Biason-Lauber, A (2010). Control of sex development. Best Practice and Research Clinical & Metabolism, 24(2):163-86. Postprint available at: http://www.zora.uzh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch Originally published at: Best Practice and Research Clinical Endocrinology & Metabolism 2010, 24(2):163-86.

Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch

Year: 2010

Control of sex development

Biason-Lauber, A

Biason-Lauber, A (2010). Control of sex development. Best Practice and Research Clinical Endocrinology & Metabolism, 24(2):163-86. Postprint available at: http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Best Practice and Research Clinical Endocrinology & Metabolism 2010, 24(2):163-86.

CONTROL OF SEX DEVELOPMENT

Anna Biason-Lauber

University Children’s Hospital, Zurich, Switzerland

Correspondence to :

Anna Biason-Lauber, MD University Children’s Hospital Div. of Endocrinology/Diabetology Steinwiesstrasse 75 CH-8032 Zurich Switzerland Tel + 41 44 266 7623 Fax + 41 44 266 7169

E-mail: [email protected]

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ABSTRACT

The process of sexual differentiation is central for reproduction of almost all metazoan, and therefore for maintenance of practically all multicellular organisms. In sex development we can distinguish two different processes¸ sex determination , that is the developmental decision that directs the undifferentiated embryo into a sexually dimorphic individual. In mammals, sex determination equals gonadal development. The second process known as sex differentiation takes place once the sex determination decision has been made through factors produced by the gonads that determine the development of the phenotypic sex. Most of the knowledge on the factors involved in sexual development came from animal models and from studies of cases in whom the genetic or the gonadal sex does not match the phenotypical sex, i.e. patients affected by disorders of sex development (DSD). Generally speaking, factors influencing sex determination are transcriptional regulators, whereas factors important for sex differentiation are secreted hormones and their receptors. This review focuses on these factors and whenever possible, references on the “prismatic” clinical cases are given.

Keywords: Sex development; sex determination, sex differentiation; Ovary; Testis; Disorders of sex development (DSD); Bipotential state.

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Abbreviations:

DSD: Disorders of sex development Pax2: Paired Homeobox 2 Emx2: Empty spiracle homolog 2 Lhx9: Lim Homeobox protein 9 WT1: Wilms Tumor 1 GATA4: GATA-binding protein 4 SRY: Sex determining Region Y SOX9: SRY- box 9 SF1/NR5A1: /Nuclear Receptor subfamily 5, group A, member 1 DAX1: Dosage sensitive sex reversal, Adrenal hypoplasia congenital critical region on the gene 1 DMRT1: Doublesex and MAB3-Related Transcription factor 1 DHH: Desert Hedgehog ATRX: Alpha Thalassemia, mental Retardation syndrome, X-linked CBX2: Chromobox homolog 2 TSPYL1: Testis-specific protein Y-like-1 MAMLD1: Mastermind-Like Domain-Containing Protein 1 PGD2S: Prostaglandin D2 synthase RSPO1: Root-plate specific Spondin 1 WNT4/FST: Wingless Type MMTV intergration site family, member 4/Follicostatin FOXL2: Forkhead transcription factor AMH/AMHR = MIS: Anti-Müllerian Hormone/ Receptor = Müllerian Inhibiting Substance HOXA: Homeobox A Lim1/LHX1: Lim Homeobox protein 1 LH/CG: /Corionic POF: Premature ovarian failure ODG: Ovarian dysgenesis FMR1: Fragile X Mental Retardation 1 DIAPH2: Diaphanous (dia) homolog 2 GDF9: Growth Differentiation Factor 9 BMP15: Bone Morphogenic Protein 15 NOBOX: Newborn Ovary Homeobox FIGLA: Factor in Germ Line Alpha

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1. PHYSIOLOGY OF SEX DEVELOPMENT.

In sex development we can distinguish two different processes¸ sex determination that is the developmental decision that directs the undifferentiated embryo into a sexually dimorphic individual and sex differentiation that takes place once the sex determination decision has been made through factors produced by the gonads that determine the development of the phenotypic sex. At the beginning of gestation (1st and 2nd week) embryos of the two sexes differ only for their . Starting at week 3 specific genes lead to the differentiation of the gonads, which in turn produce hormones inducing anatomical and psychological differences, leading to behavioral differences that are ultimately influenced by the social environment. As very nicely put by Sekido and Lovell-Badge : “Overall, sex determination is a story of opposing forces and crucial alliances but, although the winning team takes all, its rule can be surprisingly tenuous“ 1 The main steps of intrauterine sex differentiation are depicted in Fig. 1. At gestational weeks 6-7 the (Müllerian duct) develops next to the mesonephric duct. If testes develop and secrete testosterone, the mesonephric (Wolffian) duct increases in size and differentiate into epididymis, vas deferens, and prostate. A glycoprotein secreted from the testicular Sertoli cells known as Anti-Müllerian Hormone (AMH) or Müllerian Inhibiting Substance (MIS), results in Müllerian duct regression. If testes do not develop, the mesonephric duct does not grow and eventually degenerates, whereas the paramesonephric duct proliferates and the , , and upper third of the develop (Fig.2). In mammals, including humans, the differentiation of the gonads is the turning point of this whole process. The classical text-book theory says that in the presence of the sex determining region on the Y-chromosome (SRY), the default ovarian pathway of sex determination will be inhibited and therefore testes will be formed. In the XX individual due to the absence of SRY no inhibition of the default program will take place and ovaries will develop. Ovarian determining factors might help the process of differentiation. However, these factors are yet to be determined. The second model called the Z-factor theory was proposed to explain the cases where XX individuals develop testes in the absence of SRY. According to this theory, the XX gonad expresses a factor that has both anti-testis and pro-ovary function. SRY in XY individuals acts as an inhibitor of the Z-factor to lift the block on the male pathway. In this case, the bipontential gonad will differentiate into a testis 2. The Z-factor remains unknown. It is important to notice that most of the knowledge on the factors involved in sexual development came from studies of cases in whom the genetic or the gonadal sex does not match the phenotypical sex, i.e. patients affected by defects of sex development 3 and from animal models. This review focuses on these factors and whenever possible, references on the “prismatic” clinical cases are given . Figure 3 and table 1 summarize the role of such factors and the effects of their in mice and men.

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2. FACTORS INVOLVED IN SEX DETERMINATION

2.1. INDIFFERENTIATED STATE (BIPOTENTIAL)

Pax2

Pax-2 is a transcriptional regulator of the paired-box family and is widely expressed during the development of both ductal and mesenchymal components of the urogenital system. Pax- 2 homozygous mutant newborn mice lack kidneys, ureters and genital tracts. These defects might be attributed to dysgenesis of both ductal and mesenchymal components of the developing urogenital system 4.

In humans, genetic defects in PAX2 are identified in renal-coloboma syndrome, without genito-urinary abnormalities.

Emx2

The homeobox gene Emx2 is a mouse homologue of a Drosophila head gap gene empty spiracles (ems) and is essential for the development of dorsal telencephalon 5. At the same time, Emx2 is expressed in the epithelial components of the developing urogenital system and, in Emx2 mutant mice, the kidneys, ureters, gonads and genital tracts were completely missing. Degeneration of the Wolffian duct and mesonephric tubules was also abnormally accelerated in these mice without the formation of the Mullerian duct 6.

Mutations in EMX2 are a rare cause schizencephaly and somatic mutations in EMX2 have been found in endometrial carcinomas 7, but, again, no human sex developement defect due to EMX2 mutations has been described to date.

Lhx9 Transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Lhx9 mutants do not exhibit additional major developmental defects 8.

Although LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans, none of such mutations has yet been found.

WT1

The WT1 gene encodes a zinc finger DNA-binding protein that is inactivated in some Wilms’ tumors. analysis in human patients and genetic experiments in mice revealed that WT1 has a role in development besides in tumor suppression. WT1 has at least 4 splicing variants depending on the presence of exon 5 (+/- 17 amino acids in the mature protein)

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and/or 9 nucleotides coding for the 3 amino acids KTS (+/-KTS). Post-trancriptional modifications of the Wt1 pre-mRNA lead to the production of up to 24 isoforms, which increase the complexity of Wt1 action. WT1 is already expressed within the undifferentiated gonad, and its importance there has been demonstrated in Wt1-/- mice, in which the gonads undergo apoptosis. Since the gonads and adrenal cortex share a common primordium 9 it is not a surprise that adrenal glands are also affected in these mice 10. Expression of Sf1 and expression of WT1 in the gonad begin about simultaneously and might be dependent. Wilhelm and Englert demonstrated that WT1, and more specifically its DNA binding form WT1(-KTS), can activate the Sf1 promoter in vitro and in vivo 11. The apoptotic phenotype in Wt1-knockout mice might therefore be caused by a lack of Sf1 expression. Alternatively, WT1 might activate an Sf1-independent pathway required for adrenogenital survival. This could include the anti-apoptotic factor Bcl-2, which might be directly activated by WT1 12. A second role for WT1 during gonad formation occurs at the level of sex determination, through its control over Sry expression. Other potential targets during gonad formation include Wnt-4 13, Dax1 14 and AMH, all of which seem to be activated by WT1(-KTS) variants. Dax1, a gene that can interfere with male sexual development in mouse and man was proposed to be a repressor of the AMH promoter that could displace WT1 from its complex with Sf1. For more details see 15

Mutations in the WT1 gene have been identified in patients with renal disease , such as Wilms tumor and isolated diffuse mesangial sclerosis (IDMS), and complex diseases like WAGR syndrome, Frasier syndrome, Denys-Drash syndrome (DDS), and Meacham syndrome. The association of aniridia, hemihypertrophy, and other congenital anomalies with Wilms tumor was first described by Miller et al in 1964 16. The syndrome subsequently became known as the WAGR syndrome (Wilms tumor, Aniridia, Genitourinary anomalies, and mental Retardation syndrome). Aniridia is due to the PAX6 gene, whereas the other features are due to WT1 gene defects 17. Frasier syndrome is a rare disorder defined by 46, XY DSD and progressive glomerulopathy 18. Patients present with normal female external genitalia, streak gonads, and XY , and frequently develop gonadoblastoma. Wilms tumor is not a feature of the syndrome. Frasier syndrome is caused by a mutation in the donor splice site in intron 9 of WT1, with the predicted loss of the +KTS isoform 19. In contrast with Frasier syndrome, most individuals with Denys-Drash syndrome 20 21 have ambiguous genitalia or a female phenotype, an XY karyotype, and dysgenetic gonads. Renal symptoms are characterized by diffuse mesangial sclerosis, usually before the age of 1 year, and the patients frequently develop Wilms tumor. Pelletier identified mutations of various types in WT1 as the molecular cause of DDS 22. Meacham described two 46, XY individuals with a novel constellation of genital, cardiac, and pulmonary malformations. The genital abnormalities consisted of a true double vagina, retention of mullerian structures, and undervirilization of the external genitalia 23. Although phenotype and sometimes phenotype overlap with that of DDS patients 24, the Meacham syndrome patients do not have renal disease.

GATA4

GATA-4, a member of the GATA family of transcription factors is required for proper development of the heart. It is also present in the gonads and may be a regulator of gonadal gene expression. In mice of both sexes, GATA-4 specifically marked the developing somatic cell lineages (Sertoli in testis and granulosa in ovary) but not primordial germ cells.

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Interestingly, abundant GATA-4 expression was maintained in Sertoli cells throughout embryonic development but was markedly down-regulated shortly after the histological differentiation of the ovary on embryonic day 13.5. This pattern of expression suggested that GATA-4 might be involved in early gonadal development and possibly sexual dimorphism 25

In humans, mutations in GATA4 have been related to atrial septal heart defects only.

2.2. TESTES

SRY

The discovery of Sry, the mammalian Y-chromosomal sex-determining gene, promised to answer many questions relating to the genetics and developmental biology of sex determination 26 27. The simple transgenesis experiment of adding Sry function to an XX mouse to induce male phenotype confirmed the master switch role of this gene 28. But since then, little else about this gene has proven to be simple or straightforward. The fact that Sry resides on the makes it vulnerable to degradation. As a result, it is minimally conserved and shows some functional flaws, surprising indeed for a gene on which survival and propagation of mammalian species so much depends. In the mouse embryo, Sry exhibits a tightly-controlled and limited spatiotemporal profile of expression in the precursors of Sertoli cells of the XY gonad 29-31. Early studies revealed that Sry is first expressed around 10.5 days post coitum (dpc), shortly after the emergence of the genital ridges, reaches peak levels of expression at 11.5 dpc, and is extinguished shortly after 12.5 dpc in mouse 32-34. SRY clearly throws a molecular switch to engage a male-specific cascade of molecular events, but continued expression of SRY is not required for these events to unfold. Despite almost 20 years of work since the discovery of SRY, little is known about its biochemical function. The HMG domain, a DNA-binding and DNA-bending motif, plays a central role, being the only region conserved between species and the site of almost all clinical mutations causing XY . It seems that SRY acts on a single gene, Sox9, the expression of which is then rapidly reinforced by positive regulatory loops. SOX9 then drives Sertoli cell formation and, therefore, testis differentiation. If SRY is absent or fails to act in time, Sox9 is silenced and development of the follicle cell and ovary ensues, with β-catenin being one of the crucial components driving this process.

Mutations in SRY are the cause of 46,XY DSD due to pure gonadal dysgenesis, originally described as XY, female with gonadal dysgenesis 35.

SOX9

Several lines of evidence indicate that Sox9 is the best candidate for a direct SRY target gene 30 31 36. First, Sox9 expression is strongly upregulated soon after the expression of SRY begins, whereas it is downregulated in the ovary. Second, cell-fate mapping experiments show that SRY-positive cells exclusively become SOX9-positive Sertoli cells. Third, heterozygous mutations in SOX9 are responsible for the human skeletal malformation syndrome, campomelic dysplasia in which most XY patients have male to female sex

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reversal. Similarly, targeted Sox9 ablation in mice also leads to ovary development in XY embryos 37 38 However, the expression of Sox9 in the genital ridge is not entirely dependent on SRY and the normal gonadal Sox9 transcriptional regulation consists of a SRY-independent initiation a SRY-dependent upregulation; and SRY-independent maintenance in adult life . SF1 is a good candidate for initiating or sensitizing Sox9 expression because early sex- independent expression of Sox9 is abolished in Sf1 null mutant gonads 30 39. SF1 sensitizes Sox9, initiating a low level of expression in the genital ridge of both sexes at 10.5 dpc. In the male, SF1 (probably with other factors such as WT1−KTS) also activates Sry expression. Sox9 expression is upregulated by the action of SRY together with SF1, whereas it is downregulated in the female. This downregulation is unlikely to be passive, implying the presence of one or more currently unknown repressors. After the transient expression of Sry has ceased, high levels of SOX9 are maintained by its direct autoregulation and via FGF9 signaling (Fig. 4).

As mentioned, mutations in SOX9 lead to 46,XY DSD and skeletal abnormalities known as camptomelic dysplasia.

SF1/NR5A1

Human SF-1/NR5A1 is a 461 amino acid protein that shares structural homology with other members of the nuclear receptor superfamily (www.nursa.org). Critical functional domains of this protein include: an amino-terminal 2 zinc finger DNA-binding domain (DBD), an accessory DNA-binding region, a hinge region, and a ligand-binding domain (LBD) that forms an AF-2 structure. The expression pattern of SF-1 is consistent with its central role in regulating adrenal development, gonad determination and differentiation, and in the hypothalamic-pituitary control of reproduction and metabolism. In the mouse, Sf-1 is expressed in the early adrenogonadal primordium from 9 dpc and, thereafter, in the developing and gonad 40-42. In humans, SF-1 expression has been shown in the developing adrenal gland and bipotential gonad at 32-33 days post conception 43 44. Following testis determination (from around 42 days onwards in humans), SF-1 expression is consistently maintained in the somatic cells of the early testis, where it may play a crucial role together with SRY in supporting SOX9 expression 36. In Sertoli cells, SF-1 activates expression of AMH from around 7 weeks gestation, which leads to the regression of Müllerian structures in the developing male fetus. In Leydig cells, SF-1 activates the expression of steroidogenic enzyme systems from 8 weeks gestation, which results in androgenization of the external genitalia. In females, persistent expression of SF-1 has been reported in early ovarian development in humans 44, whereas Sf-1 expression may decline in the mouse. However, SF-1 is detectable in somatic cells (granulosa and theca cells) of the adult ovary 45. The exact role SF-1 plays in the ovary is unclear, as the related nuclear receptor LRH-1 (NR5A2) may also have a role in regulating aromatization and oestrogen synthesis. In addition to its expression in the adrenal gland and gonad, SF-1 is also expressed in the developing ventromedial hypothalamus (VMH), pituitary gonadotropes, and spleen 46-48.

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Deletion of the gene (Nr5a1) encoding Sf-1 in XY mice results in impaired adrenal development, complete testicular dysgenesis with Müllerian structures, and female external genitalia.

These studies in mice prompted the search for the human counterpart. Initial studies to identify SF1/NR5A1 mutations in humans focused on patients with primary adrenal failure, 46,XY gonadal dysgenesis and Müllerian structures. The first prismatic case was identified by Achermann et al in 1999 in a patient with such phenotype 49. Mutation in the human SF1/NR5A1 gene has a phenotypic spectrum that ranges from complete testicular dysgenesis with Müllerian structures, through individuals with mild clitoromegaly or genital ambiguity, to severe penoscrotal hypospadias or even anorchia 50. Although not essential for ovarian determination 51, heterozygote mutations in NR5A1 have recently been linked to ovarian insufficiency 52.

DAX1/NR0B1

DAX1 encodes a member of the orphan nuclear (NHR) family of transcriptional regulators. The C-terminal domain shows homology to the ligand-binding domain (LBD) of related NHRs, although a ligand remains unknown. The N-terminal domain represents a novel domain comprising 3.5 repeats of a 65–67-amino acid motif containing two putative zinc fingers, and possibly defining a nucleic acid-binding domain. DAX1 has a transcriptional silencing domain which has been proposed to interact directly with corepressors to mediate repression 53. At least two corepressors, Alien and NCoR, interact in vitro with the DAX1 C-terminus 54 55, one of which (Alien) is expressed in the testis 56. In cultured cells, DAX1 represses Sf1-mediated transcriptional activation of steroidogenic genes, such as Cyp11A, Cyp17 and Cyp19 essential for the synthesis of and , it interacts with the AF2 domain of ligand-bound receptors, ERα and ERβ and repress ER-mediated activation of target reporter genes. It is probable that DAX1 and SF1 interact during gonadogenesis in a similar manner to that seen during steroidogenesis.. Together, these studies suggest a common function for DAX1 as a repressor of SF1 trans- activation in both steroidal and gonadal tissues.

Inactivating mutations in DAX1 (NR0B1) can lead to adrenal insufficiency with glucocorticoid and mineralocorticoid deficiency 57. These mutations are responsible for many, but not all, patients with the cytomegalic form of adrenal hypoplasia congenita (AHC). These mutations also cause hypogonadotropic (HH) which is consistent with the expression of DAX1 in the hypothalamus and pituitary. Duplications of the region of the X chromosome containing DAX1 cause dosage sensitive sex reversal 57 58. Transgenic XY mice with additional copies of the mouse DAX1 ortholog expressed at high levels do not have male to female sex reversal, but do show delayed testicular development 59.

DMRT1

Raymond and co-workers 60 isolated the male sexual regulatory gene mab3 61 from the nematode Caenorhabditis elegans and found that it is related to the Drosophila melanogaster sexual regulatory gene 'doublesex' (dsx) 62. Both genes encode proteins with a DNA-binding motif that Raymond et al. referred to as the 'DM domain.' The same authors identified a

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human gene, which they designated DMT1, that encodes a protein with a DM domain and found that DMT1 is expressed only in testis.

Muroya et al. 63 reported clinical and molecular findings in five 46, XY and one 46, XX patients with distal 9p . Some of the XY and the XX subjects had female external genitalia, one case showed ambiguous external genitalia, and another exhibited male external genitalia with left cryptorchidism and right intrascrotal testis. Gonadal explorations at gonadectomy revealed the presence of various morphologies, from streak gonad and right agonadism, to hypoplastic testes.

DHH

In an animal model Bitgood and collaborators 64 found that male Desert Hedgehog (Dhh)-null mice were sterile and failed to produce mature spermatozoa, and that the peripheral nerves of Dhh-null mutant mice were highly abnormal. The perineurial sheaths surrounding the nerve fascicles were abnormally thin, and extensive minifascicles consisting of perineurial-like cells were formed within the endoneurium. In a patient with 46,XY partial gonadal dysgenesis (PGD) associated with minifascicular polyneuropathy, Umehara et al. 65 found a homozygous missense mutation at the initiating codon in exon 1 of the DHH gene , which predicted a failure of translation.

ATRX

The protein encoded by this gene contains an ATPase/helicase domain, and thus it belongs to the SWI/SNF family of chromatin remodeling proteins. XY patients with deletions or mutations in the ATRX gene display varying degrees of sex reversal, implicating ATRX in the development of the human testis 66. To explore further the role of ATRX in mammalian sex differentiation, Pask et al. 67 cloned and characterized the homologous gene in a marsupial. To their surprise, active homologs of ATRX were detected on the marsupial Y as well as the X chromosome. The Y-borne copy (ATRY) displayed testis-specific expression. This, as well as the sex reversal of ATRX patients, suggested that ATRY is involved in testis development in marsupials and may represent an ancestral testis- determining mechanism that predated the evolution of SRY as the primary mammalian male sex-determining gene. The authors found no evidence for a Y-borne ATRX homolog in mouse or human, implying that this gene has been lost in eutherians and its role supplanted by the evolution of SRY from SOX3 as the dominant determiner of male differentiation. The range of genital phenotypes caused by ATRX mutations together with the observation that ATRX patients always have some testicular tissue, suggest that ATRX functions in sex differentiation rather than sex determination in humans 68.

CBX2/M33

When Katoh-Fukui and coworkers ablated the M33 gene in mice, they observed sex reversal in XY animals 69. M33 is a member of the Polycomb(PcG)/Trithorax (TrxG) proteins. Polycomb group (PcG) proteins are highly conserved regulatory factors that were initially discovered in Drosophila. PcG genes are best known for their role in maintaining silent expression states of Hox genes during development, while trithorax group (trxG) proteins

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maintain. They act by regulating chromatin structure and chromosome architecture at their target loci (for more informations http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html).

M33 deficient mice have male-to-female sex reversal with perfectly normal female gonads and genitalia in 50% of the cases, but are invariably sterile. Similarly, we made the intriguing discovery of a loss-of-function double heterozygote mutation state in a 46,XY girl with normal ovaries at histology, normal uterus and external female genitalia, accidentally diagnosed because of a discrepancy between prenatal karyotype and phenotype at birth. Functional studies demonstrated that the mutated CBX2 does not properly bind to and does not adequately regulate the expression of target genes essential for sex development such as SF1/NR5A1. Our data identify CBX2 as essential for normal human male gonadal development , suggest that it lies upstream of SRY in the human sex development cascade and identify a novel autosomal recessive cause of defect of sex development 70.

TSPYL1

Testis-specific protein Y-like-1 is a member of the TSPY-SET-NAP1L1 family of chromatin modifiers 71 that are involved in nucleosome assembly 72. Besides being linked to beta globin regulation 73, mutations in TSPYL1 were found by Puffenberger e co-workers in a previously unrecognized syndrome called Sudden Infant Death with Dysgenesis of the Testes (SIDDT). Genotypic males with SIDDT had fetal testicular dysgenesis and ambiguous genitalia, with findings such as intraabdominal dysplastic testes, deficient fetal testosterone production, fusion and rugation of the gonadal sac, and partial development of the penile shaft. 46,XX sexual development was normal. Affected infants appeared normal at birth, but developed signs of visceroautonomic dysfunction early in life followed by death before age 12 months of abrupt cardiorespiratory distress 74 75. Interestingly, cases of sudden infant death without testicualr dysgenesis do not have mutations in TSPYL1 75, suggesting the testis-specific role of this protein.

MAMLD1/CXorf6

In the course of identifying the gene responsible for X-linked myotubular myopathy (MTM1), which maps to proximal Xq28, Laporte et al. (1997) identified a novel gene, Mastermind-Like Domain-Containing Protein 1 (MAMLD1, also called Chromosome X Open Reading Frame 6 :CXORF6, F18) 76, expressed in several tissues including fetal Sertoli and Leydig cells around the critical period for sex development (E12.5-E14.5) . Most patients with of this region of chromosome X also had ambiguous genitalia. The proof of the importance of MAMLD1 came when Fukami et al performed direct sequencing of the coding exons 3-6 of the CXORF6 gene and their flanking splice sites in 166 individuals with various types of 46,XY disorders of sex development. They identified 3 nonsense mutations in 4 Japanese individuals with penoscrotal hypospadias as the most evident phenotype 77. Two years later, again Fukumi et al demonstrated that MAMLD1 acts as a transcritpion factor by transactivating the promoter if a non canonical Notch target gene hairy/enhancer of split 3 (Hes3), auguments testosterone production, most likely under the regulation of SF1.78

More recently, a gain-of-function mutation in MAMDL1 was described in a 46,XX DSD woman with virilization (clitoromegaly, streak gonads, small uterus) 79, suggesting a role of this protein in ovarian development.

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PGD2S

Lipocalin-type prostaglandin D2 synthase (L-PGDS) and hematopoietic prostaglandin D2 synthase (H-PGDS) are two enzymes expressed in the testes and involved in the nuclear translocation and expression of SOX9, a central factor in testis determination during foetal life. Knockout of L- and H-PGDS in male mice induces delayed testicular organisation and a uni- or bilateral testis migration defect.

Wilhelm et al found that Pgds was expressed in embryonic mouse Sertoli cells immediately after the onset of Sry and Sox9 expression. Pgds upregulation was mediated by Sox9, but not Sry, and required the binding of dimeric Sox9 to a paired SOX recognition site within the Pgds 5-prime flanking region 80.

The process of testicular descent is not fully understood, but several factors, such as Hoxa-10, epidermal growth factor (EGF) and calcitonin gene-related peptide (CGRP), together with androgens and insulin-like hormone 3 (Insl3), have been suggested to regulate it. Philibert et al an H-PGDS gene (but not L-PGDS) abnormality was identified in one boy referred to our pediatric endocrinology clinic at 15 months with bilateral cryptorchidism and severe growth retardation (-4DS). Functional analysis of the mutations showed about a 40% decrease in glutathione transferase activity (indispensable for PGDS activity) and total prostaglandin D2 synthase abolition. Taken together, these data suggested that H-PGDS defect can be responsible for cryptorchidism in human 81.

2.3. OVARIES

RSPO1

Kamata et al 82 identified the mouse R-spondin gene, which encodes a 265-amino acid sequence with a calculated molecular mass of 29 kD. Unlike other secretory proteins of the thrombospondin family, R-spondin contains no apparent secretion cleavage site, but has a putative N-terminal nuclear localization signal. R-spondin transcripts were observed mainly in central nervous system (CNS) tissues. Outside the CNS, R-spondin transcripts were detected in forelimb buds after 10 dpc, especially in the body trunk and the proximal posterior part of the anterior limb bud. Kim and co-workers found that human R-spondin is expressed in enteroendocrine cells as well as in epithelial cells from various tissues 83. In mice, the ovarian phenotype of XX, Rspo1-/- is striklingly similar to that of Wnt4 -/- female mice, especially for the formation of coelomic vessels and the presence of functional steroidogenic cells. These animal models suggest that the ovarian phenotype in Rspo1-/- mice is at least in part due to failing overexpression of Wnt4 in XX gonads. In contrast Rspo1 -/- female mice do not show adrenal or uterine abnormalities, as Wnt4 -/- mice do, in agreement to the fact that Rspo1 is not expressed in adrenals or mesonephros, indicating that activation of Wnt4 in these organs is independent of Rspo1 (For review see 84 ).

The essential role of R-spondin 1 in human ovarian development was demonstrated by studyind individuals with palmoplantar hyperkeratosis with squamous cell carcinoma of skin

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and 46 XX, DSD in whom Parma et al. demonstrated mutations in the RSPO1 gene 85. The authors concluded that RSPO1 is produced and secreted by fibroblasts and regulates keratinocyte proliferation and differentiation. The presence of 'functional' testes in the sex- reversed individuals was confirmed by the absence of mullerian derivatives and by the masculinization of the internal and external genitalia, presumably induced by functioning Sertoli and Leydig cells, respectively. All sex-reversed individuals were sterile. Notably, the data suggested that normal RSPO1 is not required for testis differentiation and function. Parma et al speculated that RSPO1 may synergize with WNT4 in XX gonads to stabilize beta-catenin 85.

WNT4

WNT4 is a member of the WNT family of secreted molecules that function in a paracrine manner to affect a number of developmental changes. WNT proteins bind to members of the Frizzled (FZ) family of cell-surface receptors and possibly to the single-pass transmembrane protein LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6) 86. The binding of WNT to FZ ultimately leads to reduced degradation of -catenin with consequent β-catenin- dependent activation of T-cell factor/lymphocyte enhancer factor transcription factors, and induction of Wnt-responsive genes 87. Wnt proteins can also signal via a β -catenin independent non-canonical pathway involving protein kinase C (PKC) and c-jun NH2- terminal kinase (JNK) 88. (Readers interested in learning more about WNT proteins should visit the informative site http://www.stanford.edu/~rnusse/wntwindow.html). Wnt4 is produced in ovarian somatic cells (pre-granulosa cells). Wnt4 up-regulates Dax189, a gene known to antagonize SF1, and thereby inhibits steroidogenic enzymes. Vainio and collaborators 90 observed in Wnt4-deficient mice that gonadal development and steroidogenic function were affected exclusively in female Wnt4-knockout mice, whereas both male and female mice had similar defects in development and adrenal function. Wnt4-knockout female mice were masculinized, as demonstrated by the absence of Müllerian ducts and the presence of Wolffian ducts, and expressed the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase, which are required for the production of testosterone and are normally suppressed in the developing female ovary. The ovaries of these mice also had less oocytes, suggesting a role of Wnt4 in the life of female germ cells. This function is crucial for the organization of ovarian structure, since female germ cells have a central role in this process and in maintenance of the ovary, as demonstrated by the fact that when oocytes are either absent 91 or lost after follicle formation 92 ovarian follicles never form or degenerate subsequently. In contrast, testis development proceeds in the absence of germ cells. WNT4 and follicostatin 93 appear to maintain oocyte viability once germ cells have reached their final destination in the gonad.

In humans, more copies of WNT4, due to duplication of chromosome 1p31-p35, were found in a patient with ambiguous genitalia, severe hypospadia, fibrous gonads and remnants of both Müllerian and Wolffian ducts 89, i.e. male-to-female sex reversal. On the other end of the spectrum, when both copies of the gene are inactive, as in the case of homozygote mutations, a severe clinical entity, called the SERKAL syndrome results 94. The syndrome was described in three 46,XX fetuses and is characterized by female-to-male sex reversal with ambiguous genitalia, gonadal morphology ranging from ovotestis to normal testis, renal agenesis, adrenal hypoplasia, pulmonar and cardiac abnormalities.

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In the middle of such spectrum one might expect to find patients with intermediate defects of sex development. Searches for clinically relevant WNT4 mutations sometimes in large cohorts of these patients were unsuccessful 95. We described three women with absent Müllerian structures (uterine and fallopian tubes) and clinical signs of excess. Their phenotype resembles that of patients with the Mayer–Rokitansky–Küster–Hauser syndrome and is also strikingly similar to that of Wnt4-knockout female mice. This constellation of findings prompted us to search for and allowed us to find mutations in the WNT4 gene in these patients 96-99.

From a more clinical point of view, we agree with Clement-Ziza 100 that the WNT4 mutation should not be looked for in MRKH syndrome. However, MRKH-like syndrome associated with should be investigated for mutations in this gene, and may classify for a distinct clinical entity.

FOXL2

FOXL2 is a single-exon gene encoding a forkhead/winged helix (fkh) transcription factor 101. FOXL2 is a nuclear protein. In vertebrates, FOXL2 is one of the earliest known markers of ovarian differentiation 102. Thus, it may play a role in the early stage of development of the ovarian somatic compartment. As it is still strongly expressed in postnatal and adult follicular cells, it may also play a role in follicle development and/or maintenance during fertile life. Despite the importance of FOXL2 in ovarian development and maintenance, only a few transcriptional targets have been described so far 103 104 In the context of the pituitary, FOXL2 seems to stimulate the expression of the gonadotropin-releasing hormone (GnRH) receptor. Foxl2 influence the expression of aGSU, StAR and several steroidogenic enzymes.

Mutations in FOXL2 are responsible for blepharophimosis–ptosis–epicanthus inversus syndrome (BPES), which is a genetic disease leading to complex eyelid malformation and other mild craniofacial abnormalities and can be associated with premature ovarian failure 101 105

Genes and proteins responsible for maintanance of ovarian function, such as BMP15, GDF9 and NOBOX, whose mutations cause premature ovarian insufficiency, will be discussed later in this chapter.

2.4. MÜLLERIAN DUCTS

AMH

Anti-Mullerian hormone (AMH) produced by fetal Sertoli cells is responsible for regression of Mullerian ducts, the anlage for uterus and Fallopian tubes, during male sex differentiation. A member of the transforming growth factor-beta superfamily, AMH signals through two transmembrane receptors, type II which is specific and type I receptors, shared with the bone morphogenetic protein family.

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Mutations of the AMH and AMH receptor type II (AMHR-II) genes lead to persistence of the uterus and Fallopian tubes in males 106-109

HOXA

HOX genes are a family of regulatory molecules that encode conserved transcription factors controlling aspects of morphogenesis and cell differentiation during normal embryonic development. HOXA Hox genes are also expressed in the adult uterus. Hox genes are essential both for the development of müllerian tract in the embryonic period and adult function. Sex steroids regulate Hox gene expression during embryonic and endometrial development in the . EMX2 and β3- acting downstream of Hoxa10 gene are likely involved in both these developmental processes 110 111

The role of WNT4 and EMX2 in sex development has been discussed above .

Lim1/LHX1

Lim1 encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development. In the developing urogenital system, Lim1 expression has been documented in the Wolffian (mesonephric) duct, the mesonephros, metanephros and fetal gonads. Using, a Lim1 lacZ knock-in allele in mice, we identified a previously unreported urogenital tissue for Lim1 expression, the epithelium of the developing Müllerian duct that gives rise to the oviduct, uterus and upper region of the vagina of the female reproductive tract. Lim1 expression in the Müllerian duct is dynamic, corresponding to its formation and differentiation in females and regression in males. Although female Lim1-null neonates had ovaries they lacked a uterus and oviducts. A novel female mouse chimera assay was developed and revealed that Lim1 is required cell autonomously for Müllerian duct epithelium formation. These studies demonstrate an essential role for Lim1 in female reproductive tract development 112.

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3. GONADAL DYSGENESIS

Gonadal dysgenesis is characterized by a progressive loss of primordial germ cells on the developing gonads of an embryo. This loss leads to extremely hypoplastic and dysfunctioning gonads mainly composed of fibrous tissue, hence the name streak gonads.

Although most etiologies of gonadal dysgenesis have been already described in previous sections of this chapter, some causes merit a special mention.

A. Chromosomal abnormalities

1. , pure gonadal dysgenesis. 45,X0, variants and mosaics

The syndrome is named after Henry Turner, an Oklahoma endocrinologist, who described it in 1938 113. It is a chromosomal disorder in which all or part of one of the X chromosome is absent. In some cases, the missing chromosome is present in some cells but not others, a condition referred to as mosaicism. There are characteristic physical abnormalities, such as short stature, swelling, broad chest, low hairline, low-set ears, and webbed necks. Girls with Turner syndrome typically experience gonadal dysgenesis , which results in and sterility. Concurrent health concerns are also frequently present, including congenital heart disease, hypothyroidism , diabetes, vision problems, hearing concerns, and many other autoimmune diseases. Finally, a specific pattern of cognitive deficits is often observed, with particular difficulties in visuospatial, mathematical, and memory areas. A detailed review of such a complex disease is beyond the purpose of this chapter and can be read for instance in 114.

2. , seminiferous tubules dysgenesis. 47, XXY, variants and mosaics

The syndrome was named after Harry Klinefelter, an endocrinologist at Massachusetts General Hospital in Boston, Massachusetts, who first described it in 1942 115. The principal effects are development of small testicles and reduced fertility . Some degree of language learning impairment may be present, and neuropsychological testing often reveals deficits in executive functions. In adults, possible characteristics vary widely and include little to no signs of affectedness, a lanky, youthful build and facial appearance, or a rounded body type with some degree of gynecomastia. Some variations e.g. (XXYY) and mosaicism are possible with different severity of symptoms. For more details see 116 117 118.

3. Mixed gonadal dysgenesis and ovostesticular DSD (true hermaphroditism).

Mixed gonadal dysgenesis: some authors reserve the term mixed gonadal dysgenesis (MGD) for individuals who present a 45,X/46,XY karyotype with a testis on one side and a streak on the other side ( 119 and references therein); other authors apply the term to all patients who have varying degrees of asymmetrical gonadal dysgenesis with testicular differentiation on either side, bilateral streak testis, or bilateral dysgenetic testis . Ovotesticular DSD (OT-DSD or true hermaphroditism) is the rarest form of intersexuality in humans, and the term is

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applied to an individual who has both well-developed ovarian and testicular tissues. The differentiation of OT-DSD from MGD is important for three reasons. First, a bilateral gonadectomy is recommended as soon as possible in all individuals with MGD containing Y- chromosome material. There is no benefit in delaying surgery because approximately one third of patients with MGD develop gonadoblastoma during the 1st to 4th decades of life and because 30% of gonadoblastomas are overgrown by more malignant tumors, such as germinoma, endodermal sinus tumor, immature teratoma, embryonal carcinoma, or choriocarcinoma . In OT-DSD, however, only the removal of the opposite gonad from the assigned gender and a biopsy of remaining gonadal tissue for histological evaluation may be appropriate. Second, gender assignment is critical for the treatment of patients of OT-DSD because these patients usually do not have any other developmental malformations, and thus normal sexual and reproductive functions can be achieved by proper management and sex assignment at a young age . After the removal of the testicular portion of an ovotestis, about 38% of these patients with a 46,XX karyotype menstruate by age 14 years 120; ovulation 121 and successful childbearing have also been described 122 123. In contrast, it is important that bilateral gonadectomies be performed in patients with MGD before the patient reaches , not only to prevent the development of malignant tumors but also to avoid virilization if the patient is to be raised as female. Third, certain medical problems, such as deficient immunoglobulin levels, aberrant bony development of inner ear structures, and cardiovascular and renal anomalies, are more common in MGD patients than in OT-DSD patients; therefore, patients with MGD should receive more medical attention. For a differential diagnosis, chromosomal constitutions are not helpful. Various types of chromosomal abnormalities have been described in cases of OT-DSD, such as 46,XX; 46,XY; 46,XX/46,XY; 45X/46,XY; and others. MGD cases also have heterogeneous chromosomal changes but are usually characterized by a 45,X/46,XY; 45,X/47,XYY; or 46,XY karyotype 124 . The pathogenesis of OT-DSD and MGD still remains unclear. In cases of 46,XX true hermaphroditism, mosaicism with a Y-bearing cell line at low level 125 or partial inactivation of the Y-bearing X chromosome that was formed during paternal meiosis 126 127 have been suggested to explain the mechanism of abnormal sexual development. In cases of MGD, mutations in SRY genes were usually absent 128; however the cytogenetic mosaicism, mutation in the genes downstream of SRY, and unpaired or incompletely paired X chromosome have been proposed as a mechanism to cause defective formation of the follicular mantle and degeneration of oocytes. Histopathologically, there are no major differences in the testicular part of the gonads between the two entities. On the other hand a rise of testosterone levels after hCG is observed exclusively in cases of OT-DSD and will not occur in MGD patients. The presence of numerous primordial follicles containing primary oocytes with or without maturing follicles is considered well-developed ovarian tissue when making a definitive diagnosis of OT-DSD after birth. For more extensive review see 119.

B. Single gene defects

B.1. 46, XX

Besides Section 2.3. and Table 1, an additional form of gonadal dysgenesis in women is Premature Ovarian Failure (POF), defined as at age less than 40 years.

POF1 is associated to premutations (mutations that do not cause symptoms and needs a second mutation to become „full“) in Fragile X Mental Retardation 1 (FMR1) gene (Xq26-

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q28) 129. In an international collaborative study of 760 women from fragile X families, 130found that 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. In 63 (16%) of the premutation carriers, menopause occurred before the age of 40, compared with none of the full-mutation carriers and 1 (0.4%) of the controls, indicating a significant association between premature menopause and premutation carrier status.

POF2A is caused by mutations in the Drosophila Diaphanous (dia) homolog 2 (DIAPH2, Xq22). In the fruit fly mutant alleles of dia affect spermatogenesis or oogenesis and lead to sterility. Bione et al 131 identified DIAPH2, which they called DIA, as the gene disrupted by a breakpoint in a family with premature ovarian failure and stated that DIAPH2 is the first human member of the FH1/FH2 protein family. Members of this family affect cytokinesis and other actin-mediated morphogenetic processes that are required in early steps of development.

POF2B. Bione et al 131considered the POF1B gene (Xq21) a candidate for premature ovarian failure because it is located within the critical region for normal ovarian function, escapes X inactivation, and a breakpoint in the third intron was identified in a POF patient with an X;1 translocation 132 but they could not demonstrate any mutation in POF1B in their POF patients. In the same work they determined that Pof1B expression is absent from adult mouse ovary but is present between embryonic day 16.5 and postnatal day 5, suggesting that the gene may be critical for the steps required for early ovary development leading to the final number of germ cells. Based on its expression pattern in mouse development Bione et al also hypothesized that POF1B could play a role in the pairing of meiotic chromosomes and that alteration in its function could lead to cell death and a drastic reduction in the final number of oocytes created during ovarian development. Alternatively, POF1B may act as an antiapoptosis factor, to slow down the process of germ cell loss; as a consequence, loss of function of POF1B could lead to exaggerated germ cell apoptosis and POF. In 2006 Lacombe et al. 133 identified a G-to-A transition at nucleotide 1132 in exon 10 of the POF1B gene that resulted in an arg329-to-gln (R329Q) mutation in a Lebanese family in which premature ovarian failure mapped to the Xq21.1-q21.33. Affected family members were homozygous. The R329Q variant altered the arginine within an amino acid triplet, serine-leucine-arginine, that is part of a protein kinase C phosphorylation recognition site.

POF3 is caused by mutations in FOXL2 (see above)

POF4 is associated with defects of Bone Morphogenic Protein 15 (BMP15; Xp11.2). This entity is also known as Ovarian Dysgenesis 2 (ODG2). The bone morphogenetic protein (BMP) family is part of the transforming growth factor-beta superfamily, which includes large families of growth and differentiation factors. By Northern blot analysis, Dube et al 134 showed that mouse Bmp15 is expressed only in the ovary and precisely in the oocyte soon after primordial follicles are recruited, and that expression is maintained until after ovulation. Studies by Aaltonen and coworkers 135 showed that in human ovary BMP15 (which they called GDF9B) is expressed in the oocytes of late primary follicle. Otsuka et al 136 showed BMP15 expression in oocytes throughout follicular development. The importance of BMP15 for human disease was firstly proven by Di Pasquale et al 137 who reported an Italian family in which 2 sisters had hypergonadotropic ovarian failure due to ovarian dysgenesis and a dominant negative mutation in BMP15. Later reports confirmed the role of BMP15 in POF 138 139.

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POF5. Newborn Ovary Homeobox (7q35) NOBOX is a homeobox gene that is preferentially expressed in oocytes. In mice, it is essential for folliculogenesis and regulation of oocyte-specific genes 140. In a 35-year-old Caucasian woman with premature ovarian failure Qin et al 141 identified heterozygosity for a 1064G-A transition in exon 6 of the NOBOX gene, resulting in an arg355-to-his (R355H) substitution within the highly conserved homeodomain region.

POF6. Factor in Germ Line Alpha (FIGLA, 2p12) is a helix-loop-helix transcription factor expressed in all ovarian follicular stages and in mature oocytes, with less frequent expression in preimplantation embryos. Zhao and co-workers 142 analyzed the FIGLA gene in 100 Chinese women with premature ovarian failure and identified a 22-bp deletion and a 3-bp in- frame deletion in 2 patients, respectively, that were not found in controls.

POF7 is related to mutations in SF1/NR5A1 (see above).

Ovarian DysGenesis (ODG1). Hypergonadotropic ovarian dysgenesis with normal karyotype can be caused by mutations in the gene encoding follicle-stimulating hormone receptor (FSHR, 2p21-p26). Elliott et al 143 reported the condition in 3 sisters who had normal stature and sex chromatin but had never menstruated and had severe osteoporosis. Somatic features of Turner syndrome were not found. In the Finnish population in which ovarian dysgenesis with normal XX karyotype is relatively common (approximately 1 in 8,300 females), Aittomaki et al 144 demonstrated that affected females are homozygous for a C-to-T transition in exon 7 of the FSHR gene, predicting an ala189-to-val substitution in the extracellular ligand-binding domain of the protein.

FSHR mutations also appear to play a role in Ovarian Hyperstimulation Syndrome. Greb and collaborators 145 showed that the FSH receptor ser680/ser680 genotype was associated with higher ovarian threshold to FSH, decreased negative feedback of luteal secretion to the pituitary during the intercycle transition, and longer menstrual cycles.

Syndrome-associated ODG:

Perrault syndrome is characterized by ovarian dysgenesis and sensorineural deafness 146 147. Although the exact genetic cause of the disease remains unknown, there is evidence for an autosomal recessive mode of inheritance 147.

Immunodeficiency, pulmonary fibrosis, ODG. Somech et al 148described 2 sisters, born of first-cousin parents of Sri Lankan descent, who presented in infancy with immunodeficiency, gonadal dysgenesis, and fatal pulmonary fibrosis. The infants displayed no dysmorphic features. Immune studies demonstrated combined humoral and cellular abnormalities including reduced immunoglobulin production, absence of lymphoid tissue, markedly reduced T lymphocyte numbers and function, and reduced newly thymus-derived T cells. Both infants succumbed to rapidly progressive pulmonary fibrosis; autopsy revealed streak ovaries bearing no discernible oocytes. The molecular basis of the disease is not known.

Mitochondrial dysfunction. Two sisters were described by Hisama et al 149 with hypergonadotropic hypogonadism, marked short stature, and recurrent episodes of dehydration with metabolic acidosis. Both patients had normal intelligence and normal 46,XX karyotypes. Laboratory studies suggested mitochondrial dysfunction; however, results

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of mitochondrial DNA studies and of biochemical analysis of electron transport chain activity in skeletal muscle were normal.

B.2. 46, XY

Besides Section 2.2. and Table 1 Testes, an entity called alopecia universalis, XY gonadal dysgenesis with ambiguous genitalia with an XY karyotype and SRY positivity and laryngomalacia was described. El-Shanti et al. 150 reported 5 individuals from 2 unrelated families from Jordan with alopecia universalis congenita, gonadal dysgenesis, and laryngomalacia that persisted beyond infancy. An additional feature of this entity was growth delay due to growth hormone deficiency.

C. Environmental

Testicular dysgenesis syndrome. The term testicular dysgenesis syndrome (TDS) was first coined in 2001 151. It was hypothesized that many cases of abnormal spermatogenesis, cryptorchidism, hypospadias, and testicular cancer may have a common aetiology, and that all these clinical problems may result from an irreversible developmental disorder originating in early fetal life. Etiological factors that explain how TDS may arise include genetic defects (mosaicisms for sex chromosome ) and polymorphism (still unclear), environmental and lifestyle factors, and intrauterine growth disorders, which may act separately or in concert. Exposure to endocrine-disrupting compounds such as phthalates (mainly used as plasticizers), which act as anti-androgens, seem to be a central risk factors. Lifestyle such as maternal smoking during pregnancy 152 and increasing body mass index in young men also negatively influence testicular function 153. These factors lead to testicular dysgenesis with decreased Leydig cell and Sertoli cells function. Leydig cell deficiency causes androgen insufficiency and INSL3 decrease explaining cryptorchidism, hypospadia and impaired spermatogenesis. Sertoli cells dysfuntion contributes to the and to the onset of testicular cancer (for review see 154 ). Chapter 7 discusses this problem in greater details.

4. FACTORS INVOLVED IN SEX DIFFERENTIATION.

LH/CG RECEPTOR, STEROIDOGENIC ENZYMES, STEROID RECEPTORS

The role of these factors in sex development will be discussed in great detail in Chapters 4-6

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FIGURE LEGENDS

Figure 1: Embryonic development. GW: Gestational Week. Data derived from PC Sizonenko in Pediatric Endocrinology, edited by J. Bertrand, R. Rappaport, and PC Sizonenko, (Baltimore: Williams & Wilkins, 1993), pp. 88-99.

Figure 2: The undifferentiated sexual system at 6-7 week of gestation. Precursor structure and their mature counterparts have the same color. Fossum G. Atlas of Clinical Gynecology: Pediatric and Adolescent Gynecology. Edited by Morton Stenchever (series editor), Alvin F. Goldfarb.

Figure 3 : Sexual Development cascade. Mutations in various genes can lead to a variety of syndromes of dysgenesis involving the müllerian or wolffian ducts, gonads, kidneys, and adrenal glands as a result of a deficiency or excess of the proteins shown. For abbreviations see title page. Modified from McLaughlin and Donahoe, 2004. 155.

Figure 4: A time-line of events in mammalian sex determination (simplified). After the establishment of the bipotential genital ridge by several genes, the early expression of Sf1 and Wt1 initiates also that of Sox9 in both sexes. β-catenin also begin to accumulate as a response to Rspo1–Wnt4. In XX cells, β-catenin levels accumulate to repress SOX9 activity. In XY cells, with the contribuition of CBX2, increasing levels of SF1 activate Sry expression and then SRY, together with SF1, increase Sox9 expression. CBX2 influences directly SF1 expression and might do so indirectly by removing the DAX1 repression on SF1. Once SOX9 levels reach a critical threshold, several positive regulatory loops are initiated, including autoregulation of its own expression and formation of feed-forward loops via FGF9 or PGD2 signaling. If SRY activity is weak, low or late, it fails to boost Sox9 expression before β- catenin levels accumulate sufficiently to shut it down. At later stages, in XX gonads, FOXL2 increases and can maintain granulosa (follicle) cell differentiation by repressing Sox9 expression. In the testis, SOX9 promotes the testis pathway, including Amh activation, and it also probably represses ovarian genes, including Wnt4 and Foxl2. Solid lines : validated influence; broken line: possible influence.

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115. Klinefelter HJR, EC Jr; Albright (Syndrome characterized by gynecomastia, aspermatogenesis without a-Leydigism and increased excretion of follicle- stimulating hormone. J Clin Endocrinol Metab 1942;2:615–624.

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0

FIGURE 1 0 GW

Development of Wolffian ducts 4 Migration of germ cells 5 Migration of germ cells 6 Development of Müllerian ducts Differentiation of seminiferous tubules 7

Regression of Müllerian ducts. 8 Appearance of Leydig cells 9 1st meiotic prophase in oogonia

Masculinization of external genitalia begins 10 Wolffian ducts regression

Fetal testes in the internal inguinal rings 12-14 Penile urethra completed

Appearance of first spermatogonia 14

16 Appearance of first ovarian follicles

Max Leydig cells. Testosterone peak 17

Regression Leydig cells. Testosterone lower 20

1st multilayer ovarian follicles 24 Canalisation of the vagina

Descent of testes 28 Cessation of oogonia multiplication //

PC Sizonenko in Pediatric Endocrinology, edited by J. Bertrand, R. Rappaport, and PC Sizonenko 38 , (Baltimore: Williams & Wilkins, 1993), pp. 88-99.

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FIGURE 2

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FIGURE 3

Müllerian-duct-derived female reproductive tract WNT4 Steroidogenic enzymes HOXAs Lim1

Ovary Gastrulation Urogenital ridge Primitive streak Adrenal NOBOX BMP15 R-spondin1 FOXL2 GDF9 Emx2, GATA-4, SF-1 WNT4 FIGLA Fst FSHR DAX1 Lim1, Lhx9, WT1 CBX2 SRYSOX9 Notocord DHH Gonad DMRT1 Intermediate mesoderm ATRX Kidney LHR CXorf6 /MALMD1 TSPYL1 Testis Pax2 Steroidogenic enzymes Wolff-duct-derived Androgen receptor male reproductive tract AMH AMH Receptor

Modified from McLaughlin, NEJM 2004, 350: 367

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FIGURE 4

CBX2 DAX1 FGF9R Sry PGD Sf1 2 Sf1 Sertoli cells Testis Sox9 Sox9 Amh XY Sry WT1 β-cat SF1 Foxl2 Lhx9 GATA4 Rspo1 Wnt4 XX Sox9 Ovary Follicles Sox9 β-cat Foxl2 ER Wnt4 Aromatase Rspo1

TIME

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