Sex Development

Home , Genital tubercle, Virilization, XY gonadal dysgenesis

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The makings of maleness: towards an integrated view of male sexual development

Dagmar Wilhelm* and Peter Koopman*‡ Abstract | As the mammalian embryo develops, it must engage one of the two distinct programmes of gene activity, morphogenesis and organogenesis that characterize males and females. In males, sexual development hinges on testis determination and differentiation, but also involves many coordinated transcriptional, signalling and endocrine networks that underpin the masculinization of other organs and tissues, including the brain. Here we bring together current knowledge about these networks, identify gaps in the overall picture, and highlight the known defects that lead to disorders of male sexual development.

Hypospadias The differences between males and females are a source secondary effects such as infertility and gonadal tumours. Incorrect placement of the of enduring fascination. This is hardly surprising, given Sexual dysgenesis is often traumatic, stigmatized and urethral opening in males, not the mysteries surrounding these differences and their under-recognized as a medical issue in our society, at the tip of the penis. profound effect on our daily lives. For the developmen- and identifying responsible genes will be increasingly tal biologist, the morphological differences between the important for diagnosing these disorders, counselling sexes are particularly intriguing given that they arise affected patients, and making a prognosis that will inform through dichotomous differentiation of a common set gender-assignment options. However, genes responsible of precursor tissues, a unique situation in embryonic for proper sexual development are often difficult to find development. For any given species, there are not one by conventional genetic studies, which require fertility. but two developmental biologies. The discovery of Sry in 1990 (REFS 2,3) opened the In simple terms, the development of mammalian way for genetic dissection of the cascade of events lead- maleness or femaleness hinges on whether testes or ing to male development. There is a growing molecular ovaries form in the embryo from the paired, ambipo- and cellular understanding of testis determination and tent structures known as genital ridges1. This decision the early events in testis differentiation, mainly from depends on the presence and correct function of a male- analyses of mouse mutants and genotype–phenotype determining gene from the Y chromosome, Sry, which correlations in humans who are affected by disorders functions in a specific subset of genital ridge cells to of sexual development. In addition, technologies such stimulate them to differentiate as Sertoli cells — the cells as microarray analysis have identified many genes that that interact with and nurture the germ cells. The Sertoli show sex-specific expression during gonad development *Division of Molecular cells then seem to orchestrate the differentiation of other and might be important in testis development. Genetics and Development, Institute for Molecular cell types required for testis formation, such as the germ However, key questions still remain. How does SRY Bioscience, The University cells and steroid-producing cells. If Sry is absent or does induce Sertoli cell differentiation? How do Sertoli cells of Queensland, Brisbane, not function correctly, other regulatory cascades lead to orchestrate the differentiation of the other testicular cell QLD 4072, Australia. ovary development and female characteristics. lineages, including the germ line? What factors, gonadal ‡ ARC Centre of Excellence in In reality, sexual development is vastly more complex, or non-gonadal, are necessary to induce secondary sexual Biotechnology and Development, Institute for hinging on delicate networks of molecular signals that characteristics such as testicular descent and differentia- Molecular Bioscience, specify sex-specific differentiation, organogenesis and tion of the external genitalia? What are the phenotypic The University of Queensland. endocrine function. The fragility of these networks is clear: consequences of mutations in genes that are important Correspondence to P.K. disorders of sexual development are among the most com- in these processes? We focus on these questions in an e-mail: [email protected] mon human birth defects. They range in frequency and also effort to summarize the knowns and unknowns of male doi:10.1038/nrg1903 in severity, from hypospadias to complete sexual ambiguity sexual development, from testis specification, to the Published online 11 July 2006 and sex reversal (TABLE 1), and are often associated with development of the genital tract, male accessory sex

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Table 1 | Disorders of human sexual development Syndrome Phenotype Genes or other alterations known to be involved Hypospadias Incorrect placement of the urethral opening in males HOXA13, HOXD13 Cryptorchidism Failure of testicular descent INSL3, LGR8 XY true hermaphroditism Ovotestes, ambiguous genitalia Trisomy of chromosome 22 XY sex reversal Ovaries, female genitalia and secondary characteristics SRY, SOX9 Swyer syndrome XY females with complete gonadal dysgenesis SRY Denys–Drash syndrome Pseudohermaphroditism, nephropathy, predisposition to Wilms tumour WT1 Frasier syndrome Pseudohermaphroditism, progressive glomerulopathy WT1 WAGR syndrome Wilms tumour, aniridia, genito-urinary malformations, mental retardation WT1 Adrenal hypoplasia congenita Adrenal hypoplasia, hypogonadotropic hypogonadism DAX1 Campomelic dysplasia Skeletal dysmorphology and XY sex reversal SOX9 Persistent Müllerian duct Normally virilized men with a uterus and fallopian tubes, often with AMH, AMHR syndrome unilateral inguinal hernia or cryptorchidism Congenital bilateral aplasia of Absence of vasa deferentia, infertility CFTR vas deferens X-linked lissencephaly with Microcephaly, lissencephaly, agenesis of the corpus callosum, epilepsy, ARX abnormal genitalia syndrome poor temperature regulation, chronic diarrhoea, ambiguous genitalia α-Thalassaemia/mental Severe mental retardation, α-thalassaemia, genital abnormalities, facial ATRX retardation syndrome, X-linked anomalies, lung, kidney and digestive problems Congenital adrenal hyperplasia Androgen excess produced by adrenal glands, XX with varying degrees of Enzymes that are necessary virilization for hormone production (21-hydroxylase in 95% of patients) Androgen insensitivity syndrome Varying from nearly normal male to nearly normal female AR Hand-foot-genital syndrome Short first metacarpals and metatarsals, carpal and tarsal fusion, fifth-finger HOXA13, HOXD13 clinodactyly, genital abnormalities in both sexes, hypospadias in the male This table summarizes the sexual disorders mentioned in the text. For references and further reading see REF. 103. AMH, anti-Müllerian hormone; AMHR, anti- Müllerian hormone receptor; AR, androgen receptor; ARX, aristaless-related homeobox; ATRX, α-thalassaemia/mental retardation syndrome, X-linked; CFTR, cystic fibrosis transmembrane conductance regulator; HOXA13, homeobox A13; INSL3, insulin-like 3; LGR8, the INSL3 receptor; SOX9, SRY-box containing gene 9; WT1, Wilms tumour 1.

organs and external genitalia, to male-specific brain Mutational analyses in mice have shown that several features (FIG. 1). We aim to move beyond studies of testis transcription-factor genes are required for the early for- development in order to build up an integrated picture of mation of the indifferent genital ridges, including empty the breadth of issues in male sexual development. spiracles homologue 2 (Emx2) (REF. 4), GATA-binding protein 4 (Gata4) (REF. 5), Lim homeobox protein 9 Development of the testes (Lhx9) (REF. 6), steroidogenic factor 1 (Sf1; also known In eutherian mammals, the first morphological sign of as NR5A1) (REFS 7,8), and Wilms tumour 1 (Wt1) (REF. 4). male differentiation is the formation of cords in nas- Two of these, WT1 and SF1, are also crucial for the for- cent testes. Before this, both male and female embryos mation of the genital ridges in humans. Mutations in develop paired, ambipotent genital ridges that are these genes result in malformed gonads and ambiguous indistinguishable between sexes. genitalia9,10. Intriguingly, Wt1 and Sf1 have additional, sex-specific roles later in gonadal development. The indifferent gonad. The genital ridges form as linear One enigmatic gene, Dax1 (also known as Nr0b1), swellings on the ventromedial surfaces of the mesone- is expressed in the indifferent gonad of both sexes, and phroi. Each mesonephros is derived from intermediate gain-of-function and loss-of-function mutations in mice mesoderm in the aorta-gonad-mesonephros region of affect both testis and ovary determination. It may be that the trunk, flanking the dorsal aorta and bounded ven- Dax1 levels and thresholds are extremely critical for its trally by epithelium at its coelomic surface (FIG. 1a). In male versus female function11. both sexes, mesonephric mesenchyme and thickening coelomic epithelium contribute to the evagination of the Sry — the male determinant. The bifurcation in the genital ridge from the mesonephros as development pro- development of testes and ovaries is triggered by the ceeds. In mice, the genital ridges start to appear around expression of Sry. Loss-of-function and gain-of-function 10 days post coitum (dpc), and remain morphologically mutations in mice and humans indicate that this gene Eutherian mammals Mammals that have a placenta; undifferentiated until about 12 dpc, despite different is necessary and sufficient for testis development in 12–14 includes all mammals except programmes of genetic activity in males and females mammals . Its expression is tightly regulated in mice, monotremes and marsupials. beginning around 10.5 dpc (see below). occurring in a wave that starts in the centre of the genital

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a Development of external genitalia b Formation of the aorta-genital-ridge- mesonephros region Allantois Cloacal Hindgut Mesonephros membrane Cloaca Urorectal Genital septum ridge Aorta 10.5 Coelomic Wolffian duct dpc 11.5 epithelium Müllerian duct onwards dpc

Urogenital Genital sinus c Testis and genital tract differentiation tubercule 13–17.5 Seminal vesicle dpc Prostatic bud

Testis Vas Testicular cords Urethra deferens Efferent ducts Distal urethral Rete testes epithelium Penile urethra Epididymis

14.5–18 dpc

e Development of brain dimorphisms d Testicular descent 1st phase 2nd phase

Kidney

Cranial ligament Scrotum

Gubernaculum Gubernaculum Gential tubercle Figure 1 | Major steps in male sexual differentiation. The external genitalia appear as a mesenchymal swelling between the two layers of the cloacal membrane at around 10.5 dpc and develop further under the influence of testicular androgens (a). A transverse section through a mouse embryo at 11.5 days post coitum (dpc) shows the developing urogenital ridge, which develops out of the intermediate mesoderm on either side of the aorta and is composed of the mesonephros and genital ridge. Within the mesonephros the Wolffian (male) and Müllerian (female) ducts form. At 11.5 dpc males cannot be distinguished morphologically from females (b). However, expression of the male-determining gene Sry induces a cascade of gene expression that results in the differentiation of the genital ridge and the Wolffian duct into the testis and male genital tract (rete testes, epididymis, vas deferens and seminal vesicle), respectively (c). At 13 dpc the bipotential urogenital sinus develops and at 17.5 dpc the prostatic bud is formed by the urogenital sinus under the influence of testicular androgens. Between 14.5 and 18 dpc the testis migrates into the developing scrotum in two phases (d). The first phase is due to an enlargement of the gubernaculum, whereas during the second phase the gubernaculum migrates into the scrotum, guided by calcitonin-related peptide that is released by the genito-femoral nerve. The brain develops sexual dimorphisms, some of which are due to direct genetic effects, although most are caused by sex hormones (e).

ridges around 10.5 dpc, reaching peak levels and encom- However, the time at which peak levels of expression passing the whole gonad at 11.5 dpc, before declining, are attained is crucial. In a phenomenon known as first in the centre then later at the poles, to undetectable B6-YDOM sex reversal22, Y chromosomes from several levels around 12.5 dpc (REFS 15,16). However, the molec- Mus domesticus variants differ in their ability to induce ular mechanism of this regulation remains a mystery. testis formation on a C57BL/6J genetic background, Several factors have been implicated by virtue of reduced resulting in phenotypes that range from complete XY sex Ovotestes Sry expression in targeted mouse mutants; these include a reversal, to unilateral or bilateral ovotestes, to delayed testis Gonads in which ovarian and splice variant of WT1, GATA4 and its cofactor FOG2 (also formation. This has been ascribed to delayed expression testicular tissue are present 5,17,18 23,24 together. known as ZFPM2), and the insulin receptor family . of Sry from these Y chromosomes , adding to previous It is not clear in these mutants whether the level of Sry evidence that levels of Sry expression are important for XY true hermaphroditism expression per cell is affected, or the number of cells male sex-determining function25. Similar regulatory phe- This condition comprises the that express Sry is reduced, and none of these genes is nomena might explain the occurrence of ovotestes in some presence of both ovarian and XY true hermaphroditism testicular tissue either in the expressed in a wave pattern similar to Sry. cases of in humans. The current 19–21 same gonad as an ovotestis, or Differences between species imply that it is belief is that Sry expression must reach a certain threshold an ovary and a testis. immaterial how quickly Sry expression is shut down. level within a definite temporal window of competence

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in the precursors of supporting cells, otherwise the the same cell type, the pre-Sertoli cell38–40. It is clear that ovary-determining pathway will initiate in these cells. Sox9 represents an early acting and crucial component Sry encodes a nuclear, high-mobility group (HMG) of the male sex-determining pathway: loss of function of domain protein that binds and bends DNA. Outside the SOX9 results in XY gonadal dysgenesis in mice and also in HMG domain, SRY exhibits little sequence or structural the human disorder campomelic dysplasia, whereas gain conservation between species, and almost all mutations of function in transgenic mice induces XX maleness41–47. that have a clinical phenotype (for example, XY females Sox9 is present in a wide diversity of metazoans, possibly with complete gonadal dysgenesis in Swyer syndrome) fulfilling a similar role even in Drosophila melanogaster48, reside within this motif, underscoring the importance of suggesting that it is an ancient and conserved effector this domain26. These mutations generally impair SRY DNA binding and/or bending, or nuclear translocation27–30. The SRY protein in humans and mice has other domains that mediate protein–protein interaction and transcriptional transactivation in vitro. The sig- nificance of the former is suggested by some missense and frameshift mutations causing XY sex reversal in humans31–33. However, it is not clear whether SRY functions as a transcriptional activator in vivo; the frequency of SRY-negative XX maleness in the human population has been taken as evidence that SRY might repress a repressor of the male programme rather than directly activate a sex-determining cascade34. Either way, it is curious that no target genes for SRY have been identified, and the molecular cascade of events triggered by SRY activity still remains obscure.

Differentiation of Sertoli cells. The genital ridges consist of several cell lineages (FIG. 2), each of which is thought to be bipotential, generating different cell types in testes and ovaries depending on the signals received. Among these is a progenitor known as the supporting-cell precursor lineage, which gives rise to both the Sertoli cells and the ovarian granulosa cells that support the development of germ cells in males and females, respectively. Landmark experiments by Burgoyne and colleagues35 that involve mouse XX–XY chimaeras showed that all gonadal lineages were composed of similar numbers of XX and XY cells, but Sertoli cells were overwhelmingly XY. This indicates Endothelial cells that SRY functions cell autonomously to trigger the differ- Pecam1 Peritubular myoid cells Ptc entiation of Sertoli cells, and that pre-Sertoli cells (defined Germ cells as supporting cells that express Sry and/or Sox9 (SRY-box Oct4, Pecam1, E cadherin Leydig cells containing gene 9) but are not yet arranged into cords) Sertoli cells Scc, HSD3B, Ptc can signal to other lineages to induce their male-specific Sox9, Amh, Dhh, Dmrt1 XY gonadal dysgenesis differentiation and so orchestrate testis development. This can lead to pure gonadal However, up to 10% of Sertoli cells in these chimae- Figure 2 | Histological and gene-expression map of an dysgenesis, in which patients ras were found to be XX. This implies the existence of early embryonic testis. A haematoxylin and eosin have streak gonads stained section that illustrates the different cell types of (undeveloped gonadal a mechanism by which supporting cell precursors that the testis and the genes that are specifically expressed in structures), Müllerian structures do not express Sry can be recruited to differentiate into these cells: endothelial cells (red) form the male-specific (owing to insufficient AMH Sertoli cells through a secreted signal. Recent work has secretion) and a complete vasculature, germ cells (orange) that later develop into absence of virilization. shown that pre-Sertoli cells produce prostaglandin D2, sperm are enclosed by a layer of Sertoli cells (green), which Alternatively, patients can have which, by binding to its receptor and upregulating Sox9 support and nourish the germ cells, and peritubular myoid dysgenetic testes. In this case, expression, recruits other cells to the Sertoli cell fate15,36,37. cells (black), which help to maintain cord integrity and are enough AMH is produced to This might act as a backup mechanism, reinforcing male later responsible for pulsatory contractions that are regress the Müllerian duct and sex-determination when Sry expression or function required for export of sperm. The steroid-producing Leydig there might be enough is impaired. cells (purple) reside in the interstitium together with other testosterone for partial cells such as macrophages and mesenchymal cells. Amh, virilization. Downstream of Sry, other factors such as SOX9, anti-Müllerian hormone; Dhh, desert hedgehog; Dmrt1, SOX8, DAX1 and FGF9 (fibroblast growth factor 9) have doublesex and mab-3 related transcription factor 1; Campomelic dysplasia a role in Sertoli cell differentiation and function. Sox9 is A syndrome that is Pecam1, platelet/endothelial cell-adhesion molecule 1; Ptc, characterized by skeletal the best candidate for a direct SRY target gene, although patched; Scc, side-chain cleavage (also known as Cyp11a1); abnormalities and sex reversal, conclusive proof has yet to be produced. In mice, Sox9 is Sox9, SRY-box containing gene 9; HSD3B, hydroxy-δ-5- caused by mutations in SOX9. expressed shortly after the onset of Sry expression and in steroid dehydrogenase-3β.

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of male sex determination, in contrast to Sry, which associated with a block in Leydig cell differentiation68,69. is exclusively mammalian. The elusive relationship Additionally, ATRX (α-thalassaemia/mental retarda- between SRY and Sox9, and the probable nature of the tion syndrome, X-linked), also known as XNP or XH2, transcriptional cascade downstream of SRY, have been is mutated in the ATRX syndrome, which is character- reviewed extensively elsewhere49,50. ized by severe mental retardation, α-thalassaemia and Besides the differentiation of Sertoli cells, testis devel- a range of genital abnormalities that suggest that ATRX opment immediately after the onset of Sry expression could be involved in Leydig cell development70. For differs from female gonad development in its increased both genes, however, the molecular role during Leydig cell proliferation51 and the induced immigration of cell differentiation is unknown. In addition, signalling mesonephric cells52,53. Although several factors such through PDGFs and their receptor PDGFRA have been as neurotropin 3 (NT3; also known as NTF3) (REF. 54), identified in knockout mice to be essential for fetal71 and hepatocyte growth factor (HGF)55, and platelet-derived adult72 Leydig cell differentiation. growth factor-α (PDGFA)56 are able to induce this cell migration in vitro, none has been shown to have Testis differentiation — germ cells. Primordial germ cells a role in vivo. Presumably, because this migration and originate neither in the gonad nor the mesonephros, increased proliferation are male-specific events, the fac- but instead migrate from their origin at the posterior tors that mediate them are regulated either by Sry or one extremity of the embryo through the hindgut to populate of its early downstream genes such as Sox9. the genital ridges at around 10.5 dpc in the mouse. Here The immigrant mesonephric cells are thought to dif- they associate with somatic cells to form primitive sex ferentiate into Leydig, endothelial and peritubular myoid cords, the precursors of testis cords and ovarian follicles. cells (FIG. 2), depending on interactions with somatic cells Important factors in germ-cell migration include the fra- within the gonad. This raises the question of the nature gilis proteins IFITM1 and IFITM3 (interferon-induced of these interactions and how these other testicular cell transmembrane proteins 1 and 3) (REF. 73), whereas stro- types differentiate. mal cell-derived factor 1 (SDF1; also known as CXCL12) and its receptor CXCR4 are involved in the colonization Testis differentiation — peritubular myoid cells. Peri- of the genital ridges74. tubular myoid (PM) cells are flat, smooth-muscle-like Like other cell types in the gonad, germ cells are cells that ensheath testis cords and are necessary for influenced by Sertoli cells to differentiate in a male- cord development and structural integrity53,57. Little is specific fashion regardless of their sex-chromosome known about them, partly owing to the lack of a specific genotype. Germ cells in a testis enter a state of mitotic marker 58. One factor that is correlated with differentia- arrest around 12.5 dpc (REFS 75,76), a state in which they tion of PM cells is the Sertoli cell-specific, secreted factor remain until after birth, whereas germ cells in an ovarian desert hedgehog (DHH). Its receptor patched (PTC; also environment enter meiosis around 13.5 dpc, signalling known as PTCH1) is expressed on PM and Leydig cells. the onset of oogenesis77. Null mutation of Dhh in mice led to impaired differen- Interestingly, the long-standing dogma that germ cells tiation of PM and Leydig cells and subsequently to femi- enter meiosis cell autonomously is questioned by recent nized males59–61. Similarly, mutations in human DHH data showing that retinoic acid, produced by the meso- have been associated with partial and pure XY gonadal nephros, induces entry into meiosis. Male germ cells dysgenesis accompanied by impaired cord formation are protected from the effects of retinoic acid by their and reduced testosterone levels62–64. Other unknown enclosure within the testis cords. Sertoli cells, which sur- Sertoli cell-derived factors might be involved in directing round primordial germ cells in the testis cords, express PM cell differentiation. CYP26B1, an enzyme that breaks down retinoic acid78,79. However, the mechanism by which male germ cells arrest Testis differentiation — endothelial cells. Although mitotically has not been determined. The fact that after gonads of both sexes are heavily vascularized from an 12.5 dpc male germ cells are committed to develop as early stage65,66, endothelial cells in the testis form a charac- spermatogonia suggests that either mitotic arrest pre- teristic vasculature with a prominent coelomic vessel on vents entry into meiosis, or that a temporal window of the anti-mesonephric surface and branches between the competence to respond to retinoic acid signalling exists testis cords. The formation of the coelomic vessel, but within the germ cells.

Lissencephaly not the side branches, is suppressed by the secreted sig- Germ cells are crucial for the differentiation of ovar- A brain malformation that nalling molecule WNT4. Null mutation of Wnt4 in mice ian follicles and the maintenance of the ovary, presuma- is characterized by the resulted in the ectopic formation of a coelomic vessel bly secreting factors that are required for these processes. incomplete development of in XX animals67. Because blood vessels and testis cords This signalling might be a consequence of entry into the folds of the outer region occupy complementary domains, there is likely to be an meiosis. By contrast, germ cells are not required for of the brain (the cerebral cortex), which causes the interplay between testis cord formation and vascular testicular development or maintenance: fetal testes surface of the brain to appear patterning, although is not clear which directs which. in which the germ cells are chemically or genetically abnormally thickened and depleted develop normally 80. unusually smooth. Testis differentiation — Leydig cells. Fetal Leydig cells

Ovarian follicle synthesize crucial hormones for male sex differentiation. Testicular descent. An important aspect of sexual A cyst in which the oocyte The aristaless-related homeobox gene ARX, identified development is that testes and ovaries end up in different matures. in X-linked lissencephaly with abnormal genitalia, is locations in the body. The testes migrate to their final

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location in two phases. The transabdominal phase of tes- Initial Epididymis Ros1 ticular descent occurs in mice between 14.5 and 18 dpc segment Interstitium (in humans at 8 to 15 weeks of gestation), and is controlled by the enlargement of the caudal genito-inguinal Caput Bmp7,8 ligament and the gubernaculum, and by the regression Testis of the cranial ligament (FIG. 1c). Mutation studies in cords Bmp4 mice demonstrated that the insulin-like 3 (INSL3) Corpus hormone, produced by Leydig cells, using its receptor LGR8 (also named GREAT), is necessary and sufficient to mediate this phase of testicular descent 81. Mutations Cauda Bmp7,8 in these genes have been found in male patients with cryptorchidism, but these account for only a small proportion of all cases of this common disorder 82. The second, inguinoscrotal phase is usually completed by 20 days after birth in mice, and by the thirty-fifth Hoxa9,10,11 week in humans. In contrast to the first phase, which is dependent on the growth of the gubernaculum, this Hoxd9,10 phase requires the migration of the gubernaculum from Vas deferens the groin into the scrotum (FIG. 1c). This is guided by the neurotransmitter calcitonin gene-related peptide (CGRP; also known as CALCA), which is released by the genito- femoral nerve under the control of androgens. Mutations in genes that are involved in androgen signalling and those that encode transcription factors, such as homeobox A10 (Hoxa10), Hoxa11 and Desrt (developmentally and Fgf10 sexually retarded with transient immune abnormalities), Seminal cause disruption of the second stage of descent in mice, vesicle Gdf7 leaving the testes at the level of the bladder, which is in contrast to Insl3 and Lgr8 mutations that lead to a high Figure 3 | Schematic representation of the intra-abdominal location83,84. Clearly the regulatory differentiation of the male genital tract. The Wolffian mechanisms that drive testicular descent are sensitive to duct differentiates under the influence of testicular imbalance and malfunction and are often secondary androgens into the epididymis, vas deferens and seminal vesicle. The epididymis can be further divided, to other disorders — factors that might contribute to the morphologically and functionally, into the initial segment, high frequency of undescended testes in newborn boys. caput, corpus and cauda. The sperm, which are produced in the testes, mature during their passage through the Development of the male genital tract caput and corpus, whereas the cauda functions Early stages — the genital ducts. Both male and female predominantly for storage. The different segments of the embryos initially have two pairs of genital ducts. In male genital tract are marked by specific gene expression. males, one pair, the Wolffian (or mesonephric) ducts, Null mutations of these genes result in phenotypes that are generate the mature genital tract, whereas the other, the restricted to these segments. Homeobox A10 (Hoxa10) and Müllerian (or paramesonephric) ducts, disappear. In Hoxa11 knockout mice are sterile and the epididymis shows females, the Müllerian ducts survive and the Wolffian homeotic transformation. In Hoxa10 null mice the cauda epididymis seems to have transformed into the corpus, ducts disappear. This represents a different strategy to whereas in Hoxa11 knockout mice the vas deferens shows that used in gonad development, which involves dichot- partial transformation into the cauda. Mutations in bone omous differentiation of a bipotential precursor tissue. morphogenetic protein 4 (Bmp4) result in extensive However, a bipotential structure, the urogenital sinus, degeneration of the epididymal epithelium of the corpus contributes to the genital tract by forming the prostate in region, rather than in the caput and cauda regions as for males and the lower vagina in females (FIG. 1b,d). Bmp7 and Bmp8 knockout mice, whereas mice that are null In an XY embryo, the Müllerian ducts degenerate for Ros1 show defects in the differentiation of the initial in an active process, involving a TGFB (transform- segment. For the proper development of the seminal vesicle, fibroblast growth factor 10 (Fgf10) and growth Cryptorchidism ing growth factor-β)-family molecule, anti-Müllerian differentiation factor 7 (Gdf7) are required. The condition of having hormone (AMH; also known as Müllerian-inhibiting undescended testes. substance or MIS). AMH is secreted by the Sertoli cells of differentiating testes and binds to its receptor, MISRII Persistent Müllerian duct syndrome (also known as AMHR2), on the surface of Müllerian Later stages — differentiation of the Wolffian duct. In A rare form of male pseudo- duct mesenchymal cells. This induces a signalling males, the Wolffian ducts further develop under the hermaphroditism that is most cascade that results in the production and secretion of influence of testosterone, produced by Leydig cells, commonly characterized by matrix metalloproteinase 2 (MMP2), which induces into a system of organs — the epididymis, vas defer- bilateral fallopian tubes and a 85 (FIGS 1b,3) uterus combined with an apoptosis in the Müllerian duct epithelial cells . Failure ens and seminal vesicle — that connect the otherwise more or less normal in these processes in humans can lead to persistent testes with the urethra. These Wolffian duct derivatives male phenotype. Müllerian duct syndrome (PMDS)86–88. can be distinguished by their morphologies, specific

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a differentiation along the Wolffian duct. Knockout Ventral Outgrowth mouse models of Gdf7, Bmp4, Bmp7, Bmp8a and Bmp8b (which encode bone morphogenic proteins)89–92 and Genital Bmp4, Hoxa13, the homeobox genes Hoxa10 and Hoxa11 (REFS 93,94) tubercle Hoxd13, Ptc result in defects in specific areas of the epididymis and seminal vesicle, confirming the region-specific require- Cloacal Shh membrane ment for signalling molecules and transcription factors (FIG. 3). Furthermore, the phenotypic effects of a Urethral fold null mutation of Ros1 implicates the encoded tyrosine kinase receptor in the regionalization and terminal dif- Labioscrotal ferentiation of the epididymal epithelium95. Studies of swellings mouse mutants have also identified two genes that are necessary for the proliferation of epithelial cells, and therefore tube elongation: those that encode the orphan Dorsal G-protein coupled receptor LGR4 (REF. 96) and relaxin, a naturally occurring inhibitor of collagen deposition97. Subsequently, the growth factor PDGFA, expressed b Hoxa13, Hoxd13 in the epithelium and its receptor PDGFRA in the surrounding mesenchyme maintain the structural Apoptosis Bmp7 Fgf8 integrity of the tube98. Ventral Genital tubercle Proliferation Studies of the human disorder congenital bilateral Distal urethral epithelium Wnt5a aplasia of vas deferens (CBAVD) have implicated the cystic fibrosis transmembrane conductance regulator Mesenchyme Fgf10 (CFTR) in the development of the male reproductive Urethral groove tract. A high proportion of males with cystic fibrosis Shh also display CBAVD and are therefore infertile. However, many CBAVD patients with CFTR mutations do not Fusion of urethral folds show the cystic fibrosis lung phenotype. This is probably explained by differences in the tissue-specific alterna- tive splicing of CFTR between the vas deferens and the lung 99. However, it is not yet known how mutation of this membrane-bound chloride channel leads to the loss of vasa deferentia during development. Dorsal Development of the external genitalia Figure 4 | Development of the external genitalia. a | Caudal view of the indifferent A second organ system that differentiates under the anlage. The cloaca is closed off from the exterior by the cloacal membrane, which is influence of testosterone is the external genitalia. The bordered anteriorly by the genital tubercle and laterally by the urethral and labioscrotal specialized male and female genital anatomy that has swellings. Sonic hedgehog (SHH) signalling from the epithelium results in the evolved in mammals provides greatly increased repro- upregulation of bone morphogenetic protein (Bmp4), homeobox A13 (Hoxa13), Hoxd13 ductive efficiency compared with the system of external and patched (Ptc) expression in the mesenchyme. This expression pattern exhibits a fertilization found in many birds and fish100. Perhaps balance of apoptosis that is induced by Bmp4 and proliferation, which is indirectly even more so than the gonads and the duct systems, the controlled by Hoxa13 and Hoxd13 through the upregulation of fibroblast growth factor 8 vastly different morphologies of the male and female (Fgf8). This balance is necessary for correct development of the genital tubercle with mis- external genitalia belie their origins from a common set regulation resulting in hypospadias. b | The genital tubercle elongates to form the penis, while the urethral groove forms on the ventral aspect of the genital tubercle, extending of ambiguous embryonic structures. distally in a solid epithelial plate (distal urethral epithelium). The penile urethra subsequently forms by proximal to distal fusion of the urethral folds. The labioscrotal The indifferent stage. In mice, at around 10.5 dpc, the swellings migrate caudally and fuse at the midline to form the scrotum. SHH from the external genitalia first become visible as a small mesen- epithelium and FGF10 from the mesenchyme maintain their own expression in a positive- chymal swelling between the two layers of the cloacal feedback loop. HOXA13 and HOXD13 continue to signal back to the distal urethral membrane (FIG. 1d). Subsequently, the ventrally located epithelium to induce Bmp7 and Fgf8 expression. urethral groove appears, which has a solid plate of epithelial cells at the distal end. This distal urethal epithelium, which is comparable to the apical ectodermal ridge gene-expression patterns and functions, although they of the limb bud, functions as a signalling centre to stimu- are contiguous and collaborate with each other in sup- late the outgrowth and differentiation of the genital tuber- porting male gamete development. Similar to other cle through epithelial–mesenchymal interactions. The testosterone-induced, male-specific organs such as expression and relationship of the key molecules involved Anlage the prostate and external genitalia, their development in this outgrowth — Fgfs, SHH (sonic hedgehog), Wnts, A group of cells that are destined to become a specific depends on epithelial–mesenchymal interactions. It is HOXA13 and HOXD13, Bmps and their antagonist structure or tissue in the adult, believed that region-specific, inductive signals from noggin (FIG. 4; Supplementary information S1 (table)) but have not yet differentiated. the surrounding mesenchyme specify the characteristic — are remarkably similar to those involved in limb

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bud development and branchial arch outgrowth during a Initiation AR craniofacial development101. ?

Differentiation of male external genitalia. After this SHH initial, bipotential phase, at around 16 dpc in mice and Urethra 12 weeks of gestation in humans, the development of the external genitalia becomes sex-specific. 5α-Reductase is expressed in the genital tubercle mesenchyme, and in males it converts testosterone into 5α-dihydrotesto- b Growth HOXA13 SHH sterone (DHT), the biologically more potent androgen. HOXD13 DHT signals through the androgen receptor (AR) that NKX3.1 is present in cells of the developing external genitalia, which leads to a series of morphological changes. The genital tubercle elongates further with the urethral folds Urethra approaching each other, finally fusing, from proximal c Branching to distal, to form the tubular penile urethra (FIG. 4b). The morphogenesis FGF10 TGFB1 scrotum is formed by the genital swellings, which move caudally and fuse in the midline. 5α-Reductase and AR FGF7 FGFR2 are also expressed in the female external genitalia, but do not lead to male differentiation owing to the lack of SHH testosterone. However, in females with congenital adrenal hyperplasia, the adrenal glands produce abnormally high Notch levels of androgens, which lead to varying degrees of BMP4 BMP7 virilization of the female external genitalia102. In males, mutations resulting in a defective AR, or low levels of AR, cause androgen insensitivity syndrome, the most common form of XY sex reversal. Progression of This disorder is characterized by deficient or absent d Differentiation and p63 ductal lumen, maturation FOXA1 differentiation virilization of 46,XY individuals despite normal or of basal and even elevated androgen levels. More than 300 muta- luminal cell types, tions in the X-linked, single-copy AR gene have been and formation of smooth described, leading to phenotypes that range from com- muscle cells plete androgen insensitivity and female phenotype, to partial insensitivity and ambiguous genitalia, to mild Urethra forms with a male phenotype but that are infertile. The testes do not usually descend; however, there is no Figure 5 | Morphological and gene-expression changes uterus because the degeneration of the Müllerian ducts during prostate development. a | Circulating androgens is androgen-independent. initiate the development of the prostate from the Mutations in the gene encoding 5α-reductase are urogenital sinus; the androgen receptor (AR), which is another common cause of male pseudohermaphroditism. necessary for budding of the urethral epithelium (green), is Such individuals are deficient in DHT, which is required expressed in the mesenchyme (pink). An unknown factor, for the full masculinization of the external genitalia and which could be an activator or repressor, mediates a signal Branchial arches development of the prostate, but not for the normal dif- to the epithelium, which causes the upregulation of Shh A series of paired segmental ferentiation of the Wolffian ducts, epididymides, vasa expression. b | Sonic hedgehog (SHH), at least in part by structures that are composed deferentia and seminal vesicles103. upregulating the transcription factor NKX3.1, and the of ectoderm, mesoderm and mesenchymal homeobox genes Hoxa13 and Hoxd13 The high prevalence of hypospadias in humans sug- neural crest cells that are promote further growth of the prostatic ducts. c | Most of positioned on each side of the gests that urethral fusion is a delicate and finely regulated these ducts remain unbranched until birth in rodents but, developing pharynx. In process. The cell-surface molecules ephrins, and their subsequently, epithelial–mesenchymal interactions result mammals, the branchial arches receptors (Ephs), have been implicated in this process: in further elongation and branching morphogenesis. contribute to pharyngeal organs and to the connective, mice in which ephrin B2 and EphB2/EphB3 signalling Ductal branching and budding are inhibited by the skeletal, neural and vascular is disrupted show variable levels of incomplete urethral mesenchymal signalling factors BMP4 (bone tissues of the head and neck. tubularization104. In addition, mutations in many of the morphogenetic protein 4) and BMP7, and stimulated by the above-mentioned genes that control the initial phase antagonist of these Bmps, Notch. FGF7 (fibroblast growth Congenital adrenal of genital tubercle outgrowth result in hypospadias, factor 7) and FGF10, expressed in the mesenchyme, bind to hyperplasia indicating a requirement for coordinated control of FGFR2 on epithelial cells, which leads to the maintenance A condition that is in most of SHH expression. This positive regulation is limited by the cases due to CYP21 deficiency, urethral fusion and genital tubercle outgrowth. For negative-feedback loop of downregulation of Fgf and is characterized by the example, mutations in HOXA13 and HOXD13 lead to expression by SHH signalling. d | Finally, in a proximal to deficiency in the hormones hand-foot-genital syndrome, an autosomal dominant cortisol and aldosterone and distal direction, the epithelial cell types differentiate under an overproduction of disorder, which is characterized by malformation of the control of transcription factors such as p63 and 105–107 androgens, which results in the distal limbs, accompanied by hypospadias . forkhead box A1 (FOXA1), smooth muscle cells form ambiguous genitalia in females. Furthermore, experiments in rodents have suggested around the epithelium and the ductal lumen advances.

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Genital tubercle Penis Brain Wolffian duct Vas deferens, epididymis, seminal vesicle

Androgen receptor, Shh, Wnts, Androgen receptor, Bmps, Fgfs, HoxA13, HoxD13, Bmps, Sry? Other genes? Hox, Cftr, Hoxa10, Hoxa11, noggin, Ephs, ephrins Lgr4, Pdgfa, Pdgfra

Androgens Androgens External genitalia Androgens Male genital tract

Testicular descent Genital ridge Testis Urogenital sinus Prostate Androgens Sry, Sox9, Sox8, Fgf9, Sf1, Insl3, Lgr8, Hoxa10, Hoxa11, Androgen receptor, Shh, Dhh, Ptc, Wt1, Dax1, Atrx, Desrt Fgfs, Hoxa13, Hoxd13, Arx, Pod1, Pdgfa, Pdgs CD44, follistatin

Figure 6 | Overview of genetic pathways that drive male sexual differentiation in mammals. The Y-linked gene Sry is the master switch that determines the differentiation of the bipotential genital ridge into a testis (bottom middle box). Expression of Sry sets in motion a cascade of male-specific gene expression such as Sox9, Sox8 and Fgf9. Later, the testis has to descend (depicted by a broad arrow) into the scrotum for full functionality. Subsequently, testicular androgens initiate the differentiation of secondary male sexual characteristics such as the male genital tract, external genitalia and brain, which involves organ-specific, regulatory gene networks.

that some industrial chemicals, pharmaceuticals, (table)). How do the same set of control genes lead to environmental pollutants and natural products have the formation of these distinct structures? This remains anti-androgenic properties that can result in the femi- unknown, but interacting partners might be expressed nization of male external genitalia, which might explain differentially in these structures, or different thresholds the alarming increase in the occurrence of hypospadias or temporal and spatial combinations of expression of in recent decades108. these key genes might occur in different tissues.

Development of the prostate Sexual differences in the brain The prostate is an essential mammalian-specific, male Males and females have clear behavioural differences and accessory sex gland that contributes to the seminal are differentially susceptible to many behavioural, emo- plasma fluid. This important role in mammalian repro- tional and personality disorders. Sex-specific differences duction and the high incidence in humans of prostatic in brain morphology traditionally have been attributed diseases, including benign and malignant tumours, to the action of steroid hormones that are produced by make it necessary to understand prostate development the gonads. Although these are still seen as the main fac- and biology. tors, recent data suggest that genetic differences could The prostate develops from the urogenital sinus, also have a direct role. which is derived from the cloaca that is a caudal extension Microarray analysis identified more than 50 genes of the hindgut (FIG. 1b,d). Interestingly, its development that show a sex-specific expression pattern in the brain is similar to that of the external genitalia. The indif- of 10.5 dpc mice, a stage that is too early for any gonadal ferent urogenital sinus forms in both male and female hormone influence, which supports the hypothesis that mice at approximately 13 dpc (7 weeks of gestation in chromosomal constitution has a role in mammalian humans), and remains morphologically indistinguish- brain differentiation113. These genes encode proteins that able until 17.5 dpc, when testicular androgens induce range from those that regulate the cell cycle and con- the outgrowth of solid buds from the urogenital sinus trol transcription, to enzymes and structural proteins. epithelium into the urogenital sinus mesenchyme109,110. It remains unclear whether the sex-specific expres- Curiously, the androgen receptor is expressed on the sion of these genes is a cause or consequence of brain mesenchyme and induces prostatic epithelial devel- sexual dimorphism, and, if they do contribute to neural opment, which implies that there is an unidentified, differentiation, what their functions might be. secreted, mesenchymal factor that mediates the action A more restricted screen was used to test the hypoth- of androgens. After this initial hormone-dependent esis that genes encoded on the sex chromosomes have stage, the development of the prostate is characterized by a direct role in sexual differentiation of brain and sex- epithelial–mesenchymal interactions, resulting in cell dif- specific behaviours114. This study used a fully fertile ferentiation and branching morphogenesis, that involve mouse model in which Sry was moved from the Y chro- the same key molecules (FGFs, SHH, BMPs, HOXA13 mosome to an autosome. By breeding these mice with and HOXD13) as the development of the external wild-type XX females four genotypes were produced: genitalia, in addition to a few others (for example, CD44 XX females, XYSry– females, XYSry– (Sry+) males, and XX and follistatin)111,112 (FIG. 5; Supplementary information S2 (Sry+) males, a system in which sex determination is

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Substantia nigra independent of the Y chromosome. One sexual dimor- depending on secreted signals from nascent Sertoli cells. A region of the ventral phism, the density of vasopressin-immunoreactive In the developing duct system there is no bipotential midbrain that contains pigment fibres, was identified that is due to direct effects of sex precursor, but instead both sets of ducts are laid down and sends afferent dopamine- chromosomal genes114. However, this mouse model did initially, with only one surviving at the expense of the releasing neurons to the not allow the effects of the male-determining gene Sry other. The external genitalia, on the other hand, are striatum. to be investigated. constructed from basically the same cell types in males Expression of Sry mRNA has been reported in the and females, but are moulded into strikingly different mouse and human brain115–118, but the biological sig- forms through the action of elaborate endocrine and nificance has remained obscure. Recently, Dewing and paracrine cascades. co-workers demonstrated that not only the mRNA but Significant progress has been made in identifying also SRY protein is expressed in tyrosine-hydroxylase- genes and regulatory networks that drive male-specific expressing neurons of the substantia nigra, and that exper- differentiation (FIG. 6). The picture that emerges is one imentally induced knock-down of Sry expression results of a complex interplay between transcription factors, in a decrease of tyrosine hydroxylase, suggesting that the secreted signalling molecules, hormones and recep- expression of this enzyme might be directly regulated by tors that, if disturbed, can result in various phenotypes. SRY. Tyrosine hydroxylase is the rate-limiting enzyme in Interestingly, a similar network of ‘hub’ genes — includ- the synthesis of dopamine in the dopaminergic neurons ing members of the Hox, Fgf, hedgehog and Wnt fami- of the nigrostratial system that controls specific motor lies — has an important role in each system (the testes, behaviours. Interestingly, experimental, unilateral down- prostate and external genitalia). The challenge is to find regulation of Sry has led to quantifiable sensorimotor the specific members of each family that are unique to deficits in male mice, assessed by akinesia and limb-use a particular system, and the tissue-specific target genes asymmetry tests, which implicates the direct control of that are regulated by this common network. specific motor behaviour by Sry independent of hormo- Despite the advances described above, most cases of nal influences119. Although these experiments provide XY sex reversal, SRY-negative XX sex reversal and true evidence that Sry and/or other genes are directly involved hermaphroditism remain unexplained at the molecular in sexual dimorphism of the brain, more work is needed level. Either a large number of key sex-determining genes to elucidate the precise molecular mechanisms, and how await identification, or mutations outside the coding these are modified by different androgen dosages. regions of crucial known genes such as Sry and Sox9 (for example, regulatory mutations or mutations that affect Challenges for the future post-transcriptional processing) are more prevalent The mammalian embryo develops for a significant than previously suspected — or both. Approaches such period in a sexually ambiguous manner, until the sex- as microarray profiling, comparative genomic hybridiza- determining switch gene Sry is activated, testes differ- tion and mutagenesis screening will no doubt have an entiate and other organs and tissues that differ between important role in efforts to understand human disorders males and females — including the brain — set off on of sexual development. their sex-specific developmental trajectories. Therefore, In addition to explaining testis development, these in both male and female embryos, the complete set of methods will contribute to the growing picture of how genes, cell types and precursor structures must exist to other parts of the male reproductive system develop. equip the embryo for each of two possible outcomes. A This, in turn, will provide a clearer picture of how devel- remarkable set of strategies is used in different parts of opment of the seemingly disparate elements of male the embryo to achieve this plasticity. For example, cell ontogeny is coordinated. Until then, we can only wonder lineages inhabit the early genital ridges that are capable at the complexity of events between the activation of Sry of male-specific or female-specific differentiation, and the characteristics that make men — well, men.

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