Sex Determination and Differentiation and this necessitated a finer modification of the said

Dra. Gloria Marie Valerio experiment

o Several experiments were done over a period of 10
Sex Determination years and the conclusion was, if the Barr body is
- Factors that make an individual a male or a female present in AT LEAST 20 out of 100 cells tested, the
- Established at fertilization subject is a genetic female
o If the Barr body is present in 0-4 cells out 100 cells
Gametogenesis tested, the subject is a genetic male
- Oogenesis - In blood smears, the Barr body is present in the neutrophil,
o Development and maturation of the female sex cell attached to the nucleus, appearing as an appendage
o Occurs in the ovaries o If the Barr body is present in 1-5 out of 300
o An oocyte has 44 autosomal chromosomes and 2 sex neutrophils, the test is positive and the subject is a
chromosomes (XX) genetic female
o An oocyte undergoes several mitotic divisions and 2 - Vaginal smears have the highest positivity among the three
meiotic divisions, giving rise to a mature ovum with 22 smears simply because of the direct effects of estrogen on the
autosomal chromosomes and 1 chromosome (always vaginal epithelium
X)
- Spermatogenesis Clinical Sex Sex No. of Total No. of
o Development and maturation of the male sex cell Conditions Chromatin Chromosomes Autosomal Chromosomes
o Spermatocytes has 44 autosomal chromosomes and 2 Test Chromosomes
Normal (+) XX 44 46
sex chromosomes (XY)
Female
o Just like the oocyte, the spermatocytes will undergo
Normal Male (-) XY 44 46
several mitotic divisions and 2 meiotic divisions, giving Super Female (+) XXX 44 47
rise to a mature sperm cell with 22 autosomal Klinefelter’s (+) XXY 44 47
chromosomes and 1 sex chromosome (either X or Y) Turner’s (-) XO 44 45
- If during fertilization, the sperm cell that carries the X- Mongoloids (-) male XX or XY 45 47
chromosome fertilizes the ovum, the product or zygote will have (+) female
44 autosomal chromosomes and 2 sex chromosomes (XX),
producing a normal genetic female Abnormal Sexual Chromosomal Constitution
nd
- On the other hand, if a Y-chromosome bearing sperm cell fertilizes - In normal males, after the 2 meiotic division, a mature sperm
the ovum, the product or zygote will have 44 autosomal cell should only have 1 sex chromosome (X or Y)
nd
chromosomes and 2 sex chromosomes (XY), producing a normal - Similarly in normal females, after the 2 meiotic division, a
genetic male mature ovum should only have 1 sex chromosome (always X)
- Sex or gender of an individual means “genetic sex” which is - Any abnormalities in the sex chromosomal constitution of an
established during fertilization individual is a result of the non-disjunction of the sex
nd
- An individual can have only 1 of the 2 sexes: chromosomes after the 2 meiotic division
nd
o XX  female - If after the 2 meiotic division, there will be non-disjunction of
o XY  male the sex chromosome (1 sperm cell still has 2 sex chromosomes
- For instance, a newborn child is delivered, a doctor’s basis of and the other has none or 1 ovum still has XX and the other has
declaring the sex of the baby is primarily the appearance of the none), this may lead to sex chromosomal aberrations shown
external genitalia below:
o If a baby presents with a non-fused labia with a small OVUM
SPERM
clitoris, that baby will be declared as a female X XX O
o If a baby presents with a fused scrotal sac with a penis X XX XXX XO
or phallus, that baby will be declared as a male NORMAL FEMALE TRIPLE X FEMALE TURNER’S
SYNDROME
o If a newborn presents with an ambiguous external
Y XX XXY YO
genitalia, sex determination is more difficult, as in NORMAL MALE KLINEFELTER’S NON-VIABLE
pseudo-hermaphrodites SYNDROME
 In cases like this, the doctor should first sign XY XXY XXXY XY
the birth certificate and then perform a sex KLINEFELTER’S TRIPLE X MALE NORMAL MALE
test SYNDROME
O XO XX OO
Sex Chromatin Test TURNER’S NORMAL MALE NON-VIABLE
SYNDROME
- In 1949, 2 scientists: Barr & Bertram, discovered the presence of
the sex chromatin mass/body in female somatic cells
- The first experiment was done on cat neurons and was later Hair Root Test
modified in human samples of buccal smear, blood smear and - Pluck 3-4 strands of normal scalp hair with intact roots
vaginal smear - Tease the hairroots and stain with a fluorescent dye and under a
- They found the presence of the sex chromatin mass/body in microscope, look for fluorescent “F body” or “Y body”
female somatic cells and later on, this sex chromatin mass was - “Y body” is part of the Y-sex chromosome
called the Barr body - Present and positive only in genetic males
- They concluded that if the Barr body is present, the sex chromatin - Invariably absent in genetic females
test is positive and this is usually true when the sex chromosomal
constitution of the individual is XX: (+) Barr body, (+) sex Karyotyping or Chromosomal Analysis
chromatin test  normal genetic female - Most accurate but most expensive
- In a buccal smear taken from the inner cheek, the Barr body can - A tissue culture is done where autosomal chromosomes are
be found in the inner surface of the nuclear membrane paired with each other as well as the sex chromosomes
o Problems may arise with false positives where a - This test cannot be done by a mere medical technologist or a
chromatin mass is incorrectly identified as a Barr body, medical doctor, only by a scientist who specializes in Genetics
 - After all of the chromosomes have been paired up with each
other, an individual’s chromosomal constitution can be
determined
Sex Differentiation
- Gonads are the primary organs of reproduction
- Testes in males and ovaries in females
- Genital ducts give rise to the internal genitalia
o Wolffian ducts – male internal genitalia
o Mullerian ducts – female internal genitalia
- Structures that make up the external genitalia
Gonadal Differentiation
- The testes and ovaries share a common anlage, the genital ridge
(bilateral)
th
- From fertilization until the 7 week of embryonic life, the genital
ridge is indifferent, thus there is no anatomical evidence whether
it will develop into testes or ovaries
- The genital ridge is divided into 2 parts: inner medulla and outer
cortex
- So although the testes and ovaries will come from the same
structure, each will come from a different part of that structure
(medulla for testes and cortex for ovaries)
- Also genetically determined
Testicular Development
th th
- In the presence of the Y-chromosome during the 7 – 8 week of
life, the medulla develops into testes while the cortex regresses
o Present in the Y-chromosomes is a membrane protein
called H-Y antigen which is the Testes-Determining
Product
Ovarian Development
- In the absence of the Y-chromosome and in the absence of the H-
th th
Y antigen between the 8 – 10 week of life, it is the cortex that
will develop into the ovaries while the medulla regresses
- Presence of XX is a prerequisite for normal ovarian development
o XO – neither testicular nor ovarian differentiation
 Gonad will remain as a long pale streak of
connective tissue
- Hormonal treatment of the mother during pregnancy will have no
effect on gonadal development  Thus, gonadal differentiation is
genetically determined
Differentiation of Genital Ducts
- Just like the gonads, both the Mullerian and Wolffian ducts are
present in their indifferent stage during the early weeks of
embryonic life
o In male differentiation, Mullerian ducts regress and
Wolffian ducts develop
o Conversely, in female differentiation, Wolffian ducts
regress and Mullerian ducts develop
- Mullerian ducts will give rise to the female internal genitalia
o Uterus
o A pair of fallopian tubes or oviducts
- Wolffian ducts will give rise to the male internal genitalia
o Epididymis
o Vas deferens
o Seminal vesicles
o Ejaculatory duct
- Presence of functional testes
o In males (XY), the Y-chromosome indicates the
presence of the H-Y antigen  testes develop
o If the testes are functioning normally, it will stimulate
the Wolffian ducts complete their development
(Mullerian structures involute and the male internal
genitalia will fully develop)
o The mere presence of the testes will not guarantee
that it will be the Wolffian ducts that will develop. It
should also be functioning normally
- Absence of testes (in females or in males with non-functional
testes)
o Mullerian ducts will develop and Wolffian ducts will
regress or involute
o Ovaries – not needed for development of female
internal genitalia
o Genital ducts have inherent tendency to feminize in
the absence of functional testes
Male Differentiation
- Genetic male – embryonic testes secretes:
o MRF (Mullerian Regression Factor)  inhibits
development of Mullerian ducts  no female internal
genitalia (action is unilateral)
 In the case of a non-functional testes, no
substance will be secreted to inhibit the
development of the Mullerian ducts
o DOS (Duct Organizing Substance) and Testosterone 
stimulate the development of the Wolffian ducts
- In the presence of these three secretory products of the
FUNCTIONAL embryonic testes, development of the Mullerian
ducts is inhibited and development and maturation of the
Wolffian ducts is stimulated
Female Differentiation
- Without testes  No MRF  No DOS  No Testosterone 
genital ducts will have inherent tendency to feminize
- Presence of ovary is not a prerequisite for the development of the
female internal genitalia
- No gonad (e.g. Turner’s syndrome)  No MRF, DOS, Testosterone
 still normal development of uterus and fallopian tubes because
again, in the absence of functional testes, the genital ducts have
an inherent tendency to feminize
o Rudimentary gonad  only uterus and fallopian tubes
develop, regardless of chromosomal sex
Differentiation of External Genitalia
Anlage Male Female
Genital tubercle Penis Clitoris
Urethral folds Part of corpus spongiosum Labia minora
Laboscrotal swelling Fuse to form scrotal sac Labia majora
Urogenital slit Closed, complete fusion Vagina
Development of External Genitalia
- Males
o 44 autosomal chromosomes and 2 sex chromosomes
XY
o Y-chromosome  H-Y antigen  testes will develop
o Functional testes  MRF, DOS, Testosterone 
Mullerian ducts regress, Wolffian ducts develop into
male internal genitalia
o In the case of a functional testes, it will also secrete
dihydrotestosterone in addition to testosterone
 These 2 will masculinize the external
genitalia
 Because just like the internal genitalia, in
the absence of a functional testes, the
external genitalia has an inherent tendency
to feminize
 Without testosterone and
dihydrotestosterone  female external
genitalia
o In male adults, testosterone is also responsible for the
development of male secondary sexual characteristics
that will include: growth of facial hair, body hair, pubic
hair, development of a low-pitched voice and extra
protein deposition in the muscle (anabolic nature of
testosterone so males are more muscular than
females)
 - Females
o XX  no Y  ovaries develop  no MRF, DOS,
Testosterone  feminine external genitalia
o Main secretory product of ovary: estrogen
o Estrogen is responsible for development of female
secondary sexual characteristics that will include:
development of a high-pitched voice, extra fat
deposition into the subcutaneous tissues of the
breasts, buttocks and thighs
- Thus, no amount of surgical or hormonal modification in an
individual born male or female will ever change his or her genetic
makeup
Components of an Individual’s Sexual Identity
Male Female
Chromosomal/Genetic Sex XY XX
Nuclear Sex (-) Barr body (+) Barr body
Gonadal Sex Testes Ovary
Hormonal Sex Mainly androgen Mainly estrogen
Internal Genitalia Wolffian ducts Mullerian ducts
Sex Chromosomal Constitutions in Ambiguous External Genitalia
- Normal 46 XX  female
- Normal 46 XY  male
- Abnormal sex chromosome constitutions
Differential Diagnosis of Ambiguous External Genitalia
- Perform a Sex Chromatin Test
o (+) Result may suggest:
 Female pseudo-hermaphroditism
 True hermaphroditism
 Chromatin positive variants of gonadal
dysgenesis (e.g. Klinefelter’s XXY so even if
the subject is a male, he could test positive
for the sex chromatin test)
o (-) Result may suggest:
 Male pseudo-hermaphroditism
 True hermaphroditism
 Chromatin negative variants of gonadal
dysgenesis (e.g. Turner’s syndrome XO)
Male Pseudohermaphroditism
- XY sex chromosomes  Gonads: exclusively testes but the mere
presence of testes is not an actual determinant of one’s sex and
thus it should also be functional
- One cause of male pseudohermaphroditism is testicular failure 
testes is not functioning normally or if the testes is functioning
normally but there are inadequate receptors in the target cells for
the hormone secreted by the testes
o Because there is an inherent tendency of the internal
and external genitalia to feminize in the presence of a
non-functional testes, the inherent tendency to
feminize will not be overcome  resulting to both
ambiguous external and internal genitalia
- Genital ducts or external genitalia
o Lacks masculinisation
o Some degree of feminization
- Failure of fetal testes to overcome inherent tendency to feminize
o Secretory failure of testes
o Failure of target tissues to respond normally
Turner’s Syndrome
- Short stature, prominent webbing of neck, lack of breast
development, scant pubic hair, small phallus, child-like
appearance
- XO  no gonads  normal uterus and fallopian tube  external
genitalia is female due to inherent tendency to feminize  female
internal genitalia will remain infantile due to absence of ovaries
and estrogen  no maturation of internal and external genitalia
- No estrogen  no androgen  no negative-feedback  high FSH
and high LH
- High FSH and LH: more than 96 mouse units / 24 hours
- Negative sex-chromatin pattern
Female Pseudohermaphroditism
- Large phallus, labia majora resembling a scrotal sac, incomplete
closure of the urogenital sinus
- Short stature, with body hair and facial hair, masculine
distribution of pubic hair, small breasts, muscular
- XX  presence of ovaries  No MRF, DOS and Testosterone 
female internal genitalia  ambiguity is confined exclusively to
the external genitalia
- External genitalia of newborn female pseudohermaphrodite with
congenital adrenal hyperplasia
o If adrenal glands are hyperplastic, androgen secretion
is elevated and this will masculinize the external
genitalia
o If the pregnant mother is taking exogenous androgens
th
before the 12 week of gestation, it will have an effect
on the external genitalia of the developing fetus 
ambiguous genitalia
o Measurement of androgen levels is now a part of
newborn screening procedures
- Ovaries, Mullerian derivatives  normally developed
- Anatomical ambisexuality – limited to external genitalia
- Masculinzation – due to extragonadal androgens
- Even with severe masculinisation of external genitalia – uterus
and fallopian tubes remain normal
True Hermaphroditism
- Not only caused by non-disjunction of sex chromosomes, a
mutation of the sex chromosomes is also implicated in the
etiology of true hermaphroditism
- Presence of both ovarian and testicular tissues
- An ovotestis may be observed on histological examination
- External genitalia – may resemble either male or female
o Often ambiguous
o ¾ of patients reared as males because of size of phallus
- In virtually all cases  there is a uterus
- 2/3 of patients  have appreciable breast development
- 70% - are chromatin positive
o (+)  menstruate
o (-)  will not menstruate
- Note well that true hermaphrodites can never impregnate
themselves
Case
- 20 y/o – reared as female
- Physical examination (Dept. Of Ob-Gyn FEUH)
o Ht: 5’6 and ½“ - Wt: 138 lbs.
o Breasts: not developed
- External genitalia is ambiguous: 2 scrotal-like labia each containing a 2 x 3 x
2 cm rounded, slightly firm, movable mass
- A longitudinal groove in the middle of the 2 labia
o Upper end – enlarged clitoral-like structure
o Lower end – opening that enters into a vagina-like cavity – 10
cm long admits only a probe
- Internal genitalia - no grossly palpable uterus, tubes or ovaries (by rectal
exam)
- Sex chromatin test (Clinical Laboratory, Department of Pathology, FEU-
NRMF) Specimen:
o Buccal smear and blood smear
o Hair root test is (+)
- Results
o Barr chromatin bodies
 Negative / 600 cells
 Control: 2 normal female average count: 69/100
cells (+) Barr bodies
o Drumstick – 0/500 PMN
o Hair root – 2 slides prepared
 15/100 cells and 18/100 cells
 With “Y-bodies” under fluorescent
microscope
Paolo Warren (Med-IA)