Embryology – PBL 5 – sexual differentiation

Development of male and femal sexula organs in embryo

1. Internl
a. Gonads:
i. Gonadal ridges, initially appearing as undifferentiated structures, form through a series of intricate processes
involving epithelial proliferation and condensation of mesenchyme. These ridges serve as the primordial
structures from which either testes or ovaries will develop.
ii. Primordial germ cells, originating from the yolk sac endoderm
 These cells migrate through the primitive streak and dorsal mesentery before settling in the genital ridges
around the fifth week of development.
iii. Upon their arrival, primordial germ cells interact with the surrounding somatic cells, initiating gonadal
differentiation.
 The genetic sex of the embryo, determined at fertilization, dictates the fate of these gonads, whether they
will develop into testes or ovaries.
iv. The process of gonadal differentiation is heavily influenced by the presence or absence of the SRY (sex-
determining region on Y) gene located on the Y chromosome.
 The SRY gene encodes a transcription factor known as the testis-determining factor, which triggers a
cascade of downstream events leading to male sexual differentiation.

b. Testis Development:
i. In embryos with a Y chromosome and the presence of the SRY gene, the genital ridges differentiate into testes.
The SRY gene activates the proliferation of primitive sex cords within the gonadal tissue.
ii. Primitive sex cords, under the influence of SRY, continue to proliferate and invade the medullary region of the
developing gonad, forming testis cords.
 These cords consist of both germ cells and somatic cells.
iii. Concurrently, Leydig cells, derived from the mesenchyme surrounding the developing gonad, begin to
differentiate.
 Leydig cells are responsible for the production of testosterone,
iv. By the eighth week of gestation, Leydig cells become functional and start synthesizing testosterone.
 This hormone plays a crucial role in the masculinization of the embryo, influencing the differentiation of
both internal and external genitalia.
 v. The testis cords undergo further morphological changes, eventually forming seminiferous tubules (in puberty),
the site of spermatogenesis in adult males.
 Interstitial cells of Leydig, situated between the seminiferous tubules, continue to produce testosterone,
maintaining the male phenotype throughout development.
 Once the seminiferous tubules are canalized, they join the rete testis tubules, which in turn enter the
ductuli eiferentes.
a. These efferent ductules are the remaining parts of the excretory tubules of the
mesonephric system.
b. They link the rete testis and the mesonephric or Wolflian duct, which becomes the ductus
deferens.

c. Ovary development
i. Dissociation of Primitive Sex Cords:
 In the absence of the SRY gene and under the influence of the XX sex chromosome configuration, the
primitive sex cords within the indifferent gonad dissociate into irregular cell clusters.
 These clusters, composed of primitive germ cells, occupy the medullary region of the developing ovary.
ii. Formation of Ovarian Medulla and Cortical Cords:
 As development progresses, the irregular cell clusters within the medullary part of the ovary disappear.
 They are gradually replaced by a vascular stroma, which forms the ovarian medulla, the innermost
region of the ovary.
 Meanwhile, the surface epithelium of the female gonad continues to proliferate.
 By the seventh week of gestation, it gives rise to a secondary generation of cords known as cortical
cords.
a. These cortical cords penetrate the underlying mesenchyme but remain close to the surface of
the ovary.
iii. Formation of Primordial Follicles:
 By the third month of development, the cortical cords further differentiate into isolated cell clusters.
 Cells within these clusters continue to proliferate, surrounding each oogonium (precursor to ova) with a
layer of epithelial cells known as follicular cells.
 Together, the oogonia and follicular cells constitute a primordial follicle, representing the basic unit of
ovarian follicular development.
d. Overview
i. Genetic sex of an embryo is established at the time of fertilization, based on whether the spermatozoon carries an
X or a Y chromosome.
ii. In embryos with an XX sex chromosome configuration, the medullary cords of the indifferent gonad regress,
and secondary cortical cords develop, leading to the formation of ovaries.
iii. In embryos with an XY sex chromosome complex, the medullary cords develop into testis cords, and secondary
cortical cords fail to develop, resulting in testicular formation.

e. Genital Duct Development

i. Indifferent Stage:
 Both male and female embryos start with two pairs of genital ducts: mesonephric (Wolffian) and
paramesonephric (Müllerian) ducts.
 The paramesonephric duct arises as an invagination of the epithelium on the urogenital ridge, extending
cranially into the abdominal cavity and caudally running alongside the mesonephric duct before fusing
with its counterpart to form the uterine canal.

ii. Genital Duct Development in Males:

 Stimulated by testosterone, parts of the mesonephric kidney system differentiate into genital ducts.
  Excretory tubules contact cords of the rete testis to form efferent ductules, while mesonephric ducts
persist and become the main genital ducts, including the epididymis and vas deferens.
 Anti-Müllerian hormone (AMH), produced by Sertoli cells under the influence of SRY and SOX9 genes,
induces regression of paramesonephric ducts, except for the appendix testis.

iii. Genital Duct Development in Females:

 Estrogen and the absence of testosterone and AMH drive the development of paramesonephric ducts into
the main genital ducts.
 Initially, these ducts consist of cranial, horizontal, and caudal portions. With the descent of the ovary, the
cranial and horizontal parts form the uterine tube, while the caudal parts fuse to form the uterine canal.
 The fused paramesonephric ducts give rise to the uterus, cervix, and upper vagina.
 WNT4, the ovary-determining gene, inhibits SOX9 expression and promotes ovarian differentiation.
Estrogen stimulates paramesonephric ducts to form female genital structures.
f. 4. Molecular Regulation:
i. Male Development: SRY and SOX9 genes upregulate AMH production, inducing regression of paramesonephric
ducts. Testosterone, mediated by SF1, promotes differentiation of male genital ducts and external genitalia under
the influence of dihydrotestosterone.
ii. Female Development: WNT4 inhibits SOX9, allowing for ovarian differentiation. Estrogen stimulates
paramesonephric ducts to form female genital structures and influences external genitalia differentiation.

g. Vagina development
 i. 1. Formation of Sinovaginal Bulbs:
 Upon contact between the paramesonephric ducts and the urogenital sinus, solid evaginations known as
sinovaginal bulbs emerge from the pelvic part of the sinus. These bulbs initiate the formation of the
vaginal structure.
 The sinovaginal bulbs undergo proliferation, gradually extending and coalescing to form a solid vaginal
plate. This process occurs in conjunction with the cranial expansion of the plate, which contributes to the
separation of the uterus from the urogenital sinus.
ii. 2. Canalization of the Vagina:
 Over time, typically by the fifth month of development, the solid vaginal plate becomes entirely canalized,
establishing the lumen of the vagina. This canalization process is crucial for the formation of the
functional vaginal passageway.
 Notably, the upper part of the vagina, including the wing-like expansions known as vaginal fornices
around the end of the uterus, originates partially from the paramesonephric ducts.
iii. 3. Formation of the Hymen:
 As the vaginal lumen forms, it remains separated from the urogenital sinus by a thin tissue plate called the
hymen. The hymen consists of a combination of the sinus epithelial lining and a layer of vaginal cells.
 During perinatal life, the hymen typically develops a small opening, although variations exist in its
structure and presence.
iv. 4. Persistence of Excretory Tubules Remnants:
 Remnants of excretory tubules may persist in the mesovarium, giving rise to structures such as the
epoophoron and paroophoron.
a. These remnants serve as vestiges of earlier developmental stages.
 While the mesonephric duct largely regresses during female development, small portions may
occasionally persist, potentially leading to the formation of Gartner cysts later in life

2. External
a. 1. Indifferent Stage:
i. In the third week of development, mesenchyme cells migrate around the cloacal membrane, forming cloacal folds,
which unite cranially to create the genital tubercle. Urethral folds and anal folds arise from the cloacal folds, while
genital swellings emerge adjacent to the urethral folds.
ii. The genital swellings later develop into scrotal swellings in males and labia majora in females, although sexual
differentiation is not distinguishable at this stage.
b. 2. External Genitalia in Males:
 i. Male external genitalia development is driven by androgens from fetal testes, resulting in rapid elongation of the
genital tubercle, now termed the phallus. As the phallus extends, it pulls the urethral folds forward, forming the
lateral walls of the urethral groove.
ii. The urethral groove, lined by endoderm-derived epithelium, constitutes the urethral plate. By the end of the third
month, the urethral folds close over the urethral plate, creating the penile urethra, although it doesn't extend to
the phallus tip (4th month) yet.
iii. The scrotal swellings, originating in the inguinal region, migrate caudally and contribute to the formation of the
scrotum, separated by the scrotal septum.

c. 3. External Genitalia in Females:

i. Female external genitalia development lacks androgen influence and proceeds along a different pathway. The
genital tubercle remains relatively small and doesn't elongate significantly.
ii. The genital tubercle undergoes slight elongation to form the clitoris.
iii. Unlike in males, the urethral folds in females remain unfused and develop into the labia minora.
iv. Genital swellings, under the influence of estrogens, enlarge to form the labia majora, which enclose and protect the
developing structures of the female reproductive tract.
v. The urogenital groove remains open in females, forming the vestibule, which is the space between the labia
minora containing the urethral and vaginal openings.
vi. Although the genital tubercle in females does not elongate extensively, it is relatively larger compared to males
during the early stages of development.
vii. Estrogen-driven development of the female external genitalia ensures the formation of structures necessary for
reproductive and urinary functions.

d. Descent of testies (not on exam)

i. Initial Position:
  Testes develop retroperitoneally in the abdominal region.
 They need to descend caudally to reach the scrotum for proper function.
ii. Pathway:
 Passage occurs through the inguinal canal, approximately 4 cm long, just above the medial half of the
inguinal ligament.
 Entry is via the deep (internal) inguinal ring, and exit is through the superficial (external) ring near the
pubic tubercle.
iii. Developmental Process:
 By the end of the second month, the urogenital mesentery attaches the testis and mesonephros to the
posterior abdominal wall.
 The mesentery transforms into a ligamentous structure, the caudal genital ligament, facilitating gonadal
descent.
 Additionally, the gubernaculum, a band of mesenchyme, extends from the caudal pole of the testis
towards the inguinal region.
iv. Descent Mechanism:
 The gubernaculum aids in testicular migration, forming an intra-abdominal and extra-abdominal portion.
 Intra-abdominal migration occurs due to outgrowth of the gubernaculum, while increased intra-abdominal
pressure aids in passing through the inguinal canal.
 Regression of the extra-abdominal portion completes testicular descent into the scrotum.
v. Timing and Influences:
 Testes reach the inguinal region by approximately 12 weeks' gestation, migrate through the inguinal canal
by 28 weeks, and reach the scrotum by 33 weeks.
 Hormonal factors, including androgens and M18, influence the process.
 Blood supply from the aorta is retained during descent, and testicular vessels extend to the scrotum.
vi. Peritoneal Coverings:
 Peritoneal layers derived from the processus vaginalis cover the testis.
 Layers from the abdominal wall, including the transversalis fascia, internal abdominal oblique muscle,
and external abdominal oblique muscle, also encase the testis during migration.
 e. Descent of ovaries (not on exam)
i. Initial Position:
 Unlike testes descent, ovarian descent in females is considerably less pronounced.
 The ovaries settle just below the rim of the true pelvis.
ii. Suspensory Ligament:
 The cranial genital ligament forms the suspensory ligament of the ovary.
 This ligament maintains the position of the ovary within the pelvic cavity.
iii. Ligaments of the Ovary Proper:
 The caudal genital ligament contributes to the formation of the ligament of the ovary proper.
 It also gives rise to the round ligament of the uterus, extending into the labia majora.
iv. Descent Process:
 Ovarian descent is less extensive compared to testicular descent in males.
 There is no equivalent process to the gubernaculum as seen in testicular descent.
 Instead, the ligaments associated with the ovaries maintain their position within the pelvis.
v. Timing and Completion:
 Ovarian descent occurs during fetal development but is completed before birth.
 The ovaries remain within the pelvic cavity, supported by their ligaments, and do not undergo significant
migration similar to the testes.

Determinantion of phenotypic sex

1. Genetic sex:
 In mammals, genetic sex is determined at fertilization based on the chromosomal content of the gametes. If an X
chromosome-bearing sperm fertilizes the egg, the resulting embryo will be female (XX), while if a Y chromosome-
bearing sperm fertilizes the egg, the embryo will be male (XY).
 The presence of the Y chromosome triggers testes development in male embryos. The sex-determining region Y
(SRY) gene, located on the Y chromosome, plays a crucial role in this process. SRY activates the expression of
genes involved in testes differentiation, leading to the development of male sexual characteristics.
2. Dosage compensation:
 Dosage compensation mechanisms ensure that the expression of X-linked genes is balanced between males (XY)
and females (XX), despite the difference in the number of X chromosomes.
 In mammals, females undergo X chromosome inactivation, where one of the two X chromosomes is randomly
silenced in each cell. This process is initiated by the long non-coding RNA Xist, which spreads across the inactive
X chromosome, leading to its transcriptional silencing. This results in the formation of a Barr body, which is a
condensed and transcriptionally inactive X chromosome.
 Dosage compensation in Drosophila involves increasing the transcriptional activity of the single X chromosome in
males to match the level of expression from the two X chromosomes in females. This is regulated by a set of male-
specific genes known as the MSL complex.
3. Gene interactions:
 Gene interactions play a crucial role in determining the sexual phenotype by regulating the expression of genes
involved in sexual differentiation.
 In mammals, genes such as SRY, SOX9, FGF9, and WNT4 interact to orchestrate the development of male or
female reproductive organs. SRY and SOX9 promote testes development, while WNT4 is involved in ovary
development.
 In Drosophila, the Sex-lethal (Sxl) gene regulates sex-specific splicing of RNA, leading to the development of male
or female characteristics.

4. Gene-hormone-system interactions:
 Hormones produced by the developing gonads play a critical role in directing sexual differentiation.
 In mammals, testosterone produced by the testes masculinizes the developing embryo by stimulating the
development of male reproductive organs and secondary sexual characteristics.
 In females, estrogen stimulates the development of female reproductive organs and secondary sexual
characteristics.
 The molecular interactions between genes and hormones, such as SRY/SOX9 activation of testosterone production,
orchestrate the development of male sexual characteristics.
 i. Male Development: SRY and SOX9 genes upregulate AMH production, inducing regression of paramesonephric
ducts. Testosterone, mediated by SF1, promotes differentiation of male genital ducts and external genitalia under
the influence of dihydrotestosterone.
ii. Female Development: WNT4 inhibits SOX9, allowing for ovarian differentiation. Estrogen stimulates
paramesonephric ducts to form female genital structures and influences external genitalia differentiation.
Timeline of sexual developmant

Fertilization (Week 0):

 Fertilization occurs when a sperm cell penetrates and fertilizes an egg cell, resulting in the formation of a zygote.
 The zygote contains 46 chromosomes: 23 from the sperm (either X or Y) and 23 from the egg (always X).
Weeks 1-2:
 The zygote undergoes rapid cell division through mitosis, forming a blastocyst.
 Implantation of the blastocyst into the uterine lining occurs around day 6-7 post-fertilization.
Week 3:
 Gastrulation begins, leading to the formation of three germ layers: ectoderm, mesoderm, and endoderm.
 The primitive streak appears, marking the beginning of bilateral symmetry.
 The embryonic disc forms, consisting of two layers: epiblast and hypoblast.
Weeks 4-5:
 The gonadal ridges appear as paired thickenings of the mesoderm near the developing kidneys.
 Primordial germ cells (PGCs) migrate from the yolk sac to the genital ridge, which will later differentiate into ovaries
or testes.
 Sexual differentiation begins under the influence of genetic factors, particularly the presence or absence of the SRY
gene on the Y chromosome.
Weeks 6-7:
 In embryos with XY chromosomes and presence of the SRY gene, the gonadal ridges develop into testes.
 In embryos with XX chromosomes or absence of the SRY gene, the gonadal ridges develop into ovaries.
  The bipotential gonads differentiate into either testes or ovaries depending on the genetic and hormonal environment.
Weeks 8-12:
 Testes differentiation: Sertoli cells in the developing testes produce anti-Müllerian hormone (AMH), causing the
regression of Müllerian ducts (which would otherwise form the female reproductive tract).
 Ovaries differentiation: Absence of AMH allows Müllerian ducts to develop into the fallopian tubes, uterus, and upper
vagina.
Weeks 13-16:
 Development of external genitalia begins.
 In males, the genital tubercle elongates to form the penis, and the urogenital folds fuse to form the scrotum.
 In females, the genital tubercle becomes the clitoris, and the urogenital folds remain separate, forming the labia
minora.
Weeks 17-20:
 Sexual differentiation of external genitalia continues.
 In males, the testes descend into the scrotum.
 In females, the labia majora develop from the fusion of the genital swellings.
Weeks 21-24:
 Sexual characteristics become more distinct.
 Sexual differentiation is mostly complete, but development continues until birth and beyond.
Puberty (Around ages 8-13 in females, 9-14 in males):
 The hypothalamus begins secreting gonadotropin-releasing hormone (GnRH), initiating the onset of puberty.
 The pituitary gland responds by releasing follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which
stimulate the gonads to produce sex hormones (estrogen in females, testosterone in males).
 Secondary sexual characteristics such as breast development in females and facial hair growth in males emerge.

What is AIS and how it is developed

1. Definition:
a. Androgen Insensitivity Syndrome (AIS) is a complex disorder that affects sexual development in individuals
with male chromosomes (XY) but incomplete or absent masculinization of external genitalia.
i. AIS results from a lack of androgen receptors or a failure of tissues to respond to androgen hormones,
particularly dihydrotestosterone (DHT), which is essential for the development of male genitalia.
2. Complete Androgen Insensitivity Syndrome (CAIS):
 In CAIS, individuals have a complete lack of functional androgen receptors, rendering them unresponsive to
androgens like testosterone and DHT.
 Despite having XY chromosomes and testes that produce normal levels of testosterone, external genitalia develop
along a predominantly female pattern due to the absence of androgen signaling.
 The paramesonephric (Müllerian) ducts are suppressed in response to Müllerian Inhibiting Substance (MIS)
secreted by the testes, resulting in the absence of uterus and uterine tubes.
 Typically, individuals with CAIS have a female appearance at birth, with normal female external genitalia, a short
or poorly developed vagina, and undescended testes found in the inguinal or labial regions.
 While they lack internal female reproductive organs, individuals with CAIS often identify and are raised as females.
However, they do not undergo menstruation and are infertile.
3. Partial Androgen Insensitivity Syndrome (PAIS):
 PAIS represents a spectrum of conditions where there is partial responsiveness to androgens, resulting in varying
degrees of masculinization.
 External genitalia may appear ambiguous, with features ranging from mild clitoromegaly in females to a small
penis with hypospadias in males.
 Testes may be undescended, and individuals may exhibit varying degrees of virilization, depending on the extent
of androgen insensitivity.
 PAIS individuals may undergo a more complex gender assignment process due to the variability in genitalia and
hormonal responses.