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Genitalsex differentiation involves a series of events whereby the sexually indifferent fetus progressively acquires male or female characteristics in the gonads, genital tract and external genitalia. Normal sex development consists of several sequential stages. Genetic sex, as determined by the chromosome constitution, drives the primitive gonad to differentiate into a testis or an ovary. Subsequently, internal and external genitalia will follow the male pathway in the presence of specific testicular hormones, or the female pathway in their absence. Since the presence of the fetal testis plays a determining role in the differentiation of the reproductive tract, the term "sex determination" has been coined to designate the differentiation of the gonad during early fetal development.

THE BIPOTENTIAL GONAD

The Gonadal Ridge

Theprimordia of the gonads develop at approximately day 32 post-fertilization (Table 1) in the human embryo at the ventral surface of the cranial mesonephroi, in the intermediate mesoderm. The mesonephroi also give rise to the adrenal glands and the urinary system. The gonadal primordia are initially formed exclusively by somatic, mesoderm-derived cells: mesenchymal cells of mesonephric origin and coelomic epithelial cells that cover the coelomic surface of the gonadal ridge.

Table1. Chronology of sex differentiation.

Age from conception	CR length (mm)	Event
32 days	5	Gonadal primordia develop Growth of Wolffian ducts Primordial germ cell differentiation
37 days	10	Primordial germ cells reach gonadal ridge Differentiation of Müllerian ducts
42-50 days	15-20	Seminiferous cord differentiation
55-60 days	30	Beginning of secretion of AMH Leydig cell differentiation Cranial part of Müllerian ducts begins to regress
9 weeks	40	Leydig cells produce testosterone Beginning of masculinization of urogenital sinus and external genitalia
10 weeks	45-50	Meiotic entry of oocytes in the medulla Beginning of degeneration of female Wolffian ducts Male Müllerian ducts have disappeared Prostatic buds appear
12 weeks	55-60	The vaginal cord is formed Primordial follicles appear Seminal vesicles develop Testis at internal inguinal ring
14 weeks	70	Completion of male urethral organogenesis
16 weeks	100	Primary follicles appear
20 weeks	150	Testosterone serum level is low Formation of prostatic utricle
22 weeks	180	Vagina reaches perineum
24 weeks	200	Graafian follicles appear Beginning of penile growth
27-30 weeks	230-265	Inguino-scrotal descent of the testis
36 weeks	300	Secondary and tertiary follicles produce AMH

Afew general transcription factors belonging to the large homeobox gene family play an important role in the stabilization of the intermediate mesoderm and the formation of the urogenital ridges (Table 2). Mice in which Lhx1 (1), Emx2 (2)or Pax2 (3)has been inactivated fail to develop urogenital derivatives. However, these ubiquitous factors are essential for the development of other vital embryonic structures. Lhx9, although also expressed in central nervous system structures of the early embryo (4), only seems to be essential for the proliferation of somatic cells of the gonadal ridge (5). Several other factors are involved in cell proliferation in the gonadal primordium both in XX and XY embryos. For instance, when signaling of the insulin/insulin-like growth factor family was impaired in a mouse model by a triple knockout of insulin receptor tyrosine kinase family comprising Ir, Igf1r and Irr, both XX and XY gonads were significantly reduced in size (6). Also in Pod1 knockout mice, gonads are severely hypoplastic in both XX and XY fetuses (7). Since cell proliferation is more important in the male than in the female early developing gonad (8-10), sex-reversal is often observed in XY embryos with an alteration of gonadal cell proliferation. It has been suggested that this is due to a reduction in the number of SRY-expressing pre-Sertoli cells, resulting in very low levels of SRY expression that are insufficient to trigger testicular differentiation (11, 12).

Table2. Factors involved in early gonadal ridge development.

Gene	Chromosomal localization	Expression	Function	References
LHX1 (LIM homeobox gene 1)	11p12-p13	Primitive streak, prechordal and intermediate mesoderm, brain, thymus, tonsil	Differentiation and development of the head, neural and lymphoid tissues and urogenital structures	(1)
LHX9 (LIM homeobox gene 9)	1q31-q32	Central nervous system, forelimb and hind limb mesenchyme and urogenital system	Activation of SF1 in gonadal primordia	(4, 5)
EMX2 (homolog of empty spiracles homeobox gene 2)	10q26.1	Telencephalon and epithelial components of the urogenital system	Arealization of the neocortex and induction of the mesenchyme	(2, 13)
PAX2 (Paired box gene 2)	10q24.3-q25.1	Mesonephros, metanephros, adrenals, spinal cord, hindbrain and optic and otic vesicles	Regulation of WT1 expression and of mesenchyme- to- epithelium transition	(3, 14, 15)
M33 (CBX2) (Chromobox homolog gene 2)	17q25	Unknown	Regulation of homeotic genes	(16, 17)
ATRX (XH2) (Helicase 2, X-Linked)	Xq13	Widespread	Nucleotide excision repair and initiation of transcription	(18, 19)
Insulin receptor (Ir) Insulin growth factor receptor 1 (Igfr1) Insulin receptor-related receptor	19p13.2 15q25-q26 1q21-q23	Widespread Widespread brain, heart, lung, liver, small intestine, kidney, thymus, spleen, muscle, adipose tissue and cartilage	Metabolic, cell proliferation	(6, 20)
Pod1	6q23-q24	Epithelium of the developing gastrointestinal, genitourinary, and respiratory systems	Basic helix-loop-helix transcription factor	(7)
WT1 (Wilms tumor associated gene 1)	11p13	Urogenital ridge derivatives	DNA- and RNA-binding protein with transcriptional and post-transcriptional regulating capacity	(21, 22)
SF1 (Steroidogenic factor 1)	9q33	Gonadal ridges, adrenal gland primordia, hypothalamus and pituitary	Transcriptional regulation of STAR, steroid hydroxylases, gonadotropins, aromatase, AMH and DAX1, and stabilization of intermediate mesoderm	(23-25)

Thedifferentiation of the gonadal ridge from the intermediate mesoderm requires the expression of sufficient levels of WT1 and SF1. WT1 was initially isolated from patients with Wilms' tumor, an embryonic kidney tumor arising from the metanephric blastema. By alternative splicing and alternative translation initiation, WT1 encodes at least 8 different zinc-finger proteins acting as transcriptional and/or post-transcriptional regulators owing to their ability to bind both DNA and RNA (21). The -KTS splicing variant, lacking the three amino acids lysine (K), threonine (T) and serine (S) at the end of the third zinc finger, is required for cell survival and proliferation in the indifferent gonad (26). The first indication of a role for WT1 in gonadal and renal development was its expression pattern in the urogenital ridges (22). During gonadal differentiation, WT1 is expressed in the coelomic epithelium and later in Sertoli and granulosa cells (27-30). In mice with a knockout of WT1, neither the kidneys nor the gonads develop (31). In humans, mutations in the WT1 gene do not completely prevent urogenital ridge development but may result in gonadal dysgenesis associated with nephroblastoma (Wilms' tumor) (32, 33)and/or nephrotic syndrome owing to glomerular diffuse mesangial sclerosis (34).

SF1, initially described as a regulator of steroid hydroxylases, is an orphan nuclear receptor expressed in the hypothalamus, the pituitary, the gonads and the adrenal glands (24, 35). In mice with a knockout of the SF1 gene, the intermediate mesoderm is not stabilized and the gonadal and adrenal primordia soon degenerate (36). SF1 also plays an important role in spermatogenesis, Leydig cell function, follicle development and ovulation, as demonstrated by a gonad-specific disruption of SF1 (37).

WT1 (38, 39), through interaction with CITED2 (40, 41), and LHX9 (5)regulate the expression of SF1 upstream of the gonadal development cascade. In humans, the phenotype resulting from SF1 mutations does not exactly match that of SF1 knockout mice: testes develop, although severely dysgenetic, in XY individuals (42-45). whereas ovaries do not seem to be affected in XX patients (46). Sex reversal is usually, but not always, associated with adrenal insufficiency (44, 45, 47). SF1 is one of the increasing number of examples of dosage-sensitive mechanisms in human sex differentiation, since mutations at the heterozygous state are sufficient to induce sex reversal in XY individuals (42).

TheGerm Cells

Initiallyformed exclusively by somatic cells, the gonads are subsequently colonized by the primordial germ cells (PGCs). PGCs derive from pluripotent cells of the proximal epiblast, which move, at a very early stage of embryonic life, through the primitive streak into the extra-embryonic region at the base of the allantois (48). Not all of these cells are committed to a germ cell lineage since they also give rise to extra-embryonic mesoderm cells (49). In the 4^thweek, some cells present in the yolk sac near the base of the allantois differentiate into PGCs, which can be identified by their expression of PRDM1 (50), and later alkaline phosphatase, Oct4 and the tyrosine kinase receptor c-kit (Fig. 1A) (49). Subsequently, PGCs become embedded in the wall of the hind gut, gain motility and migrate through the dorsal mesentery to reach the gonadal ridges in the 5^thweek (Fig. 1B).

The mechanisms responsible for the epiblast cells competency to become PGCs involve several extraembryonic ectoderm-derived factors, including bone morphogenetic protein 2 (BMP2) (51), BMP4 (51-53)and BMP8B (52, 54). PGC specification require PRDM1 and PRMD14 expression in competent cells of the proximal epiblast (reviewed by ref. (48). Early migration of PGCs is dependent on the expression of interferon-induced transmembrane proteins 1 and 3 (IFITM1 and IFITM3) in the surrounding mesoderm (55). During migration, PGCs proliferate actively but do not differentiate (49). Germ cell migration through the dorsal mesentery to the gonadal ridges and survival/proliferation in both XX and XY embryos is driven by signaling between kit ligand (also known as Stem cell factor [SCF], Steel factor or mast cell growth factor [MGF]), which is expressed in somatic cells of the gonadal ridge and the hind gut along the pathway of PGC migration, and its receptor present in germ cells, c-kit (Fig. 1) (56-58). PGC migration and genital ridge colonization is also dependent on stromal cell-derived factor 1 (SDF1, also known as CXCL12) and its receptor CXCR4 (59), and on interactions with extracellular matrix proteins, like fibronectin and laminin, while proliferation and/or survival involve many other factors (49, 60, 61)(48).

Interactionwith somatic cells of the gonadal ridges

Uponarriving in the undifferentiated genital ridge, by the end of the 5^thweek, germ cells continue to proliferate by mitosis and maintain bipotentiality for approximately one week. Then germ cells in the male gonad become enclosed in the seminiferous cords and differentiate into the spermatogonial lineage, which does not enter meiosis until the onset of puberty. Gonocyte proliferation in the fetal testis is inhibited by androgens (62). In the female gonad, germ cells continue to proliferate by mitosis and enter meiosis in the 10th week (Table 1). Prevention of entry into meiosis was first thought to be a specific effect of male somatic cells since germ cells entering a prospective ovary or those which have failed to enter gonads of either sex enter meiosis at approximately the same time and develop into oocytes, irrespective of their chromosomal pattern (63). The existence of a testicular meiosis-preventing factor and an ovarian meiosis-inducing factor was initially postulated (64), but these factors could not be isolated. Recent studies have shed light on the sexually dimorphic evolution of gametogenesis in the fetal gonads. The mesonephros from the indifferent gonad, as well as the lung and adrenal gland, synthesize retinoic acid that acts as a meiosis inducer (65, 66). The fetal ovary continues to produce retinoic acid, which binds to the retinoic acid receptor (RAR) present in the germ cells and induces the expression of STRA8 (66), a transcription factor that upregulates DAZL and SYCP3, two proteins involved in the formation of the synaptonemal complex essential for the onset of meiosis (for review, see ref. (48). Conversely, germ cells embedded in the seminiferous cords do not enter meiosis because they are protected from retinoic acid action: Sertoli cells express CYP26B1, an enzyme that catabolizes retinoic acid, thus acting as a meiosis-preventing factor (65, 66). NANOS2 is another meiosis-preventing protein, since it also represses STRA8 expression in the fetal testis (67).

Chromosomal constitution does not influence sex differentiation of germ cells: XX germ cells surrounded by Sertoli cells differentiate into spermatogonia, whereas XY germ cells in an ovarian context differentiate into oogonia and then enter meiosis (68). However, germ cells whose karyotype is discordant with the somatic lineages fail to progress through gametogenesis and enter apoptosis later in life. This is also true for germ cells with an abnormal sex chromosome complement, e.g. 45,X (Turner syndrome) and 47,XXY (Klinefelter syndrome).

The influence of germ cells on the developing gonad is sexually dimorphic: Germ cells are essential for the maintenance and differentiation of the fetal ovary, otherwise prospective follicular cells degenerate and streak gonads result. In contrast, the development of the testes is not hindered by the lack of germ cells (68, 69).

No sexual difference can be observed in the gonads until the 6th week of embryonic life in humans and 11.5 days post-coitum (dpc) in mice. Undifferentiated gonads of XX or XY individuals are apparently identical and can form either ovaries or testes. This period is therefore called indifferent or bipotential stage of gonadal development.

SEXDETERMINATION

Thepioneering experiments of fetal sexual differentiation carried out by Alfred Jost in the 1940’s clearly established that the existence of the testes determines the sexually dimorphic fate of the internal and external genitalia (Fig. 2) (70, 71). Irrespective of their chromosomal constitution, when the gonadal primordia differentiate into testes, all internal and external genitalia develop following the male pathway. When no testes are present, the genitalia develop along the female pathway. The existence of ovaries has no effect on fetal differentiation of the genitalia. The paramount importance of testicular differentiation for fetal sex development has prompted the use of the expression “sex determination” to refer to the differentiation of the bipotential or primitive gonads into testes. Compelling evidence for the importance of the Y chromosome for the development of the testes, irrespective of the number of X chromosomes present, has existed since 1959 (72, 73). However, the identification of the testis-determining factor (TDF) on the Y chromosome did not prove easy and several candidates (e.g. HY antigen, ZFY) were successively proposed and rejected until the SRY (Sex-determining region on the Y) gene was cloned in 1990 in man (74)and mouse (75). Experimental (76, 77)and clinical (74, 78)evidence clearly established that SRY was the testis determining factor. Considerable progress has been made since SRY was identified, and it has become clear that sex determination is a far more complex process, regulated by competing molecular pathways in the supporting cell lineage of the bipotential gonad. In the next section, we describe the morphological aspects of fetal testicular and ovarian differentiation and the underlying molecular mechanisms, involving genes mapping to sex-chromosomes (Fig. 3) and autosomes (Table 2).

Testicular versus Ovarian Differentiation: the early divergent pathways

Duringthe bipotential stage, many genes are expressed at similarly low levels in XY and XX gonadal ridges. A non-exhaustive list includes WT1, SF1, SOX9, FGF9, PGD2, DAX1, WNT4, FOXL2, RSPO1 and β-catenin. While SOX9, FGF9 and PGD2 have more testis-promoting activity, DAX1, WNT4, FOXL2, RSPO1 and β-catenin are predominantly ovary-promoting genes. However, probably all are necessary for normal gonadal development in both sexes, with gene dosage and resulting relative expression levels playing an important role in the sexually divergent fate of the gonads.

SRY,the master switch for testis determination

SRYis a member of a family of DNA-binding proteins bearing a high mobility group (HMG) box and maps to the short arm of the Y chromosome Yp11.3, very close to the pseudoautosomal region 1 (PAR1). PAR1 on Yp and PAR2 on Yq are the only regions of the Y chromosome that undergo meiotic recombination with homologous sequences of the X chromosome during male spermatogenesis (Fig. 3). The proximity of SRY to PAR1 makes it susceptible to translocation to the X chromosome following aberrant recombination and provides an explanation for 80% of XX males (79)and for a low proportion of XY females. Surprisingly, mutations and deletions of the SRY locus only account for 15% of XY females (80). The physiologically relevant mechanism of action of SRY in gonadal differentiation still remains to be completely elucidated. There are reports suggesting that it may act as a transcriptional activator, but also as a repressor, or as an architectural factor allowing DNA bending for other factors to regulate target gene transcription (for review, see ref. 81). Proteins that interact with SRY and could have a relevant function in gonadal differentiation include SIP-1/NHERF2 (82, 83)and KRAB-O (84).

A tight regulation of SRY expression is essential for fetal gonadogenesis: both timing and level of expression are determinant, as revealed by experiments in mouse showing that SRY levels has to reach a certain threshold at a certain stage of fetal development to induce testis differentiation (85). SRY expression commences between days 41 and 44 post-fertilization in humans (86). The mechanisms underlying the initiation of SRY expression begin to be unraveled (for review, see refs. (87, 88). The +KTS splice variant of WT1 (26, 89, 90), SF1 (91), GATA4/FOG2 (92)and Sp1 (93, 94)are able to activate SRY transcription. The transcriptional co-factor CITED2 acts in the gonad with WT1 and SF1 to increase the expression of SRY levels to attain a critical threshold to efficiently initiate testis development (41). The +KTS isoform of WT1 might also act as a posttranscriptional stabilizer of SRY mRNA (88). Whether members of the insulin receptor family have a direct implication on SRY expression still needs further investigation (6). Sox9 seems to downregulate Sry expression in the mouse (95-97); in man, however, SRY expression persists after testis differentiation (86).

Owingto its Y-chromosome localization, SRY can only be expressed in the XY gonadal ridge, thus playing a paramount role in breaking the expression level equilibrium existing hitherto between testicular and ovarian promoting genes in the bipotential gonads.

SOX9:a target of SRY

Oncethe importance of SRY as a mammalian testis-determining gene was established, scientists have been prompted to search for a testis-specific SRY-target gene. SOX9, an autosomal member of the HMG-box protein superfamily mapping to 17q24.3-q25.1, has become a firm candidate (98). In the mouse, Sox9 is expressed at low levels in the bipotential gonads of both sexes under SF1 regulation (81, 99), but persists only in testicular Sertoli cells after Sry expression has peaked (Fig. 4) (100, 101). Sox9 is a direct target of Sry in mice (97). However, it is also possible that indirect mechanisms mediate Sox9 activation, in line with the hypothesis indicating that SRY might act as a repressor of a negative regulator of the male cascade(102). For instance, targeted disruption of Foxl2 leads to Sox9 upregulation in the XX gonad (103), and prostaglandin D2 has been shown to upregulate Sox9 in the absence of Sry (104, 105).

SOX9mimics SRY effects independently of SRY expression. In fact, overexpression of Sox9 during early embryogenesis induces testicular differentiation in two different models of transgenic XX mice (106, 107). Functional analysis of Sox9 during sex determination, by conditional gene targeting in mice, has shown that homozygous deletion of Sox9 in XY gonad interferes with sex cord development and with activation of testis specific markers (96). Further evidence for the role of SOX9 in testicular development comes from observations in humans, where a double dose of SOX9 expression is required. Heterozygous mutations result in haploinsufficiency resulting in campomelic dysplasia, a polymalformative syndrome that includes sex-reversal due to gonadal dysgenesis in XY individuals (98). Sox9 also affects differentiation of the reproductive tract by upregulating the expression of anti-Müllerian hormone (AMH) (108), a Sertoli cell-specific factor involved in male differentiation of the internal genitalia (see below).

Sox8 is another member of the Sox family. Like Sox9, Sox8 is expressed in the central nervous system, limbs, kidneys, gonads and craniofacial structures. During mouse embryo development, Sox8 expression can be observed in the developing testis around the time of sex determination (110). Sox8 can bind the canonical target DNA sequences and activate AMH transcription acting synergistically with SF1, but with less efficiency than Sox9 (111).

Observations made in XY intersex patients with normal SRY together with the discovery of proteins showing a sexually dimorphic pattern of expression in the gonads following SRY peak have helped to identify other loci, likely to be involved in testicular differentiation, which are discussed hereafter.

FGF9and PGD2: maintaining SOX9 expression levels after SRY has vanished

SOX9expression is maintained at high levels in the male gonad despite down-regulation of SRY soon after testicular determination (99). Recent work has shown that SOX9 is capable of autoregulating its expression (99). Additionally, SOX9 upregulates the expression of FGF9 and PGD2. FGF9 interacts with its receptor FGFR2, initiating a feed-forward loop that maintains SOX9 expression and also results in downregulation of WNT4 expression (112){Kim, 2007 18713 /id}. PGD2 induces SOX9 expression (105)and nuclear translocation (113), thus increasing its availability to target genes.

Somatic cell proliferation appears to be critical for early testicular differentiation (9). FGF9 (Fibroblast growth factor 9) and WNT4 act as antagonistic signals in early gonadal differentiation (112). FGF9 controls cell proliferation in a sexually dimorphic fashion: the disruption of FGF9 expression by targeted deletion in transgenic mice does not affect XX gonads but prevents testicular differentiation and results in sex reversal in XY mice (114). In the mouse, Fgf9 and Wnt4 are expressed in the undifferentiated XX and XY gonads at the same levels: Fgf9 near the coelomic surface and Wnt4 near the mesonephric border (112). As already mentioned, when Sry expression is initiated and upregulates Sox9 in the XY gonadal ridge, the balance between Fgf9 and Wnt4 is disrupted: Sox9 enhances Fgf9 expression which in turn maintains high Sox9 expression thus resulting in a feed-forward loop that accelerates commitment to the male pathway. Wnt4 expression is downregulated when a threshold level of Fgf9 is reached (112). Fgf9 controls the proliferation of a population of cells that give rise to Sertoli progenitors (10). In Fgf9 knockout mice, initial Sertoli cell differentiation is not hindered: Sry and Sox9 expression is observed but soon weakens resulting in an aborted differentiation of Sertoli cell precursors (112). Although in experimental conditions, FGF9 is capable of inducing proliferation of XX coelomic epithelium cells, this does not result in Sertoli cell differentiation, clearly indicating that increasing cell proliferation is not sufficient to induce testicular differentiation, and that other pro-testicular signals are also required (114).

The insulin-receptor tyrosine kinase family

Another example of the importance of cell proliferation in the early stages of testicular gonadogenesis is the observation that XY mice with a triple targeted deletion of Ir (insulin receptor), Igf1r (Insulin-like growth factor receptor 1) and Irr (Insulin-related receptor) fail to develop testes resulting in complete sex reversal (6). These members of the insulin-receptor tyrosine kinase family are involved in the control of cell proliferation.

ATRXand CBX2

ATRX,also known as XH2, is an X-encoded DNA-helicase whose mutation results in mental retardation, α-thalassemia and gonadal dysgenesis in XY individuals (18, 19). M33, whose homolog in human is CBX2 (16)is a transcriptional regulator whose disruption in mice causes several defects including retardation of embryonic gonadal formation and gonadal sex reversal in XY fetuses (17). Like SRY, ATRX and CBX2 might be involved in chromatin remodeling, which seems to play an important role in sex determination (115).

DAX1,FOXL2, WNT4, RSPO1 and β-catenin: anti-testicular, pro-ovarian, or both?

DAX1,encoding for an orphan nuclear receptor and mapping to the DSS (Dosage Sensitive Sex-reversal) region on Xp21 was the first putative testis repressor and/or ovarian determining gene. A duplication of DSS results in sex-reversal in 46,XY patients (116), and Dax1 overexpression in transgenic XY mice impairs testis differentiation (117). However, the disruption of Dax1 in XX mice does not prevent ovarian differentiation (118). Furthermore, DAX1 is essential for normal testicular cord formation (119, 120). These observations in rodent models, together with DAX1 expression pattern in the human fetus showing persistently low levels in both XX and XY gonads from 33 days post-fertilization (i.e the bipotential stage) through 15 fetal weeks (86), strongly suggest that low DAX1 levels are necessary for gonadal development in both sexes. Abnormally low or high DAX1 expression seems to result in abnormal gonadal differentiation (121).

WNT4is a secreted protein that functions as a paracrine factor to regulate several developmental mechanisms. Wnt proteins bind to the frizzled (Fz) family of membrane receptors, leading to the activation of the phosphoprotein dishevelled (Dsh) and a subsequent increase in β-catenin levels owing to an inhibition of its degradation rate (for review, see ref. 122). Wnt4 is expressed at similar levels in the XY and XX bipotential gonads. When Sry upregulates Sox9 in XY gonads, and the feed-forward loop with Fgf9 is established, Wnt4 is silenced (112). In XX gonads, Wnt4 dominates and results in an induction of β-catenin and silencing of Fgf9 and Sox9 (112). Wnt4 also up-regulates Dax1 (123), which antagonizes SF1 and thereby inhibits steroidogenic enzymes. Wnt4-deficient XX mice express the steroidogenic enzymes 3-hydroxysteroid dehydrogenase and 17-hydroxylase, which are required for the production of testosterone and are normally suppressed in the developing female ovary (124, 125). In humans, a duplication of 1p31-35, where human WNT4 maps, causes ambiguous genitalia of XY patients, probably due to low testosterone production (123). Wnt4 is also involved in sex differentiation of the internal genital tract (see below).

R-spondinsare secreted activators of the transcriptional activity of β-catenin, probably via upregulation of Wnt4 (126). In mouse gonads, Rspo1 is specifically up-regulated in XX somatic cells from E11.5 onwards and controls the differentiation of the ovary (127), probably by upregulating β-catenin with the consequent silencing of Sox9 (reviewed in ref. 81). Loss of function mutations in the human RSPO1 gene and Rspo1 gene ablation in mice result in the formation of ovotestes in the XX fetus (127-129), probably owing to SOX9 upregulation.

Ingoats, XX males develop in the event of a deletion in the autosomal PIS locus (130, 131). A winged helix/forkhead transcription factor gene, FOXL2, has been identified at this locus. FOXL2 is expressed in the mouse and human ovary (132), where it is involved in granulosa cell differentiation (133). In the XY fetus, Sox9 represses Foxl2 expression in the gonad (134). On the other hand, Sox9 expression is upregulated in XX Foxl2^-/-gonads, but only postnatally. Therefore, β-catenin and FOXL2 are both required to maintain Sox9 in a repressed state during normal ovary development: β-catenin is crucial during embryonic stages whereas FOXL2 takes over postnatally (reviewed in ref. 81). While FOXL2 mutations result in a variety of phenotypes, from adult ovarian failure to development of streak gonads (132, 135-137), no XX males have been reported.

Insummary, DAX1 is necessary for both testicular and ovarian development, with a need for precise gene expression dosage. WNT4, RSPO1 and β-catenin seem to have both pro-ovarian and anti-testicular activities from early embryonic life, while FOXL2 may also have similar actions postnatally. Antagonism between SOX9 and -catenin seems to underlie the fate of the gonads. RSPO1 and WNT4, acting to establish -catenin as the dominant signal in XX gonads, drive this competition toward the ovarian pathway. Stabilization of -catenin counteracts the effects of SRY by destabilizing its target, SOX9 (138).

Stabilizationof Testis Differentiation: Vascular, Cellular and Molecular Pathways

Inthe XY fetus, the initially amorphous cluster of gonadal cells becomes segregated in two compartments: testicular cords and interstitial tissue. These architectural changes are heralded by gonadal ridge vascularization, a highly dynamic and sexually dimorphic process. At variance with the differentiating ovary that recruits vasculature by typical angiogenesis, the XY gonad recruits and patterns vasculature by a remodeling mechanism: pre-existing mesonephric vessels disassemble and generate a population of endothelial cells that migrate to the gonad, below the coelomic epithelium, where they reaggregate to form the coelomic vessel, an arterial vessel that runs the length of the testis at its antimesonephric margin (139, 140). The formation of this vessel is one of the earliest hallmarks of testis development that distinguishes it morphologically from the developing ovary (139, 141). Evidence now exists for a close spatial relationship between testis vascularization and cord formation (140). Furthermore, all of the cells migrating from the mesonephros to the coelomic zone of the differentiating testis express endothelial markers like VE-cadherin, which indicates that endothelial, rather than peritubular myoid cells, underlie the dependence of cord formation on cell migration. Moreover, disruption of vascular development blocks formation of testis cords (40). Subsequently, Sertoli cells aggregate and enclose germ cells (142). The interaction between differentiating peritubular myoid cells and Sertoli cells results in the formation of basement membrane of the testicular cords. Mesenchymal cells and matrix and blood vessels fill the interstitial space, in which Leydig cells will soon appear. Beyond vascularization, , which is necessary to allow efficient export of testosterone(143), cell migration from the mesonephros largely contributes to testicular organogenesis (144, 145)and is antagonized by the initiation of meiosis in germ cells (146).

Differentiation of Sertoli and Leydig cells

SRY is expressed in pre-Sertoli cells having delaminated from the coelomic epithelium in the central part of the indifferent gonad and induces their differentiation (147, 148). SRY-expressing pre-Sertoli cells lying beneath the coelomic epithelium play a central role in the migration of cells from the mesonephric mesenchyme into the differentiating gonad (142). Experimental work using XX-XY chimeras has shown that not 100% of Sertoli cell precursors need to express SRY to differentiate along the male pathway: in fact up to 10% of Sertoli cells were XX. However, a threshold number of SRY expressing –i.e. XY– cells seems to be essential in order for Sertoli cell differentiation, and thus testicular development, to be guaranteed (149).

Along with SRY, FGF9 as well might have a role in inducing mesonephric cell migration into the developing fetal testis and Sertoli cell differentiation. FGF9 is expressed in Sertoli cells of the fetal testis and Fgf9 null mice have dysgenetic gonads (114)(see below).

Testicularcord formation can be detected in human fetuses 13-20 mm crown-rump length (43-50 days) beginning in the central part of the gonad (122, 150). Cord formation is heralded by the development of a new type of cell, the primitive Sertoli cell, characterized by a polarized, large and clear cytoplasm with abundant rough endoplasmic reticulum and complex membrane interdigitations (151), a downregulation of desmin and an upregulation of cytokeratins (152), and the expression of AMH and DHH (153). Sertoli cells aggregate around large, spherical germ cells, with a large nucleus and pale cytoplasm, called gonocytes at this stage, which can be observed in the center of testicular cord cross-sections. The structural basis of cord formation seems to be dependent on basal lamina deposition between Sertoli and peritubular cells with myofibroblastic characteristics (145). In the interstitial compartment, connective tissue, blood vessels and Leydig cells can be observed. One particular feature of testicular vasculature is the formation of the coelomic vessel, a large vessel that appears below the coelomic epithelium very early in testicular differentiation (139). Surrounding the gonad, the basement membrane layer underlying the coelomic epithelium thickens to form the tunica albuginea.

Morphologically and functionally distinct from testicular cords, the interstitial compartment contains developing Leydig cells, the most important androgen producing cells in the male. Leydig cells derive from the coelomic epithelium and also from mesenchymal cells of mesonephric origin (8, 154, 155). They differentiate in the interstitial tissue by the beginning of the 8th week in the human embryo, after testicular cords have completely formed, and soon begin to produce testosterone, which plays an essential role in the stabilization of Wolffian ducts and the masculinization of external genitalia. Leydig cells also produce insulin-like growth factor 3 (INSL3), a growth factor responsible for the transabdominal phase of testicular descent (156). Fetal Leydig cells are large, eosinophilic cells with an abundant smooth endoplasmic reticulum and numerous mitochondria, but no Reinke's crystalloids, which are restricted to adult Leydig cells. Although the initial differentiation of fetal Leydig cells may occur independently of gonadotropin action in rodents (157), further Leydig cell differentiation and proliferation depends on placental hCG in the first and second trimesters of fetal life and on fetal pituitary LH thereafter (158). Leydig cell number peaks at mid-gestation (Fig. 5) and then slightly decreases. After birth, Leydig cells disappear from the interstitial tissue of the testis until puberty (159).

The morphology of the testes changes very little during fetal life and after birth, until pubertal onset. Testicular cords maintain the same appearance, with only slight changes. In the perinatal period, gonocytes are progressively replaced by spermatogonia, which are smaller and located in contact with the basement membrane. It is not until puberty, when germ cells enter meiosis and Sertoli cells acquire adult characteristics, that a lumen develops and testicular cords become seminiferous tubules.

Vanin-1, a cell-surface molecule involved in the regulation of cell migration, might also be responsible for differentiating Sertoli cell association with, and adhesion to, migrating peritubular cells (160, 161). Nexin-1, expressed by early Sertoli cells, could act to maintain the integrity of the basal lamina (160).

DHHand itsreceptor Patched2might also play a role in Sertoli-peritubular cell interaction and basal lamina deposition (162, 163). DHH is a protein secreted by fetal Sertoli cells, but not by somatic components of the fetal ovary, immediately after testicular determination (164). Patched2 is expressed in germ, peritubular and interstitial cells of the testis (165). Testes develop abnormally during fetal life in Dhh null mice, resulting in XY sex-reversal. Seminiferous cords are disorganized owing to defects in the basal lamina and peritubular cells, with germ cells occasionally lying in the interstitial tissue, and Leydig cells are hypoplastic (162, 163). Homozygous mutations of DHH in 46,XY patients are associated with gonadal dysgenesis (166, 167).

XY sex-reversal due to gonadal dysgenesis has also been reported in patients with partial deletions of chromosome 9p (168-170). The geneDMRT1, mapping to 9p in the human (171), may be involved since it is expressed in the fetal testis (172, 173)and its inactivation in XY mice causes defects in testis differentiation (174). The exact mechanisms whereby DMRT1 acts during testicular differentiation have not yet been clarified.

Sertoli cells express growth factors, like nerve growth factors (NGFs), hepatocyte growth factor (HGF) and platelet derived growth factors (PDGFs), which can induce cell migration from the mesonephros under experimental conditions (11). However, evidence for an in vivo role has only been provided for NGFs and their receptors TrkA (Ntrk1) and TrkC (Ntrk3) (175, 176)and PDGFs and their receptor Pdgfr-α (177).

In Leydig cell development, not only hCG but also growth factors play a role. FGF9 (114), DHH (162)and PDGFs (177)are Sertoli cell-secreted signals involved in Leydig cell differentiation. SF1 action, essential for steroidogenic protein expression and function (178), is suppressed by WNT4-activated DAX1 expression (123). By counteracting WNT4, and thus downregulating DAX1 in interstitial cells of XY gonads, SRY might indirectly enhance SF1 action (179, 180). Finally, ARX is an X-chromosome gene identified in patients with X-linked lissencephaly and genital abnormalities probably associated with a block in Leydig cell differentiation (181).

Genes involved in male sex determination are shown in Fig. 6.

Timingof testicular differentiation

Inorder for the fetal testis to adequately differentiate and secrete masculinizing hormones, not only do all these factors need to be present at sufficient levels in the right cell lineage, but their expression must also be initiated within a narrow time window. In XY mice, the delay of Sry expression has been postulated to result in higher Dax1 levels in the genital ridge at the time of gonadal differentiation. Consequently, ovarian development proceeds and primordial follicles develop in the center of the gonad, resulting in an ovotestis (85, 117), or in the whole gonad, with the formation of an ovary (182). These ovarian structures subsequently degenerate since germ cells, which are essential for follicular development, fail to enter meiosis and undergo apoptosis owing to the lack of two X chromosomes.

Stabilizationof Ovarian Differentiation: Cellular and Molecular Pathways

Ovarianmorphogenesis

Inthe XX fetus, the gonad remains indifferent after the 7th week from a histological standpoint, but a functional differentiation already develops: XX gonads become capable of estradiol production at the same time as XY gonads begin to synthesize testosterone (183). PGCs proliferate by mitosis and differentiate to oogonia. Ovarian maturation proceeds from the center to the periphery. At week 10, oogonia in the deepest layers of the ovary enter meiotic prophase, the first unequivocal sign of morphological ovarian differentiation. Subsequently, oogonia become surrounded by a single layer of follicular cells, most probably derived from supporting cell precursors of coelomic epithelial and mesonephric origin (184), they enter meiosis and become oocytes and form primordial follicles (Fig. 7). The earliest primary follicles appear at 15-16 weeks and the first Graafian follicles at 23-24 weeks (185). By the end of the 7^thmonth of gestation, mitotic activity has ceased and almost all germ cells have entered meiotic prophase. Oocytes proceed to the diplotene stage, where they remain until meiosis is completed at the time of ovulation in adult life. However, not all oocytes undergo meiosis: from 6-7 million ovarian follicles at 25 weeks, only 2 million persist at term (186). Most oocytes undergo apoptosis and follicles become atretic. AMH is produced, albeit in low amounts, after the 36th week of development (187)by granulosa cells from primary to antral follicles, but not by primordial follicles (188, 189).

Theinvolvement of germ cells in the organization of gonadal structure is one major difference between the ovary and the testis. In fact, germ cells appear to be a critical regulator in organizing and maintaining the ovarian structure. While fetal testis development progresses normally in the absence of germ cells (190), ovarian follicles do not form when germ cells are absent (191). Furthermore, if germ cells are lost after formation of follicles, these rapidly degenerate (192, 193).

Molecularpathways of ovarian differentiation

InXX gonads, very few endothelial cells migrate from the mesonephros to the gonad, which suggests that cortical and medullary domains of the ovary are already established in early gonadogenesis, although no morphological boundaries are evident, consistently with molecular evidence of discrete gene expression domains specified by 12.5 dpc in the mouse ovary (194). Wnt4 induces the expression of follistatin (Fst): both molecules are involved in the inhibition of the coelomic vessel formation (anti-testis effect) but also in the survival of meiotic germ cells (pro-ovarian effect) (194, 195).

DAX1,WNT4 and FOXL2

Themechanisms involved in the differentiation of ovarian structures are still poorly understood (Fig. 8), and no ovary-determining gene has been characterized. DAX1, an initial candidate, does not seem to be involved, since ovarian development occurs in knockout mice (118).

Asalready mentioned, WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids (anti-testis effect) (112, 123, 124, 195, 196). Follistatin (Fst) seems to be a gene downstream of WNT4 (194). Wnt4- and Fst-null ovaries develop the male-specific coelomic vessel and Leydig cell-like androgen producing cells. Wnt4 and Fst are also involved in the survival of meiotic germ cells (pro-ovarian effect) (194, 195). However, some of the abnormalities in gonadal development might not be sex-specific, but rather derive from the lack of expression of Wnt4 in the undifferentiated gonadal ridge, since testes from Wnt4 knockout XY mice also show disorganized testicular cords (195).

FOXL2is a transcription factor expressed in germ and somatic cells more strongly in the female than the male fetal gonad from the 8^th. fetal week (133, 137, 197). Mutations in the human FOXL2 gene have been found in females with premature ovarian failure associated with eyelid abnormalities characterized by blepharophimosis, ptosis and epicantus inversus (BPES)(132, 135). Although important to granulosa cell differentiation, FOXL2 does not seem essential to early ovarian development (103, 137).

Oogenesisand folliculogenesis

Twomajor events can be distinguished in ovarian development: germ cell migration, proliferation and meiosis, and folliculogenesis. For a long time, it has been known that two intact X chromosomes are required in the human -in contrast to the mouse- for ovarian differentiation and development (198). The lack of two X chromosomes results in germ cell loss and, subsequently, absent folliculogenesis. Therefore, all the factors involved in the proliferation and migration of PGCs in early embryogenesis (see “The germ cells” section) are essential for ovarian formation. A number of genes are upregulated in the human ovary before and during primordial follicle formation; their functional implications still need to be elucidated (199). In mice, neurotrophins (NTs) and their NTRK tyrosine kinase receptors facilitate follicle assembly and early follicular development (200). Factors involved in germ cell meiosis are also important. Stabilization of oocytes requires the expression of DAZLA, a cytoplasmic protein present in female and male germ cells (201), and Msh5, a protein involved in DNA mismatch repair (202). In Msh5 null mice, oocytes are lost before the diplotene stage resulting in ovarian dysgenesis. Stra8 gene, an early marker of female germ cells, is upregulated in embryonic germ cells of XX gonads prior to meiotic entry and is not expressed in male embryonic germ cells. Stra8 expression in germ cells of XX gonads takes place in an anterior-to-posterior wave and is followed by the upregulation of the meiotic gene Dmc1 (203). A number of other factors involved in oocyte development have been described (for review, see ref. 204).

Although not essential to ovarian differentiation, several factors are involved in the development of ovarian follicles. FIGα is crucial for the formation of primordial follicles (205). AMH regulates the recruitment of primordial follicles into subsequent steps of folliculogenesis (206), NOBOX, SOHLH1 and SOHLH2 are critical transcription factors during the transition from primordial to primary follicles (reviewed in ref. (48), and GDF9 is important for follicle growth beyond the primary stage (207, 208). An increasing number of factors are involved in later steps of folliculogenesis (for review, see ref. (48).

THEINTERNAL REPRODUCTIVE TRACT

The Indifferent Stage

Upto 8 weeks in the human fetus, the internal reproductive tract is similar in fetuses of both sexes and consists of a set of two unipotential ducts, the Wolffian and Müllerian ducts (Fig. 9).

Wolffianducts

TheWolffian duct is the excretory canal of the primitive kidney, the mesonephros. In the human embryo, Wolffian ducts originate dorsolaterally to the segmented pronephric blastema and laterally to somites 8-13 in embryos 25 to 32 days old (Table 1). In normal embryogenesis, a single ureteric bud evaginates from the Wolffian duct and grows dorsally, in response to inductive signals from the metanephric mesenchyme (209). Growing caudally, Wolffian ducts progressively acquire a lumen and reach the hindgut, which caudally becomes the cloaca. The Wolffian ducts become incorporated into the male genital system when renal function is taken over by the definitive kidney, the metanephros. As pointed out by Dorothy Price (210), the duration of the ambisexual stage of Wolffian development is determined by the duration of mesonephric activity.

Müllerianducts

Müllerianducts arise in 10-mm human embryos as a cleft lined by the coelomic epithelium, between the gonadal and mesonephric parts of the urogenital ridge. This coelomic opening will later constitute the abdominal ostium of the Fallopian tube. The cleft is closed caudally by a solid bud of epithelial cells, which burrows in the mesenchyme lateral to the Wolffian ducts and then travels caudally inside their basal lamina.Initially, these cells are mesoepithelial, ie they exhibit characteristics of both the epithelium and the mesenchyme, they will become completely epithelial only in the female, at the time male ducts begin to regress (211). At 8 weeks of development, the growing solid tip of the Müllerian duct, now in the pelvis, lies medial to the Wolffian duct, having crossed it ventrally in its downward course. For a while, the two Müllerian ducts are in intimate contact, then they fuse, giving rise to the uterovaginal canal (Fig. 10), which makes contact with the posterior wall of the urogenital sinus, causing an elevation, the Müllerian tubercle, flanked on both sides by the opening of the Wolffian ducts (Fig. 9).

Developmentof the Müllerian duct occurs in three phases (Fig. 11) (211). First, cells of the coelomic epithelium are specified to a Müllerian duct fate. These can be identified by a placodelike thickening of the coelomic epithelium and by the expression of Lim1. During the second, Wnt4-dependent phase, these primordial Müllerian cells invaginate from the coelomic epithelium to reach the Wolffian duct. Upon contact with the Wolffian duct, the third or elongation phase begins, consisting in proliferation and caudal migration of a group of cells at the most caudal tip. As could be expected, integrity of protein kinase pathways is required for cell proliferation (212). Close contact with the Wolffian duct is also necessary to Müllerian growth, indeed the lack of transcription factors required for Wolffian development, such as Lim1 or Pax2, leads to Müllerian truncation (see Table 3). Wolffian ducts do not contribute cells to the elongating Müllerian tip (211, 213), but act by supplying Wnt9b, secreted by Wolffian epithelium (214).

MaleDifferentiation

Maledifferentiation of the internal genital tract is characterized by regression of Müllerian ducts and differentiation of the Wolffian duct into male accessory organs.

Müllerianduct regression

Müllerianregression, the first sign of male differentiation of the genital tract, occurs in 55 to 60 day-old human embryos in the area where the ducts are closest to the caudal pole of the testis (216)(Fig. 12). Once initiated, the regression of the Müllerian duct extends caudally as well as cranially, sparing the cranial tip which becomes the Morgagni hydatid, and the caudal end, which participates in the organogenesis of the prostatic utricle. Müllerian regression of the cranial part of the Müllerian duct begins while the duct is still growing caudally towards the urogenital sinus (217)and is characterized by a wave of apoptosis spreading along the Müllerian duct (218, 219). Shortly afterwards, the peri-Müllerian mesenchyme condenses to form a fibrous whorl, which progressively strangles the Müllerian duct and finally remains the only witness of its former existence. Mesenchymal changes are preceded by the dissolution of the basement membrane, which precipitates apoptosis and allows extrusion of epithelial cells and their transformation into mesenchymal cells (219, 220). Epithelial-mesenchymal transformation is an important factor of epithelial cell loss during Müllerian regression. From a molecular standpoint, Müllerian regression is marked by the migration of anti-Müllerian hormone type II receptors from the coelomic epithelium to the peri-Müllerian mesenchyme (215), the deposition of peri-epithelial extracellular matrix (221)increased expression of Mmp2 (222), and accumulation of ß-catenin in the nucleus (219).

Stabilizationand differentiation of Wolffian ducts

Thesecond aspect of male differentiation of the internal genital tract is the stabilization and differentiation of the Wolffian ducts (223). After the loss of mesonephric functional activity, the mesonephric nephrons and caudal tubules degenerate but the cranial tubules persist to form the male efferent ducts. The connections between the mesonephric tubules and the gonadal primordium are permanently established in the sixth week; in the male, they give rise to the rete testis, while in the female, they form the rete ovarii. Between weeks 9 and 13 in the human embryo the upper part of the Wolffian duct differentiates into the epididymis. Below, it is surrounded by a layer of smooth muscle and becomes the vas deferens, which opens into the urogenital sinus at the level of Müllerian tubercle. In sexually ambiguous individuals, in whom Wolffian and Müllerian ducts coexist, the vas deferens is embedded in the uterine and vaginal walls (224, 225). The seminal vesicle originates from a dilatation of the terminal portion of the vas deferens in 12 wk-old fetuses.

Testiculardescent

Duringhuman fetal development, the testis migrates from its initial pararenal position to its terminal location in the scrotum. Testicular descent is a complex process that has been subdivided into several phases. Transbdominal movement brings the testis down to the internal inguinal ring at 12 weeks (Fig. 13). As the testis approaches the inguinal ring, the epididymis is pulled into the canal and serves as a wedge for opening it (226). The gubernaculum testis, a jelly-like structure which connects the lower pole of the testis and the tail of the epididymis to the scrotum, swells and pulls the testis down to the inguinal ring. while the cranial suspensory ligament dissolves. In the female, the cranial ligament holds the ovary in a high position and the gubernaculum remains long and thin as the round ligament (227). After 25 weeks, the gubernaculum bulges beyond the external inguinal ring and is hollowed out by a peritoneal diverticulum called the processus vaginalis. The second –inguinoscrotal- phase of testicular descent occurs between 27 and 30 weeks after conception (228). « Physiological » cryptorchidism is frequent in premature infants.

FemaleDifferentiation

Femaledifferentiation of the internal genital tract is characterized by lack of stabilization of the Wolffian ducts, which have completely disappeared at 90 days of human fetal development, except for vestiges such as the Rosenmüller organs or Gartner canals.

Müllerianducts persist, establish apico-basal characteristics and develop into an epithelial tube which will give rise to the endometrium (211)while the surrounding mesenchyme differentiaties into the myometrium of the uterus and Fallopian tubes (229). The loss of mesoepithelial characteristics signals the end of the AMH-sensitive window of Müllerian ducts. Tubal differentiation involves formation of fimbriae and folds in the ampullary region (Fig. 14) and acquisition of cilia and secretory activity by the high columnar epithelium. The uterotubal junction is demarcated by an abrupt increase in the diameter of the uterine segment and by the development of epithelial crypts.. The early endometrium is lined by a closely packed columnar epithelium in which gland formation and vacuolated cells can be recognized as gestation advances. Functional changes at the level of the endometrium and endocervix resemble those elicited by estrogen stimulation in the adult uterus. The cervix occupies the distal two-thirds of the fetal uterus (185).

THEUROGENITAL SINUS AND EXTERNAL GENITALIA

The Indifferent Stage

Upto approximately 9 weeks, the urogenital sinus and external genitalia remain undifferentiated (Fig. 15). The urogenital sinus is individualized in 7-9 mm (~5 week) human embryos, when a transverse urorectal septum divides the cloaca into the rectum dorsally and the primitive urogenital sinus ventrally, closed by the genital membrane which disappears in 20-22 mm (~8 week) embryos (216). The Müllerian tubercle demarcates the cranial vesicourethral canal from the caudal urogenital sinus.

At 12 weeks in males and females alike, the vaginal primordium is formed by the tips of the Müllerian ducts, and medial and lateral outgrowths, the sinovaginal bulbs, which fuse to form the vaginal cord or plate. When the cells of the vaginal plate desquamate, the vaginal lumen is formed.

In embryos 8-15 mm long (~6 weeks), the genital membrane is surrounded by the labioscrotal swellings. These are connected to the caudal poles of the genital ridges by fibrous bands which later develop into the gubernaculum testis in males. The genital tubercle emerges as a ventral medial outgrowth between the genital folds. After the corpora cavernosa and glans have differentiated, the ventral surface of the genital tubercle is depressed by a deep furrow, the urethral groove. The external genitalia remain undifferentiated up to approximately 9 weeks (216)(Fig. 15).

MaleDifferentiation

Urogenital sinus and prostate

Maleorientation of the urogenital sinus is characterized by prostatic development and by the repression of vaginal development. Prostatic buds appear at approximately 10 weeks at the site of the Müllerian tubercle and grow into solid branching cords. Maturation of the prostatic gland is accompanied by development of the prostatic utricle. Two buds of epithelial cells, called the sino-utricular bulbs in the male, develop from the urogenital sinus close to the opening of the Wolffian ducts and grow inwards, fusing with the medial Müllerian tubercle, to form the sino-utricular cord, enclosed within the prostate gland, which canalizes at 18 weeks to form the prostatic utricle, the male equivalent of the vagina (230).

Externalgenitalia

Masculinizationof the external genitalia begins in human male fetuses 35-40 mm long (~9 weeks) in human male fetuses by lengthening of the ano-genital distance (216)(Fig. 15). Fusion of the labioscrotal folds, in a proximal to distal fashion, forms the epithelial seam (231), which closes the primary urethral groove. The urethral plate, an extension of the urogenital sinus present within the genital tubercle from the earliest stages of development (232), lies in the roof of the primary urethral groove and extends to the tip of the phallus. The literature concerning penile development is controversial. Most textbooks describe it as a two-step process, with the proximal urethra forming by fusion of the urethral folds around the urethral plate and the distal urethra arising from a invagination of the apical ectoderm. However, some authors (231, 233)believe that the entire human male urethra is of endodermal origin, formed by the urethral plate dorsally and the fused urethral folds ventrally. The seam is remodeled into the tubularized urethra without connection to the epidermis. The ventrally discarded excess epithelial cells migrate into the ventral skin of the penis. Abnormalities of seam formation or remodeling could explain the vast majority of cases of hypospadias in which defects of androgen synthesis or metabolism cannot be demonstrated (234).

Urethral organogenesis is complete at 14 weeks, apart from a physiological ventral curvature, which can persist up to 6 months of gestation (235). However, surprisingly, no size difference exists between penile or clitoral size before 14 weeks (236, 237), in spite of the fact that serum testosterone levels peak at that time (238). Maximal phallic growth occurs during the third trimester of fetal life, at a time when male testosterone levels are declining. At that time, the fetal pituitary has taken over control of Leydig cell production of testosterone in the fetus; indeed, microphallus is a classical sign of congenital gonadotropin deficiency. The insensitivity of the male genital tubercle to high levels of androgens during the second trimester is not due to a low expression of the androgen receptor or of 5α-reductase type 2 in the corpora cavernosa (239).

FemaleDifferentiation

Femaleorientation of the urogenital sinus is characterized by lack of prostatic differentiation and the acquisition of a separate vaginal opening on the surface of the perineum (Fig. 15). At the end of the ambisexual stage, the vaginal anlagen is located just underneath the bladder neck. In females, the lower end of the vagina slides down along the urethra until the vaginal rudiment opens directly on the surface of the perineum at 22 weeks (240). The hymen marks the separation between the vagina and the diminutive urogenital sinus, which becomes the vestibule.

Theembryological origin of the vagina is still hotly debated. According to the generally accepted work of Koff (241), the upper part of the vagina derives from the Müllerian ducts and the lower part from the sinovaginal bulbs, which by fusion form the vaginal plate, derived from the urogenital sinus. It is now thought that the Wolffian ducts do not contribute to the sinovaginal bulbs but have a helper function during downward movement of the vaginal bud in the female. In the male the caudal ends serve as androgen operated switch for the negative control of vaginal development. The rudimentary vagina in the complete androgen insensitivity syndrome corresponds to caudal ends of the Müllerian ducts. Atresia of the vagina in the Mayer-Rokitansky-Küster-Hauser syndrome could be explained by the failure of Wolffian and Müllerian ducts to descend caudally (242).

Developmentof female external genitalia is essentially static (243). The anogenital distance does not increase, the rims of the urethral groove do not fuse, the labioscrotal swellings give rise to the labia majora. The dorsal commissure forms at their junction. The genital folds remain separate and become the labia minora. When the vagina acquires a separate perineal opening, the diminutive pars pelvina and the pars phallica of the urogenital sinus become the vestibule.

CONTROLOF SEX DIFFERENTIATION

Growth factors

Genital duct formation

Moleculargenetic studies in the mouse have contributed to the identification of growth factors essential for the formation of the sexual ducts (Table 3) [see refs. (244)and (223)for review]. Since Wolffian ducts are required for the elongation of Müllerian ducts, absence of growth factors necessary to Wolffian development will also induce Müllerian truncation. Many growth factors, such as Lim1, Emx2, Hoxa-13, Pax2 and Pax8 are essential for the development of other organs, in contrast the role of Wnt4a and Wnt7a, a subset of the Wnt family homologous to the Drosophila wingless gene, is restricted to reproductive organs. Mutations of WNT4 have been reported in three cases of Müllerian aplasia associated with hyperandrogenism in girls (reviewed in ref. 245)but Wnt4 mutations do not appear to be associated with classical forms of the Rokitansky-Küster-Mayer syndrome (246). Members of the dachsung gene family, dach 1 and 2 also play a role by regulating the expression of Lim1 and Wnt7 (247). The ribonuclease III endonuclease, Dicer1, is required for normal female reproductive tract development (248).

Congenitalbilateral absence of the vas deferens is responsible for 1-2 % of cases of male infertility and is present in 95% of patients affected with cystic fibrosis (249), a bronchial and pancreatic disease due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Whether efferent duct maldevelopment is a primary defect of cystic fibrosis or a secondary degenerative change resulting from obstruction by mucus is not known at the present time. Isolated absence of vas deferens in otherwise healthy men is often associated with the presence of a single CFTR allele mutation (250).

Table3. Consequences of null mutations of growth factors on morphogenesis of genital ducts.

Growth factors	Wolffian ducts	Müllerian ducts	Gonads	Reference
β-catenin	Normal	Lack of oviduct coiling	Loss of germ cells in the ovary. Testes normal	(251, 252)
Dach1/Dach2	Normal	Hypoplasia of female reproductive tract	Normal	(247)
Dicer1	Normal	Hypoplasia of female reproductive tract	Reduced ovulation rate	(248)
Emx2	Early degeneration	Do not form	Absent	(2)
Hoxa-13	Rostral ureteral junction	Agenesis of caudal portion	Normal	(253, 254)
Igf1	Agenesis of caudal portion	Infantile uterus	Abnormal Leydig cells. No ovulation	(255)
Lim1 (Lhx1)	Do not form	Do not form	Normal	(256)
Pax2	Early degeneration	Early degeneration	Normal	(3)
Pax8	Normal	Endometrium does not form	Normal	(257)
Retinoic acid receptors	Agenesis of vas deferens and seminal vesicles	Agenesis of uterus and cranial vagina	Normal	(258)
Wnt4	Persist in females	Do not form	Virilized ovary	(124)
Wnt7a	Normal	Persist in males		(259)

Externalgenitalia, urogenital sinus

Developmentof the fetal external genitalia begins with outgrowth of the genital tubercle from paired genital swellings. The early genital tubercle masculinizes if exposed to androgens but early patterning is androgen-independent and regulated by a cascade of signaling molecules which orchestrate interaction between tissue layers and mesenchymal/epithelial tissues (Table 4). External genitalia are appendages emerging from the caudal body trunk, hence many genes which pattern distal limb development also play a predominant role during genital tubercle formation, for example BMPs (260), FGF-8 and 10, Hox gene families (for reviews, see refs.(261-263)and Wnt/ßcatenin required for normal genital tubercle outgrowth (264). Sonic hedgehog (Shh) signaling plays a crucial role by regulating many of the mesenchymal genes involved (263, 265, 266)(Fig. 16). The homeotic genes Hoxa13 and Hoxd13 act in a partially redundant manner since double null mutants, though more likely to survive than Hoxa13 null ones, show more severe urogenital abnormalities than those with at least one functional allele (253). Sox9 (267)and Fgf10 (268)both play a role in early prostate bud differentiation.

Correctvaginal development requires Wnt, Pax and Ltap genes. Vaginal abnormalities similar to those elicited by diethylstilbestrol (DES) administration, i.e. vaginal clear-cell adenocarcinoma, vaginal adenosis, transverse vaginal ridges and structural malformations of the cervix and uterus, occur in transgenic mice deficient in Wnt7a, a signaling molecule expressed by the Müllerian epithelium (269, 270), suggesting that DES exposure acts by deregulating Wnt7a during uterine morphogenesis (270). Wnt7a deficiency could act by interfering with normal mesenchymal-epithelial signaling, which is required for correct morphogenesis of the reproductive tract. Vaginal opening is regulated by Pax8 (257)and Ltap (loop-tail), a component of the frizzled pathway (271). Females heterozygous for the semi-dominant mutation Ltap have tail loops and an imperforate vagina (272).

Table4. Growth factors in urogenital development

Growth factors	Role in urogenital development	References
BMP4	Restricts prostate ductal budding	(273)
BMP7	Closure of the distal urethra	(262)
FGF8	Initiation of genital swellings ;	(274)
FGF10	Development of the glans penis and clitoridis, and prostate	(263, 268, 274)
HOXA10	Atrophic seminal vesicles in null mice	(275)
HOXA13	In mice, semi-dominant mutations lead to limb defects, vaginal hypoplasia and deficiency of the os penis (Hypodactyly syndrome) In humans, an autosomal dominant mutation produces limb and uterine abnormalities and urinary tract malformations (Hand-Foot-Genital syndrome)	(276) (277)
HOXD13	Hoxd-13 null mice display decreased ductal branching in the prostate and seminal vesicle and agenesis of bulbourethral gland	(254)
HOXA13/HOXD13 null mutants	No genital tubercle, no partition of the cloaca in double mutants	(253)
Ltap	Vaginal opening	(271, 272)
MSX2	Disruption of vaginal epithelium and lack of caudal Wolffian regression	(278)
PAX8	Vaginal opening	(257)
SHH	Outgrowth and patterning of external genitalia and urogenital sinus Development of prostatic ducts Inhibition of apoptosis in penile smooth muscle	(263) (279) (266)
SOX9	Lack of ventral prostate development	(267)
Wnt/β-catenin	Masculinization of external genitalia	(280)
WNT5a	Genital tubercle agenesis in null mice	(260)

HormonalControl of Male Sex Differentiation

Theclassical experiments of Jost (70, 71)have taught us that an embryo, whatever its genetic sex, will develop along female lines provided it is not exposed to testicular secretions (Fig. 2).

Testicular hormones are the main forces driving male sex differentiation (Fig. 17). Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance (MIS), a member of the TGF-β family produced by Sertoli cells, triggers Müllerian regression, the first step of male sex somatic differentiation. Testosterone, produced by fetal Leydig cells, is responsible for the maintenance of Wolffian ducts and their differentiation into male sex accessory organs. Virilization of urogenital sinus and external genitalia is under the control of dihydrotestosterone (DHT), a reduced metabolite of testosterone. A Leydig cell product, insulin-like factor3 (Insl3) promotes testicular descent.

Anti-Müllerianhormone

AMHis secreted in high levels by Sertoli cells from the time of testicular differentiation until puberty (Figs. 15 and 16) and at lower levels thereafter (for reviews, see refs. (281-283). In the female, AMH is produced by granulosa cells of growing follicles from birth to menopause (187, 284-286). Low expression of AMH or its receptor has also been identified in the endometrium (287), and in the brain (288)and in motor neurons (289)where it acts as a survival factor (289).

The AMH protein is a 140-kD glycoprotein homodimer belonging to the TGF-β family (290)and as such is cleaved to generate N-terminal and C-terminal homodimers (291). The homology of AMH to other members of the transforming growth factor-β (TGF-β) family is restricted to the bioactive C-terminus (292, 293), for which a molecular model has been built, through analogy to crystallized members of the family (294)(Fig. 20). The human 2.8-kb gene has been cloned (290)and mapped to chromosome 19p13.2-p13.3 (295). It consists of five exons, the last one coding for the C-terminal fragment.

The AMH gene has been cloned in other eutherian mammals such as the mouse (296)and rat (297)and in the marsupial tammar wallaby (298). AMH is also present in the chick (299, 300)and American alligator (301), both of which carry Müllerian ducts which regress in the male. More surprisingly, AMH orthologs (302)and the AMH type II receptor (303)have been cloned from the gonads of modern teleost fish, which do not possess Müllerian ducts. In fish, AMH appears to be involved essentially in germ cell proliferation and gonadal development (304, 305), which suggests that AMH acquired its anti-Müllerian activity during the course of evolution. Indeed, in higher vertebrates, AMH continues to play a role upon gonadal differentiation and steroidogenesis both in males (306)and in females (286).

In the human fetal male gonad, AMH mRNA and protein can be detected from the 8th week, when Sertoli cells become identifiable and begin to arrange in cord-like structures, the future seminiferous tubules (307)(Fig. 18). High AMH production by Sertoli cells continues until puberty (Fig. 19), irrespective of the disappearance of Müllerian ducts.

AMHis measurable in human serum by ELISA. Currently, two kits are commercially available: one from Immunotech (308)and one from Diagnostic System Laboratories (DSL) (309). Each uses different sets of antibodies and different AMH standards, but the results are now very similar (310), in contrast to earlier versions of the kits (311). Measurement of AMH in serum has diagnostic applications in patients with disorders of sex development (308), as a marker of prepubertal testicular function in boys (312)and of follicular reserve in women (313-316), in follow-up of granulosa cell tumors (284, 285, 317)and perhaps in prediction of successful in vitro fertilization (314), particularly when levels are measured in follicular fluid (318). Variations during the menstrual cycle are minimal (319). By contrast, the clinical usefulness of AMH in seminal fluid in men with non-obstructive azoospermia is less clear-cut due to dispersion of normal values (320-324). Further discussion of the diagnostic and potentially therapeutic value of AMH in the adult ovary and testis is outside the scope of this review.

Thechronological expression of AMH is of greatest importance in sex differentiation (Fig. 19). In the male, AMH must be expressed before Müllerian ducts lose their responsiveness, i.e. before the end of the 8th week in the human fetus (325, 326). Thus, the AMH gene is likely to be under tight transcriptional control. SOX9, a member of the SRY gene family essential for testis determination, independentlyplays an essential role in the initiation of AMH transcription in the mammalian male fetus. Mutation of the SOX9 binding site of the AMH promoter completely abolishes initiation of AMH production in transgenic mice, while that of SF-1 decreases AMH transcription, but nevertheless allows Müllerian regression to take place (108). SF-1 and another transcription factor, GATA4, bind to response elements present on the AMH promoter to enhance SOX9-activated AMH expression (327, 328). The Wilms tumor associated gene WT-1 (329)and the transcription factor GATA-4 (330)synergize with SF-1 to increase AMH transcription, a synergy antagonized by the X-linked gene DAX-1 (38, 331)(Fig. 21). Many genes regulating AMH gene transcription play a similar role in sex determination (81).

Inmales, Sertoli cells continue to produce AMH until puberty when expression is curtailed by the synergistic negative action of androgens and meiotic entry (332). In humans, lack of expression of the androgen receptor in Sertoli cells before 6 months after birth accounts for the high levels of AMH during fetal life and in the neonate (333, 334). Conversely, early expression of the androgen receptor in the tammar wallaby pouch male mediates repression of AMH at the time of initial virilization of the male reproductive tract (335). In the absence of androgen action, e.g. in patients with defects of androgen synthesis or action, AMH levels are abnormally elevated during neonatal and pubertal periods (336), probably due to stimulation by FSH (337, 338)(Fig. 22).

AMHtransduction

Likeother members of the TGF-β family, AMH signals through two distinct membrane-bound receptors, both serine/threonine kinases (Fig. 23). AMH receptor type II (AMHR-II) binds specifically to AMH. Its mature form in the human, expressed at the cell surface, has a mass of 82 kDa (339). Truncated forms of the receptor are not secreted, unless the signal sequence is replaced by the TGF-β one, suggesting that the AMH-RII signal sequence is defective (340)A monoclonal antibody against AMH-RII is now available (341). A three-dimensional model of extra- and intracellular domains has been built by analogy with crystallized receptors of the TGF-β family (Fig. 23) (340).

Thegene for AMHR-II, located on chromosome 12 q13, is 8 kbp long and divided into 11 exons. Exons 1-3 code for the signal sequence and extracellular domain, exon 4 for most of the transmembrane domain, and exons 5-11 for the intracellular serine/threonine kinase domains (342). AMHR-II is expressed in the mesenchymal cells which surround the Müllerian duct, and also on Sertoli, granulosa (343, 344), Leydig cells(306), endometrium (287)and neurons (288, 289). Expression of the receptor in the peri-Müllerian mesenchyme requires the presence of the signaling molecule Wnt-7a. In its absence,Müllerian ducts cannot respond to AMH and persist in XY individuals (259). WT1 (345)and SP600125, an inhibitor of the c-Jun N-terminal Kinase Inhibitor (346)also activate AMH-RII.

Activationof AMHR-II by its ligand results in phosphorylation and nuclear translocation of the BMP-specific cytoplasmic effectors Smads 1, 5 and 8 (Fig. 24) (348-350), suggesting that signaling occurs through BMP type I receptors, ALKs 2, 3 and 6. ALK3 is absolutely required since its targeted inactivation leads to the persistence of Müllerian derivatives in transgenic mice (347, 351). In a Sertoli cell line, ALK3 is also the most potent receptor, ALK2 can compensate for its absence while ALK6 has an inhibitory effect (352).

Thepersistent Müllerian duct syndrome

Mutationsof AMH (294)and AMHR-II in man (340)(Fig. 25) and gene knockout in mice (353, 354)are associated with a rare form of 46,XY disorder of sex development (DSD), the persistent Müllerian duct syndrome (PMDS), transmitted according to an autosomal recessive pattern.

XY individuals are externally normally virilized, and persistence of Müllerian duct derivatives is discovered at surgery for either inguinal hernia or cryptorchidism (Fig. 26). Transverse testicular ectopia, ie presence of both testes in the same scrotal sac, is not unusual. No mutations of type I receptors have been detected in human PMDS patients (Belville, unpublished), probably because type I receptors are shared by AMH and BMPs and embryos with defective BMP function cannot complete gastrulation (355). Women homozygous for AMH or AMHR-II mutations are normally fertile but it is too early to know whether they will undergo early menopause since “AMH-null” female mice exhibit early follicular depletion (206). Treatment should be as conservative as possible, aiming to remove the Müllerian remnants only if they impede testicular descent due to the inclusion of the vas deferens in the uterine and cervix wall (356-358).

Testosteroneand dihydrotestosterone

Testosteroneand its reduced metabolite dihydrotestosterone (DHT) are the main factors involved in maintenance of the Wolffian duct, development of prostate and male sex accessory organs and virilization of the external genitalia.

Testosteronebiosynthesis

Beginningat 9 weeks, testosterone is produced from cholesterol by gonadotropin stimulation of fetal Leydig cells, through the coordinated action of steroidogenic enzymes, most of which are also expressed in the adrenal gland, which explains why certain steroidogenic disorders are common to the testis and adrenal (Fig. 27). P450 side-chain cleavage enzyme, which is responsible for the initial step in the steroidogenic pathway, is coded by CYP11A1 on chromosome 15q23-q24 and is located at the inner mitochondrial membrane. Translocation of cholesterol into the mitochondrion is dependent on steroidogenic acute regulatory protein (StAR), a phosphoprotein coded by a gene located on chromosome 8p11.2 (359). StAR, steroidogenic P450 enzymes, and AMH are positively regulated by SF-1, whose action is opposed by DAX-1 (38). DAX-1 per se is required for proper testicular determination (see section on Male sex determination: Genetic pathways).

Pregnenolone is subsequently metabolized by another cytochrome P450, P450c17, in the smooth endoplasmic reticulum. The cytochrome P450c17, encoded by gene CYP17 located on chromosome 10q24 (360), bears two distinct activities: a 17α-hydroxylase activity responsible for the conversion of pregnenolone to 17α-hydroxypregnenolone and a 17-20 desmolase activity, capable of converting 17α-hydroxypregnenolone to dehydroepiandrosterone (DHEA). This pathway, commonly known as the Δ⁵pathway, is predominant in humans and higher primates (361).

TheΔ⁴compound progesterone, is successively converted to 17α-hydroxyprogesterone and Δ⁴-androstenedione by P450c17. In humans, two isoforms of 3ß-HSD have been identified : 3ß-HSD type 1, expressed mainly in the placenta, mammary gland and skin, and 3ß-HSD type 2, expressed in the gonads and adrenal glands (362). Both genes (named HSD3B1 and HSD3B2, respectively) are located on chromosome 1p11-13. Only mutations in the type 2 gene result in congenital adrenal hyperplasia and/or DSD.

The final step in testosterone biosynthesis is reduction of Δ⁴-androstenedione to testosterone through the activity of 17ß-hydroxysteroid dehydrogenase (17ß-HSD), formerly known as 17-ketosteroid reductase (17-KSR). Three isoforms of 17ß-HSD have been identified. The type 3 isoform, present in the testis and encoded by the HSD17B3 gene, located on chromosome 9q22, seems to be the only one involved in fetal male sexual differentiation (363). XY patients with impaired 17ß-HSD type 3 activity usually develop with female or ambiguous external genitalia; however, Wolffian ducts derivatives are present in most, probably due to accumulation of the 17ß-HSD substrates Δ⁴-androstenedione and Δ⁵-DHEA, with weak androgen activity. Type 1 isoform is encoded by gene HSD17B1 located on chromosome 17q21, and type 2, by gene HSD17B2 on chromosome 16q24. While 17ß-HSD types 1 and 2 are essentially expressed in the placenta and the ovary and involved in estrogen metabolism, 17ß-HSD type 2 expression has been reported in the liver with the capacity for testosterone synthesis. The extragonadal expression of these 17ß-HSD isoforms, mainly the type 2 isoform, may explain the mechanism of virilization observed at puberty in XY patients with 17ß-HSD type 3 deficiency (364).

Gonadotropincontrol of testosterone production

Testosteroneproduction by the human fetal testis is detectable at 9 weeks, peaks between 14 and 17 weeks and then falls sharply, so the serum concentrations of testosterone overlap in males and females in late pregnancy. Gonadotropin stimulation is not required for the initiation of steroid synthesis (365)but is necessary to maintain Leydig cell function subsequently. Testicular and serum levels of testosterone are closely correlated with chorionic gonadotropin concentration, the peak of fetal testicular steroidogenic activity coincides with the acme of concentrations of hCG in the circulation. In adult Leydig cells, the capacity to respond to sustained gonadotropic stimulation by increased androgen production is curtailed by the development of a refractory state, due to receptor down-regulation (366). Fetal Leydig cells apparently escape desensitization, allowing them to maintain a high testosterone output during the several weeks necessary to male differentiation of the genital tract.

When chorionic gonadotropin declines in the 3rd trimester, the hypothalamic-pituitary axis takes over (238, 367)(Fig. 28). Impaired pituitary gonadotropic function in 46,XY fetuses does not result in major signs of impaired virilization because the most important steps of sexual differentiation take place during the fourth month, when Leydig cells are controlled by hCG. Usually, these patients present with cryptorchidism and micropenis, owing to reduced Leydig cell number and low testosterone production during the third trimester. LH and hCG signal through a common seven-transmembrane domain receptor coupled to G proteins, the LH/CG receptor, present on testicular Leydig cells. The human gene encoding the LH receptor, located on chromosome 2p21 (368), contains 11 exons. The first 10 exons encode a long N-terminal extracellular domain responsible for hormone binding, while the 11th exon encodes the whole transmembrane domain, involved in the cAMP/PKA signal transduction pathway. A functionally normal LH/CG receptor is absolutely necessary in fetal life to achieve a normal development of the Leydig cell population and androgen production. Loss of function mutations affecting the transmembrane domain or the extracellular domain result in Leydig cell aplasia or hypoplasia in the XY fetus (368). Although they do not affect XX fetal development, amenorrhea and infertility are observed in women (369).

Theandrogen receptor

Testosteroneand DHT exert their action on androgen-dependent tissues by binding to the androgen receptor, a member of the steroid receptor family (Fig. 29). Mutations of this receptor lead to the androgen insensitivity syndrome, a relatively common disorder of sex development characterized by a female external genital appearance in XY patients with normal production of testicular hormones; milder phenotypes are also possible (370). The androgen receptor is encoded by a single-copy gene located on the long arm of the X chromosome, locus Xq11-12 (371). It spans 75-90 kb and its open reading frame of 2.75 kb comprises 8 exons. Exon 1 is the longest and codes for the amino-terminal transactivation domain (372). A highly polymorphic CAG triplet containing 14-35 repeats towards the 5’-end of exon 1, is useful as a genetic marker for inheritance of X chromosomes. Interestingly, expansion of the trinucleotide repeat which encodes this long tract of glutamine residues segregates with X-linked spinal and bulbar atrophy (373, 374). Exons 2 and 3 code for sequences containing two zinc fingers implicated in DNA binding. Most mutations, however, occur in exons 4 to 8, which encode the steroid hormone binding domain (375). The 5’-portion of exon 4 codes for the hinge region between the DNA- and steroid-binding domains, and plays a regulatory role (376).In contrast to receptors for other steroid sex hormones, which reside in the nucleus even in the absence of ligand binding, the androgen receptor resides mainly in the cytoplasm in the absence of hormone (377). The androgen receptor binds to specific DNA motifs, the androgen response elements (ARE), present in the promoter regions of androgen-activated genes(378). Transcriptional activity can be enhanced or repressed by ligand-dependent coactivators or corepressors (379, 380).

Testosteronemetabolism : 5α-reductase, dihydrotestosterone, aromatase

Themechanisms whereby androgens virilize the male embryo have been elucidated mainly by the early work of Wilson and his associates (381). Testosterone is the major steroid released by fetal testes in the blood stream and enters cells by passive diffusion. Organs such as the Wolffian duct, which are adjacent to the fetal testis, take up testosterone locally (382, 383). This source of androgen, crucial for Wolffian duct development, is not available if testosterone is supplied only via the peripheral circulation, as in 46,XX DSD due to adrenal hyperplasia.

Inside the cell, testosterone is converted to dihydrotestosterone (DHT) by the enzyme 5α-reductase. The conversion of testosterone to DHT amplifies the androgenic signal through several mechanisms. DHT cannot be aromatized to estrogen, and thus its effects are purely androgenic. Testosterone and DHT do bind to the same androgen receptor but DHT binds with greater affinity which results in a stabilization of the hormone-receptor complex for a longer period of time (384). Therefore, in tissues equipped with 5α-reductase at the time of sex differentiation, such as the prostate, urogenital sinus and external genitalia, DHT is the active androgen (Fig. 30). Patients with 5α-reductase deficiency virilize very poorly at these levels (384). Wolffian duct stabilization takes place between weeks 9-13 while 5α-reductase is not expressed there until about week 13, but since it is exposed to high local concentrations of testosterone, the Wolffian duct differentiates nevertheless.

Two functional isoenzymes of, with different pH optima, have been characterized (386): 5α-reductase type 1, encoded by gene SRD5A1 located on chromosome 5p15, is not expressed in the fetus, but is transiently active in newborn skin and scalp and permanently expressed in liver after birth and in skin from the time of puberty (387). Type 2 is encoded by gene SRD5A2, which maps to chromosome 2p23. This isoenzyme is expressed in fetal genital skin and in the urogenital sinus from early fetal development (387). Tissue distribution and ontogeny of both isoforms as well as mutation studies in humans with 46,XY DSD clearly indicate that type 2 plays a major role in sexual differentiation of male external genitalia but the emergence of type 1 probably accounts for the pubertal virilization of the type 2-deficient patients. Testosterone is also the source of estrogen, due to the irreversible action of the P450 enzyme aromatase, encoded by Cyp19, located on chromosome 15p21.1.

Androgensand the male urogenital tract

Androgensare required for stabilization and subsequent differentiation of the Wolffian ducts. Testosterone is delivered directly from the testis down the lumen of the Wolffian duct (383)reaching a higher concentration there than in organs which rely on their blood circulation for their androgen supply. Thus patients with androgen insensitivity whose AR retain very low but significant residual activity insensitivity have a female phenotype but retain an epididymis or vas deferens (388, 389). Wolffian duct differentiation is more susceptible to androgen deficiency than is its stabilization and is programmed during a critical time window, between 15.5 and 17.5 pc in the rat fetus. Because the androgen receptor is expressed in the WD stroma but not in the epithelium during this time, Wolffian duct differentiation is likely to be dependent on androgen-mediated signaling from the stroma to the epithelium (390)and may also involve non-androgenic factors (391). In marsupials, β-androstanediol, not testosterone, is responsible for the differentiation of Wolffian ducts (392)and other components of the male reproductive tract (393). In addition to their role in the initial outgrowth and pattering of the external genitalia (see above), growth factors also play a role in the androgen-dependent masculinization phase. For instance, the Wnt/β-catenin pathway is indispensable for the development of male external genitalia (280), contrasting with the feminizing role of β-catenin in gonadal differentiation (138).

Controlof testicular descent

Controlof testicular descent is still a hotly disputed issue, which we will not attempt to resolve here. AMH has been considered because persistence of Müllerian derivatives in intersex patients is frequently associated with cryptorchidism and because AMH serum levels are sometimes decreased in cryptorchid patients but this could due to testicular dysgenesis, a frequent cause of testicular malposition. Insulin-like factor 3 (INSL3), a member of the insulin/relaxin hormone superfamily secreted by fetal Leydig cells which maps to chromosome 19p13.2-p12 (394)has been shown to affect gubernaculum development in mutant mice (395); mutations of this gene have been detected in cryptorchid patients (396). Prenatal DES treatment, which is associated with cryptorchidism, impairs Insl3 expression in mouse testis and interferes with gubernacular development (397).

Androgens mediate the disappearance of the cranial suspensory ligament (398)and are required for the inguinoscrotal phase of testicular descent, perhaps acting through the genitofemoral nerve and the neuropeptide calcitonin gene-related peptide, as postulated by Hutson et al. (227)(Fig. 13). Because testicular descent is controlled by testicular hormones, the positive correlation between cryptorchidism and testicular dysgenesis is not surprising but a direct causal relationship is difficult to establish.

Estrogensand xenoestrogens

Althoughestrogen deficiency is not directly implicated in normal prenatal sexual development, unregulated exposure to female hormones may play a role in the pathophysiology of 46,XY DSD. Male fetuses exposed to diethylstilbestrol develop gross abnormalities of the reproductive tract (399-401). More recently, xenoestrogens present in food or the environment have been held responsible for the increasing incidence of cryptorchidism, hypospadias and testicular cancer (401-403)although genetic factors should not be disregarded (404). In non-human primates, fetal exposure to phtalate does not result in reproductive abnormalities (405).

HormonalControl of Female Differentiation

Estrogens, diethylstilbestrol

The conclusion that ovarian hormones are not necessary to female fetal development was first reached by Jost (70, 71)and is supported by the female phenotypic development of subjects with bilateral gonadal aplasia regardless of are expressed in fetal mouse--their karyotype. Estrogen receptors α and Müllerian ducts (406), yet their targeted inactivation does not prevent female sex differentiation (407). The same is true of aromatase knockout mice which are unable to synthesize estrogens (408). Yet, while null mouse models have demonstrated the innocuity of estrogen deficiency for reproductive tract development, inappropriate estrogen exposure is clearly detrimental. The most tragic illustration of estrogen toxicity is the « DES story ». Diethylstilbestrol (DES), a synthetic estrogen, was widely administered to pregnant women beginning in the 1940s in the hope of preventing abortion. It was later discovered that female progeny exhibited severe abnormalities of the reproductive tract : vaginal clear-cell adenocarcinoma, vaginal adenosis and squamous metaplasia, transverse vaginal ridges and structural malformations of the cervix and uterus (409). The DES phenotype is more severe in mice mutant for Msx2, a homeotic gene involved in vaginal epithelial differentiation (278).

CONCLUSION

Abewildering number of hormones and growth factors is involved in sex determination and differentiation, making it one of the best studied developmental processes.due to the fact that even the most severe disorders of sex determination/differentiation are not life-threatening. Yet, more than half of patients with incomplete male sex differentiation are not properly diagnosed, even in competent endocrine centers (410). Much remains to be done...