Sexual development is one of the significant traits in an organism’s life because it is closely related to its genetic fitness. The only legacy we pass into subsequent generations are germ cells in the developing gonads.[1] In sex development, there are two distinguishably different processes, namely sex determination and sex differentiation. Sex determination is the developmental decision that directs the bipotential gonad into a sexually dimorphic individual.[2] Sexual differentiation is how the male and female sexual organs develop from bipotential embryonic structures. It involves sequential stages, namely genetic, gonadal, hormonal, phenotypic, and psychological. At the genetic stage, chromosomal sex is determined by the chromosomal constitution after fertilization, where XY denotes male and XX indicates female. Until the sixth week of embryonic life, no sexual difference is observable in the fetus. Undifferentiated gonads of XX or XY are similar in morphology and can form either ovaries or testes. Differentiation of bipotential gonad into either ovaries or testes occurs in the gonadal stage. Followed by gonadal differentiation, the internal genital tract and the external genitalia develop into male or female structures in the phenotypic stage.[3][4]

Sex determination is a dynamic and complex process regulated by a wide variety of genetic and environmental causes. This process determines the development of a bipotential gonad into the testes or ovary. In humans, sex is determined by the constitution sex chromosomes, in first and the second week of gestation, where the embryos of both the sexes differ only in their karyotype-males are XY and females are XX. In the third week of gestation, specific genes induce differentiation of gonads.[3][5][6]

The initial event in the sex differentiation is the development of gonadal primordium or gonadal/urogenital ridge. They appear as a pair of longitudinal gonadal ridges at the 4th to 5th week and are formed by intermediate mesoderm and covered by coelomic epithelium. The gonadal primordia derive from mesonephros, which is the primary embryonic kidney that functions for a short time in the early fetal period (4th week). Urogenital ridges are common precursors of the urinary and genital systems and the adrenal cortex. Each urogenital ridge divides into urinary and adrenogonadal ridge in the 5th week.  Epithelial cells of gonadal primordium enter the mesenchyme and result in the formation of primitive sex cords. Later these sex cords separate from the epithelial surface are called bipotential gonads. At the 6th to 7th gestational weeks, the paramesonephric ducts (Mullerian ducts) develop next to the mesonephric ducts (Wolffian ducts). Both sets of ducts form on the surface of the mesonephric kidneys. In males, Wolffian duct differentiates into the epididymis, vas deferens, and seminal vesicle, mediated by testes derived androgens while it regresses in females. In females, the Mullerian duct differentiated into fallopian tubes, uterus, and upper part of the vagina. And they regress in males under the influence of testis-derived anti-Mullerian hormone.[7]

Testes

If an embryo is a genetic male (46 XY), the bipotential gonad differentiates to testes under the influence of the SRY gene. The sex-determining region Y (SRY) is a single exon gene, and it encodes a transcription factor called the testes-determining factor (TDF); this induces male sex determination. SRY gene expression aids in the differentiation of Sertoli cells, which later results in the production of Mullerian inhibiting substance. SOX9 also helps in the Sertoli cell differentiation, which is upregulated by the SRY gene. It is the critical step in the initiation of testis development.[8]

Initially, gonadal cells segregate into two compartments by invading the gonadal medulla by primitive sex cords and forms testicular cords and interstitial tissue. Testicular cords are composed of germ cells and sustentacular cells of Sertoli. Interstitial Leydig cells lie between the testes cords, which derive from the original mesenchyme of gonadal ridge. Gonadal ridge vascularisation is a dynamic process. The XY gonad recruits and patterns vasculature by a remodeling mechanism, whereas developing ovary recruits vasculature by normal angiogenesis. In differentiating testes, pre-existing mesonephric vessels dissociate and form a cluster of endothelial cells that migrate and reach below the coelomic epithelium of gonad, where they assemble to form the coelomic vessel, a vessel that runs the length of the testes at its antimesonephric margin. The formation of this vessel is one of the earliest hallmarks of testes development that distinguishes it morphologically from the developing ovary. Mesonephric ducts form the main genital duct of the male embryo. The remaining parts of the excretory tubules form the efferent ductules—these link rete testis and mesonephric duct, which becomes the ductus deferens. The seminiferous tubules and rete testis tubules enter into the ductules efferents.[9][1]

Ovary

In female embryo with 46 XX, gonads become indifferent after the 7th week of gestation. Ovary-specific transcription factors induce ovarian differentiation, which are FOXL2 (forehead transcription factor 2 ), WNT4 (wingless-type MMTV integration site family member 4), RSPO1 (R spondin 1), and the beta-catenin pathway.[9] Therefore differentiation of ovary is an active process.[10] Initially, primitive sex cords disassemble into irregular cell clusters, and these clusters occupy the medullary part of the ovary. Later, these irregular cell clusters are replaced by vascular stroma, which forms the ovarian medulla. Primitive germ cells proliferate by mitosis and develop onto oogonia. At the 10th week, oogonia in the center enter the meiotic prophase; it is the first unequivocal sign of morphological ovarian differentiation. Maturation of the ovary proceeds from the center to the periphery. Subsequently, oogonia become surrounded by a layer of granulosa cells, later they enter meiosis and become oocytes and form primordial follicle. The primary follicles initially appear at 15-16th weeks and the first Graffian follicles at 23th to 24th weeks. By the end of the 7th month of gestation, mitotic activity has stopped, and almost every germ cell has entered meiotic prophase. Then they are arrested at the diplotene stage and meiosis completed at the time of ovulation in adults, whereas meiosis starts only at puberty in the male gonad.

The fetal testis development progresses in the absence of germ cells, while ovarian follicles require germ cells for their development. So the involvement of germ cells in the stabilization of gonads is one of the major differences between ovary and testis. Also, the coelomic vessel formation, which is the characteristic of the testis differentiation, does not occur in the case of the ovary.[9][11][10]

Mullerian Duct

 The Mullerian ducts (paramesonephric duct) are the progenitor of the upper female genital tract. They develop from intermediate mesoderm. The Mullerian and Wolffian ducts develop on the surface of the mesonephric kidneys. Mullerian ducts formation involves three phases; specification, invagination, and elongation.[12] Mullerian ducts grow as a coelomic epithelial cleft on the surface of the gonadal ridges at 5 to 6 weeks of gestation. Those coelomic invaginations finally become the abdominal Ostia of fallopian tubes. The paired Mullerian duct migrate caudally along with Wolffian duct to reach urogenital sinus. It is a critical step in female reproductive tract development. If Mullerian ducts fail to merge with urogenital sinus, it can lead to lower vaginal agenesis. Sometimes it causes uterine and vaginal agenesis in females, which in turn leads to primary amenorrhea in girls (MRKH syndrome). In females, Mullerian ducts develop into fallopian tubes at their cranial end and fuse in midline to form the uterus and upper aspect of the vagina at their caudal ends.

In a male fetus, during the 3rd month of fetal development, the Sertoli cells of the testes begin to secrete a substance called anti-Mullerian hormone. lt is mediated by beta-catenin, causing Mullerian ducts to atrophy instead of developing into the female reproductive organs. organs.[7][12][13][14]

Wolffian Duct    

Wolffian ducts/mesonephric ducts arise from the intermediate mesoderm. WDs play a significant role in the fetus, and it induces the formation of three kidney primordial, namely pronephros, mesonephros, and metanephros. Initially, WD drains the first kidney or pronephros and subsequently functions as the excretory duct of the mesonephros. After the development of a definitive kidney, it acquires reproductive function.[15][16] The most cranial portion of WDs persist, and form appendix epididymis and the remaining part of the cranial portion form the rete testis. The part of the mesonephric duct just below this, elongate and convoluted to form epididymis and the remainder of the duct gradually develops a thick muscular coat and forms the vas deferens. Seminal vesicles develop from the caudal end of each mesonephric duct as a lateral diverticulum. A part of the mesonephric duct, between seminal vesicle and urethra, becomes a common ejaculatory duct.

During the 9th and 10th weeks of embryonic life, SRY induced Leydig cell differentiation occurs, which leads to testosterone production. Testosterones plays an important role in the stabilization of the mesonephric duct. Locally produced testosterone from the testis is essential for virilization of Wolffian duct, and they act directly to virilize it, not their derivatives. The absence of testosterone in females leads to a regression of the Wolffian duct.[17][18][19]

External Genitalia

External genitalia in male and female are ambisexual initially, then it undergoes sex differentiation and leads to the formation of male and female forms of external genitalia. Up to the 9th week, the urogenital and the external genitalia are identical in both the sexes. In the 5th week,  mesenchymal cells migrate to the perineum as cloacal folds. These mesenchymal cells assemble in the midline and form genital tubercle. The genital tubercle is located just above the urogenital ostium, and it is covered laterally by urogenital folds and labioscrotal folds. Epithelial cells from urogenital sinus invade genital tubercle results in the formation of the epithelial urethral plate. These stages are identical in both male and female fetuses, and it occurs between the gestational age of 8 and 12 weeks.[20]

Male

In males, the genital tubercle undergoes growth and elongation under the influence of testicular androgens and form penis—the scrotum forms by the fusion of labioscrotal folds at the midline. The solid epithelial plate undergoes canalization and forms a groove on the surface of the genital tubercle, which is bounded by urethral folds. Penile urethra is formed by the fusion of urethral folds in the midline, thus converting groove into the penile urethra. Failure of this fusion results in hypospadias (abnormal urethral opening at the tip of the penis proximal to its original location). The penile foreskin forms from the nearby ectoderm. The development of male external genitalia complete by 14 weeks of gestation. But the testes start to descend from the abdominal cavity only at ten weeks of gestation. The descend is aided by gubernaculum, which is a fold of peritoneum and is attached to the testes. The descent of testes is completed by the 25th to 35th weeks.[20][21][22] Undescended testes are more common in premature and low birth weight infants. Those with undescended testes have an increased chance of getting testicular tumors, especially germ cell tumors. Dihydrotestosterone, which forms from the testosterone with the help of 5-alpha reductase induces the differentiation of male external genitalia and development of urogenital sinus.[16]

Female

Female external genitalia development is regulated by the absence of androgens and maternal estrogens. The caudal end of the fused paramesonephric ducts come in contact with the posterior part of the urogenital sinus, where the sinovaginal bulb develops, and it will form the lower two-thirds of the vagina. Initially, it was solid and lumen forms later. Hymen separates the vagina and the remaining part of the urogenital sinus.

In the absence of testosterone, the genital tubercle forms the clitoris. The urethral folds and labioscrotal folds fail to fuse and become labia minora and majora. The differentiation of female external genitalia starts by 11 weeks and completes by 20 weeks of gestation.[20]