncompetitively
inhibited human and rat testicular 3β-HSD with IC50 val-
ues of 53.2 and 46.15 μM, respectively. Hydroxychlor is
more potent than methoxychlor to inhibit 3β-HSD with
IC50 values of 8.2 and 13.8 μM.181 Hydroxychlor also inhib-
ited human and rat 17β-HSD3 with IC50 values of 12.1 and
32.0 μM and inhibited human and rat CYP17A1 with IC50
values of 1.13 and 6.87 μM, respectively.182 Methoxychlor
and hydroxychlor competitively inhibited human 11β-
HSD1 with the IC50 values 1.917 and 8.88 μM, respec-
tively,183 and hydroxychlor inhibited rat 11β-HSD1 with an
IC50 value of 9.15 μM.183 Hydroxychlor inhibited human
and rat 11β-HSD2 activities with IC50 values of 55.57 and
12.96 μM.183
Lindane is an organochlorine insecticide that
impaired rat reproductive system.184,185 Lindane inhib-
ited LH-stimulated testosterone production by rat Leydig
cells,186,187 suggesting that the compound might affect tes-
ticular steroidogenesis.188 Indeed, lindane inhibited mouse
CYP11A1 activity.189
Endosulfan is an organochlorine insecticide that is
being phased out globally. It is still used extensively in
India, China, and a few other countries. Endosulfan is a
potent human androgen receptor antagonist in an in vitro
screening assay and inhibited testosterone production.190
Triclosan is an antibacterial and antifungal organochlo-
rine used for consumer products. Treatment of triclosan
(0.001–10 μM) resulted in a significantly decreased activity
of adenylyl cyclase enzyme and lower testosterone section
in isolated rat Leydig cells, and this effect can be prevented
by the treatment of forskolin, indicating that triclosan tar-
gets adenylyl cyclase enzyme.191
Carbamates
Carbamates are used as insecticides and fungicides.
Maneb is a widely used fungicide in agriculture. Exposure
of maneb (1 and 4 mg/kg BW/day, i.p.) for 9–18 days to
male rats reduced testosterone production and CYP11A1
activity in Leydig cells.192 Carbendazim is a metabolite
of benomyl, one of the most widespread environmental
contaminants. Exposure to carbendazim (25 mg/kg BW/
day) to male rats for 48 days significantly reduced testos-
terone levels and 3β-HSD and 17β-HSD3 activities without
affecting serum LH levels.193 Exposure to carbendazim to
rats also caused an increase in ROS production and the
downregulation of Star mRNA levels. The addition of a
flavonoid can prevent this, suggesting an ROS-inducing
mechanism.194
Molinate is a thiocarbamate that is a selective herbi-
cide to control weeds. Exposure to molinate (40 mg/kg
BW/day) to rats for 7 days inhibited testosterone levels.195
Ziram is a widely used thiocarbamate fungicide for crops.
Ziram competitively inhibited rat 11βHSD1 with an IC50 of
87.07 μM. Ziram noncompetitively inhibited both rat and
human 11β-HSD2 with IC50 values of 90.26 and 34.93 μM,
possibly interacting the cysteine residues of 11β-HSD2.196
Thiram is a thiocarbamate fungicide and animal repellent.
It inhibited human 11β-HSD2 with a similar mechanism
to ziram.197
Polycyclic aromatic hydrocarbon
Polycyclic aromatic hydrocarbons (PAHs) are a family of
hydrocarbon compounds containing multiple aromatic
rings. They are produced by incomplete combustion of coal,
gas, and other organic matters. There are over 100  PAH
chemicals. One of the PAHs is benzo[a]pyrene (B[a]P), a
ubiquitous environmental pollutant. Oral exposure to B[a]
P reduced serum and intratesticular fluid testosterone
levels in rats and downregulated the expression levels of
Star and Hsd3b1 in Leydig cells.198 7,12-dimethylbenz[a]
anthracene (DMBA) is a PAH metabolite and Leydig cells
are involved in PAH and DMBA metabolism.199 Exposure
to DMBA to MA-10 Leydig cells and rat primary Leydig
cells inhibited cAMP-stimulated steroid production.200
Organophosphates
Organophosphates include all the insecticides, herbicides,
and some flame retardants and plasticizers.
Chlorpyrifos, dimethoate, dichlorvos, and malathion
are organophosphate insecticides. Piperophos is an
organophosphate herbicide. An in vitro human androgen
receptor screening assay showed that chlorpyrifos and
piperophos are potent androgen receptor antagonists.190 In
vitro treatment of piperophos and chlorpyrifos decreased
testosterone secretion and downregulated Cyp11a1,

Hsd3b1, Hsd17b3, and Star in rat Leydig cells.190
Chlorpyrifos also decreased in LH-stimulated stimulated
cAMP production.190
In utero and lactational exposure to dimethoate (4, 8,
and 16 mg/kg BW) from gestational day 6 to postnatal
day 21 to mice significantly reduced Leydig cell number,
size, and lowered plasma LH and testosterone levels in
the neonates.201 Dimethoate inhibited testosterone bio-
synthesis in isolated rat Leydig cells and this effect can
be restored by the treatment polyunsaturated fatty acids,
which increased the mitochondrial cholesterol pool avail-
able for testosterone biosynthesis.202 Dimethoate inhibited
testosterone biosynthesis in Leydig cells may be caused
by downregulating Star expression due to the stimulation
of COX-2 because the treatment of rofecoxib (a selective
COX-2 inhibitor) prevented the effect of dimethoate.203
Dimethoate also decreased CYP11A1 activity and choles-
terol transportation in MA-10 mouse Leydig cells.204
Dichlorvos205,206 and malathion206 treatment disrupted
Leydig cell numbers and lowered testosterone production
in rats. Exposure to dichlorvos (30 mg/kg/day through
dermal painting) for 90 days reduced rat Leydig cell
numbers.207
Tricresyl phosphate is an organophosphate compound
that is used as a plasticizer and for diverse other applica-
tions. Tricresyl phosphate in vivo and in vitro inhibited
testosterone production in rat Leydig cells.208
Many organophosphates—for example, triphenyl phos-
phate and tris-(2-chloroethyl)—phosphate, are also used
as flame retardants. Triphenyl phosphate and tris-(2-­
chloroethyl) phosphate in vitro significantly downregu-
lated the expressions of Cyp11a1, Cyp17a1, Hsd3b1, and
Hsd17b3 genes and inhibited testosterone production in
TM3 mouse Leydig cells.209
Azole compounds
Azole compounds refer to any of the heterocyclic com-
pounds with a five-membered ring of carbon atoms and
two to three nitrogen atoms. Azole compounds include
drugs, plant fungicides, and plant growth retardants.
Many azole compounds such as ketoconazole inhib-
ited cytochrome P450 to exert their effects. They inhibit
cytochrome P450-dependent 14α-demethylase activity
that catalyzes the conversion of lanosterol to ergosterol,
an essential component of fungal cell membranes used to
kill fungi.210 Some of these drugs may also have side effects
that disrupt Leydig cell functions. Ketoconazole inhibited
testosterone production in rat Leydig cells by more than
50% at 0.1 μg/mL. The imidazole antifungal drugs econ-
azole and miconazole are used for skin or vaginal fungal
infections. They decreased cAMP-stimulated StAR pro-
tein expression posttranscriptionally in Leydig cells to
inhibit testosterone production.211
Etomidate is an intravenous anesthetic agent. In intact
Leydig cells, 30-μM etomidate inhibited androgen syn-
thesis in isolated rat Leydig cells. Etomidate competitively
inhibited both CYP11A1 and 3β-HSD1 activities with IC50
values of 12.62 and 2.75 μM, respectively. In addition,
Environmental toxicants targeting Leydig cells  255
etomidate downregulated the expression levels of Cyp11a1,
Hsd17b3, and Srd5a1.212 At 1 μM, etomidate inhibited tes-
tosterone and progesterone production in isolated rat
Leydig cells.213
Rosiglitazone is a thiazolidinedione drug used to treat
diabetes. Exposure to rosiglitazone (5 mg/kg BW/day) for
15 days to rats inhibited testosterone production, possibly
via inhibiting CYP11A and 3β-HSD activities.214
Many azole chemicals, such as prochloraz, epoxicon-
azole, tebuconazole, and iprodione are used for fungicides
to protect plants in the agriculture field. Prochloraz is
classified as an antiandrogen. Maternal exposure to pro-
chloraz caused malformation of male reproductive tracts
in fetal male rats and reduced steroidogenesis in the tes-
tis.215 Prochloraz decreased serum testosterone levels and
delayed puberty in males during pubertal exposure.216
Exposure to prochloraz (50 and 150 mg/kg BW/day) to
rats from gestational day 7 to postnatal day 16 inhibited
testicular testosterone levels but increased progester-
one levels without affecting Cyp17a1, suggesting a direct
inhibition of CYP17A1 in the Leydig cells.217 Indeed, pro-
chloraz inhibited rat testicular CYP17A1 activity with Ki
around 1 μM.215 Using human adrenal H295R cells, pro-
chloraz also concentration-dependently inhibited human
CYP17A1 activity,218 and the inhibition was more selective
since it did not inhibit another CYP enzyme CYP11B1,
which is required for glucocorticoid biosynthesis.218
Exposure to epoxiconazole (15 or 50 mg/kg BW/day)
or tebuconazole (50 or 100 mg/kg BW/day) from gesta-
tional day 7 to postnatal day 16 to rats reduced testos-
terone production in the testis.219 Exposure to iprodione
(50, 100, or 200 mg/kg BW/day) from postnatal days 23
to 52 decreased testosterone, 17α-hydroxyprogesterone,
and androstenedione whereas serum LH was unaffected.
It also reduced ex vivo testosterone and progesterone pro-
duction, suggesting that it affects steroidogenesis through
enzyme inhibition of the steroidogenic pathway before
CYP17A1.220
DBCP
DBCP is a pesticide that has been used for over 20 years to
control plant worms. It was banned in the United States in
1977 because it induced infertility in male workers.221–223
After exposure to DBCP, males may develop oligospermia
and hypogonadism; however, the cause is reversible.224–226
Exposure to DBCP (1–5 mg/kg BW/day) to adult rats for 6
months significantly reduced intratesticular testosterone
levels and LHCGR numbers in adult Leydig cells with the
elevation of LH levels indicating direct impairment in the
testis.227 Kelce et al.228 demonstrated that DBCP also had a
direct inhibitory effect on the 17α-hydroxylase activity of
CYP17A1 but not the 17,20-lyase activity.228
Metals
Many heavy metals (cadmium, chromium, arsenic, and
organotins) are present in the environment because of
industrial wastes or the widespead use of metal-containing
materials.
256  Environmental toxicants on Leydig cell function
Cadmium is used as the stabilizer in the manufacture
of polyvinyl chloride polymer, phosphate fertilizers, pre-
servative pigments, and batteries. Cigarettes are also a
major source of cadmium. Each cigarette contains 5 mg
of cadmium. Cigarette smokers had significant levels of
blood cadmium and the levels of cadmium in blood of
smokers was 4–5 times higher than those in nonsmok-
ers.229 Cadmium has a very long elimination half-life of
20–40 years and could lead to accumulation in the human
body.230 When cadmium was exposed, it strongly accumu-
lated in the interstitial cells in rat testis.231 Cadmium is a
Leydig cell toxicant. Exposure to cadmium (0.015g/L in
drinking water) for 1, 3, 6, and 12 months to rats reduced
Leydig cell numbers and cell size.232 A low dose of cad-
mium reduced Leydig cell numbers in mice.233 Cadmium
exposure to rats induced an increase in ROS level and
decreased Star and Hsd3b1, and vitamin C and vitamin
E were able to prevent the impairment.234 Exposure to
cadmium (3 mg/kg BW/week, subcutaneous) to rats for
4 weeks inhibited serum testosterone levels, and alpha-
tocopherol was able to prevent it. Cadmium could induce
ROS in rat Leydig cells to inhibit testosterone production
and 3β-HSD activity, and treatment with N-acetylcysteine
was capable of preventing the damage.235 These results sug-
gest a cadmium-mediated ROS-inducing mechanism236,237
that could decrease intracellular levels of cAMP and dihy-
drolipoamide dehydrogenase activity to lower testoster-
one production in Leydig cells.230,238
Lead is heavy and often causes the intoxication. Lead
environmental contamination includes mining, smelting,
manufacturing and recycling activities, and industrial uses
(leaded paint and leaded gasoline). In MA-10 cells, treat-
ment with lead suppressed the cAMP-induced expression
of StAR and progesterone production.239 Moreover, cad-
mium, a calcium channel blocker, abolished the inhibitory
effect of lead on MA-10 cell steroid production. This indi-
cates that lead might act on calcium channel to regulate
MA-10 cell steroidogenesis.239
Chromium is a heavy metal that is commonly used in
industrial processes such as leather tanning operations,
metal processing, stainless steel welding, chromate pro-
duction, and chrome pigment production. When chro-
mium was exposed, it also strongly accumulated in the
interstitial cells in rat testis.231 Chromium may also be a
Leydig cell toxicant. Exposure to hexavalent chromium
(20, 40, and 60 mg/kg BW/day) for 90 days reduced Leydig
cell numbers, downregulated Hsd3b1, and decreased tes-
tosterone levels.240 In vitro treatment of chromium to TM3
Leydig cells inhibited steroid production in these Leydig
cells.241,242
Mercury is a heavy metal. Mercury pollution in indus-
try is often in an inorganic form. However, organic
mercury compounds, such as methylmercury, are con-
centrated in the food chain because it can be produced
inside fish when they are exposed to inorganic mercury.
Exposure to methylmercury (20 mg/L) via drinking
water to rats for 8 weeks reduced plasma testosterone
levels.243 Oral exposure to mercuric chloride at doses of
0.01, 0.05, and 0.1 μg/mL for 1–7 months caused Leydig
cell death.243
Arsenic chemicals are present in the environment and
they may impair Leydig cell function. Exposure of arse-
nic (0.15, 0.3, 1.5, and 3.0 mg/kg BW/2 days) for 3 weeks
reduced LH and testosterone levels and downregulated
Cyp11a1, Hsd3b, and Cyp17a1 mRNA levels.244 Exposure
to sodium meta-arsenite (30–40 mg/L) via drinking water
to adult mice for 30, 45, and 60 days induced Leydig cell
atrophy.245 Exposure to arsenic (3 mg/kg BW/day) for
30 days to adult mice led to the reduction of 3β-HSD and
17β-HSD3 activities and testosterone levels and decreases
in glutathione S-transferase activity and glutathione lev-
els. Coadministration of α-tocopherol and ascorbic acid
prevented the damage, suggesting that arsenic damages
Leydig cells via increasing testicular ROS.246
Cobalt and copper are also heavy metals that can inhibit
steroid production in TM3 Leydig cells.242
Organotins are organic metals. Organotins are widely
used as agricultural fungicides, rodent repellents, and anti-
fouling biocides for ships and fishing nets.247 Increasing
pollution due to organotins in the environment has been
found. Organotins may be Leydig cell toxicants. Organotins
can directly inhibit many androgen-biosynthetic and
metabolizing enzyme activities. Tributyltin inhibited
rat 3β-HSD with a Ki of 2.4 μM.248 Tributyltin inhibited
pig and rat CYP17A1 with IC50 values of 117 and 50 μM,
respectively.249 Tributyltin inhibited pig 17β-HSD3 with
the IC50 value of 48 nM.249 Tributyltin inhibited human
SRD5A1 and SRD5A2 with IC50 values of 19.9 and 10.8 μM,
respectively.250 However, the modes of action are different,
being a competitive inhibition for SRD5A1 and irrevers-
ible for SRD5A2.250 Tributyltin and triphenyltin directly
inhibited pig CYP17A1 with an IC50 value of 117 μM.249
Triphenyltin directly inhibited pig CYP17A1 with an IC50
value of 117 μM.249 Triphenyltin inhibited pig 17β-HSD3
with an IC50 value of 148 nM.249 Triphenyltin also inhibited
human 3β-HSD2, 17β-HSD3, and SRD5A2 with IC50 values
of 4.0, 4.2, and 0.95 μM, respectively.251 Triphenyltin pos-
sibly targeted the critical cysteine residues of SRD5A2 to
irreversibly inhibit SRD5A2 activity.251
Plant toxicants
Gossypol is a polyphenolic compound isolated from cotton
seeds.252 Gossypol exposure could come from the inges-
tion of cotton-seed oils and materials. Gossypol was once
developed as a male contraceptive,253 but it apparently has
some severe side effects, including an effect on Leydig cell
function. Men with long-term ingestion of crude cotton
seed oil that contains gossypol had abnormal Leydig cell
function.254 Gossypol inhibited hCG-induced testosterone
production in cultured canine Leydig cells.255 Gossypol at
10–20 μM in vitro also inhibited basal and LH-stimulated
testosterone production in mouse Leydig cells.256 Gossypol
contains enantiomers, (+) gossypol and (-) gossypol, and
only the (-) gossypol was found to have the antifertility
function.257 However, at ≥21 μM both enantiomers inhib-
ited LH-stimulated testosterone production in mouse

Leydig cells.257 At higher concentrations, gossypol inhib-
ited cAMP production in Leydig cells.258 Further studies
showed that gossypol directly inhibited some steroido-
genic enzymes in Leydig cells. Gossypol inhibited bovine
CYP11A1 activity at 30 μM.259 Gossypol inhibited human
and rat Leydig cell 3β-HSD activities with IC50 values of
3 and 0.2 μM, respectively.260 Gossypol inhibited human
and rat 17β-HSD3 in a clear enantiomer-specific manner,
with (−)-gossypol in inhibition of human and rat enzyme
activities (IC50 values of 0.36 and 3.43 μM, respectively)
and (+)-gossypol inhibition of human and rat enzyme
activities (IC50 values of 1.13 and 10.93 μM, respectively).260
Gossypol inhibited SRD5A1 more potently than SRD5A2
activity.261 As well, gossypol inhibited the glucocorticoid
metabolizing enzyme, human and rat 11β-HSD2, with
IC50 values of about 1 μM.262
Several flavones and isoflavones might also target Leydig
cells. One of them is genistein. Genistein is a soy isofla-
vone and a phytoestrogen that is widely distributed in the
human and animal diet. Several studies showed that iso-
flavones could inhibit testosterone production in Leydig
cells.263 Indeed, genistein competitively and potently
inhibited human and rat Leydig cell 3β-HSD activity with
IC50 values of 90 and 640 nM, respectively.264 Genistein is
a much more potent inhibitor of SRD5A2 than SRD5A1.261
Considering that there is increasing consumption of soy-
based food products and their potential effects on Leydig
cells, these findings are greatly relevant to public health.
Many fungi produce toxins. Zearalenone is one of them.
Zearalenone is a mycotoxin produced by some Fusarium
and Gibberella species. Studies have pointed to the pos-
sible toxicity of zearalenone in Leydig cells. Zearalenone
inhibited LH-stimulated testosterone production in mouse
Leydig cells.265 However, it did not affect the expression
and the binding activity of LHCGR.265 It also downregu-
lated Cyp11a1, Cyp17a, and Hsd17b3 mRNA levels, possibly
via suppressing Nur77 (a transcription factor) expres-
sion in mouse Leydig cells.265 Zearalenone inhibited cell
growth and increased autophagy in rat Leydig cells.266 It
was found that zearalenone induced Leydig cell apoptosis
via an endoplasmic reticulum stress-dependent signaling
pathway.267 Proteomics analysis showed that zearalenone
increased energy production through promoting fatty acid
uptake and beta-oxidation and increased excessive oxida-
tive stress, thus possibly leading to lower steroidogenic
enzyme expression levels.268 Prevention of ROS did not
seem to restore steroidogenesis in MA-10 Leydig cells,269
suggesting that its ROS-inducing effect does not con-
tribute to the inhibition of steroidogenesis. α-Zearalenol
(0.01–100 μM), a zearalenone metabolite, also suppressed
hCG-induced testosterone production and downregulated
Star, Cyp11a1, and Hsd3b1 in mouse Leydig cells,270 indi-
cating a similar mechanism to that of zearalenone.
Deoxynivalenol is a type B trichothecene and is a myco-
toxin that occurs predominantly in grains. Deoxynivalenol
inhibited Leydig cell steroidogenesis and oxidative stress,
and prevention of oxidative stress also cannot restore its sup-
pression of steroidogenesis in MA-10 mouse Leydig cells.269
References 257
Aflatoxins are chemicals that are produced by certain
molds. Aflatoxin B1 reduced the responsiveness of rat
Leydig cells to LH/hCG for stimulating testosterone pro-
duction.271 Aflatoxin B1 downregulated Star, Hsd3b1, and
Hsd17b3 mRNA levels, possibly via increasing ERK phos-
phorylation and suppressing p38 MAPK and JNK activa-
tion in rat Leydig cells.272
T-2 toxin is one of the mycotoxins produced by several
fungi.273 T-2 toxin (1–100 nM) inhibited hCG-stimulated
testosterone production and downregulated Star, Cyp11a1,
and Hsd3b1 mRNA levels in mouse Leydig cells.274 T-2
toxin inhibited LH-stimulated testosterone biosynthesis
in primary mouse Leydig cells.275
Citrinin is a mycotoxin that is often found in food.
Citrinin (50 and 100 μM) inhibited hCG-stimulated
testosterone production and downregulated StAR,
CYP11A1, and 3β-HSD1 levels via inducing rat Leydig cell
apoptosis.276
Enniatin B is a mycotoxin that may be found in con-
taminated cereals. Enniatin B (0.1–100 μM) reduced tes-
tosterone production in Leydig cells.277
Cytochalasins are fungal metabolites that have the abil-
ity to bind to actin filaments. Cytochalasin B (0.1–50 μM)
inhibited LH-, AMP-, and pregnenolone-stimulated tes-
tosterone production and LH-stimulated cAMP forma-
tion.278 Cytochalasin B also blocked glucose uptake in rat
Leydig cells.279
SUMMARY AND CONCLUSIONS
Testicular Leydig cells are the place for the biosynthesis
and secretion of androgens and insulin-like 3, which are
critical for developmental and reproductive function in
the male. Disruption of testosterone and insulin-like 3
biosynthesis and androgen metabolic activation by envi-
ronmental toxicants can cause malformity of the male
reproductive tract, sexual dysfunction, infertility, or ste-
rility. Many toxicants are shown to impair Leydig cells via
one or more multiple pathways. Our knowledge on the
different Leydig cell toxicants for disruption of particular
target molecules involved in steroidogenesis is still lim-
ited, and therefore further studies are warranted to assess
the effects of environmental toxicants on male fertility.
ACKNOWLEDGMENTS
Supported by NSFC (81102150 to LY, 30871434 and
31171425 to RSG, 81601266 to XL), Zhejiang Provincial
NSF (LY15H310008 to RSG, LQ16H040005 to XC), and
Health & Family Planning Commission of Zhejiang
Province (11-CX29 and 2013ZDA017 to RSG, 2015103197
and 2017KY483 to XL, 2016KYB199 and 2017KY466 to
XC), Science and Technology Department of Zhejiang
Provincial Government (2012C13016-1 to QL).
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Index
Page numbers followed by f and t indicate figures and tables, respectively.

A androgen receptor functional HIV and, 185 Autophagy-related gene 7 (Atg7),

Abdominal examination, 139 domains, 40 Antisperm antibodies (ASA), 15
Acquired immune deficiency cell- and temporal-specific 63, 126 Autoregulatory UBA domain,
syndrome, 63 expression of AR, 43 Antithyroid agents, 235–236; 227
Acrosome development, 193 spermatogenesis, 40 see also Endocrine Axial elements (AE), 107
Acrosome reaction, 127 testis gene expression disruption Axl receptor, 55; see also Tyro3,
Actin, 191 is regulated by mechanisms Axl, and Mer (TAM)
crossing linking proteins, 160 testosterone, 46–47 Antiviral proteins, 57–58 receptors
microfilaments, 215 testosterone and AR are AP1, 24 α-Zearalenol, 257
monomers, 191 required to complete AP2, 24 Azole compounds, 255
Actin-based cytoskeletons, meiosis, 44–45 Apale spermatogonia, 124 Azoospermia; see also Fertility/
174f, 175; see also testosterone and number of Apermatogenesis associated 3 infertility in humans,
Spermiation gonocytes, 43–44 (SPATA3), 221 regulation of
Actin-like protein 7A (ACTL7A), testosterone is required for Apical tubulobulbar complex about, 125–126
222 spermatid attachment (apical TBC), 172 acrosome reaction/
Actin-like protein 7B (ACTL7B), and release of sperm, Apoptotic germ cells, capacitation, 127
222 45 phagocytosis of, asthenozoospermia,
Actin-related protein 3 (Arp3), testosterone is required to 53–54 126–127
224 maintain the blood- Aquaporin 1 and 9 (AQP1 and DNA fragmentation, 127
Activator protein-2 (AP-2), 123 testis barrier, 45–46 AQP9), 119 etiology of, 144t
Active spermatid transport, 172 testosterone production, 42 5α-reductase (SRD5A1), 246, oligozoospermia, 126
Acyline, 85 testosterone signaling 246f, 247 pretesticular, 144
ADAM3, 10 pathways, 42–43 Argonaute proteins (AGO), 128 teratozoospermia, 127
Adark spermatogonia, 124 testosterone supports ARKO mice, 43, 44 treatment, 126
Adenovirus, 42 the expansion of Aromatase (Cyp19a1), 116, 117 Azoospermia factor (AZF), 127,
Adherens junction (AJ) spermatogonia, 44 Aromatase inhibitors (AI), 125, 128
hybrid, 175 Androgen response elements 145
proteins, 160 (AREs), 40, 234 Arpc1b, knockdown of, 196 B
testis-specific actin-rich, 171 Androgens, 116 Arp2/3 complex, 191 Barrier functions, classical JAMs
Adhesion proteins, 194 Anejaculation(AE), 147; see also Arsenic chemicals, 256 and CAR, 75–76
Adjudin 1-(2,4-dichlorobenzyl)- Male infertility ART, see Assisted reproductive Basal compartment, 184
1H-indazole-3- about, 149 techniques (ART) Basal ectoplasmic specialization
carbohydrazide, 225 and failure of emission, Arteriole smooth muscle (basal ES), 205
Adluminal compartment, 184 management of, 149 AR expression, 43 Basal inter-Sertoli cell junctions,
Adrenal chromaffin, 85–86 Angiotensin, regulation of JAMs Aryl hydrocarbon receptor 194
Adult Leydig cells, 245 and CAR, 77 (AhR), 157 Basic fibroblast growth factor
17α-ethinylestradiol (EE2), 157 Animal early development, Assisted reproductive techniques (bFGF), 97
Aflatoxins, 257 epigenetic changes (ART), 130, 130t; Β-COP (coatomer protein
AGFG1, 14 on, 157 see also Chemicals, complexers), 5
Agouti gene, 157 Annexin A1, 24 environmental Benzophenones, 253
AIP1, actin disassembly factor, Annexin A6, 11, 12, 13f, 19 motile sperm counts in, 130t 17β-estradiol (E2), 206
193 Antero-posterial (A/P) axis, 211 oligozoospermia and, 126 concentrations, 116, 117
Air pollution, on fertility, 129 Antiandrogenic chemicals, Asthenospermia; see also Male 17β-estradiol (E2) in male
A-kinase anchor protein 4 235; see also infertility reproduction, role of
(AKAP4), 221, 222, 223 Endocrine disruption causes, 142–143 environmental estrogens
Albumin, 24, 28f mechanisms management on human testicular
Amino acid sequence, 191 Antiandrogens, 252, 255 semen volume and pH, development,
Aminoglycosides, 129 Antibiotics, 129, 141t 143 119–120
AMP-activated kinase (AMPK), for fertility/infertility in postejaculate urinalysis, knockouts of ESR2 or GPER,
201 humans, 129 143 119
Anaerobic glycolysis, 101 Antibodies, to spermatic concentration and sperm loss of ESR1 causing male
Analytical ultracentrifugation antigens, 63 count, 143 reproductive defects,
(AUC), 75 Antigen-presenting cells (APCs), Asthenozoospermia, 126–127; 118
Anaphylatoxins, release of, 86 86, 88 see also Azoospermia loss of membrane ESR1
Androgen Antihypertensives, 129, 141t defined, 126 expression causing
endogenous, 235 for fertility/infertility in etiologies of, 126 male reproductive
Androgen, luteinizing hormone humans, 129 male infertility, 222 abnormalities,
(LH) and, 239; see also Anti-Ifnar1 MAb (IFNα receptor Atlastin 2, 24 118–119
Endocrine disrupting 1 monoclonal α-tubulin, 24 3β-HSD, see 3β-hydroxysteroid
chemicals (EDC) antibody), 186 Autoantigens, 184 dehydrogenase (HSD)
Androgen-binding protein (ABP), Anti-JAM-A Fab BV11, Autocrine regulation of 17β-HSD3, see
124, 237 monoclonal antibody, spermatogonia, 97–99 17β-hydroxysteroid
Androgen biosynthesis, 245–246 73, 75 Autoimmune orchitis, 59 dehydrogenase 3
Androgen receptor antagonist, Anti-Müllerian hormone experimental models of, (HSD3)
252 (AMH), 124, 63–64 3-β-hydroxysteroid
Androgen receptor- (AR-) 170, 239; see pathogenesis, 64, 65–69 dehydrogenase
mediated activity, 234 also Endocrine BTB, 65–66 (3β-HSD), 225, 245,
Androgen receptors (ARs), 156 disrupting chemicals germ cell apoptosis 246–247
Androgen regulation of (EDC) mediators, 67–69 17β-hydroxysteroid
spermatogenesis, Antioxidants, 127 immune cells, 66, 67f dehydrogenase
40–47 Antiretroviral therapy (ART) Autonomic dysreflexia (AD), 150 (17β- HSD), 225, 253

268
 Index 269

17β-hydroxysteroid BTB, see Blood-testis barrier antiandrogenic Clathrin assembly protein

dehydrogenase 3 (BTB) chemicals, 235 (CALM), 16
(HSD3), 247 β-TrCP, 99 antithyroid agents, Clathrin heavy chain (CHC), 21
11β-hydroxysteroid 235–236 Claudin, 84, 85
dehydrogenase C endocrine disrupting Claudin-3, 171
isoform 1 (11β- Cadmium, 256 chemical (EDC) Claudin-11, 171, 206, 214, 215, 225
HSD1), 248 Cadmium (batteries and affection of Clusterin (CLU), 223
Binding immunoglobulin protein pigments), 129 neuroendocrine Coat protein complex II (COPII),
(BIP), 21, 24 Cadmium-based model systems, control of testicular 16
Bioinformatics 225 function, 236 Cobalt, 256
for biomarkers identified Caenorhabditis elegans (C. estrogenic chemicals, Cofilin, 191
using omics elegans), 202 234–235 Cohesins, 109
approaches, 226–227, Caenorhabditis elegans (C. expression of steroid Colony-stimulating factor 1
227t–229t elegans) model, 158 hormone receptors in (CSF-1), 97, 170
in translational research of Calmodulin protein kinase IV testicular cells, 234 Complement cascade activation,
male reproduction, (CaMKIV), 221 paracrine regulation of 86
220–221 Calnexin, 21, 24 testicular cells, 240f Computational characterization/
Biomarkers and drug targets, Calpain 11 (CAPN11), 221 Chemical pollutants on integrative analysis of
227t–229t Calspermin (CAS), 221 spermatogenesis and proteins
Biomarkers identification; see Calyx, 194 male infertility bioinformatics for
also Computational Capacitation, 127 EDC, mechanistic actions of biomarkers
characterization/ CAR, see Coxsackievirus and epigenetic changes identified using
integrative analysis of adenovirus receptor on animal early omics approaches,
proteins (CAR) development, 157 role of, 226–227,
bioinformatics for, using Carbamates, 254 receptor mediated 227t–229t
omics approaches, Carbendazim, 254 pathways, 156–157 bisphenol-A (BPA)-
226–227, 227t–229t Carbonic anhydrase II (CAR2), 119 environmental pollution and based model for
exogenous compounds-based Carboxypeptidase D, 19 reproductive health identification of
models for, 223–224 Catsper1-4, 172 human reproductive crucial drug targets,
genomic, transcriptomic, and CBAVD, 128 health, declining, 224–225
proteomic approaches C-C chemokine receptor 5 155–156 cadmium-based model
in, 221 (CCR5), 185 overview, 155 systems, 225
BIP, see Binding immunoglobulin CCDC136 (coiled-coil domain PFC, human exposure risk, exogenous compounds-
protein (BIP) containing 136), 16 and reproductive based models
Bisphenol A (BPA), 130, 156, 224, CD147, 96, 97 health for biomarkers
225, 252 Cell adhesion, function of about, 158–160 identification,
Bisphenol-A (BPA)-based model classical JAMs and perfluoroalkyl chemicals/ 223–224
for crucial drug CAR, 76 mechanistic activities, genomic, transcriptomic, and
targets identification, Cell map, 5 160 proteomic approaches
224–225 Cell mediated immune response, reproductive toxicity of EDC in biomarkers
Bisphenols, 252–253 88 from laboratory identification, 221
2-bis-(p-hydroxyphenyl)- Cell polarity, 211 animal and cell studies genomic and transcriptomic
trichloroethane function of classical JAMs hypothalamic-pituitary biomarkers, 221–222
(HPTE), 238 and CAR, 76 gonadal axis, 157–158 male contraceptive-based
Blood-testis barrier (BTB), 40, Cell signaling for testosterone spermatogenesis, 158 model, 225–226
42, 95, 124, 158, 168, biosynthesis in Leydig Chemicals, environmental; omics/bioinformatics
171, 185, 194, 205; cells, 247–248 see also Fertility/ methods in
see also Germ cell Cellular metabolism, 101 infertility in humans, translational research
development Celsr, 211 regulation of of male reproduction,
defined, 83 Central element (CE), 107 about, 129–130 220–221
germ cell development, 83–84 Central nervous system (CNS), 185 assisted reproductive overview, 220
integrity, disruption of, 55 Ceruloplasmin, 124 techniques (ART), proteomic markers and drug
JAMs, 73, 75 CFTR gene mutations, 128 130, 130t targets, 222–223
maintenance of testicular CGN (cis Golgi network), 5 Chemokine receptor type 4 Congenital bilateral absence
immunoprivilege, 63, Chemical exposure effects, 236 (CXCR4), 98 of vas deferens
65–66 Chemical exposures on testis cell- Chemotherapeutic agents, 129 (CBAVD), 126, 146
male germ cells, localization, cell interactions and fertility/infertility in humans, Connexin-43, 65, 160, 194, 196,
58 endocrine function 129 224
movement of germ cells EDC, classes of, 234, 235t Chlamydia spp., 89 Constitutive androstane
across, 84–85 EDC disruption of paracrine Chlamydia trachomatis, 59, 63 receptors (CAR), 156
permeability, 55 relationships between Chlorpyrifos, 254 Copper, 256
restructuring of junctions, testicular cells Cholesterol Cortactin, knockdown of, 196
73, 75 anti-Müllerian hormone chemical structures of, 247f Corticoids, for orchitis, 69
spermatogonia and (AMH), 239 transporters, 246f Coxsackievirus and adenovirus
spermatocytes on, 58 estrogen, 239 Cholesterol-transporting protein, receptor (CAR)
structure and function, 65–66 follicle-stimulating 248 functions
testis immune privilege, 85 hormone (FSH), Chromatids, 106 barrier, 75–76
testosterone to maintain, 45–46 238–239 Chromium, 256 cell adhesion, 76
Body mass index (BMI), 129 growth factors, 239 Chromosomal synapsis, 109 cell polarity, 76
Bone morphogenetic protein 4 LH and androgen, 239 Chromosomal synapsis, 107, 109; molecular structure, 73–75
(BMP4), 98, 124, 170 paracrine regulators of see also Mammalian overview, 73, 74f
Bordetella pertussis, 64, 85 testicular cells, 238t meiosis, genetics of tissue distribution and
Bortezomib, 99 EDC targeting of testicular Chromosome movement, 106 localization in testis,
Brain-derived neurotrophic cells Cigarette smokers, 256 74t, 75
factor (BDNF), 170 about, 236 cis Golgi network (CGN), 5 overview, 73
Branched actin polymerization, germ cells, 237 Citrinin, 257 regulation, in testis, 77
194 Leydig cells, 237–238 c-Kit genes, 44 CREB transcription factor, 42
Breast cancer related protein Sertoli cells, 237 c-Kit receptor, 98 Crossover formation, 110;
(BCRP), 225 endocrine disruption, Classical pathway, testosterone see also Meiotic
Breastfeeding, 159 mechanisms of signaling, 42 recombination
270 Index

Crucial drug target identification, Defensin, beta 119 (DEFB119), Ejaculatory duct obstruction Environmental estrogens on
bisphenol-A (BPA)- 222 (EDO), 143, 146 human testicular
based model for, Deleted-in-azoospermia-like Ejaculatory dysfunction; see also development, 119–120
224–225 (DAZL) gene, 222 Male infertility Environmental pollution and
Cryptorchidism, 145 Dendritic cells (DCs), 52, 64, 66 about, 147 reproductive health
CXCL12, 98 Deoxynivalenol, 257 anejaculation, 149 human reproductive health,
Cyclic-AMP-(cAMP)-dependent Desmoglein 2 (DSG2), 222 anejaculation/failure declining, 155–156
protein kinase A, 223 Desmosome (DS), 205 of emission, overview, 155
Cyclin-dependent kinases (CDK), Deubiquitinating enzymes management of, 149 Environmental toxicants on
107 (DUB), 99 retrograde ejaculation, Leydig cell
Cyclin-related protein CNTD1, 110 Diabetes mellitus (DM), 129 147–148 about, 248, 249t–251t, 250
CYP11A1, see Cytochrome P450 obesity and, 129 Electroejaculation, 150 benzophenones, 253
cholesterol side chain Diagnosis, of human Electrophoresis, two-dimensional bisphenols, 252–253
cleavage enzyme autoimmune orchitis, (2-DE), 220 carbamates, 254
(CYP11A1) 69 EM analysis, 5 1,2-dibromo-3-
CYP17A1, see Cytochrome P450 1,2-dibromo-3-chloropropane Embrel , 67–68
® chloropropane
17α-hydroxylase/17,​ (DBCP), 129, 255 Emp70 (endomembrane protein (DBCP), 255
20-lyase (CYP17A1) Dibutyl phthalate (DBP), 235 70), 7 metals, 255–256
cyp11a1 expressions, 253 Dichlorodiphenyltrichloroethane End-binding protein 1 (EB1), 175 organochlorines, 253–254
cyp19 gene, 175, 176 (DTT), 155, 253 Endocrine disrupting chemicals organophosphates, 254–255
cyp19KO, 118 Dichlorvos, 255 (EDC); see also perfluoroalkyl substances
Cysteine 451 (C451A), 118 Di(2-ethylhexyl) phthalates Chemical exposures (PFAS), 248–249
Cystic fibrosis transmembrane (DEHP), 158, 235, on testis cell-cell phthalates, 251–252
regulatory (CFTR) 251, 252 interactions and plant toxicants, 256–257
protein, 126, 146 Diethylstilbestrol (DES), 118, 156 endocrine function polycyclic aromatic
Cytidine 5-monophosphatase Differential-in-gel electrophoresis affection of neuroendocrine hydrocarbons (PAH),
(CMPase), 5 (DIGE), 220 control of testicular 254
Cytochalasin D, 196 Digital rectal examination, 143 function, 236 Enzymes for testosterone
Cytochalasins, 257 Dihydrotestosterone (DHT), 42, classes of, 234, 235t biosynthesis and
Cytochrome b5, 247 234, 245, 246 disruption of paracrine metabolism; see also
Cytochrome c oxidase subunit 6B Dimethoate, 255 relationships between Leydig cell function,
(COX6B), 223 7,12-dimethylbenz[a] anthracene testicular cells environmental
Cytochrome P450 (DMBA), 254 anti-Müllerian hormone toxicants on
17α-hydroxylase/​ Diphtheria toxin, 83 (AMH), 239 3β-HSD, 246–247
17,20-lyase Disordered spacer region, 227 estrogen, 239 17β-HSD3, 247
(CYP17A1), 245, 247 DMC1, 45, 110 follicle-stimulating CYP11A1, 246
Cytochrome P450 cholesterol DMRT, 45 hormone (FSH), CYP17A1, 247
side chain cleavage DNA-binding domain (DBD), 40 238–239 SRD5A, 247
enzyme (CYP11A1), DNA-break detection, 127 growth factors, 239 Epidermal growth-factor (EGF),
246 DNA fragmentation, 127, 128; see LH and androgen, 239 52, 53f
Cytochrome P450 (CYP) also Azoospermia; paracrine regulators of Epidermal growth factor receptor
isoforms, 160 Male infertility testicular cells, 238t substrate 8 (Eps8), 224
Cytoplasmic tails, of classical about, 150 effects of, 155 Epididymal obstruction,
JAMs, 73 management, 150 mechanistic actions of posttesticular
Cytoskeletal reorganization DNA integrity, 150 epigenetic changes azoospermia, 146, 147
genetic regulation, 176 DNA replication, 7 on animal early Epididymis, 85, 139
hormonal regulation, 175–176 Docosahexaenoic acid (DHA), 16 development, 157 Epididymitis, 59
Cytoskeletons, actin- and Doublesex- and mab-3-related receptor mediated Epithelial cycle of
MT-based, 174f, 175; transcription factor 1 pathways, 156–157 spermatogenesis1, 211
see also Spermiation (DMRT1), 222 reproductive toxicity of, from Epithelial-mesenchymal
Cytoskeletons (F-actin) and Double-strand breaks (DSB) laboratory animal transition (EMT), 97
spermatogenesis repair, 109–110; and cell studies Epoxiconazole, 255
F-actin biogenesis, dynamics, see also Meiotic hypothalamic-pituitary Epoxyeicosatrienoic acids (EET),
and function, 191–192 recombination gonadal axis, 157–158 160
F-actin in Sertoli cells Dpy19l2, 17 spermatogenesis, 158 ER-associated protein degradation
in ectoplasmic Drosophila, 99, 193, 202 targeting of testicular cells (ERAD), 10
specializations, Drosophilia, 211 about, 236 Erp44 (endoplasmic reticulum
194–195 Drug targets, proteomic markers germ cells, 237 resident protein), 10
in seminiferous tubule and, 222–223 Leydig cells, 237–238 ERP57 (ER protein 57), 23
contraction, role of, Dynamic light scattering (DLS) Sertoli cells, 237 Escherichia coli, 58–59, 63
197 analysis, 75 Endocrine disruption Esr1 knockout (Esr1KO) mouse,
Sertoli cell F-actin- mechanisms 118
containing structures, E antiandrogenic chemicals, Esr2 knockouts (Esr2KOs), 119
dynamics of (during E1 activating enzyme, 99 235 Esr1KO, 119
spermatogenesis), EAO (experimental autoimmune antithyroid agents, 235–236 Estradiol, 129
196–197 orchitis) model, 55, EDC affection of treatment, 196
in tubulobulbar complex 63–64, 66, 84 neuroendocrine Estrogen
(TBC), 195–196 Ebola virus, 88 control of testicular in EDC disruption
F-actin in spermatogenic cells ECM protein, 96 function, 236 of paracrine
in spermatogonia and E2 conjugating enzyme, 99 estrogenic chemicals, relationships, 239;
meiotic cells, 192–193 Ectoplasmic specialization (ES), 234–235 see also Endocrine
during spermiogenesis, 75, 171 Endocytic vesicle-mediated disrupting chemicals
193–194 adherans junction, 45 protein trafficking (EDC)
Cytosolic DNA sensors, 55, 57 apical, 225 event, 172 environmental, on
core PCP proteins regulating, Endoplasmic reticulum (ER), 3 human testicular
D 216f Endosomalsorting complex development, 119–120
Daf-2 gene, 202 F-actin in, 194–195 required for treatment in males, early, 118
Database for Annotation, EDC, see Endocrine disrupting transport-0 ­(ESCRT-0), Estrogenic chemicals, 234–235;
Visualization and chemicals (EDC) 13 see also Endocrine
Integrated Discovery EF1α, elongation factor, 21 Endosulfan, 254 disruption
(DAVID), 221 EGF receptor (EGFR), 42 Enniatin B, 257 mechanisms
 Index 271

Estrogen receptor 1 (ESR1) in spermatogenic cells Fibroblast growth factor 9 Germ cells (GCs), 183, 237;
loss of, causing male in spermatogonia and (FGF9), 106 see also Endocrine
reproductive defects, meiotic cells, 192–193 Fibroblast growth factor receptor disrupting chemicals
118 during spermiogenesis, 3 (FGFR3), 222 (EDC)
in male reproduction, role of, 193–194 Flagella biogenesis, 194 across BTB/SCB, movement,
see 17β-estradiol (E2) F-actin biogenesis Flame retardants, 156 84–85
in male reproduction, dynamics, and function, Flavones, 257 apoptosis mediators, 67–69
role of 191–192 Fluorescence in situ hybridization generation of, 1, 2f–3f
Estrogen receptor 1 (ESR1), 117, F-actin disorganization, 214 (FISH), 127 Germ cell tumors (GCTs), 88
120 F-actin elongation, 191 Focal adhesion kinase (FAK), gfra1 gene, 101
Estrogen receptor 2 (ESR2), 117 Fads2 gene, 16 196 GIANTIN, 8–9
knockouts of, 119 FAM3C, see Family with Focal contact, 174 GL54D, 8–9, 12
Estrogen receptor 1 (ESR1) sequence similarity Focal orchitis, 64 Glial cell-derived neurotrophic
expression, loss of 3, member C Follicle-stimulating hormone factor (GDNF), 44
membrane (FAM3C) (FSH), 44, 123, production, 44
causing male reproductive Family with sequence similarity 124, 143, 157, 206, Glial cell line-derived
abnormalities, 3, member C 236, 238–239; see neurotrophic factor
118–119 (FAM3C), 11, 12, 14f also Endocrine (GDNF), 192, 193,
Estrogen receptors (ESRs), 116, Family with sequence similarity disrupting chemicals 202
156, 234 71, member F1 (EDC) Glial-derived neurotrophic factor
Estrogen receptors (ER) (FAM71F1), 221 FSCN3, 194 (GDNF), 95
expression in male Fascin protein (Fascin 1-3), 225 Functions, classical JAMs and about, 95, 96f, 97
reproductive organs, F-box protein, 99, 100 CAR role of SC and, 124
117–118 Fbxw11, 99 barrier, 75–76 treatment of, 98
Estrogen response elements Fertility cell adhesion, 76 Globozoospermia, 14
(ERE), 234 as function of age, 128 cell polarity, 76 Globular actin (G-actin), 191
Estrogens history of, 123 Fungicide (vinclozolin), 157 Glucocorticoid metabolizing
in males, 116 and pollution, 129 Fungicides, 254 enzymes, 248
Estrogen sources in Fertility/infertility in humans, Fz/Dgo/Dsh complex, 213 Glucocorticoid receptors (GR),
males; see also regulation of 156
Spermatogenesis, azoospermia G Glucose metabolism, 223
membrane/nuclear about, 125–126 Galectin-1, 88 Glucose-regulated protein 170
estrogen receptors in acrosome reaction/ Galnt3, 16 (GRP170), 23
about, 116–117 capacitation, 127 GALNTL5, 16 Glucose transporter 3 (GLUT-3),
early estrogen treatment in asthenozoospermia, Galntl5, 16 20–21
males, effects of, 118 126–127 Gametogenetin (GGN), 221 Glucose transporter 8 (GLUT-8),
estrogen receptors (ER) DNA fragmentation, 127 Gamma-aminobutyric acid 225
expression in male oligozoospermia, 126 (GABA), 124 GLUT-4, 21
reproductive organs, teratozoospermia, 127 Gap junction (GJ), 171, 205 Glutathione S transferase (GST)
117–118 treatment, 126 Gap junction (GJ) protein isoforms, 24
Etanercept (Embrel®), 67–68 environmental chemicals connexin-43 (Cx43), Glutathione S-transferase Mu 3
Ethylene dibromide (gasoline), about, 129–130 160 (GSTMu3), 223
129 assisted reproductive Gba2, 172 Glyceraldehyde- 3-phospahte
Etomidate, 255 techniques (ART), GBF1, 7–8, 12, 19 dehydrogenase
E3 ubiquitin ligase, 99 130, 130t GC1-spg cells, 101 sperm-specific, 172
Eukaryotic initiation factor 4E fertilization, requirements GDNF, see Glial cell-derived Glyceraldehyde-3-phosphate
binding protein-1 for, 125, 125t neurotrophic factor dehydrogenase
(4E-BP1), 201 genetic factors, 127–128 (GDNF) (GAPDHS), 222, 223
European Chemicals Bureau, history of fertility, 123 Gdnf transgene, 44 Glyceraldehyde-3-phosphate
224 hormonal regulation of Gene array analysis, 99 dehydrogenase, testis-
Exogenous compounds- spermatogenesis, 125 Gene knockout, 96 specific (GAPDS), 223
based models leydig cells, role of, 124–125 Gene ontology, 221 Glycolysis, 101
for biomarkers medications Generation of germ cells, 1, 2f–3f Glycoproteins, 125
identification, antibiotics, 129 Genes/nucleic acid, 100 GnRH antagonist, 42
223–224 antihypertensives, 129 Genetic anomalies, male treatment, 44
Exonuclease 1 (EXO1), 110 chemotherapeutic agents, infertility, 173t Golgi apparatus regulation of
Experimental autoimmune 129 Genetic mutations, in TAM differentiation, 1–31
orchitis (EAO) model, fertility and pollution, receptors, 52 data from other sources,
55, 63–64, 66, 84 129 Genetic regulation, cytoskeletal 14–16
Extracellular matrix (ECM) hormonal, 129 reorganization, 176 fragmentation of the Golgi
proteins, 95 psychiatric-related, 129 Genetics of mammalian meiosis, apparatus in late
Extracellular regulated kinase obesity and diabetes mellitus, see Mammalian spermatids, 18–19
(ERK), 237 129 meiosis, genetics of Golgi apparatus
pregnancy and infertility, Genistein, 257 and acrosome formation,
F epidemiology of, 123 Genomic approaches in 3–5
F-actin Sertoli cells (SC), role of, 124 biomarkers molecular dissection of, 5
in Sertoli cells spermatogenesis and identification, 221 structure and function, 5
in ectoplasmic spermiogenesis, Genomic biomarkers, 221–222 Golgi proteins
specializations, 123–124 Germ cell aromatase expression, coincidental temporal
194–195 varicoceles 117 expression of non-
in seminiferous tubule about, 128 Germ cell arrest (GA) Golgi and, 21, 23–24
contraction, role of, fertility as function of phenotypes, 221 defining in spermatids,
197 age, 128 Germ cell development; 17–18
Sertoli cell F-actin- Fetal Leydig cells, 237, 245 see also Human in early spermatids
containing Fetal male germ cells, 106 spermatogenesis during acrosome
structures, FG repeats-containing protein 1 blood-testis barrier (BTB), formation, 9–10
dynamics of (during (HRB), 14 171 forming Hermes body at
spermatogenesis), Fibroblast growth factor 2 from undifferentiated step 19 spermatids,
196–197 (FGF2), 97 spermatogonia to 19–20
in tubulobulbar complex Fibroblast growth factor 8 haploid spermatids, localized to
(TBC), 195–196 (FGF8), 98 168–171 spermatocytes, 7–9
272 Index

matching protein GPR30, 234 Human reproductive health, Immunoelectron microscopy, 107
expression profiles of G protein-coupled estrogen declining, 155–156 Immunoglobulin A (IgA), 126
Golgi and non-Golgi receptor (GPER), 117 Human Sertoli cells, 206 Immunoglobulin G (IgG), 126
proteins during knockouts of, 119 Human spermatogenesis Immunoglobulin M (IgM), 126
spermatogenesis, G-protein-coupled receptor germ cell development Immunoglobulin superfamily
20–21 (GPR), 154 blood-testis barrier (IgSF), 73
overlapping expression Granulomas, 64, 65 (BTB), 171 Immunoregulation, testicular,
profiles of non-Golgi Granzymes, 86 from undifferentiated see Testicular
and, 24–25 GRASP55, 9–10, 12 spermatogonia to immunoregulation
proteins involved in Growth and differentiation factor haploid spermatids, Immunoregulatory mechanisms,
acrosome binding to 3 (GDF3), 123 168–171 183
the nuclear surface, Growth-arrest–specific gene 6 overview, 168 Immunosuppression, in testis, 55
16–17 (Gas6), 52, 53 round spermatids to Indoleamine-pyrrole
proteins of unknown Growth factors, endocrine functional 2,3-dioxygenase
function, 20 disrupting chemicals spermatozoa, (IDO), 88
for residual bodies at step (EDC), 239 development of Infection and inflammation, 63
19 spermatids, 20 Guillain-Barré syndrome, 186 cytoskeletal Infectious orchitis, 59
segregation to acrosome, reorganization, Infertility
12 H 175–176 etiological factors, 63, 69
Golgi role in chromatoid Haploid spermatids, 168–171; spermiation, 172, 174–175 in humans, regulation of, see
body and MVB see also Germ cell spermiogenesis, 171–172, Fertility/infertility in
formation, 13–14 development 173t humans, regulation of
introduction, 1 Heat shock proteins (HSPs), 54 Human T-cell leukemia virus male, see Male infertility
spermatogenesis, 1–3 Heat shock-related 70 kDa type 1 (HTLV-1), 188 pregnancy and, epidemiology
defined, 1 protein 2 (HSPA2), Human tubal fluid (HTF), 150 of, 123
spermiogenesis, 3 223 Humoral immune response, Inflammation, spermatogenesis
unexpected ER resident Heavy metals, 255 86–88 and, 63–69
proteins with Hedgehog signaling, 98 HUWE1 protein, 100 antibodies to spermatic
Golgi/ acrosome HEI10, 110 Hydroxychlor, 254 antigens, 63
localizations, 10 Hemivasotomy, 147t Hydroxyeicosatetraenoic acids autoimmune orchitis
unexpected proteins with Hepatitis virus, 58 (HETEs), 160 BTB, 65–66
Golgi/ acrosome Hermes body, 5, 6f, 10, 19, 20 Hypophysectomy, 44 experimental models,
localizations, 11–12 Heterophilic trans-interaction, Hypoplastic seminal vesicles, 143 63–64
Golgi cisternae, 8, 9 JAMs, 73, 74t, 75 Hypospermatogenesis, 126, 144, germ cell apoptosis
Golgi markers, 7, 8 2,5-hexanedione and 145 mediators, 67–69
Golgi proteins, 5 carbendazim, 173 Hypothalamic kisspeptin (KiSS1), immune cells, 66, 67f
coincidental temporal High endothelial venules (HEVs), 157 pathogenesis, 64, 65–69
expression of non- 75 Hypothalamic pathways, 236 human orchitis, 63
Golgi and, 21, 23–24 High-intensity focused Hypothalamic-pituitary gonadal perspectives, 69
defining in spermatids, ultrasonography axis, 157–158 treatment of orchitis, 69
17–18 (HIFU), 146 Hypothalamic-pituitary-testes Inhibin, 125
in early spermatids during High-mobility group box 1 (HPT) axis, 143 Inhibin B, 124
acrosome formation, (HMGB1), 54 Innate defense system
9–10 High-risk chemotherapeutic I PRR-initiated innate immune
forming Hermes body at agents, 129 ICA69, 16 responses
step 19 spermatids, H4K44 acetylation (H4K44ac), IHO1, protein complex, 110 Leydig cells, 57–58
19–20 110 Imatinib, 141t male germ cells, 58
localized to spermatocytes, Homophilic trans-interaction, Immotility (IM), 142 in testis, PRRs, 55–57
7–9 JAMs, 73, 74t, 75 Immune cells TLR-initiated innate immune
matching protein expression Homozygous mutations, 111 autoimmune orchitis, 66, 67f responses in Sertoli
profiles of Golgi HORMAD1, 109 in promoting germ cell death, cells, 57
and non-Golgi Hormonal medications, 129, 140t 67 TLR signaling in testicular
proteins during fertility/infertility in humans, Immune homeostasis, TAM macrophages, 58–59
spermatogenesis, 129 receptors in Innate immune responses
20–21 Hormonal regulation regulating, 52–53 by microbial pathogens, 52
overlapping expression cytoskeletal reorganization, Immune privilege, 183 PRR-initiated
profiles of non-Golgi 175–176 potential downside of, 88–89 Leydig cells, 57–58
and, 24–25 of spermatogenesis, 125 of testis, 183–184, 184f; see male germ cells, 58
proteins involved in acrosome Hotspot, meiotic recombination, also Testis, HIV/ Sertoli cells, TLR-initiated, 57
binding to the nuclear 110; see also Meiotic ZIKV viruses in testicular cells, TAM
surface, 16–17 recombination Immune regulation within testis, inhibition, 54–55
proteins of unknown HSP70.2 (HspA2), 11, 12, 14f SCs, 81–89 Insecticides, 254
function, 20 Human chorionic gonadotropin BTB/SCB, 83–85 Insulin-like 3, 245
for residual bodies at step 19 (hCG), 125 movement of germ cells Insulin-like growth factor, 124
spermatids, 20 Human genes encode type 1, 247 across, 84–85 Insulin-like peptide (ILP), 202
segregation to acrosome, 12 Human HSD3B1, 246 testis immune privilege, 85 Insulin receptor, 235
Golgi ribbon, 5, 10, 18, 20 Human immunodeficiency virus cell mediated immune Insulin receptor subtype-2 (IRS-2),
Golgi trafficking, 7 (HIV), 58, 59, 63, response, 88 225
GOLPH3 (Golgi phophoprotein), 88, 185–186; see also humoral immune response, Integrative analysis of proteins,
see GPP34 Testis, HIV/ZIKV 86–88 see Computational
Gonadotropin, 157 viruses in overview, 81 characterization/
Gonadotropin-releasing Human infertility, implications testis immune privilege, 81, integrative analysis of
hormone (GnRH), in, 111; see also 82f, 83f proteins
125, 144, 236 Mammalian meiosis, Immune responses Interferons (IFN), 185
Gonocytes, 94 genetics of cell mediated, 88 IFN-gamma (IFNγ),
testosterone and number of, Human menopausal humoral, 86–88 regulation of JAMs
43–44 gonadotropins innate, see Innate immune and CAR, 77
GOPC, 15 (hMG), 125 responses IFN-stimulating gene 15
Gopc, 172 Human orchitis, 63 Immune tolerance to male germ (ISG15), 58
Gossypol exposure, 256–257 Human primordial germ cells cells, TAM receptors Interleukin (IL), 65, 66, 68, 86
GPP34, 9–10, 11, 19 (hPGC), 168 in, 55 Interleukin-1alpha (IL-1α), 124
 Index 273

Intermediate cellular Levonorgestrel (LNG), 171 Long noncoding RNA (lncRNA), Male infertility
compartment theory, Leydig cell function, 128 causes of, 220
85 environmental Low-risk chemotherapeutic Male reproductive organs,
Intermediate chemotherapeutic toxicants on agents, 129 estrogen receptors
risk agents, 129 androgen biosynthesis, Luteinizing hormone (LH), 123, (ER) expression in,
Intracytoplasmic sperm injection 245–246 143, 157, 236 117–118
(ICSI), 126, 130t, cell signaling for and androgen, 239; see also Male reproductive system,
139, 172 testosterone Endocrine disrupting knockout phenotypes
Intratesticular testosterone, 175 biosynthesis in chemicals (EDC) in, 76–77
Intrauterine insemination (IUI), Leydig cells, 247–248 Lymphomonocyte infiltration, Mammalian genome, 100
128, 130, 130t, 142 environmental toxicants 63 Mammalian meiosis, genetics of
In vitro fertilization (IVF), 123, targeting Leydig cell chromosomal synapsis, 107,
130t, 139, 158 about, 248, 249t–251t, M 109
In vitro meiosis, 110–111, 111t; 250 Macrophages human infertility,
see also Mammalian benzophenones, 253 antigen-presenting cells, 66 implications in, 111
meiosis, genetics of bisphenols, 252–253 testicular, TLR signaling in, meiotic initiation, 106
Ionomycin-stimulated carbamates, 254 58–59 meiotic recombination
conditions, 86 1,2-dibromo-3- Madin-Darby canine kidney crossover formation, 110
Ionotropic P2X receptors chloropropane (MDCK) cells, 75 DSB repair, 109–110
(P2XRs), 99 (DBCP), 255 MAJIN, DNA-binding meiotic recombination
Islet cell auto antigen 1-like metals, 255–256 transmembrane hotspot, 110
protein (ICA1L), 16 organochlorines, protein, 106 programmed meiotic DSB
Isoaspartyl peptidase/ 253–254 Malathion, 255 formation, 109
lasparaginase organophosphates, Male contraceptive-based model, telomere-led meiotic
(ASRGL1), 223 254–255 225–226 chromosome
Isoflavones, 257 perfluoroalkyl substances Malectin, 24 movement, 106–107,
(PFAS), 248–249 Male fertility, connecting mTOR 107f, 108t
J phthalates, 251–252 signaling to, 202, 203t in vitro meiosis, 110–111,
JAMs, see Junctional adhesion plant toxicants, 256–257 Male germ cells 111t
molecules (JAMs) polycyclic aromatic division, 193 Mammalian target of rapamycin
Junctional adhesion molecules hydrocarbons (PAH), immune tolerance to, TAM (mTOR), 101
(JAMs), 73–78 254 receptors, 55 Man2á1, 12
functions enzymes for testosterone innate immune responses in ManIIX, 12
barrier, 75–76 biosynthesis and testicular cells, 52 differential expression, 17–18
cell adhesion, 76 metabolism PRR-initiated innate immune MARK4, structure of, 226, 227
cell polarity, 76 3β-HSD, 246–247 responses in, 58 Matrix-assisted laser desorption
future perspectives, 77–78 17β-HSD3, 247 TAM receptors in, 53–54 mass spectrometry
general properties, 73–77 CYP11A1, 246 Male gonadal development, 168 (MALDI-MS), 220
knockout phenotypes in male CYP17A1, 247 Male infertility Matrix metalloproteinases
reproductive system, SRD5A, 247 asthenospermia (MMP), 96, 188
76–77 Leydig cell development, causes, 142–143 MAX, 106
molecular structure, 73–75 248 management, 143 MCT2, 24
homophilic and overview, 245 postejaculate urinalysis, Mechanistic target of rapamycin
heterophilic trans- Leydig cell hyperplasia, 118 143 (mTOR) signaling in
interaction, 73, 74t, 75 Leydig cells, 7, 9, 40, 41f, 42 azoospermia spermatogenesis
overview, 73, 74f AR expression, 43 pretesticular, 144 in maintaining
tissue distribution and and EDC targeting of chemical pollutants on spermatogonial
localization in testis, testicular cells, spermatogenesis function, role of
74t, 75 237–238; see also and, see Chemical about, 202, 204–205
overview, 73 Endocrine disrupting pollutants on mTOR signaling in
regulation, in testis, 77 chemicals (EDC); spermatogenesis and meiosis, 205
Junctional restructuring theory, Environmental male infertility mTOR signaling in Sertoli
85 toxicants on Leydig DNA fragmentation cells, 205–206
cell about, 150 mTOR complexes and
K development, 248 management, 150 inhibitor rapamycin,
Kallman syndrome, 126, 128, 144 innate immune responses in ejaculatory dysfunction 201
Kartagener’s syndrome, 126, 142 testicular cells, 52 about, 147 mTOR signaling to male
KASH5, 106 PRR-initiated innate immune anejaculation, 149 fertility, connecting,
Keratin, 223 responses in, 57–58 anejaculation/failure 202, 203t
Ketoconazole, 255 source of androgens, 234 of emission, rapamycin and rapalogs
Kisspeptin (KiSS1), 157 testis immune privilege, 81, management of, 149 in human disease,
Klinefelter syndrome (KS), 126, 82f, 83f retrograde ejaculation, pharmacological
127, 145 testosterone production, 42 147–148 applications of,
Knockout phenotypes, in male TLRs in, 55 evaluation 201–202
reproductive system, Tyro3 in, 53 history, 139, 140t–141t Medications; see also Fertility/
76–77 Ligand-binding domain (LBD), physical exam, 139 infertility in humans,
Kyoto Encyclopedia of Genes and 40 investigations regulation of
Genomes (KEGG), Light microscope (LM) semen analysis, 139–142, antibiotics, 129
221 observations, 1, 4 142t antihypertensives, 129
Lindane, 254 oligospermia chemotherapeutic agents, 129
L Liquid chromatography and mass causes, 143 fertility and pollution, 129
Lactacystin, 99 spectrometry (LC–MS), management, 143 hormonal, 129
Lactate dehydrogenase C 220 testicular azoospermia psychiatric-related, 129
(LDHC), 24 Listeria monocytogenes, 64 causes, 145 MEIOB, 110
LAMAN, 12 Lkb1, 202 management, 145–146 MEIOC complex, 106
Laminin, 96 LncRNA, 100 posttesticular Meiosis, 7, 205
Lateral elements (LE), 107 Localization, in testis, 74t, 75 azoospermia, Meiosis
Lead, 256 Longitudinal intussusception 146–147, 147t mTOR signaling in, 205
Lead (paint), 129 vasoepididymostomy varicocele in vitro, 110–111, 111t; see also
Leptotene spermatocytes, (LIVE) technique, about, 150–151 Mammalian meiosis,
movement, 84–85 147, 148f management of, 151 genetics of
274 Index

Meiosis-specific genes in human Microtubule (MT)-based Myosin-based vesicles, 194 Nuclear localization signal (NLS),
infertility, 111t cytoskeletons, 174f, Myosins, 191, 192 40
Meiosis-specific proteins, 106 175, 216; see also Myxoma virus, 64 Nuclear-only estrogen receptor 1
Meiotic cells, F-actin in Spermiation (NOER), 118, 119
spermatogonia and, Minichromosome maintenance N Nuclear receptors (NR), 156
192–193 (MCM) helicase N-acetylgalactosaminyl­ N-WASP, 175, 194
Meiotic chromosome family, 110 transferase 3, 16 Nω-Nitro-L-arginine methyl
movement, telomere- MiR-145, overexpression, 77 NADPH for electron transfers, ester hydrochloride
led, 106–107, 107f, MiR-106b-25, 101 246, 247 (L-NAME), 67
108t Mitogen-activated protein NADPH oxidase 1 (Nox1), 101
Meiotic division, 1 kinases (MAPK), 235 NADPH oxidase 3 (Nox3), 101 O
of spermatocytes, 3 pathways, 118 National Health and Nutrition Obesity and diabetes mellitus,
Meiotic double-strand Mitotic phase of spermatogenesis Examination 129
breaks formation autocrine/paracrine Survey (NHANES) Obesity-associated
programmed, 109; regulation of 2011–2012, 252 asthenozoospermic
see also Meiotic spermatogonia, 97–99 National Institute on Aging patients, 223
recombination interaction between Interventions Testing Obstructed ejaculatory ducts, 143
Meiotic initiation, 106; see also spermatogonia and Program, 202 Obstructive azoospermia (OA),
Mammalian meiosis, niche, 96–97 Natural fertility, 126 125
genetics of metabolism of Natural fertilization, 125 Occludin, 65, 66, 84, 85, 224, 225
Meiotic recombination, spermatogonia, 101 Natural killer (NK) cells, 86, 88 Oligoasthenoteratospermia
106, 109; see also noncoding RNA in N-butyldeoxynojirimycin (OAT), 151
Mammalian meiosis, spermatogonia (NB-DNJ), 13 Oligoastheoteratozoospermia
genetics of regulation, 100–101 N-Cadherin, 224 (OAT), 128
crossover formation, 110 origin and subpopulations Nectin-2, 175, 194, 196 2’5’-oligodenylate synthetase1
DSB repair, 109–110 of spermatogonia, Neisseria gonorrhoeae, 59, 63 (OAS1), 58
meiotic recombination 94–95 Neonatal Leydig cells, 245 Oligospermia; see also Male
hotspot, 110 overview, 94 Neonatal pig islets (NPIs), 86, 88 infertility
programmed meiotic DSB perspectives, 102 Neonatal pig SCs (NPSCs), 86 causes, 143
formation, 109 spermatogonia, niche of, 95 Nerve growth factor (NGF), 170 management, 143
Meiotic recombination hot spot ubiquitin proteasome system Neuroendocrine control of Oligozoospermia, 63, 126; see also
locus (mrhl), 101 in spermatogonia testicular function, Azoospermia
Meiotic sex chromatin silencing regulation, 99–100 EDC affection of, 236 defined, 126
(MSCI), 109 Mitotic/proliferation phase, 1 Neurogenin 3-Cre, 205 Omics methods in translational
MEI4-REC114, 109 Mixed atrophy (MA), 221 Neurotrophin (NT), 170 research of male
Melanoma differentiation- Molecular characterization of Nicotinamide adenine reproduction,
associated protein 5 proteins, 221 dinucleotide 220–221
(MDA5), 57 Molecular structure, classical phosphatase Oncologic agents, 141t
Membrane attach complex JAMs and CAR, (NADPase), 5 Oocytes, 125
(MAC), 86 73–75 Nifedipine, 150 Orchidometer, 139
Membrane-bound Fas L (mFasL), homophilic and heterophilic NIMAlike kinase-1 (NEK1), 109 Orchitis, 59
68 trans-interaction, 73, NIPBL, 109 autoimmune
Membrane ESR1 (mESR1), 118 74t, 75 Nitric oxide synthase (NOS) experimental models of,
Mercury pollution, 256 overview, 73, 74f activity, 67 63–64
Mer receptor; see also Tyro3, tissue distribution and Nonclassical (nongenomic) pathogenesis, 64, 65–69
Axl, and Mer (TAM) localization in testis, pathway, testosterone focal, 64
receptors 74t, 75 signaling, 42–43 human, 63
immune tolerance to male Molinate, 254 Noncoding RNA (ncRNA), 100, perspectives, 69
germ cells, 55 Mono-butyl phthalate (MBP), 128 treatment of, 69
for triggering phagocytotic 251 Noncoding RNA in Organochlorines, 253–254
signaling, 53, 54f Monocarboxylate transporters spermatogonia Organohalogen chemicals, 236
Mesenchymal stem cells (MSC), (MCTs), 21 regulation, 100–101; Organophosphates, 254–255
237 Mono-ethylhexyl phthalate see also Mitotic phase flame retardants, 156
Metabolism of spermatogonia, (MEHP), 251, 252 of spermatogenesis Organotins, 256
101; see also Monomers, 191 Nongenomic (nonclassical) Orotidine 50-monophosphatase, 5
Mitotic phase of Motile sperm counts in ART, pathway, testosterone Outer dense fiber protein 2
spermatogenesis 130t signaling, 42–43 (ODF2), 223
Metabotropic P2Y receptors Motility and progressive motility Non-Golgi proteins, 20 Ovaries, 116
(P2YRs), 99 (PM), 142 Non-obese diabetic (NOD) mice,
Metals, 255–256 Mouse genes in meiosis, 108t 88 P
Methanol, 251 Mouse models Nonobstructive azoospermia Pachytene piRNA, 128
Methoxychlor, 253, 254 Leydig cells, 55, 57 (NOA), 125, 126, Pachytene stage, 111
3-Methylsulfonyl-DDE, 253 male germ cells, 58 144, 171 Pancreatic islets, for diabetes,
MFSD14A, 16 NOD mice, 88 Non-progressive motility (NPM), 85–86
MG160, 7–8 Sertoli cells, 54–55 142 P antigen family member 1
Microbial pathogens, innate TAM receptors, 52–55 Nonsister chromatids, 106 (PAGE1), 222
immune responses Movement, of germ cells across Normal sperm morphology, 127 Paracrine regulation
by, 52 BTB/SCB, 84–85 Notch gene homologue 1 of spermatogonia, 97–99
Microdissection testicular sperm Multidimensional protein (NOTCH1), 222 of testicular cells, 238t, 240f
extraction (mTESE), identification N-terminal domain (NTD), 40 Paracrine regulators of testicular
145, 145f technology N-terminal gamma-carboxylated cells, 238t, 240t
MicroRNA (miRNA), 77, 100, (MUDPIT), 220 glutamic acid (GLA), Paracrine signaling, 98
128, 170 Multidrug resistance gene 52, 53f Parathyroid allografts, 81
Microsurgical epididymal (MDR1), 225 N-terminal kinase domain, 227 Pathogen-associated molecular
sperm aspiration Multidrug resistance-related Nuclear ESR1 (nESR1) patterns (PAMPs), 55
(MESA), 126, 147, protein 1(MRP1), 225 expression, 118 Pathogenesis, of autoimmune
147f Mumps virus (MuV), 58, 59, Nuclear factor kappa B (NF-κB) orchitis, 64, 65–69
MicroTESE, 126 63, 64 MyD88-dependent pathway BTB, 65–66
Microtubule affinity regulating Mx GTPase1 (MX1), 58 and, 57 germ cell apoptosis
kinase 4 (MARK4), Myeloid differentiation protein 88 TLR signaling in testicular mediators, 67–69
225 (MyD88), 57 macrophages, 59 immune cells, 66, 67f

Pattern recognition receptors
(PRRs), 55–59
activation, 52
families, 55, 56f
immune responses in
testicular cells, 52
overview, 55–57
PRR-initiated innate immune
responses
Leydig cells, 57–58
male germ cells, 58
signaling pathways, 57
PCB126, 253
P450 cytochrome cholesterol
side chain cleavage
enzyme (CYP11A1),
245, 246f
PDILT, 10
Pelvic surgery, 126
Penile examination, 139
Pentachlorophenol (PCP), 253
Percutaneous epididymal sperm
aspiration (PESA),
126, 146, 146f
Perfluorinated compounds (PFC);
see also Chemical
pollutants on
spermatogenesis and
male infertility
human exposure risk, and
reproductive health
about, 158–160
perfluoroalkyl chemicals/
mechanistic activities,
160
Perfluorinated compounds (PFC),
156
Perfluoroalkyl chemicals/
mechanistic activities,
160
Perfluoroalkyl substances (PFAS),
248–249
Perfluorohexane sulfonate
(PFHS), 156, 159, 160
Perfluorooctane sulfonate
(PFOS), 156, 250
Perfluorooctanesulfonic acid
(PFOS), 237
Perfluorooctanoic acid (PFOA),
156, 159, 160, 250
Perforin, 86
Perinatal exposure to BPA, 158
Periodic acid-Schiff (PAS)
reaction, 172
Periodic acid Schiff (PAS)
technique, 1, 3–4, 4f
Peritubular cell-specific AR
knockout (PTM-
ARKO) mice, 44
Peritubular myoid cells
(PTMC), 116, 123,
124, 168
AR expression, 43
Peroxiredoxin 1, 24
Peroxiredoxin 6, 24
Peroxisome proliferator-activated
receptors (PPAR),
130, 156, 160
Peroxisome proliferators, 160
Pesticide (methoxychlor), 157
Peutz-Jeghers syndrome (PJS),
201, 206
P-glycoprotein, 225, 226
Phagocytosis of apoptotic germ
cells, TAM receptors
in, 53–54
Phorbol 12-myristate 13-acetate
(PMA), 86
Phosphati-dylinositol- 3-kinase
(PI3K), 118
Phosphofructokinase, 225
Phosphoinositide 3-kinase
(PI3K)-related kinase
family, 201
Phospholipid hydroperoxide
glutathione peroxida­
semitochondrial
(PHGPx), 223
Phosphorylated ERK kinase
(p-ERK), 42
Phospho-S6, 206
Phthalates, 130, 251–252
Piperophos, 254
Pituitary adenylate cyclase-
activating
polypeptide (PACAP),
170
PIWI-interacting RNA (piRNA),
100, 128
Planar cell polarity (PCP) core
proteins, 211, 213f,
214t
Planar cell polarity (PCP) matter
PCP proteins and
spermatogenesis
background, 211–213, 214t
study in vitro, 213, 214–215
study in vivo, 215
Planar cell polarity matter and
spermatogenesis
overview, 211
Plant toxicants, 256–257
Plasma membrane receptors,
234
Plasminogen activator, 124
Plasticizers, 251
Plzf, 202, 205
Polarity, cell, 76
Polarity proteins, 158
Polo-like kinase 1 (PLK1), 109
Polychlorinated biphenyls
(PCBs), 155, 156, 253
Polycomb repressive complex 1
(PRC1.6), 106
Polycyclic aromatic hydrocarbons
(PAH), 254
Polyglandular autoimmune
syndrome, 63
Polyubiquitination of K48 chain,
99
Postejaculate urinalysis; see also
Asthenospermia
causes, 143
concentration and sperm
count, 143
management, 143
Postoperative hydroceles, 151
Posttesticular azoospermia
management of
epididymal obstruction
and vasal obstruction,
146
reconstruction, 147, 147t
sperm retrieval in OA,
146–147
obstructive causes, 146
PRDM9, 110
Prdm9 gene, 110
Pregnancy and infertility,
epidemiology of, 123
Pregnane X receptors (PXR), 156
Pregnenolone, 245
Preleptotene spermatocytes, 124
movement, 84–85
Prepachytene piRNAs, 128
Prespermatogonia, 94, 168
Pretesticular azoospermia, 125
Primary immotile-cilia
syndrome, 126
Primary spermatocytes, 126
Primordial germ cells (PGC),
123, 237
Index 275
Privileged sites, 183 phagocytosis of apoptotic
Privileged tissues, 183 germ cells, 53–54
Prochloraz, 255 in regulating immune
Profilin, 191 homeostasis, 52–53
Progressive motility (PM), 142 in testis, 53–54
Proline-rich region (PRR), 40 Recombinant soluble JAM-A
Properties, JAMs, 73–77 (rsJAM-A), 73, 75
Prosaposin, 10 Redd1 expression, 101
Prospermatogonia, 94 Regulation of JAMs and CAR in
Prostaglandin (PG), 88 testis, 77
signaling pathway, 160 Reproductive health,
Prostate medications, 140t environmental
Protein c-Src, 226 pollution and
Protein disulfide isomerase human reproductive health,
ER60, 63 declining, 155–156
Protein interacting with C kinase overview, 155
1 (PICK1), 15, 16 Rete testis (RT), 116
Protein kinase C (PKC), 235 Retinoic acid (RA), 98, 106, 111,
Protein-protein docking software, 124, 170
221 Retinoic acid-inducible gene
Protein S (ProS), ligand of TAM (RIG-I)-like receptors
receptors, 52, 53f (RLRs), 55, 57
Proteomic approaches in Retinoid signaling, 106
biomarkers Retinoid X receptors (RXRs), 156
identification, 221 Retrograde ejaculation, 147–148;
Proteomic markers and drug see also Ejaculatory
targets, 222–223 dysfunction
Proteomics analysis, 257 causes, 148
Proteomics methods, 220, 223 management, 148–149
PRRs, see Pattern recognition Retroperitoneal lymph node
receptors (PRRs) dissection (RPLND),
Pseudoephedrine, 143 126
P204 signaling, 58 Rho-associated kinase (ROK), 213
Psychiatric-related medications, Ribosomal protein S6, 206
129, 141t Ribosomal protein S6 kinase
fertility/infertility in humans, (S6K), 201
129 Rictor depletion, 202
PTEN, 201, 204f, 206 RNA, noncoding, in
Purinergic signaling, 98 spermatogonia
regulation, 100–101
Q RNA virus sensor signaling, 59
Quantitative proteomics, 5 Rosiglitazone, 255
Quiescent stem cells, 170 Round spermatids, 193
Round spermatids to functional
R spermatozoa,
RAB14, 21 development of;
RAD51, 45 see also Human
RAD51/DMC1, 109 spermatogenesis
Radiotherapy, 223 cytoskeletal reorganization,
RanBP5, 23, 24 175–176
Rapamycin, 201 spermiation, 172, 174–175
inhibitor, mTOR complexes spermiogenesis, 171–172, 173t
and, 201 RPA, 109, 110
and rapalogs in human RTKs, see Receptor tyrosine
disease, kinases (RTKs), TAM
pharmacological
applications of, S
201–202 Sapreticulin, 10
treatment, 204 Scarb1 gene, 248
Raptor-deficient testes, 205 Scavenger receptor class B
RA receptors (RAR), 98 member 1(SCARB1),
RA response element (RARE), 246f
98 Schiff reaction, 2f, 3
Rattus, 99 Scrotal examination, 139
Rbfox2 (RNA binding protein), SCs, see Sertoli cells (SCs)
101 Seager Electroejaculator, 150
Reactive oxygen species (ROS), Sec23, 16, 21
101, 253, 257 SEC23IP, 16
Real-time polymerase chain Secondary spermatocytes, 126
reaction (RT-PCR), Selective estrogen receptor
220 modulators (SERM),
REC8, 109 125, 143
Rec8, 106 Selective 5-hydroxytryptamine
Receiver operating characteristics reuptake inhibitors,
(ROC), 142 129
Receptor mediated pathways, Semen analysis; see also Male
156–157 infertility
Receptor tyrosine kinases about, 139, 142, 142t
(RTKs), TAM, 52–53 morphology, 142
categories, 52 motility and progressive
overview, 52, 53f motility (PM), 142
276 Index
Semen analysis, World Health
Organization 2010,
125t
Semen analysis abnormalities,
defined, 142
Semen volume and pH, 143
Seminal proteomes, 128
Seminiferous epithelium, 1, 3, 3f
Seminiferous tubule contraction,
F-actin in, 197
Seminiferous tubules (STs), 1, 40,
63, 81
about, 111
cross-sectional profiles, 2f, 3f
structure of, 183, 184
Sendai virus, 64
Septin 4, 221
Sertoli cell AR knockout
(SCARKO) mice, 44,
45, 46, 47
Sertoli cell F-actin-containing
structures,
dynamics of (during
spermatogenesis),
196–197
Sertoli cell only (SCO), 126
Sertoli-cell-only (SCO)
syndrome, 221, 222
Sertoli cells (SCs), 3, 7, 9, 12, 19,
20, 40, 42, 45
and androgen receptor (AR),
116
AR expression, 43
BTB function, 65–66
in EDC targeting of testicular
cells, 237; see also
Endocrine disrupting
chemicals (EDC)
immune regulation within
testis, 81–89
BTB/SCB, 83–85
cell mediated immune
response, 88
humoral immune
response, 86–88
overview, 81
testis immune privilege,
81, 82f, 83f
JAMs in, 75
mTOR signaling in, 205–206
phagocytosis of apoptotic
germ cells, 53–54
potential downside of
immune privilege,
88–89
role of, 124
SC androgen receptor
knockout (SCARKO)
mice, 84
SC conditioned media
(SCCM), 88
TAM receptors in testis, 52, 53
testosterone signaling
pathways, 42–43
TLR-initiated innate immune
responses in, 54–55,
57
Sertoli cells, F-actin in
in ectoplasmic
specializations,
194–195
in seminiferous tubule
contraction, role of,
197
Sertoli cell F-actin-
containing structures,
dynamics of (during
spermatogenesis),
196–197
tubulobulbar complex (TBC),
195–196
Sertoli cells barrier (SCB), 83–85
defined, 83
germ cell development, 83–84
movement of germ cells
across, 84–85
testis immune privilege, 85
Sertolionly syndrome, 144
Sertoli-spermatid adhesion
complex, 45
Sertoli valve, 95
Serum antisperm antibodies, 146
Serum/glucocorticoid induced
kinase (SGK), 201
Sex chromosomes, 109, 127
Sex-hormone-binding globulin
(SHBG)-like domains,
52, 53f
Sexual transmission of viruses,
185
Short ncRNA, 100
Signaling pathways, PRRs, 57
Simian immunodeficiency virus
(SIV), 185
Single-cell gel electrophoresis,
127
Single-stranded DNA (ssDNA),
109
Sirolimus, 201
Sirt1, 15
S6 kinase 1 (S6K1), 202
Skp1-Cullin-F-box (SCF)
complex, 99
Slc9a3 expression, 119
Smad signaling, 77
Small interfering RNA (siRNA),
128
Small RNAs, 128
sequencing methods, 220
SMAP2, 16
Sodium/hydrogen exchanger
(NHE3; SLC9A3), 119
Soluble form Fas L (sFasL), 68–69
Solvents, 251
Somatic cells, 95
Somatic Sertoli cell, 191
Sororin, 109
SORTILIN, differential
expression, 17–18
Sox9 genes, 168
SPACA4, 222
SPATA16 protein, 16
Sperm acrosome associated 1
(Spaca1), 16
Sperm acrosome associated 4
(SPACA4), 221
Spermatic antigens, antibodies
to, 63
Spermatic cord, 139
Spermatid-associated protein
(SPERT), 222
Spermatids, 3, 67, 81, 84, 172
and actin monomers, 193
Spermatocytes, 1, 58, 67, 81, 84,
124
meiotic division of, 3
Spermatogenesis, 123
cytoskeletons (F-actin) and,
see Cytoskeletons
(F-actin) and
spermatogenesis
hormonal regulation of, 125
human, see Human
spermatogenesis
inflammation and, see
Inflammation
JAMs, see Junctional
adhesion molecules
(JAMs)
mitotic phase of, see
Mitotic phase of
spermatogenesis
PCP proteins and; see also Spermatogonial niches in mouse
Planar cell polarity testis, 96f
(PCP) matter Spermatogonial progenitor cells
background, 211–213, (SPC), 202
214t Spermatogonial stem cells (SSC)
study in vitro, 213, about, 94
214–215 fertility and infertility
study in vivo, 215 regulation, 123–124
regulation of, 174 functional activity of, 95
reproductive toxicity of EDC human, 171
and, 158 within seminiferous tubule,
SC immune regulation within 116
testis, see Sertoli cells transplantation of, 96
(SCs) Spermatogonial transplantation,
and spermiogenesis, 123–124 192
testicular immunoregulation, Spermatozoa, 1, 3, 124, 191
see Testicular identification of, 123
immunoregulation Sperm chromatin, 158
Spermatogenesis, membrane/ Sperm chromatin dispersion
nuclear estrogen (SCD), 127, 150
receptors in Sperm chromatin structure assay
E2/ESR1 in male (SCSA), 150
reproduction, role of Sperm count, concentration and,
environmental 143
estrogens impact Sperm DNA fragmentation, 127;
human testicular see also Azoospermia
development, 119–120 Sperm DNA integrity, 150
knockouts of ESR2 or Spermiation
GPER, 119 about, 172
loss of ESR1 causing male actin- and MT-based
reproductive defects, cytoskeletons, role
118 of, 175
loss of membrane ESR1 regulation of
expression causing spermatogenesis, 174
male reproductive Spermiogenesis, 1
abnormalities, defined, 124
118–119 F-actin during, 193–194
estrogen sources in males round spermatids to
about, 116–117 functional
effects of early estrogen spermatozoa,
treatment in males, development of,
118 171–172, 173t
estrogen receptors (ER) spermatogenesis and,
expression in male 123–124
reproductive organs, Sperm mitochondria-associated
117–118 cysteine-rich protein
Spermatogenic cells, F-actin in (SMCP), 222
F-actin during Sperm morphology, 142
spermiogenesis, Sperm motility, 126, 142
193–194 defects, 77
F-actin in spermatogonia and Sperm protein associated
meiotic cells, 192–193 with nucleus on
Spermatogonia; see also X chromosome B
Mitotic phase of (SPANXB), 223
spermatogenesis Sperm release, 173
autocrine regulation of, Sperm retrieval in OA, 146–147;
97–99 see also Posttesticular
F-actin in/meiotic cells, azoospermia
192–193 Spinal cord injury, 126
metabolism of, 101 SPO11, 109
and niche, interaction src kinase, 40
between, 96–97 SRD5A, see 5α-reductase
niche of, 95 (SRD5A1)
origin and subpopulations of, sry genes, 168
94–95 STAG1/STAG2/STAG3, 109
paracrine regulation of, 97–99 Stem cell factor (SCF), 123, 170,
regulation 202
ubiquitin proteasome Stem cell renewal theory, 1
system in, 99–100 Steroid hormone receptors
undifferentiated, 168–171 expression in
Spermatogonia (SPG), 1, 58, 123, testicular cells, 234
125 Steroid hormones, 234
type B, 168 receptor knockout, 118
Spermatogonial function, role of Steroid metabolizing enzyme
mTOR signaling in activities, toxicants
maintaining on, 251t
about, 202, 204–205 Steroidogenic acute regulatory
mTOR signaling in meiosis, protein (StAR), 158,
205 225, 236, 246f, 248
mTOR signaling in Sertoli Stimulator of IFN gene (STING),
cells, 205–206 57
 Index 277

STING-dependent pathway, 58 PRRs, see Pattern Thiram, 254 Trimethylation of lysine 36

Stra8, 106 recognition receptors Thyroid hormone receptors (TR), of histone H3
Strep-Tag molecule, 68 (PRRs) 156, 235 (H3K36me3), 110
Streptomyces bygroscopicus, 201 TLRs, see Toll-like Thyroid hormone response Triphenyltin, 256
Stress-70 protein, 223 receptors (TLRs) elements (TRE), 236 tsc2-deleted testes, 204
Stromal cell derived factor 1 orchitis, 59 Tight junctions (TJs), 75–76, tsc1 knockout testes, 204
(SDF-1), 123 overview, 52–55 83, 84 T-2 toxin, 257
Structural maintenance of TAM receptors, see Tyro3, actin-based, 171 Tuberous sclerosis (TSC) 1/TSC2
chromosome (SMC) Axl, and Mer (TAM) proteins, 160 complex, 201
proteins, 109 receptors with Sertoli cells, 205, 213 Tubulin beta 2B (TUBB2B), 223
Study in vitro, planar cell polarity Testicular macrophages, TLR Tissue distribution, in testis, Tubuli rectus, 95
(PCP) proteins, 213, signaling in, 58–59 74t, 75 Tubulobulbar complex (TBC),
214–215 Testicular sperm aspiration TLRs, see Toll-like receptors 173
Study in vivo, planar cell polarity (TESA), 144, 144f (TLRs) F-actin in, 195–196
(PCP) proteins, 215 Testicular sperm extraction TMED4/P25, 5–7, 12 Tumor necrosis factor alpha
Subfertility, etiological factors, (TESE), 144 TMED4/p25, 21 (TNF-α)
63 Testis TMED7/P27, 5–7, 12 germ cell apoptosis, 55
Subinguinal varicocelectomy, immune privilege, Sertoli TMED7/p27, 21 regulation of JAMs and
151f cells, 81, 82f, 83f, 85 TM9SF3, 5–7, 12, 21 CAR, 77
Substrate S6 kinase 1 (S6K1), 202 regulation of JAMs and CAR Toll-like receptors (TLRs) testicular tight junction
Subtilosin, 129 in, 77 initiated innate immune function, 66
Sulfated glycoproteins (SGP), 237 Sertoli cell immune responses in testis, Tyro3, Axl, and Mer (TAM)
Sulfoglycolipids, 10 regulation within, see 52–53, 54–55 receptors, 52–55
SUN1, 106, 107 Sertoli cells innate defense system, 55, 57 in immune tolerance to male
Superresolution imaging, 107 TAM receptors in, 53–54 pattern recognition receptors, germ cells, 55
Surface- enhanced desorption/ tissue distribution and 88 inhibition of innate immune
ionization mass localization in, PRR signaling pathways, 57 responses in testicular
spectrometry 74t, 75 TLR-initiated innate immune cells, 54–55
(SELDI-MS), 220 Testis, HIV/ZIKV viruses in responses in Sertoli overview, 52, 53f
Surgical sperm retrieval, 144 immune privilege of testis, cells, 57 phagocytosis of apoptotic
SYCP1, 107 contributing factors, TLR signaling in testicular germ cells, 53–54
SYCP2, 107 183–184, 184f macrophages, 58–59 in regulating immune
SYCP3, 107 overview, 183 TopoVI DNA topoisomerase homeostasis, 52–53
Synapsis, 107 viruses and testis subunit A (TopoVIA), schematic structure, 52, 53f
Synaptonemal complex (SC) about, 184–185 109 in testis, 53–54
about, 107 human TOR pathway in reproduction, Tyro3 receptor, 55
tripartite structure of, 107 immunodeficiency 203t Tyrosine kinase receptors, 235
Syncytial state, 94 virus (HIV), 185–186 TOR/Raptor-dependent
Syntaxin 2, 16 Zika virus (ZIKV), 186, mechanism, 202 U
Syntaxin 13, 24 187t Total motile count (TMC), 126 Ubiquilin 3 (UBQLN3), 221
Synthetic fluorinated Testis-expressed 11 (TEX11) Toxoplasma gondii, 58, 59 Ubiquitin, 21
hydrocarbons, 158 gene, 128 Transcriptomic approaches Ubiquitin-activating enzyme E1
Synthetic organochlorine Testis gene expression, 46–47; in biomarkers (Uba1), 15
pesticide, 253 see also Androgen identification, 221 Ubiquitination, 99
Systemic immune tolerance, 183 regulation of Transcriptomic biomarkers, Ubiquitin-directed AAA-ATPase
spermatogenesis 221–222 P97, 21
T Testis-specific profilins, 194 Transepithelial resistance (TER), Ubiquitin-proteasome system
TATA element modulatory factor Testis-specific serine kinase 6 76 (UPS)
(TMF), 17 (TSSK6), 222 Transferrin, 24, 28f, 124, 237 about, 99
Tbc1d20 gene, 14 Testosterone; see also Androgen Transforming growth factor- functional role of, 99
Tek4 (Tektin 4), 172 regulation of alpha (TGF-α), 124 Ubiquitin proteasome system
Telomere-led meiotic spermatogenesis Transforming growth factor-β in spermatogonia
chromosome biosynthetic enzyme (TGF-β), 124, 170 regulation, 99–100
movement, 106–107, activities, 249t–250t regulation of JAMs and Ubiquitin-specific peptidase 8
107f, 108t concentrations, 116, 125 CAR, 77 (USP8), 13
Telomeres, 106 downregulation, 59 testicular tight junction Ubiquitous environmental
Teratozoospermia, 127; see also to maintain the blood-testis function, 66 contaminants, 156
Azoospermia barrier, 45–46 Trans Golgi networks (TGNs), 5 UBQLN3, 222
TERB1, 106 and number of gonocytes, Trans-interaction, homophilic UBXD8, 10
TERB2, 106 43–44 and heterophilic, 73, UDP-glucose: glycoprotein
Terminal deoxynucleotidyl production, 42, 245 74t, 75 glucosyltransferase
transferase-mediated signaling pathways, 42–43 Transition protein 2 (TNP2), (UGGT), 23
dUTP nick end for spermatid attachment and 222 Ultrastructural defects (USD),
labeling (TUNEL), release of sperm, 45 Transition protein 1 (TNP1) gene, 142
127, 150 testis gene expression, 46–47 222 UniProtKB/Swiss-Prot, 8
Terminal kinase-associated Testosterone biosynthesis Transrectal ultrasound (TRUS), United Nations Environment
domain, 227 in Leydig cells, 143 Programme/World
Testicular azoospermia; see also cell signaling for, Transurethral resection of Health Organization
Male infertility 247–248 ejaculatory duct (UNEP/WHO)
causes, 145 Testosterone signaling pathways (TURED), 143, 146 report, 155
management, 145–146 classical pathway, 42 Transverse elements (TF), 107 UPEC (uropathogenic Escherichia
posttesticular azoospermia, nonclassical (nongenomic) Treg cells, 66, 67f coli), 58–59
146–147, 147t pathway, 42–43 Tributyltin, 256 Uridine 5’-diphospho (UDP), 236
Testicular cells, paracrine Tetrachlorodibenzo-p-dioxin, Tricellulin, 84 Uropathogenic Escherichia coli
regulators of, 238t 129 Triclosan, 254 (UPEC), 58–59
Testicular dysgenesis syndrome 2,3,7,8-tetrachlorodibenzo-p- Tricresyl phosphate, 255
(TDS) theory, 120 dioxin (TCDD), 253 Tridecyl alcohol, 251 V
Testicular immunoregulation, Tetraiodothyronine, 235 Triiodothyronine, 235 Vaccination, MuV orchitis, 59
52–59 TGNs (trans Golgi networks), 5 Trimethylation of lysine 4 Valsalva, 128, 151
innate defense system, Thiamine pyrophosphatase of histone H3 Vangl, 211–216, 214t
55–59 (TPPase), 5 (H3K4me3), 110 Vang/Pk complex, 213
278 Index
Varicocele; see also Fertility/
infertility in humans,
regulation of; Male
infertility
about, 128, 150–151
fertility as function of age,
128
management of, 151
Varicocelectomy, 150
subinguinal, 151
Varicocele repair (VR), 151
Vasal obstruction
posttesticular azoospermia,
146
vasovasostomy for, 147
Vas deferens, 139
Vasoepididymostomy (VE), 147
VASP proteins, 191, 193
Vectorial asymmetry, 211
Vesicle-associated membrane
protein 3 (VAMP3),
21
Vibrostimulation, 150
Vinclozolin, 158
Viral orchitis, 59
Viruses and testis; see also
Testis, HIV/ZIKV
viruses in
about, 184–185
human immunodeficiency
virus (HIV), 185–186
Zika virus (ZIKV), 186, 187t
Vitamin C
in DNA fragmentation, 150
Vitamin E
in DNA fragmentation, 150
Voltage-dependent anionselective Z
channel protein 2 ZBTB16 (PLZF), 44
(VDAC2), 223 Zearalenone, 257
VPS54, 14 Zika virus (ZIKV), 88, 186, 187t;
see also Testis, HIV/
W ZIKV viruses in
WAPL, 109 infection in male
Wnt6, 98 genitourinary tract,
Wnt5a, 98 187t
Wt1 gene, 170 Zinc metallopeptidase, 221
ZIP9, 43
X Zipper theory, 85
Xenoestrogens, 234 Ziram, 254
Zonadhesin, 64
Y Zona pellucida, 125, 127
Y-chromosome microdeletion, Zonula occludens (ZOs), 84
126, 127, 145
YTHDC2 complex, 106