Gonadal development.

Concepts Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 1–16 (DOI: 10.1159/000363608)

Gonadal Development Angela K. Lucas-Herald a · Anu Bashamboo b

a

b

Department of Child Health, Royal Hospital for Sick Children, Glasgow, UK; Human Developmental Genetics, Institut Pasteur, Paris, France

Abstract

The initial events of mammalian sex determination are genetically determined, resulting in translation of the sex chromosome complement (XX or XY) into the development of reproductive structures. It is a tightly controlled and highly complex process where, depending on the chromosomal constitution, the bipotential gonad anlage becomes either a testis or an ovary. This is a sequential process initiated by the establishment of the chromosomal sex followed by formation of the gonadal ridge, migration of the primordial germ cells and the sexually dimorphic differentiation of the gonads.

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The commitment of the embryonic gonad towards the male or female fate is a sequential and complex developmental process. The sex-independent growth and development of the adrenogonadal primordium into the bipotential gonadal ridge is committed to the formation of testis in the presence of the SRY gene on the Y chromosome. SRY upregulates the expression of SOX9 that sets into motion a cascade of complex genetic interactions for the formation of male internal and external genitalia whilst repressing the formation of female genitalia. The initiation and maintenance of somatic sex of the gonad as either male or female is achieved by suppression of the alternate fate. However, at least in mice, the primary sex-determining decision is not final but is maintained in adulthood by a mutually antagonistic double-repressive pathway. In the human, any imbalance between these two antagonistic genetic and physiological pathways results in inappropriate gonad differentiation and function leading to disorders of sex development (DSD). Genetic analysis of individuals presenting with DSD and sex-reversed mice has revealed a number of sexually dimorphic genes that are involved in the formation of mammalian gonads, which are discussed in this chapter. Despite an increase in the knowledge of genes involved in mammalian sex determination, the molecular mechanisms remain by and large undetermined. The use of novel ‘omics’ technologies for analyzing a large number of patients with DSD, and careful assessment of the resulting datasets may result in the identification of novel genetic factors in human sex determination and lead to the development © 2014 S. Karger AG, Basel of novel ex vivo cellular models.

In mammals, the gonad primordium arises from the urogenital ridge comprising of the pronephros (gives rise to the adrenal primordium), the mesonephros (gives rise to the gonadal primordium) and the metanephros (gives rise to the urinary organ system). In human embryos the gonadal precursors appear at 32 days postconception (dpc), and in mice at 10.5 dpc. Until the beginning of differentiation, there are no morphological differences between XX and XY mesonephros or the gonads. In the developing gonads the supporting cell lineage, Sertoli (XY gonads) or granulosa cells (XX gonads), is the first to adopt a sex-specific fate. This triggers the differentiation of the steroidogenic cells, Leydig (XY gonads) or theca cells (XX gonads), which will produce sex-specific hormones. Depending on the presence or absence of the SRY (sex-determining region on Y) gene, the gonads secrete male and female sex steroids, which induce the phenotypes of the internal and external genitalia and other reproductive tissues. The steroid hormones responsible for the development of male and female characteristics are called androgens and estrogens, respectively. The differentiation of the germ cells is dependent on the surrounding niche. Therefore, the cell fate chosen by the supporting cell lineage initiates a differentiation cascade that culminates in the formation of a fully functional gonad. In recent years, there has been a considerable increase in our knowledge of the genes and mechanisms associated with the development and function of the mammalian gonad. This information has been incurred by analysis of mutations in mice and humans presenting with disorders of sex development (DSD).

Molecular Determinants of the Bipotential Gonad

Wilms’ Tumor Suppressor 1 WT1, located on 11p13, encodes a key developmental regulator with an N-terminal proline/glutamine-rich protein interaction domain and four C-terminal zinc fingers. WT1 has been shown to be important for the regulation of numerous genes involved in urogenital development, including anti-Müllerian hormone receptor 2, Sox9, Nr5A1, Wnt4 and SRY [1]. The role of WT1 in testis development was first recognized in XY patients, with heterozygous germ-line WT1 mutations, presenting with testicular dysgenesis. In the human, heterozygous WT1 gene deletions are associated with mild genitourinary anomalies and a predisposition to Wilms’ tumor, whereas heterozygous missense mutations give rise to Denys-Drash syndrome [2]. The observation of two sisters with Frasier syndrome and WT1 mutations, one 46,XY with complete

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Lucas-Herald · Bashamboo Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 1–16 (DOI: 10.1159/000363608)


The genes identified as vital for the initial formation of the bipotential genital ridge include Wilms’ tumor suppressor 1 (WT1), nuclear receptor subfamily 5, group A, member 1 (NR5A1), chromobox homolog 2 (CBX2), LIM homeobox gene 9 (LHX9), empty-spiracles homeobox gene 2 (EMX2) and GATA-binding protein 4 (GATA4).

gonadal dysgenesis and the other 46,XX with apparently normal ovarian development and function, suggests a model whereby a critical threshold level of wild-type WT1 protein is required for normal testis determination and development, whereas ovarian development may be less sensitive to gene dosage. Two main WT1 isoforms are defined by the presence or absence of three amino acids (KTS) between zinc fingers three and four. The –KTS isoforms bind preferentially to DNA, whereas the +KTS isoforms have a higher binding affinity for RNA. Wt1–/– mice of both sexes lack kidneys and gonads due to the apoptosis of the corresponding primordium, suggesting a role for this gene in the establishment of the bipotential gonad, prior to the differentiation of either male or female gonads. A mouse model of Frasier syndrome suggests that WT1(+KTS) is involved in the cell-autonomous regulation of Sry expression, which in turn influences cell proliferation and Sertoli cell differentiation via FGF9 signaling [3]. Nuclear Receptor Subfamily 5, Group A, Member 1 NR5A1, encoding steroidogenic factor 1 (SF1) or Adrenal 4-binding protein (AD4BP), is a nuclear receptor localized on 9q33 [2]. In mice, Nr5a1 is expressed in the developing urogenital ridge, hypothalamus, and the anterior pituitary gland, indicating an essential role in the development of the hypothalamic-pituitary-gonadal axis. Mice lacking Nr5a1 have gonadal agenesis resulting in male-to-female sex reversal, obesity, and abnormalities of the ventromedial pituitary and hypothalamus gonadotropes. NR5A1 plays key roles in multiple reproductive processes (see the second section entitled ‘Nuclear Receptor Subfamily 5, Group A, Member 1’, below).

Lim Homeobox Gene 9 The LIM homeobox 9 (LHX9) encodes a protein of the LIM family, characterized by the presence of two amino-terminal domains, predominantly involved in proteinprotein interactions, and a DNA-binding homeobox domain [6]. The phenotype of the Lhx9–/– mice is similar to that of the Nr5a1–/– mice (XY female) and the expression of SF1 is considerably reduced in their genital ridge, suggesting Lhx9 as a regulator of

Gonadal Development Hiort O, Ahmed SF (eds): Understanding Differences and Disorders of Sex Development (DSD). Endocr Dev. Basel, Karger 2014, vol 27, pp 1–16 (DOI: 10.1159/000363608)

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Chromobox Homolog 2 Chromobox homolog 2 (CBX2) is a component of the polycomb group complex of regulatory proteins. In mice, disruption of Cbx2 results in posterior transformation of the vertebral columns and sternal ribs, failure of T cell expansion and sex reversal in XY animals. The XX animals displayed an absence or reduction in size of ovaries compared to wild-type littermates. Abrogation of Cbx2 results in a reduction of the expression levels of Nr5a1, suggesting it to be an upstream regulator of SF1 [4]. In the human, two heterozygous mutations of CBX2 were identified in a 46,XY female with normal external and internal genitalia and histologically normal ovaries [5]. A recent study has indicated that Cbx2 plays a role in testis differentiation through regulating Sry gene expression [4].

Nr5a1 expression. In vitro studies demonstrate that Lhx9 binds directly to the Nr5a1 promoter and has an additive effect on the WT1-induced activation. So far, no mutations in LHX9 have been described in human DSD patients.

GATA-Binding Protein 4 GATA4 belongs to the evolutionarily conserved GATA family of six tissue- and organ-specific vertebrate transcriptional regulators, and the protein consists of two conserved type IV zinc finger motifs of the distinctive form Cys-Xaa2-Cys-Xaa17-CysXaa2-Cys [8]. The C-terminal zinc finger is required for DNA recognition and DNA binding, whereas the N-terminal zinc finger contributes to the stability of this binding. The zinc fingers are also crucial for protein-protein interactions with other transcription cofactors. In the mouse and human, GATA4 is strongly expressed in the somatic cell population of the developing gonad prior to and during the time of sex determination. Mice lacking Gata4 die in utero due to profound abnormalities in ventral morphogenesis and heart tubes. The critical role for GATA4 in gonadal development (see the section entitled ‘GATA4 and FOG2’) is highlighted by Gata4ki mice that have a p.Val217Gly mutation in the N-terminal zinc finger domain and show abnormal testis development. In the human, mutations in GATA4 are associated with congenital cardiac defects and a heterozygous GATA4 p.Gly221Arg mutation has been described in a familial case of 46,XY DSD [9]. Recently, a conditional knockout of Gata4 was reported that lacked gonadal initiation or differentiation, unlike the previously reported mutants (Lhx9–/–, Sf1–/–, Wt1–/– and Emx2–/–) where the genital ridge is formed but then degenerates. The authors suggest a role for Gata4 in gonadogenesis earlier than Lhx9, Nr5a1, Wt1 and Emx2 [10]. The bipotential gonad is balanced between the male and female fate. SRY is the master regulator of sex determination. In the XY embryo, expression of SRY between embryonic day 10 (e10.0) and e12.5 in mouse and 42 dpc in human drives the initial differentiation of pre-Sertoli cells. These pre-Sertoli cells proliferate, polarize and aggregate around the germ cells to define the testes cords, and the region in between the cords form the interstitial spaces. The signals from the pre-Sertoli cells induce the migration of cells from the mesonephros or the coelomic epithelium and the enclosure of the cords by the peritubular myoid cells delimit the basal lamina to further define the testis cords. The somatic cells lying outside the cord differentiate into the fetal Leydig cells, as a result of signals emanating from the pre-Sertoli cells, and initi-

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Empty-Spiracles Homeobox Gene 2 Emx2, the mouse homolog of Drosophila empty spiracles (ems), is expressed in the epithelium of the developing urogenital system and dorsal telencephalon [7]. Emx2–/– mutants show a complete absence of the gonads and genital tract. Although little is known about the possible targets or partners of Emx2 during gonadal development, the Emx2 protein contains a pentapeptide motif that may facilitate interaction with other TALE homeobox proteins.

ate testosterone production. The endothelial cells associate to form the coelomic vessel, capable of exporting testosterone [11]. In the absence of SRY the indifferent gonad follows the ‘default’ female pathway. There are 3 essential steps in ovarian follicle development: specification of the granulosa cells, formation of the follicles and compartmentalization of the somatic cell environment. The process of ovarian development is slower than testes development, with the ovary being histologically identifiable only by the 11th week of gestation in humans. The ovarian follicle consists of a single oocyte surrounded by granulosa and theca cells. The granulosa cells are the female equivalent of the Sertoli cells and are thought to share a common precursor. Ovarian development relies on the presence of germ cells, without which granulosa cells may differentiate into Sertoli cells. Primordial follicles are the precursor to mature follicles and are formed around the time of birth when pre-granulosa cells invade clusters of germ cells. Finally, a thin layer of extracellular matrix is deposited around each follicle and the granulosa cells secrete factors involved in follicle development, resulting in the formation of the mature follicle [12].

Sex-Determining Region on the Y Chromosome SRY is the founding member of the SRY-related high-mobility group (HMG) box (SOX) family of transcription factors that is characterized by the presence of a HMG box DNA-binding domain (DBD) and act as genetic switches promoting cell differentiation. The HMG domain is conserved throughout species and, in contrast, the other regions of the SRY protein are less conserved. Interestingly, XX transgenic mice that express human or goat SRY develop as males, suggesting that the non-HMGdomain regions may be conserved in function but not in sequence. Approximately 80% of 46,XX individuals with testicular DSD and 10% with ovotesticular DSD carry SRY in their genome, which explains the phenotype. The remaining SRY-negative cases of 46,XX DSD may be explained by either hidden mosaicism of a 46,XY cell line in the gonad, mutations in a sex-determining gene downstream from SRY, or neomorphic mutations [13]. It still is not clear how Sry expression is regulated but several potential activators, including CBX2, WT1(+KTS) and FOG2-GATA4 complex, have been proposed. Recently, it was shown that Jmjd1a directly controls Sry expression by regulating H3K9me2 marks [14]. Cbx2 may regulate the expression of Sry expression in mice by upregulating the expression of positive regulators of Sry, such as Dax1, Gata4, Wt1 and Nr5a1. In mice, Sry functions within a critical window of time in individual somatic cells of the developing gonad. The onset of Sry expression begins at 10.5 dpc in mouse, reaches a peak at 11.5 dpc and fades at around 12.5 dpc. In the human, SRY is expressed in pre-Sertoli cells at 7 weeks in the XY gonad. Although


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Molecular Determinants of Testis

cerebellin 4 (Cbln4) and β-catenin have been suggested as the putative downstream targets of Sry, conclusive evidence exists only for Sox9 as the definitive target of Sry [15] (discussed in the section entitled ‘SRY-Related HMG Box 9’). When Sry is absent, Sox9 is downregulated and the development of ovaries takes place. Although SRY triggers the formation of the testes in mammals, the plasticity of the system is highlighted by some rodent species that have neither a Y chromosome nor an SRY gene.

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SRY-Related HMG Box 9 In XY human and mouse gonads, SOX9 expression is upregulated in pre-Sertoli cells immediately after the onset of SRY gene expression. SOX9 can effectively replace SRY as the master sex-determining gene since transgenic Sox9 XX mice develop testes, and a duplication of SOX9 in a 46,XX human resulted in male development. Sox9 plays an essential role in the specification and differentiation of mesenchymal cells toward the chondrogenic lineage in all developing skeletal elements and SOX9 was first identified as the causative gene for campomelic dysplasia, a condition characterized by skeletal defects and typical facies (hypoplasia, hypertelorism with a small nose and mandibular hypoplasia). Approximately 75% of XY individuals with campomelic dysplasia present with complete or partial gonadal dysgenesis, highlighting the key role of SOX9 in the sex-determination process [13]. Flanking regions that regulate gonad-specific SOX9 expression have recently been identified in both the mouse and human. The 3.2-kb testis-specific enhancer (TES) located between 10- and 13-kb upstream of mouse Sox9 has been identified, which is sufficient for gonad-specific expression of Sox9. TES activity was further narrowed down to a conserved critical region of 1.4 kb – the TES core enhancer (TESCO). Both SRY and NR5A1 (see below) bind to the TESCO enhancer sequence in vivo, probably through a direct physical interaction to activate SOX9 expression [15]. Sry expression leads to an increased Sox9 production that synergizes with Nr5a1 and regulates its own expression ensuring a positive feedback loop. The Sox9/Nr5a1 complex is a more potent activator of TESCO than the Sry/Nr5a1 complex. Sox9 can directly interact with the enhancer, but also indirectly while bound to Nr5a1. It is likely that transcriptional regulation of Sox9 during testis development involves an Sry-independent initiation, Sry-dependent upregulation and the maintenance of Sox9 expression in the absence of Sry. Once SOX9 levels reach a critical threshold, several positive regulatory loops are initiated for its maintenance, including autoregulation of its own expression and formation of feed-forward loops via FGF9 or PGD2 signaling. Other cofactors are likely to be involved in this process [15]. In vitro studies demonstrate that homologous human SRY-responsive enhancer upstream of SOX9 can also be activated by human SRY and SOX9 together with NR5A1. This suggests a conserved mechanism for male-specific upregulation and maintenance of SOX9 expression in pre-Sertoli cells in human and mouse.

GATA4 and FOG2 GATA4 cooperatively interacts with several proteins, including NR5A1, to regulate the expression of critical genes known to be involved in testis determination and differentiation (SRY, SOX9, AMH, STAR, CYP19A1, INHA, HSD3B2) [17]. Gata4 is expressed in the genital ridge. The expression pattern is conserved across species and has been linked to the role of Gata4 in testis differentiation by transcriptional activation of Sry, in synergy with WT1. Homozygous Gata4ki mice, where the mutation abrogates the interaction of GATA4 with the cofactor FOG2, can form genital ridges. However, further differentiation of the Gata4ki genital ridges into testes is blocked and this is associated with a reduction of Sox9 but not Sry expression. Mouse embryos heterozygous for Gata4ki on specific genetic backgrounds also show sex reversal of XY males to phenotypic females. Conditional ablation of Gata4 from XY genital ridges after E10.5 resulted in disruption of subsequent testis differentiation associated with upregulation of ovarian somatic markers. These studies show a necessity for Gata4 in testis development. FOG2 (friend of GATA 2; also known as ZFPM2) is a zinc finger cofactor that binds to the N-terminal zinc fingers and regulates the transcriptional activity of GATA factors. Fog2 is coexpressed with Gata4 in the heart, brain and gonads during mouse development, with high expression in the XY mouse gonad at the moment of Sertoli


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Nuclear Receptor Subfamily 5, Group A, Member 1 NR5A1 encoding steroidogenic factor-1 is an essential transcriptional regulator of genes in the steroidogenic tissues of the hypothalamic-pituitary-adrenal/gonadal axis [16]. SF1 encodes a 461-amino acid protein comprising of a DBD with two zinc fingers, an ‘A’ box (a 30-amino acid extension of the DBD) that mediates specific DNA binding, a flexible hinge region, a ligand-binding domain, and two activation function domains: AF-1 and AF-2. NR5A1 is a positive regulator/cofactor of a number of factors essential for specification and differentiation of the male gonad, including SOX9 and anti-Müllerian hormone (AMH; see the section entitled ‘Anti-Müllerian Hormone’). NR5A1 also modulates the expression of multiple genes involved in endocrine development and function, such as steroidogenic acute regulatory protein (STAR), and several cytochrome P450 (CYP) steroid hydroxylases. Nr5a1–/– mice lack the adrenals and gonads in both the sexes and the first human cases carrying mutations in NR5A1 resembled the mouse phenotype with gonadal dysgenesis and adrenal insufficiency. To date, about 60 different NR5A1 mutations have been reported in humans in association with 46,XY DSD. These cases have a range of phenotypes, including ambiguous genitalia or hypospadias with no adrenal insufficiency, vanishing testis syndrome, isolated hypoplastic penis and male infertility. Mutations in NR5A1 are also associated with premature ovarian failure and primary ovarian insufficiency in 46,XX females [16]. So far, not much is known about the regulation of NR5A1 expression itself, although it has been proposed that WT1 and Cbx2 may regulate the enhancer or promoter of NR5A1.

Anti-Müllerian Hormone AMH or Müllerian-inhibiting substance is a member of the transforming growth factor-β family that encodes a 560-amino acid glycoprotein. The AMH signaling pathway is both essential and sufficient for Müllerian duct regression. XY mice lacking Amh develop normal female reproductive tracts, including oviducts, uterus and vagina. In female mice, ectopic expression of human AMH is sufficient to cause the regression of the Müllerian structures. Both human and murine mutations in AMH or its receptor result in the persistent Müllerian duct syndrome. Sertoli cells are the only embryonic source of AMH where its expression starts in the pre-Sertoli cells and is maintained throughout male fetal development. Except for a transient decline in the perinatal period, AMH secretion remains high until puberty, when Sertoli cells are finally mature and secret lower levels of AMH. In adult females, the granulosa cells express AMH. However, its expression only starts during the perinatal period, remains low throughout reproductive life and becomes undetectable after menopause [19]. A 180-bp minimal promoter region with conserved regulatory elements in both human and mouse is sufficient for cell-specific expression of AMH, both in vivo and in vitro. Several transcription factors, such as NR5A1, SOX9, GATA4 and WT1, have been shown to regulate AMH transcription. A highly conserved nuclear receptor half site identified within the minimal promoter region of AMH is critical for DNA-protein interaction and for proper AMH expression. SOX9 and NR5A1 recognize and bind to this element to regulate AMH expression. This binding by SOX9 is essential for the initiation of AMH expression in early fetal development. SOX9 and NR5A1 physically

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cell formation. Fog2–/– mice die in utero at e13.5 due to cardiac anomalies. Fog2–/– mice fail to develop testes, and the expression of key genes involved in testis formation (Sry, Sox9 and Amh) are reduced. Mutations in FOG2 in humans have been associated with congenital heart disease and antipsychotic-induced Parkinsonism in schizophrenia, with no reported gonadal anomalies. However, a boy with a balanced t(8;10) (q23.1;q21.1) translocation was recently reported with congenital secundum-type atrial septal defect and 46,XY gonadal dysgenesis. The breakpoint on chromosome 8 mapped within the FOG2 gene. Another individual with multiple somatic anomalies and gonadal dysgenesis carried a de novo chromosomal translocation: 46,XY t(8;18) (q22; q21). The region was comprised of a 16-Mb de novo deletion within chromosomal region 8q23.1–8q24.1 that includes the FOG2 gene. Recently, using the exomesequencing approach, we have for the first time described point mutations in the coding sequence of FOG2 associated with 46,XY DSD in humans [18]. Although the exact molecular mechanism remains unknown, a physical interaction between Gata4 and Fog2 proteins is required for normal testis development. The mutation in Gata4ki results in reduction of Sry levels suggesting Gata4/Fog2 as a regulator of Sry in the developing testis. Although this has not been proven yet, multiple GATA sites have been identified in mouse, pig and human SRY promoters.

interact to further modulate AMH expression. NR5A1 can also associate with WT1KTS (but not WT1+KT) through its DBD and synergistically upregulate AMH expression. NR5A1 can interact and form a heterodimer with DAX1, albeit with a lower affinity than the WT1/NR5A1 interaction. This direct interaction between NR5A1 and DAX1 represses the AMH expression. GATA4 regulates cell and sex-specific expression of the AMH by directly binding to its site on the AMH promoter. GATA4 can also bind to NR5A1, when either NR5A1 alone is bound to its site or when both factors are bound to their respective sites on the AMH promoter to upregulate the AMH expression, which is further increased when two molecules of GATA4 simultaneously interact with NR5A1. In vitro FOG-2 can repress GATA4-dependent activation of AMH [20].

Desert Hedgehog Desert hedgehog (DHH), located on 12q12, encodes a 396-amino acid protein of the hedgehog family of signaling molecules. Dhh is expressed in the Schwann cells, vascular endothelium, endocardium and seminiferous epithelium of the mouse embryo.


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Doublesex and mab-3-Related Transcription Factor 1 Doublesex and mab-3-related transcription factor 1 (DMRT1), located on 9p24.3, has a structurally and functionally conserved zinc finger-like DNA-binding motif DM domain and is related to the mab-3 in Caenorhabditis elegans and doublesex (dsx) in Drosophilia melanogaster, which play a pivotal role in sex differentiation [21]. Dmrt1 is expressed in the developing genital ridge of both XX and XY mice, but by 12.5 and 13.5 dpc it is limited to the testis cords in XY embryos, whereas in XX animals the ovary shows a more diffused expression. After 13.5 dpc, Dmrt1 is upregulated in the testis while its expression declines in the ovary. In humans at 7 weeks of gestation, DMRT1 expression is restricted to the developing seminiferous tubules and is not detected in the developing female gonads. Dmrt1–/– mice show abnormal postnatal testis differentiation accompanied by germ cell death, whereas the ovarian development remains unaffected. 46,XY individuals with 9p monosomy develop as females with ambiguous external genitalia and a variety of gonadal anomalies, ranging from streak gonads to hypoplastic testis. Loss of Dmrt1 results in premature onset of meiosis in the developing spermatogonia, suggesting its involvement in the decision-making process of male germ cells to undergo mitosis, spermatogonial differentiation or meiosis. Dmrt1 is also required to maintain Sertoli cell proliferation and viability. Although DMRT1 may determine sex in some vertebrates, such as birds and fish, its precise role in mammalian testis development remains largely unexplored. However, recent data suggest that it may function by repressing female development. XY Dmrt1–/– mutant mice are born as males with testes, but the gonads later undergo abnormal differentiation. Further studies have shown that the loss of Dmrt1 in mouse Sertoli cells, even in adults, results in the activation of Foxl2, and Sertoli cells transdifferentiate into granulosa and theca cells, with production of estrogen and germ cells that appear feminized [22].

In the fetal testis, expression of Dhh is initiated at 11.5 dpc in the Sertoli cell precursors, shortly after the onset of Sry. On the other hand, no expression is detected in fetal ovaries. Dhh–/– female mice show normal reproduction whereas male mice are sterile and the severity of the phenotype varies with the genetic background of the animals [23]. In humans, homozygous mutations in DHH are associated with complete or partial 46,XY gonadal dysgenesis [13]. Dhh is the only hedgehog protein to be expressed by developing Sertoli cells and a hedgehog receptor, patched 1 (Ptch1), is expressed in the interstitial cells. Dhh–/– mice show a reduction in expression of Ptch1. It has been suggested that the Dhh/Ptch1 signaling pathway triggers Leydig cell differentiation by upregulating the expression of Nr5a1 and Cyp11a1 in the interstitial precursor cells. The differentiation of peritubular myoid cells and consequent formation of testis cords are also dependent on Dhh expression [23].

α-Thalassemia/Mental Retardation X-Linked Syndrome The α-thalassemia/mental retardation syndrome X-linked (ATRX) gene, located on the mammalian X chromosome, is a member of the SNF-2-like helicase superfamily subgroup that contains genes involved in DNA recombination, repair and regulation of transcription [13, 26]. The N-terminal of ATRX protein is an ADD (ATRXDMNT3L-DNMT3A) domain comprising of a GATA-type zinc finger and a plant homeodomain often found in chromatin-associated proteins, whilst the C-terminal

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Mitogen-Activated Protein Kinase Pathway A role for the phylogenetically ancient mitogen-activated protein kinase signaling pathway in mammalian sex determination was indicated by the identification of a mutation in murine Map3k4 in a forward genetic screen. XY embryos lacking functional MAP3K4 on a predominantly C57BL/6J background exhibit embryonic gonadal XY sex reversal associated with a failure to transcriptionally upregulate Sry [24]. In the human mutations in another MAP kinase, MAP3K1 have been identified in cases of 46,XY DSD. These mutant versions of MAP3K1 behave like gain-of-function alleles, enhancing functionality of the encoded protein. Two of the eleven sporadic cases of 46,XY DSD carried mutations in MAP3K1, suggesting a frequency of 18% [25]. Our own analyses of MAP3K1 also shows a frequency of mutations of 15% in otherwise unexplained cases of 46,XY DSD [unpubl. data], which is a similar mutation frequency to that reported for NR5A1 mutations in this group of patients. Interestingly, mice lacking Map3K1 do not show male-to-female sex reversal. This suggests that either the MAP kinase signaling pathways in humans and mice have diverged or that the difference in phenotype is caused by an intrinsic difference in the type of mutation. Mice generated by gene targeting have loss-of-function Map3k1 alleles, whereas the MAP3K1 mutations that are associated with 46,XY DSD are either splice site mutations, missense mutations or in-frame deletions that can appear to act as gainof-function alleles.

is a switch/sucrose non-fermenting-like ATPase domain which displays nucleosomeremodeling activity. ATRX provides a link between chromatin remodeling, DNA methylation and gene expression in developmental processes. Mutations in the gene cause ATR-X syndrome, a sex-linked condition characterized by α-thalassaemia, severe psychomotor retardation, characteristic facial features, microcephaly, short stature, cardiac, skeletal and urogenital abnormalities. Nearly all ATR-X syndrome mutations fall within these ADD and plant homeodomain domains, highlighting their importance for ATRX protein function. Anomalies of testis development are common in XY individuals with this syndrome, and 80% of XY patients have urogenital anomalies that range from complete gonadal dysgenesis that is commonly associated with truncations of the C-terminus of the protein, to relatively mild hypospadias or micropenis. Phenotypic variability has also been observed within families carrying the same ATRX mutation. The precise role of ATRX in gonadal development in mammals remains unclear. In mice lacking Atrx specifically in Sertoli cells, small testis developed due to apoptosis of proliferating Sertoli cells during fetal life. Within cells, the ATRX protein is found at heterochromatic repeats, at rDNA repeats, at telomeric repeats and within PML bodies, which themselves are often associated with heterochromatic structures, including telomeres. These repeat elements are critical for ATRX transcriptional regulation. Recently, it was demonstrated that ATRX modulates gene expression by binding to G-rich tandem repeat sequences. Differences in the size of the tandem repeats were associated with differences in the level of gene expression and this could explain the variation of the phenotype associated with identical ATRX mutations. In contrast to the testis, development of the ovary is more labile, with the potential to switch to a testis state. Ovarian somatic sex determination requires both Rspo1/ Wnt4/β-catenin and Foxl2 signaling pathways, although the degree to which these factors are instructive or permissive is not entirely clear.

Wingless-Type MMTV Integration Site Family, Member 4 Wingless-type MMTV integration site family, member 4 (Wnt4), is a member of the Wnt family of secreted molecules that function in a paracrine manner to effect several developmental changes. It encodes a protein that acts as a repressor of male differentiation. Wnt4–/– XX mice show partial masculinization of the gonad, resulting in the formation of coelomic blood vessels and the differentiation of some Leydig-like cells in the gonad. Signaling by WNT4 and R-spondin1 both lead to stabilized β-catenin, suggesting that these molecules play a key role in ovarian determination (or suppression of testis formation) by ensuring appropriate levels of stable β-catenin [27]. In the human, four dominant heterozygous mutations in WNT4 have been reported in association with androgen excess, abnormal development of Müllerian


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Molecular Determinants of the Ovary

Forkhead Transcription Factor Forkhead transcription factor (FOXL2) encodes a 376-amino acid protein, containing a 110-amino acid DNA-binding forkhead domain and a polyalanine tract of 14 residues of unknown function. Expression studies in human have shown that the FOXL2 transcript and protein are expressed in the mesenchyme of developing eyelids, in fetal and adult granulosa cells of the ovary, and in the embryonic and adult gonadotrope and thyrotroph cells of the pituitary. In mice lacking Foxl2 the male differentiation program is initiated in the ovaries, with male markers such as Sox9 sharply upregulated [28]. The ovaries contain cords that enclose cells with male Sertoli-like features. These observations suggest that FOXL2 represses male development and it is the counterpart of DMRT1 that maintains male fate by the suppression of female development. Targeted ablation of Foxl2 in the adult ovary leads to a molecular transdifferentiation of the supporting cells of the ovary, which acquire the characteristics of the Sertoli cells, including SOX9 expression [29]. The TESCO element that regulates Sox9 expression is reactivated after Foxl2 ablation, and chromatin immunoprecipitation experiments show that TESCO is bound to Foxl2 [29]. Foxl2 is also capable of attenuating TESCO activation by NR5A1, SRY/NR5A1 and SOX9/NR5A1, perhaps via direct interaction. A recent study showed that FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice [30]. FOXL2 is shown as the female sex-determining gene in the goat [31]. Unlike in mice, FOXL2 is important in the goat for fetal development, as opposed to postnatal maintenance in the former. In the human, heterozygous mutations of the FOXL2 gene are associated with blepharophimosis syndrome (BPES). Type I BPES has eyelid malformation (blepharophimosis, ptosis, epicanthus inversus and telecanthus) associated with ovarian insufficiency, whilst type II BPES is isolated. The vast majority of mutations reported in

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ducts but normal female genitalia. These heterozygous missense mutations show various degrees of virilization with no overt evidence of testicular development. These mutations significantly increased expression of the enzymes involved in androgen biosynthesis and showed dominant negative activity on the wild-type allele. The homozygous WNT4 mutation detected in a consanguineous family resulted in an embryonic lethal syndrome encompassing female-to-male sex reversal and dysgenesis of kidneys, adrenals and lungs (SERKAL syndrome – sex reversion, kidneys, adrenal and lung dysgenesis). The gonads of XX fetuses consisted of a normal-looking testis with seminiferous tubules and numerous Leydig cells throughout the interstitium. In one fetus, the testicular structure contained isolated primary follicles. A homozygous p.Ala114Val was found in affected individuals that resulted in markedly reduced WNT4-dependent inhibition of β-catenin degradation. The heterozygous carriers were normal but showed increased activity of 5 α-reductase, suggesting that WNT4 may also play a role in prostate development [13].

FOXL2 are dominant and heterozygous. Two recessive mutations have been reported, associated with BPES and ovarian failure, a phenotype identical to the dominant heterozygous mutations with no evidence of virilization. However, in both cases it is likely that the mutant alleles are hypomorphic, retaining some biological activity, so the phenotype of a true FOXL2 null mutation in the human remains to be determined [13]. R-Spondin Family 1 R-spondin family 1 (RSPO1) belongs to a family of secreted furin-like domain-containing proteins that enhance Wnt/β-catenin signaling and have pleiotropic functions in development. R-spondins bind to the cell surface receptors LGR4 and LGR5, and trigger downstream canonical Wnt signals through associated frizzled-Lrp5/6 complexes [32]. The genes Lgr4, Lgr5 and Lgr6 encode orphan 7-transmembrane receptors that relate to the receptors for follicle-stimulating hormone and luteinizing hormone. β-Catenin is the downstream effector molecule of WNT4/RSPO1 signaling and appears to be essential for initiating the development of the female gonad. Mice lacking Rspo1 show partial XX female-to-male sex reversal with a downregulation of Wnt4 expression indicating that Rspo1 positively regulates WNT4 signaling, possibly by antagonizing dickkopf-1 (Dkk1)-dependent internalization of LRP6, thereby resulting in a stabilization of cytoplasmic β-catenin [33]. In humans, mutations in RSPO1 are associated with a recessive syndrome characterized by XX sex reversal, palmoplantar hyperkeratosis and predisposition to squamous cell carcinoma of skin [34]. However, in the non-syndromic cases of testicular and ovotesticular DSD, mutations in RSPO1 have not been reported.

The choice of somatic cell fate in the developing gonad in mammals occurs following the activation of Sox9 expression by Sry on the Y chromosome, and the subsequent maintenance of gonadal fate can be viewed as a battle for dominance between male (Dmrt1, Sox9) and female (Foxl2 and Wnt/b-catenin) regulatory gene networks. Thus, the development and maintenance of the mammalian gonad is a unique biological process that is regulated by a double repressive system where equilibrium of mutually antagonistic pathways must be attained for normal development of either the testis or ovaries. Changes in this delicate balance may result in DSD or infertility in the human. DSD are defined as congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical and they encompass a range of gonadal phenotypes. Some of these are common, such as undescended testis or genital ambiguities, whereas others are rare and include a total failure of testis determination, 46,XY complete or partial gonadal dysgenesis or testis formation on a female chromosomal background – XX testicular DSD. Although mutations in a number of


13


Conclusions

testis-determining genes, such as SRY, NR5A1, WT1, MAP3K1, GATA4, NR5A1, WT1, DHH, CBX2, ATRX, FGF9 and SOX9, are associated with both syndromic and non-syndromic forms of DSD, in the majority of cases the etiology remains unknown. Although many of the early genetic, cellular and morphological events during gonadal development have been characterized, the molecular mechanisms involved in human sex determination are poorly understood. There are several reasons for this. First, there are no appropriate cellular models of sex determination. No cell line has been established with all the properties of Sertoli cells. Primary immature and mature Sertoli cells as well as established cell lines lose their characteristics during prolonged culture. Second, familial cases with errors in sex determination XY females or XX males are very rare. This has hampered classical genetic studies to identify genes involved in the process. Third, sex-determination is poorly conserved in evolution. The use of novel ‘omics’ technologies for analyzing a large number of patients with DSD and careful assessment of the resulting datasets may result in the identification of novel genetic factors in human sex determination and lead to the development of novel ex vivo cellular models. This approach will aid the understanding of the complex mechanisms associated with the development of human reproductive processes.

Acknowledgements Support was provided by March of Dimes Foundation Research Grant 1-FY07-490, EuroDSD in the European Community’s Seventh Framework Programme FP7/2007–2013 under Grant 201444, Projets Blanc Institut Pasteur/Assistance Publique-Hopitaux de Paris 2011, grant No. 295097, GM_ NCD_in_Co – Reinforcing IPT Capacities in Genomic Medicine, Non-Communicable Diseases Investigation and International Cooperation as part of the EU call FP7-INCO-2011-6. The work was also partially funded by a Franco-Egyptian AIRD-STDF grant.

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