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Hormonal Control of Teleost Reproduction:

Gonadal Events

Craig V. Sullivan
Department of Zoology
College of Agriculture and Life Sciences
Hypothalamus-pituitary-gonad axis
Photoperiod Physical environment Temperature

Sensory receptors
+/- Feedback Feedback +/-
Hypothalamus

Gonadotropin releasing hormones

+/- +/-
Pituitary
Vitellogenin

Liver Gonadotropins (FSH, LH)


Ovary Testis
Sex steroids
+
Sex steroids
(estradiol) (Testosterone [T], 11-keto-T)

+ +
Ova Secondary sex characteristics Sperm
Sex determination

Ovary Sex differentiation Testis

Oogonium Spermatogonium
OH OH
O
Oocyte Spermatocyte
HO O

Estradiol-17b CH 3 Spermatid 11-Keto-


H C OH
OH testosterone
Sperm
O

17a,20b-Dihydroxy-4-
pregnen-3-one (MIH)

Final maturation

Fertilization (MIH, Maturation-inducing hormone)


Steroidogenic pathways in gonads of salmonids
Cholesterol Steroidogenic acute regulatory protein
P450scc
3 b -HSD
Pregnenolone Progesterone
P450c17 P450c17
3 b -HSD
17 a -Hydroxypregnenolone 17 a-Hydroxyprogesterone
20 b -HSD
P450c17 P450c17
3 b -HSD
Dehydroepiandrosterone Androstenedione 17,20b-DHP
17 b -HSD 17 b -HSD
3 b -HSD
Androstenediol Testosterone 11b-hydroxy-
testosterone
P450arom P45011b
side chain cleavage
3b-hydroxysteroid dehydrogenase 11b-HSD
C17 hydroxylase/lyase Estradiol-17 b
17bhydroxysteroid dehydrogenase
Aromatase 11b- hydroxylase 11-keto-
20b-hydroxysteroid dehydrogenase 11b- hydroxysteroid dehydrogenase testosterone
Gonadal Sex Differentiation in Tilapia
Formation of gonadal anlagen

Oogenesis
meiosis
P450scc
3b-HSD
P450c17 Ovary
P450arom Ovarian cavity
Estrogen

Spermatogenesis
Sperm duct

meiosis
Testis
Androgen
P450scc
3b-HSD
Days after hatching P450c17
0 5 10 15 20 30 40 70
Gonadotropin Receptors in Teleosts
Follicle Stimulating Luteinizing Hormone Affinity
Hormone (FSH) (LH)

FSH receptor LH receptor


FSH>>> LH LH only?
Female Male
Vitellogenic Preovulatory
Spermatogonial Spermato- Spermiation
Proliferation genesis
Theca

Granulosa
Maturing
Aromatase
- Leydig cell?
17a-OH-P
Estrogen
Capaci-
tation
20b-HSD MIH 17a 20b-Progesterone (MIH)
Steroidogenesis During Oocyte Growth and Maturation

Theca Cell Granulosa Cell

Cholesterol
P-450scc
3b-HSD
20b-HSD
DHP
Oocyte
17a P 20b-S
maturation
P-450
17b-HS D
aromatase
Oocyte
Testosterone Estradiol-17b
growth

DHP, 17a 20b-Progesterone (MIH in salmonids et al.);


20b-S, 17a 20b-21-Progesterone (MIH in perciforms et al.)
Reproductive Hormones in Maturing
Salmonids

DHP DHP

LH LH
Estradiol (E2) Testosterone

FSH FSH
Testosterone 11- Keto-
testosterone

M A M J J A S O N M A M J J A S O N
Vitellogenesis Spermatogenesis
Ovulation Spermiation

Female Male
Stages of Oocyte Development in Teleosts and
Hypothesized Endocrine Control

Oogonial Entry Primary oocyte Completion of Entry into


mitosis into growth cortical alveoli vitellogenesis
meiosis stage

FSH IGF-I IGF-I FSH FSH


( 3b-HSD) ( 3b-HSD, P450arom,
IGF-I Androgens Vg uptake)
(11-KT) IGF-I
( P450arom) IGF-I
Ribosomal ( P450arom)
components Steroids
(mRNA & protein) Zona radiata (ZR) ( E2) E2
components
( Vg synthesis)

Lipids
(droplets) G. Young (2003), unpublished
mRNAs
(yolk process-
Glycoproteins ing receptors IGF-I, Insulin-like growth factor-I
(cortical granules) & enzymes)
E2, Estradiol-17b
Teleost Vitellogenesis and Oocyte Growth

Endocrine regulation Environmental control


E2
FSH

Follicle growth & maturation


Endogenous Factors & Rhythms
•Nutritional factors
•Innate biorhythms
Liver

Vitellogenin(s) Proteomic & Chemical Influences


•Enzymes
Direct regulation: •Substrates
Vitellogenins (Vgs) & their receptors (VgRs)
Endocrine Control of Vitellogenesis and Choriogenesis

Liver Sex steroid Pituitary


hormone
binding globulin

FSH
Follicle
Estrogen receptor Cells

Vitellogenin Choriogenin Estrogen


gene gene Vitellogenin receptor
Chorion

Phosvitin
Recycling
Lipovitellin

b’-component
Vitellogenin C-terminal
component

Choriogenin Endocytosis
Blood Yolk protein
H and L accumulation
Oocyte Enzymatic
processing
Maturation of Teleost Ovarian Follicles: Endocrine Control

GtH MIS

GV GV GV
Y Y Y

E2 20b-S and DHP Oil droplets GV breaks down


coalesce
Vg uptake Gap junctions GV migrates Meiosis resumes
Oocyte growth MIS receptors
Y
MPF activated

Acquisition of maturational Genomic and cytoplasmic


competence maturation
GtH, gonadotropin (LH); MIS, maturation inducing steroid (20b-S or DHP, also called MIH );
GV, germinal vesicle; MPF, maturation-promoting factor
Activation of Maturation-promoting Factor (MPF)
During MIH-induced Meiosis

17a, 20b-Dihydroxy-4-pregnen-3-one (MIH)

Oocyte plasma membrane


Receptor

35 kDa Cyclin B synthesis 35 kDa


cdc2 cdc2

35 kDa
mRNA cdc2

Cyclin B Cyclin B
Cyclin B Translation

T P T P
Serine kinase Threonine kinase
34 kDa (cdc kinase) 34 kDa (cdk7/cyclin H)
Active cdc2 cdc2
MPF
Cyclin B P S Cyclin B
IGF-I Induces Maturation Via a GtH- & MIS-Independent Pathway

IGF-I GtH MIS

GV GV GV
Y Y Y

E2 20b-S and DHP Oil droplets GV breaks down


coalesce
Vg uptake Gap junctions GV migrates Meiosis resumes
Oocyte growth MIS receptors
Y
MPF activated

Acquisition of maturational Genomic and cytoplasmic


competence maturation
Weber and Sullivan 2000, Biol. Reproduction
Cytoplasmic Maturation: MIH-induced Proteolysis
of Yolk Proteins in Striped Bass
GnRHa
Initial 12-h 6-h Ovulated
implant 12-h 9-h 6-h 0-h

GVBD
X 2.4

110 110 100 100 Oocyte volume


75 75 75 75
X 1.4
33 33
28 28
X1
20 20 20 20

- Lv heavy chain: 110 kDa 100 kDa MIH production


- Lv light chain: 33 kDa 28 kDa (King et al., 1994)
- b’-component: 20 kDa

N. Hiramatsu, A. Hara and C.V. Sullivan, Fisheries Science


Regulation of Hydration During Cytoplasmic Maturation:
Endocrine, Enzymatic, and Other Factors
GtH

MIS
Yolk formation: Final maturation
Cathepsin D&B (?)
GVBD (nuclear maturation) Cathepsin L
Lipidation: Clarification of ooplasm Cathepsin D
LP lipase Coalescence of oil droplet
Sire et al. ‘94 Bafilomycin-sensitive pathway Enzyme
Carnevali et al. ‘99 activation
Kwon et al. ‘01 H+ pump activation Float (good)
Hiramatsu et al. ‘02 H+ Cl- Yolk Cathepsin L
H+ acidification Cathepsin B Sink (bad)
MIS-dependent
yolk processing pH
3b-HSD Cl-
inhibitor
VATPase Cl- channel Trimmed Cathepsin L
C + - yolk proteins Cathepsin D
Free amino
110 100 Nutrient acid pool Carnevali et al.
pool Osmotic ‘99, ‘01
effector Hydration
Carnevali et al. ‘99; Selman et al. ‘01; H2O
Hiramatsu et al. ‘02 Hiramatsu et al. ‘02; Matsubara et al. (unpublished)
Hormonal Control of Ovarian Follicle Maturation
and Ovulation: an Integrated Model

LH MIH MIH
LH receptor mMIHR
Granulosa cell Oocyte Somatic cell
+ cAMP granulosa, thecal?
- PKA nMIHR
+ PKA
+ GVBD
+ Cx synthesis + LOX products
+ Gap junctions + PKC
+ MIH enzymes + Prostaglandins
Oocyte + Ovulation
+ Gap junctions mMIHR= Membrane MIH nMIHR= Nuclear MIH
+ MIH receptor receptor receptor

Stage 1: Induction of OMC Stage 2: MIH-mediated Stage 3: MIH-mediated


(ovulatory competence) & release from meiotic induction of ovulation
MIH production ability arrest
Spermatogenesis in Teleost Fish

A B st C
sz
sg

sc
sz

A. SPERMATOGONIAL PROLIFERATION
B. MEIOSIS AND SPERMIOGENESIS
C. SPERMIATION
sg, stem cells; sc, spermatocytes; st, spermatids; sz,
spermatozoa
Spermatogenesis and Spermiation
Spermatogenesis

Mitosis Meiosis I Meiosis II Spermiation Capacitation

Motility
Mitotic Paired
proliferation of chromosomes in
spermatogonia primary
to produce spermatocytes Before After
millions of
spermatocytes
that enter
meiosis
Maturation of
Formation of Reduction spermatozoa in
secondary division to form Restructuring of seminal plasma
spermatocytes haploid spermatids into
spermatids flagellated
spermatozoa
Hypothalamus-pituitary-gonad axis
Photoperiod Physical environment Temperature

Sensory receptors
+/- Feedback Feedback +/-
Hypothalamus

Gonadotropin releasing hormones

+/- +/-
Pituitary
Vitellogenin

Liver Gonadotropins (FSH, LH)


Ovary Testis
Sex steroids
+
Sex steroids
(estradiol) (Testosterone [T], 11-keto-T)

+ +
Ova Secondary sex characteristics Sperm
Sex determination

Ovary Sex differentiation Testis

Oogonium Spermatogonium
OH OH
O
Oocyte Spermatocyte
HO O

Estradiol-17b CH 3 Spermatid 11-Keto-


H C OH
OH testosterone
Sperm
O

17a,20b-Dihydroxy-4-
pregnen-3-one (MIH)

Final maturation

Fertilization (MIH, Maturation-inducing hormone)


Gonadal Sex Differentiation in Tilapia
Formation of gonadal anlagen

Oogenesis
meiosis
P450scc
3b-HSD
P450c17 Ovary
P450arom Ovarian cavity
Estrogen

Spermatogenesis
Sperm duct

meiosis
Testis
Androgen
P450scc
3b-HSD
Days after hatching P450c17
0 5 10 15 20 30 40 70
Reproductive Hormones in Maturing
Salmonids

DHP DHP

LH LH
Estradiol (E2) Testosterone

FSH FSH
Testosterone 11- Keto-
testosterone

M A M J J A S O N M A M J J A S O N
Vitellogenesis Spermatogenesis
Ovulation Spermiation

Female Male
The Sertoli/Germ Cell Unit
Germ cells only survive and develop
when associated with Sertoli cells
The germ cell supporting capacity of
Sertoli cells is finite

Number of Sertoli cells sets the


capacity of spermatogenesis
(e.g.,Sertoli cell proliferation)
FSH-regulated in mammals
Hemicastration model

inhibin

testis pituitary

+ FSH
Hemicastration Effects: Sertoli Cell
Proliferation & Androgen Release
1.5 LH-R 4 FSH-R

Expression
control **
3 ULG

Ex
1.0
*

Relative
2

Relative
0.5
1

0.0 0
testis seminal vesicle testis seminal vesicle

8 Androgen Release
*

tissue)
6 control

per mg
ULG

pg(ng/mg
7.5 4 *
control 4 days
# BrdU-labelled cells

OHA
2 *
*
# BrdU labeled

5.0 * 0
0 10 100 1000
LH (ng/ml)

2.5
Increased FSH-R expression
Increased Sertoli cell proliferation
0.0 Increased androgen production
Sertoli cells single paired four(+)
spermatogonia FSH-mediated?
Androgens in Fish A B

C 11 KT
(+ bovine insulin)

spermatogonia
% late B-type
Feedback effects Spermatogenesis,
in BPG axis SSC & behaviour
T stimulates
gonadotroph 11KT induces proliferation
differentiation and differentiation of eel
- Cell size spermatogonia in vitro
- LH content (Miura et al., 1991 PNAS)
- GnRH GnRH
SSC, secondary sex characteristics;
innervation LHb BPG, brain-pituitary-gonad axis
Androgens, FSHb and FSH-R 4
LHb
LHb

Relative Expression
in Juvenile Males 3

2
T (E2)
stimulatory 1

0
control KT T KT + T
T, but not KT,
1.0
reduces FSHb
FSHb

Relative Expression
steady-state
mRNA levels
control T 0.5

Androgen-induced inhibition T, but not KT,


0.0
via reduced Leydig cell activity reduces control KT T KT + T

- loss of mitochondria steady-state 1.5


FSH-R
FSH-R

Relative Expression
- steriodogenic enzyme activity mRNA levels
1.0

T might inhibit spermatogenesis by


*
compromising FSH-dependent signaling on 0.5 **
the pituitary and testicular level
0.0
control KT T KT + T
Le, Leydig cell; T, testosterone; KT, 11-ketotestosterone
Insulin-Like Growth Factor I (IgF-I) Stimulates All Stages of
11-KT- Induced In Vitro Spermatogenesis in the Japanese Eel
Control rhIgF-I (100 ng/ml) rhIgF-I + 11-KT

15 d

rhIgF-I + 11-KT

45 d
rhIgF-I, recombinant human IgF-I; 11-KT, 11-ketotestosterone; Nader et al. (1999)
GA, A-type spermatogonia (Sg); L-GB, “Late” B-type Sg;
T, spermatids; C, spermatocytes; lower left. spermatozoan Biology of Reproduction
Inhibition of 11 KT-induced eel spermatogenesis by eSRS21

11KT 11KT + eSRS21 Miura et al. (2002) Development

Northern blots
Anti-sense Sense 1 2 3
eSRS21
2.4 kb
50 b
BrDU index (%)

40
30 EF-1
c
20
a a eSRS21 (an AMH)
a
10 expressed by Sertoli Expression of
0 cells eSRS21
IC 0 5 0 5 (ng/ml) suppressed by
r-eSRS21 r-eSRS21 11KT
+11KT
AMH, Anti-Müllerian
Spermatogenesis inhibition by hormone 1 = Initial control
eSRS21 2 = Control after culture
3 = Cultured with 11 KT
Activin and some newly-discovered hormonal
mediators of teleost spermatogenesis
Gonadotropin

Spermatocyte
R
Meiosis
Leydig Cell

11 KT Nucleus

- Mitosis
AMH
+

Spermatogonium
AMH
Sertoli Cell

R
- CDK/Cyclin
RII
Nucleus
Nucleus
+ Activin B Activin B RI
(dimer)

Miura et al. (2002) Development; Nader et al., (1999) BOR IGF-I ?


Gonadotropin-stimulated Sperm Capacitation

Follicle Stimulating Luteinizing Hormone Affinity


Hormone (FSH) (LH)

FSH receptor LH receptor


FSH>>> LH LH only?

MIH-dependent Sperm Capacitation Spermatogonial Spermato- Spermiation


Proliferation genesis
% Motile

Leydig cell?
17a-OH-P
Capaci-
tation
Concentration 20bS (nM) 17a 20b-Progesterone (MIH)
Proposed Model of 20b-S (MIH) Stimulation
of Sperm Motility in Fish
LH Leydig cell

20b-S (MIS) 17a,20b-Dihydroxy-4-


pregnen-3-one

Sperm membrane !
Vamos!
receptor for 20b-S

Sperm activation
( *Ca++ influx & cAMP )

Sperm motility
*via L-type Ca ++ channels Detweiler and Thomas (1998) JEZ
Acknowledgements
Mentors

~
Reynaldo Patino Peter Thomas Graham Young Rudiger Schulz
Texas Tech. Univ. Univ. Texas Univ. Idaho Univ. Utrecht

NCSU Research Associates Graduate Students

Greg Weber Naoshi Hiramatsu Amber Garber Ben Reading Charlene Couch
USDA-ARS NCCCWA NCSU NCSU NCSU NCSU