Embryology of the male and female reproductive systems
Embryonic sexual differentiation is a very delicate process ( Fig. 1.1 ) that starts with the genotypic determination at the time of fertilization (i.e., XY or XX) and then concludes in a manner that depends on how the gametes influence the phenotype (i.e., culminating in final pubertal and brain development events). It is worth noting that most information—especially information pertaining to genetics—have been derived from mouse models since studies on human tissues are few in number and also since it has been pointed out that the developmental stages and global gene expression of both species are comparable [i.e., few exceptions exist like how SOX2 (sex-determining region Y-box 2) and SOX17 are two different transcription factors utilized by the primordial germ cells (PGCs) of the mouse and human species, respectively]. Sex determination has long been thought to have a predetermined default path toward a final female phenotype in the absence of the influence of the male pathway steering factors; however, recent data indicate that not only does the alternative sex development pathway need continuous active repression during the embryonic life but also that it might need to remain actively repressed for life. Table 1.1 outlines the possible genetic errors that can disrupt such a delicate process at different stages.
| Gene name or pseudonyms | Human gene locus | Protein name | Protein type | Genetic or cellular targets of factors | Action of factors | Effects of overexpression or underexpression of gene |
|---|---|---|---|---|---|---|
| Factors involved in both ovary and testis sex determinations | ||||||
| SF1* NR5A1 FTZF1 Ad4BP | 9q33 | SF1 | Orphan nuclear receptor/zinc finger transcription factor | WT1, SRY, SOX9, DAX1, GNRHR, LHβ, ACTHR, AMH, AMHR, STAR, CYP11A1, CYP21A2, CYP11B1, OXT, and others | Activates transcription of many genes in the development of gonads, adrenal glands; regulates steroidogenesis; synergizes with WT1; antagonizes; DAXi. Dose-dependent activity. | KO mice (XX and XY): no gonads or adrenals; retained Müllerian structures; abnormal hypothalamus. Haploinsufficient mice: reduced but not absent adrenal function Homozygous human mutation: 46,XY sex reversal and adrenal hypoplasia Heterozygous human mutation: 46,XX normal ovary, partial adrenal insufficiency |
| WT1* | 11p13 | WT1 | Zinc finger transcription factor; tumor repressor | DAX1, AMH, SRY, IGF2I, IGF1R, PDGFA, PAX2 | Represses transcription; activates transcription of SRY; dose-dependent effects | XY homozygous deletion of WT1 + KTS isoform: male-to-female sex reversal; XY homozygous deletion of WT1 + KTS isoform: streak gonads in XX and XY. Human Denys–Drash syndrome: gonadal dysgenesis, congenital nephropathy, Wilms’ tumor |
| GATA4* | 8p23.1-p22 | GATA4 | Zinc finger transcription factor | “GATA” DNA motif; AMH; genes encoding steroidogenic enzymes | Expressed early in both ovary and testis; interacts with FOG2 | KO mice: embryonic lethal, no gonadal phenotype reported. Most human mutations cause isolated cardiac defects. Rare cases are reported with both cardiac defects and 46,XY gonadal dysgenesis |
| CBX2* | 17q25.3 | CBX2 | Transcription repressor | Possibly SRY | Mediates changes in chromatin structure | KO mice (XX or XY) have retarded development of gonadal ridges; XY mice have male-to-female sex reversal. Compound heterozygous mutations cause 46,XY gonadal dysgenesis |
| Factors involved in testis sex determination | ||||||
| SRY* | Yp11.3 | SRY | HMG-box-containing transcription factor | SF1, SOX9, CYP19A1, AMH | Bends DNA; may antagonize SOX3 | XX mice expressing transgenic Sry, female-to-male sex reversal. Human SRY mutations: 46,XY sex reversal, gonadal dysgenesis Translocation of SRY to X chromosome: 46,XX female-to-male sex reversal or ovotesticular disorder of sex development |
| SOX9* | 17q24-q25 | SOX9 | HMG-box-containing transcription factor of SRY family | Supporting cells of gonadal primordium; WNT4, FGF9 | Simulates differentiation of Sertoli cells; dosage-sensitive effects | Odsex mice: derepression of Sox9 expression in XX gonads → testis development; Human mutation: 46,XY sex reversal; gene duplication or deletion in coding and/or noncoding regions results in 46,XX or 46,XY DSD |
| ATRX* XH2 | Xq13.1–q21.1 | ATRX | Helicase; transcription factor | Widespread expression early in mouse embryogenesis, more restricted expression later | Gene regulation at interphase and chromosomal segregation at mitosis | Human mutations: α-thalassemia, mental retardation, genital anomalies → male-to-female sex reversal in 46,XY |
| DHH* | 12q13.1 | DHH | Signaling molecule | Expressed only in testis | Involved in interactions between Sertoli cells and germ cells. May regulate mitosis and meiosis in male germ cells | Strain-specific effects: XY null mice have defective Leydig cell development and are feminized. Human mutations cause 46,XY gonadal dysgenesis with peripheral neuropathy |
| FGF9 | 13q11–q12 | FGF9 | Growth factor | SOX9 | Promotes Sertoli cell differentiation and germ cell survival | KO mice: XY sex reversal |
| LHCGR* | 2p21 | LH/CG receptor | G-protein-coupled, seven-transmembrane-peptide hormone receptor | Not applicable | Transduces LH signal to activate G sα → cAMP. Required for Leydig cell testosterone production | Human mutation: Leydig cell hypoplasia → male hypovirilization; mouse: normal sex differentiation Males and females infertile |
| STAR* | 8p11.2 | STAR | Mitochondrial transport protein | Not applicable | Transports cholesterol to inner mitochondrial membrane | Human mutation: congenital lipoid adrenal hyperplasia; 46,XY undervirilization |
| SRD5A2* | 2p23 | 5α-Reductase 2 | Mitochondrial enzyme | Not applicable | Converts testosterone → DHT | 5α-Reductase deficiency; 46,XY undervirilization |
| AR* | Xq11.2–q12 | AR | Ligand-dependent nuclear receptor | AMHR, CYP19 | Regulates transcription | XY mice: undervirilization of internal and external genitalia; 46,XY human mutation: androgen insensitivity syndrome |
| AMH* | 19p13.3 | AMH | Glycoprotein homodimer of TGF-β family | Mesenchymal and epithelial cells of Müllerian ducts | Ligand for AMHR; stimulates apoptosis of Müllerian duct | 46,XY human mutation: Persistent Müllerian duct syndrome |
| AMHR2* | 12q13 | Type II AMH receptor (AMHR2) | Transmembrane serine/threonine kinase receptor | Mesenchymal and epithelial cells of Müllerian ducts | Receptor for AMH; stimulates apoptosis of Müllerian duct | Persistent Müllerian duct syndrome |
| Factors involved in ovary/female sex determination | ||||||
| RSPO1* | 1p34 | RSPO1 | Thrombospondin-like secreted activator of β-catenin | WNT4, CTNNB1 | Expressed in testis, ovary, adrenals, thyroid, trachea, kidney, skin Controls expression of CTNNB1 (gene for β-catenin) in developing ovary | Mouse: XX null show partial female-to-male sex reversal, with the development of seminiferous tubules; Human: 46,XY sex reversal and palmoplantar keratosis |
| WNT4* | 1p36.23–p35.1 | WNT4 | Cysteine-rich signaling molecule/secreted growth factor | Mesonephric mesenchyme | Directs initial Müllerian duct formation in both sexes; “antitestis” factor in ovarian development | XX and XY null mice: Müllerian duct agenesis. Overexpression in XY: male-to-female sex reversal. Human 46,XY: duplication of WNT4 associated with male-to-female sex reversal |
| FOXL2* | 3q23 | FOXL2 | Transcription factor | Not reported | Expressed predominantly in ovary; earliest known marker of ovarian differentiation in mammals | Goat: deletion associated with XX sex reversal. Human mutation: 46,XX gonadal dysgenesis |
| HOXA13* | 7p15–p14 | HOXA13 | Homeodomain transcription factor | FGF8, BMP7 | Involved in epithelial–mesenchymal interactions required for morphogenesis of terminal gut and urogenital tract, including Müllerian structures | Mouse: XX null have hypoplasia of cervix and vagina. 46,XX human mutation: hand–foot–genital syndrome with uterine malformation |
| Factors with possible roles in either or both sexes | ||||||
| DAX1* NR0B1 AHCH | Xp21.3–p21.2 | DAX1 NROB1 | Orphan nuclear receptor transcription factor | RAR, RXR, STAR, CYP17A1, HSD3B2 | Represses SF1 transcription; antagonizes SF1; regulates testis cord organization. Presents premature cell differentiation Dose-dependent effects | Strain-specific defects in XY mice: overexpression → testis maldevelopment and sex reversal; homozygous deletion → adrenal hypoplasia, normal testes. Human mutations: adrenal hypoplasia congenita, hypothalamic hypogonadism |
| WNT7A | 3p25 | WNT7A | Signaling molecule | Mesenchymal and epithelial cells of Müllerian ducts | XY: involved in Müllerian duct regression; XX: stimulates the development of Müllerian duct | Male mice with homozygous deficiency of Wnt7a have retained Müllerian ducts; female Wnt7a-deficient mice have defective, though not absent, development of oviducts and uterus |
| DMRT1/2* | 9p24.3 | DMRT1 DMRT2 | DM-domain transcription factors | Expressed only in genital ridge Dose-dependent effect on postnatal testis development | XY null mice have normal prenatal testis development but abnormal postnatal testis differentiation. Human monosomy 9p: 46,XY testis maldevelopment; 46,XX primary hypogonadism | |
Germ cell specification
Three weeks after fertilization, from the proximal epiblast-derived mesodermal portion located near the extraembryonic endoderm—at the posterior part of the primitive streak—arises a group of cells (i.e., at least six in number) that display unique qualities from their surrounding tissues (i.e., larger size, clearer cytoplasm, and fewer organelles) and will later carry on their role as the PGCs. This process has been termed “specification” and is influenced by a number of factors—that belong to the transforming growth factor-β ( TGF-β ) superfamily—produced by extraembryonic ectoderm although the cascade response of the cells depends on the expression of other factors like the wingless-type MMTV integration site member 3 ( WNT3 ) (i.e., in this case to specifically respond to the bone morphogenetic proteins ( BMP ) of the TGF-β family). Genes like PR-domain containing-1 (i.e., Prdm1 or Blimp1 as previously called), Prdm14 , and transcription factor AP-2 gamma ( Tfap2c ) are downstream activation targets that should be properly expressed for a successful specification to take place since—for example— Prdm1 prevents the expression of genes that can pull the germ cell toward the somatic cell-line development. Rodriguez and colleagues provide an excellent discussion of growth factor and genes related to sex differentiation and development that can be found in their published work.
Germ cell migration and the concurrent genital ridge development ( Fig. 1.2 )
For the next 1–2 weeks post specification, PGCs acquire changes in their morphology (i.e., pseudopodia) in order to carry out active amoeboid movement and successfully migrate to the hindgut and then to the genital ridge (i.e., and into the developing sex cords). Their path goes through the dorsal mesentery and the journey seems to require the influence of various genes (i.e., homeobox protein Lim-1 ( LHX1 or Lim1 ) for proper hindgut localization and WNT family member 5A ( WNT5A ) for the PGCs to receive a proper migration cue; the mutation of the latter can partly explain findings in Robinow syndrome), the action of ligand/receptor pathways (i.e., stromal cell-derived factor 1 (Sdf1 or C–X–C motif chemokine 12 (CXCL12)/CXC receptor 4 (CXCR4) pathway), an optimum collagen-I deposition in the extracellular matrix (i.e., induced by TGF-β through the activin receptor-like kinase 5 (alk5)), and the help of autonomic nerve fibers migrating along with them to the same destination.
The development of the genital ridge commences and proceeds in conjunction with the migration. The urogenital ridge arises as a dorsal body wall thickening that harbors populations from both the primitive mesoderm and the coelomic epithelium (viz., adrenogenital primordium). The central portion—the mesonephros—of the urogenital ridge gives rise to the gonadal ridge along its ventromedial surface—which starts bulging after germ cells infiltrate it to form the primordial indifferent gonad by the end of the 5th week of intrauterine life. Certain gene mutations can affect the development of genital ridge (i.e., and consequently risk the lack of the development of the gonads themselves in addition to some malfunctions in the adrenal and renal systems) and they include Wilms’ tumor 1 ( Wt1 ), steroidogenic factor-1 ( SF1 ), Lhx1 , Lhx9 , empty spiracles homeobox 2 ( Emx2 ), and GATA-binding protein 4 ( GATA4 ).
The indifferent stage, the bipotential gonad, and the gonadal differentiation
Sustainment of the morphological development of the gonads requires the presence and the influence of germ cells only in the case of the ovary (i.e., normal testicular development continues in their absence) ; however, both the cortex and the medulla of the indifferent gonads change according to the genotype of the fetus (i.e., cortex develop and medulla regresses in the case of the XX genotype and the medullary sex cords proliferate toward seminiferous tubules formation in the case of the XY genotype). Interestingly, PGCs differentiate into male or female gametes according to the cellular environment they are placed in. For instance, female genotypic PGCs become spermatogonia when they are placed in gonads with testicular somatic cell environment (i.e., of Sertoli cell lineage) while those migrating to aberrant locations give rise to oogonia—that degenerate afterward—irrespective of their sexual genotype (i.e., though rarely they form teratomas in certain locations as the mediastinum). Assumptions about the sexual identity of the gonad grow stronger as intrauterine life progresses over the weeks depending on a number of clues—which when working toward an assumption of the presence of an ovary—that include the following: lack of testicular development beyond the 7th week; the presence of oogonial meiosis around the time of the 8th week; and visualization of primordial follicles by the 16th week.
Gonadal differentiation seems to rely on intricate genetic interactions between multiple upstream and downstream factors ( Fig. 1.3 ), many of which are yet to be fully elucidated. The long-held belief that the female differentiation pathway is the default one in the absence of male factors’ influences has recently started to be challenged. Consequently, for example, and in the case of female gonad determination and the hypotheses behind it, two possibilities are now entertained for the directions factor takes to achieve the determination: repression of somatic testis-inducing genes in addition to the activation of ovary stimulating ones as one possibility; downregulation of the same male genes while removing the repression on ovary stimulating ones as an alternate possibility. It is worthy of note that no definite hypothetical model has been adopted yet even when the knowledge about possible factors keeps-on expanding.
At about the 7th week and under the influence of the autosomal gene SRY (sex-determining region Y)-box 9 ( SOX9 ) (i.e., which gets inhibited mainly by β-catenin or catenin beta-1 gene ( CTNNB1 ) in the case of ovary determination pathway and is upregulated by the SRY gene), Sertoli cells differentiate, proliferate, and surround the arriving PGCs to form primitive sex cords while protecting the PGCs from entering meiosis prematurely (i.e., protection is vital for commitment to spermatogenesis and is mediated by degrading retinoic acid—and possibly other products—by the cytochrome p450 enzyme (Cyp26b1); mitotic arrest continues until puberty). Sertoli cells promote differentiation of peritubular myoid cells and—by the 8th week—Leydig cells (i.e., both arising from primitive interstitial cells) by secreting anti-Müllerian hormone (AMH) as a paracrine factor. SRY and other factors (i.e., transmembrane ligand ephrin-B2) influence to proper vascularization of the testes—which is characterized by early distinct arterial specification and is necessary for further transportation of testicular hormones. From the second trimester thereafter, Sertoli cells double in number fortnightly (i.e., coupled by a parallel increase in numbers of germ and Leydig cells) resulting in a rapid increase in testicular sizes. It is evident that Y-chromosome genes are necessary to maintain spermatogenesis while having extra X-chromosomes (i.e., as is the case with Klinefelter’s syndrome) can result in compromised testicular development and size.
An alternate developmental path is generally followed in the case of the ovary and differs from that of the testis in the following points:
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Failure of migration of PGCs results in a degenerated fibrous streak being formed (i.e., Turner syndrome). After their successful arrival and infiltration of the secondary cortical sex cords, the medulla starts to regress and the ovaries start to become distinguishable by the 6–8th weeks. PGCs undergo vigorous mitotic duplication during that time (i.e., reaching 600,000 in number as oogonia by the 8th week); subsequently, meiosis (i.e., which is facilitated by the action of retinoic acid and possibly nonretinoic acid factors), oogonial atresia, and the ongoing mitosis regulate their number until they reach a peak of at least 6 million cells by mid-gestation—at the time of formation of the first primordial follicle and breakdown of oocyte cell nests—and then decline afterward (i.e., cells entering meiosis freeze their progress at the meiosis-I phase by the 17th week to resume meiosis later in puberty). By the 7th month, mitosis is terminated and oogonial atresia is replaced by follicular atresia which withers their number to only 700,000 primordial follicles at birth (i.e., and 300,000 by the time of puberty). Fig. 1.4 provides more details about oogenesis throughout the intrauterine period.
Fig. 1.4
Intrauterine fetal oogenesis timeline.
Reproduced with permission from Hartshorne GM, Lyrakou S, Hamoda H, Oloto E, Ghafari F. Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection? Mol Hum Reprod . 2009;15(12):805–819 [Figure 1].
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Pregranulosa cells lack SRY expression that allows SOX9 to be antagonized properly and—thus—the commitment to granulosa cell lineage can occur under the influence of the WNT4/β-catenin pathway.
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The forkhead box 2 ( Foxl2 ) gene maintains such differentiation by continuous suppression of SOX9 .
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Nuclear receptor subfamily 0 group B member 1 ( NR0B1 ) gene (formerly called dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 ( DAX1 )) is a dosage-sensitive gene that needs two copies to antagonize SOX9 and SF1 . Conversely, as suggested by its formal name, the absence of NR0B1 gene (i.e., low dose) can potentially result in testicular dysgenesis.
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Theca cells are the Leydig cells’ female counterparts and developed from the interstitial cells under the influence of WNT4 and Foxl2 genes and in the absence of the effect of high levels of testosterone.
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Vascularization of the ovary follows the classic angiogenic route rather than recruiting endothelial cells by breaking down mesonephric vessels.
Gonadal relocation and descent ( Fig. 1.5 )
Testicular migration—and descent—begins around the 12th week of intrauterine life, goes through two major phases (viz., transabdominal and inguinoscrotal phases), and ends with the successful intra-scrotal positioning by the 32nd gestational week. In addition to the increase in the intraabdominal pressure and the passive change in position that occurs as a result of elongation of the trunk and enlargement of the pelvis (i.e., both phenomena are common to both sexes), the descent is aided in males by the secretory products of Leydig cells (including testosterone and insulin-like factor 3 (Insl3) and anti-Müllerian hormone (AMH)) that result—first—in the simultaneous regression of the craniosuspensory ligament and differentiation of the gubernaculum (i.e., to develop muscular tissue) and then the contraction of the latter to guide the testes through the inguinal canal (i.e., that contracts around the spermatic cord afterward preventing inguinal hernia) and into the scrotum—a process that the genitofemoral nerve is hypothesized to mediate through the action of calcitonin gene-related peptide (CGRP). Mutations in the INSL3 gene or its receptor gene (viz., relaxin/insulin-like family peptide receptor 2 [ RXFP2 ], aka leucine-rich repeat-containing G protein-coupled receptor 8 [ LGR8 ] or G protein-coupled receptor affecting testis descent [ GREAT ]), deficient Insl3 production, or fetal androgen insufficiency/resistance are associated with cryptorchidism in humans.
In females, the craniosuspensory ligament remains intact (i.e., which later gives rise to the ovarian suspensory ligament) while the gubernaculum remains undifferentiated (i.e., which later gives rise to the round ligament of the ovary [superior part] and that of the uterus [inferior part] which is anchored to both labia majora). Therefore, the ovaries only undergo a caudal shift to become positioned lateral to the kidneys.
Sexual duct differentiation ( Fig. 1.6 )
At week 4 of gestation, the indifferent stage of sexual duct differentiation begins as the mesonephric duct (aka Wolffian duct) forms from the lateral nephrotomes (i.e., intermediate mesoderm segments), followed 2 weeks later by the emergence of the paramesonephric duct (aka Müllerian duct) as an invagination of the urogenital ridge’s surface epithelium. After another 2 weeks, the testosterone secreted by the Leydig cells induces the differentiation of the Wolffian ducts into the epididymides (cranial segment), vasa deferentia, and seminal vesicles (lower segment) over the succeeding 4 weeks (i.e., dihydrotestosterone (DHT) does not influence the process as evident from the lack of 5α-reductase 2 enzyme expression in Wolffian ducts during differentiation). A simultaneous process of defeminization takes place where the Müllerian ducts regress under the effect of the AMH to completely disappear by the 11th week (i.e., AMH—or Müllerian inhibitory substance (MIS)—produced by Sertoli cells and the mechanism is mainly an apoptotic one). Both hormones might be primarily acting in a paracrine manner since unilateral testicular absence leads to ipsilateral persistence of the Müllerian structures and residual development of Wolffian ones ; inactivating mutations of AMH gene or AMH type II receptor defects can also negatively impact the functions of AMH (i.e., persistent Müllerian duct syndrome (PMDS) while overexpression in females leads to blind vagina and the absence of oviducts. Conversely in normal 46,XX females, the absence of the AMH allows the Müllerian ducts to continue their differentiation into fallopian tubes (cranial portions), uterus, cervix, and the upper third of the vagina (i.e., the lower portion originates from the urogenital sinus) while the absence of the testosterone prevents the differentiation of the Wolffian ducts (i.e., thus only nonfunctioning remnants can persist).
