Normal sexual development

The biological sex of an individual is determined by genetic factors that determines the development of the gonad - testis or ovary. The production of hormones by the gonads and the receptors and post receptor functions then determines the phenotype of the individual.

Early embryonic formation of the genital ridge

The earliest changes of sexual development identified in embryos is the formation of the primitive bipotent gonad as a genital ridge on the ventromedial surface of the mesonephros. Cells of the coelomic epithelium and mesenchymal cells of the mesonephros interact. Mesonephric tubules form continuous bridges with the overlying epithelium. At this stage the gonadal primordium is bipotent. Supporting cells, precursors of Sertoli cells (in males) and granulosa cells (in females), and the precursors of the steroid producing cells are present. The latter are derived from pericytes and perivascular smooth muscle cells of the blood vessels that invade and form in the gonadal primordium.

Wilms tumour suppressor 1 (WT1), steroidogenic factor 1 (SF1), Lim homeobox 9 (LHX9), empty spiracles homobox gene 2 (EMX2) and member of polychrome group (M33) are important genes in the development of the gonadal primordium. WT1 is a critical gene and results in activation of others including SF1. Wingless-type MMTV integration site family 1 (WNT1) is involved with all stages of gonadal and tubular development also.

Primordial germ cells arise from the inner cell mass at the base of the allantois at the posterior end of the primitive streak. In mice they are a population of about 45 cells that are in the region of the formation of the hindgut. They pass into the urogenital ridges, which are nearby. As hindgut development continues, any additional cells must pass through the mesentry. Survival of the primordial germ cells (PGC) is dependent on Stem Cell Factor and the presence of the tyrosine kinase receptor cKIT. Division of the PGC continues untill there are about 3000 cells. They continue to proliferate in females but enter arrest in males - once the sex determination phase begins.

The sex determination phase now begins when the sex determining region of the Y chromosome (SRY) is activated, or not.

Normal female sex development

Ovarian development

In the absence of SRY the primordial gonad doesnt change until female specific genes are activated. It has long been held that development of the ovary is a default situation but formation of an ovary requires activation of particular genes. The earliest changes are an aggregation or remodelling of the stroma and the presumptive oocytes form interconnected cysts joined by cytoplasmic bridges.A dense network of blood vessels forms and there is formation of ovigerous cords. At about the time of birth there is further reorganisation of the ovary into cortex and medulla, and the breakdown of the cytoplasmic bridges within ovigerous cords, and individual oocytes become surrounded by a somatic epithelial layer of pregranulosa cells. During this process there is considerable oocyte apoptosis. This heralds the formation of primordial follicles. Subsequently the oocyte produces a zona pelucida that forms a barrier between oocyte and granulosa cells, and the theca cells form - based on factors produced from the granulosa cells.

There is no specific ovary determining gene like SRY in males, although there are genes known to play important roles. These are called pro-ovarian genes and involve RSPO1-WNT4-FOXL2 pathways. DAX1 and FOXL2 occurs early in development and at the same time or before SRY is expressed in males.

One of the earliest events in ovarian differentiation is the activity of R-spondin1 (RSPO1). It activates the WNT/Beta catenin signalling pathway. How this activation occurs is as yet unknown. If these genes and pathways are activated in a male embryo, there is a male to female sex reversal.

WNT4/Beta catenin signalling pathway is important in early ovarian and other female reproductive organ differentiation. WNT4 is an important gene in ovarian and therefore female development. It is active in gonadal primordia and is downregulated in males. Formation of the ovary, paramesonephric duct system and ovarian vasculature is under the control of this gene. In the ovary, it is expressed during the indifferent phase of gonadal development. It inhibits the male type vascularisation of the gonad and it upregulates the follistatin gene (FST). Follistatin is a glycoprotein required for female sex determination and early ovarian development. It binds to activin (a member of the TGF beta family) and neutralises it. Follistatin is expressed in multiple organs so is not ovary specific.

Nuclear receptor subfamily 0, group B, member 1 (NROB1), also known as DAX1 is an important gene in the development of females - and it is female specific. It is upregulated by WNT4 and it is inhibited by SRY. It inhibits DMART1 which regulates SOX9, the gene that is important in the male development. It is not, by itself, an ovarian determining gene however.

Forkhead/winged helix transcription factor 2 (FOXL2) is also female specific, like NROB1 (DAX1). It, in combination with bone morphogenetic protein 2 (BMP2) gene (BMP2) upregulates FST expression. FOXL2 also inhibits DMRT1 (that regulates SOX9) so it is an antitestis gene.

SF1 is a gene that is activated at the beginning of the sex determination period in both males and females, but it reduces in amount dramatically at the end of the sex determination period in males, but remains high in female embryos. It is also involved in the development of adrenal and hypothalamic - pituitary axis.


Paramesonephric (Mullerian) duct development

The paramesonephric ducts develop after the mesonephric ducts. Both arise within intermediate mesoderm and PAX2 is involved. The caudal end of the paramesonephric duct is closely associated with the mesonephric duct and they are both enclosed in a common basal membrane. In the earliest stage, the paramesonephric duct is a short tube from a fold in the coelomic epithelium to the mesonephric duct. It is craniad in the embryo and it forms by caudal extension. The paramesonephric duct develops from invagination of the coelomic epithelium but it requires the presence of the adjacent mesonephric duct. The developing fold of the coelomic epithelium continues until the duct is formed. The two paramesonephric ducts then fuse together. The basement membranes of the paramesonephric and mesonephric ducts first disappears and then a new basement membrane forms around the 2 paramesonephric ducts. The epithelial cells merge to form a single duct. This subsequently divides again caudally to form 2 paramesonephric ducts, within one paramesoneprhic tubercle that elongates caudally to reach the urogenital sinus.

The development of the paramesonephric ducts requires expression of WNT4 and occurs in both male and female embryos. The paramesonephric duct goes on to form the uterine tube, uterus, cervix and cranial portion of the vagina, unless inhibited by AntiMullerian hormone (AMH) (also called Mullerian inhibitory substance [MIS; from the MIS gene activated in Sertoli cells])

Gonadal movement

The primitive gonad is suspended by the gubernaculum. In females, the cranial gonadal ligament remains as the cranial suspensory ligament and the posterior part becomes the round ligament of the uterus. In those species where the ovaries move into the pelvic location, the cranial gonadal ligament is removed, allowing for transabdominal migration.

Development of external genitalia.

The caudal vagina, vestibule and vulva develops from the urogenital sinus unless there is circulating testosterone. The clitoris is derived from the genital tubercle.

Normal male sex development

Testicular development

The development of the gonadal primordium is dependent on the presence of the sex chromosomes. XX individuals develop ovaries and XY individuals develop testes. The main protesticular genes pathway is SRY-SOX9-FGF. It was known that there were genes on the Y chromosome that was the testis determining region. This was called the TDF. It was subsequently found that the gene was a Y linked gene that is the sex determining region of the Y chromosome, and the gene in mice is designated Sry. Sry encodes a transcription factor that contains a high mobility group (HMG)- box DNA binding domain. This binds to specific DNA sequences, causes bending of the DNA and results in Sertoli cell differentiation. In mice, Sry expression occurs on day 10 and 36 hours later there are transcriptional events that result in the formation of Sertoli cells.WT1+KTS, GATA4 and FOG2 are part of the transcriptional or post transcriptional regulaton of Sry. SRY, the product of SRY, regulates transcription by altering chromatin structure. It is thought that it binds to promoter sites of SRY-like HMG-box protein 9 (Sox9). Like SRY, SOX9 has a HMG box that binds to specific DNA sequences. SOX9 also has 2 transcriptional activation domains that act on the testis determining pathway. M33 (Cbx2) and ATRX are involved in chromatin regulation and are involved in gonadal development. SRY, M33 and ATRX either activate testis determining genes or inhibit pro-ovarian genes.

SRY is very important in Sertoli cell development and testes dont develop unless there are sufficient Sertoli cells. Formation of sustentacular (Sertoli) cells is influenced by many genes and products including fibroblast growth factor 9(FGF9), Insulin like growth factors (IGF) and others.SOX9 maintains a positive feedback loop with FGF9 and prostaglandin D2 to inhibit the female pathway. DAX1 is a gene that appears to act downstream of SRY and is important in early Sertoli cell differentiation. DAX1 inhibits the male sex determining pathway, and if DAX1 is activated in males, a female develops.

SF1 is a gene that is activated at the beginning of the sex determination period in both males and females, but it reduces in amount dramatically at the end of the sex determination period in males, but remains high in female embryos. It is also involved in the development of adrenal and hypothalamic - pituitary axis. It activates AMH, the gene that produces the hormone Anti Mullerian hormone (prevously known as Mullerian inhibitory substance (MIS). Activation of AMH marks the end of the sex determination period in males.

As part of testicular development, cells migrate from the mesonephros to the testis. These cells are endothelial cells. Without this migration, testicular cords will not form. The formation of extra vasculature is important in testis development, especially cord formation. At this time the formation and function of the interstitial endocrine (Leydig) cells is acquired. Thus the formation of the sustentacular cells, Interstitial endocrine cells and vasculature combined is necessary for testis development. Intersititial endocrine cells develop from the same cell population as the adrenal cortex. They expand along the mesonephros to the developing testis. SF1, desert hedgehog gene (DHH) and PDGFA are involved with this. Also ARX (the homobox gene aristaless-related) has a function. Interstitial endocrine cells appear to arise from vascular smooth muscle cells and pericytes of testicular capillaries - vascular cell types act as stem cells.

Sustentacular cells originate from coelomic lining cells (called coelomic epithelium), thus they have a similar origin to the ovarian epithelium and the lining of the mesonephric and paramesonephric ducts. Their presence orchestrates the formaton of the testis - it is within pre Sertoli cells that activation of SRY and SOX9 is seen. Differentiation to sustentacular cells is accompanied by the formation of testis cords and the primitive structure of testis. Once this occurs, cells migrate from the mesonephros to form the peritubular myoid cells and the testis specific blood vessels. From these, interstitial endocrine cells develop. Neurotropin 3 (NT3), hepatocyte growth factor (HGF) and platelet derived growth factor (PDGF) are probably involved in this process.

Primordial germ cells (PGC) migrate to the gonads and there are many genes and gene products that can be detected. They are not necessary for testicular development. Testicular cord development results in the blocking of PGC proliferation in the G0/G1 phase of mitosis and they differentiate into prospermatogonia. Retinoic acid (or rather the lack of exposure of PGC by protection from the testicular cords) is important in determining whether the PGC differentiate into prospermatogonia or enter meiosis as occurs in females.

During fetal development, at about the half way point of pregnancy, the fetal testis starts to be regulated by the hypothalamic pituitary gonadal axis. Prior to this, the testis is independent. Masculination of the fetus occurs during this independent period. As the pituitary takes over, interstitial endocrine cells decline in function and Se

rtoli cells increase in number.

Mesonephric (Wolffian) duct development

The duct system of the male develops from the mesonephric ducts. Early in embryogenesis, even prior to the formation of the genital ridge and gonadal differentiation, the primitive kidney, the pronephros develops and the pronephric duct forms by infolding/invagination of coelomic epithelium from the location of the pronephros caudally. The pronephros is replaced by the mesonephros, and the pronephric duct becomes the mesonephric duct.

WNT/β catenin signaling pathways are required for development of the pronephric duct.

Development of the epididymis and deferent duct requires androgens. Postnatally there is lumocrine factors that play a role. In early embryogenesis, cell proliferation and coiling is related to androgens. It appears to be regulated by the extracellular
signal-regulated kinase (ERK) pathway, the src family of kinases, and the phosphatase and tensin homolog phosphatase Dusp6.

Interstitial endocrine cells secrete androgens and INSL3 that cause retension of the mesonephric ducts and development of male genitalia from the urogenital sinus and genitial tubercle. Control of secretion is by placental gonadotrophins in the human and horse - rodents dont have any control during gestation. There are several types of interstitial endocrine cells, including fetal and neonatal/adult types.

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Masculinisation of the fetus

SOX9 and SF1 regulates expression of AMH that is the gene that codes for antiMullerian hormone (AMH)/Mullerian inhibitory substance (MIS). When Sox9 is activated, AMH is activated and AMH production in sustentacular (Sertoli) cells causes regression of the paramesonephric duct.

Interstitial endocrine cells secrete androgens and INSL3 that cause retension of the mesonephric ducts and development of male genitalia from the urogenital sinus and genitial tubercle. Control of secretion is by placental gonadotrophins in the human and horse - rodents dont have any control during gestation. There are several types of interstitial endocrine cells, including fetal and neonatal/adult types.


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External genital development

Early in embryonic development, there is a cloaca where the intestinal, urinary and reproductive tracts converge. This cloaca divides into the urogenital sinus and an anorectal canal. The primitive urethra forms in 2 portions - the pelvic portion and the phallic portion. This is a flat membrane that forms folds that fuse to form a canal. The phallic portion forms on the genital tubercle, which becomes the penis.


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