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Sex determination and sex reversal Giovanna Camerino, Pietro Parma, Orietta Radi and Stella Valentini Sex determination in mammals is based on a genetic cascade that controls the fate of the gonads. Gonads will then direct the establishment of phenotypic sex through the production of hormones. Different types of sex reversal are expected to occur if mutations disrupt one of the three steps of gonadal differentiation: formation of the gonadal primordia, sex determination, and testis or ovary development. Addresses Dipartimento di Patologia Umana ed Ereditaria, Università di Pavia, Via Forlanini 14, 27100 Pavia, Italy Corresponding author: Camerino, Giovanna ([email protected]) Current Opinion in Genetics & Development 2006, 16:289–292 This review comes from a themed issue on Genetics of disease Edited by Andrea Ballabio, David Nelson and Steve Rozen Available online 2nd May 2006 0959-437X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2006.04.014 Introduction Gonadal sex determination and sex reversal: the general design In mammals, sex is genetically determined and is based on the chromosomal constitution of the embryo. Presence or absence of the Y chromosome, or of the Y-encoded SRY (sex-determining region on the Y chromosome) gene, determines the sex of the gonads: XY embryos develop testes, whereas XX embryos develop ovaries. In turn, the phenotypic sex of the embryo (i.e. the development of secondary sex characteristics, including internal and external genitalia) depends on the sex of the gonads. However, there is not a perfect evenness in the two pathways. Whereas functional testes are necessary for male differentiation of the embryo, female differentiation does not require the presence of functional ovaries. Testes produce hormones that are necessary and sufficient to bring about male differentiation in the embryo: these include the anti-Müllerian hormone (AMH), which induces regression of the female internal genitalia; the androgens testosterone and dihydrotestosterone, which direct the differentiation of the internal and external male genitalia; and insulin-like factor 3 (INSL3), which, together with testosterone, directs the descent of the testis into the scrotum. Conversely, sex hormones are not required for the ‘default’ female differentiation www.sciencedirect.com pathway. Thus, a phenotypic female will be formed if the gonads develop into ovaries but also if the gonads do not develop correctly (i.e. gonadal dysgenesis or gonadal agenesis) and if AMH and androgens do not reach the threshold level. Accordingly, XY sex-reversal (genetic males develop as females) in humans is relatively frequent (approximately 1 in 3000 newborns) and genetically heterogeneous, whereas XX sex-reversal (genetic females develop as males) is more rare (1 in 20000 newborn) and is usually caused by translocations of SRY to the X chromosome or to an autosome. Excellent reviews on sex determination and gonadal differentiation have been published [1,2,3,4]. Far from being comprehensive, this review only focuses on the process of gonadal sex determination and on the effects of its disruption on the establishment of the phenotypic sex of the embryo. The review is also very schematic; for example, we do not discuss ambiguous genitalia, generally caused by inadequate levels of testicular hormones. Formation of the gonadal primordia In the mouse, gonadal development begins at embryonic day 10.0 (E10), with the thickening of the coelomic epithelium adjacent to the ventro-medial surface of the mesonephros. Gonadal primordia are morphologically indistinguishable in male and female embryos. Primordial germ cells are specified in the epiblast and migrate to the developing gonads between E10 and E11 [5]. Loss-of-function mutations in a number of transcription factor genes (including Emx2, Lhx9, Sf1, Wt1–KTS, Pod1 and M33) result in the degeneration of gonads before the period of sex determination, suggesting that these proteins are required for the specification and maintenance of the gonadal primordia [1,2,3]. However, many of these genes have been shown to play important roles both in the development of other organs and in later stages of gonadal differentiation. If compatible with life, these mutations cause gonadal dysgenesis (or agenesis), and embryos develop a female phenotype irrespective of their chromosomal sex (i.e. XY sex-reversal). To date, only two of these transcription factors have been linked to an abnormal sexual development in humans. Heterozygous mutations in SF1 (Online Mendelian Inheritance in Man [OMIM] database, 184757) cause XY sex-reversal associated with adrenal failure. Different types of mutations in WT1 (OMIM, 607102) have been associated with various syndromes: WAGR, Denys– Drash, and Frasier, each characterized by different Current Opinion in Genetics & Development 2006, 16:289–292 290 Genetics and disease degrees of gonadal dysgenesis associated with kidney anomalies or predisposition to kidney tumors. Sex determination in XY gonads Testes begin to differentiate earlier than ovaries, suggesting that the initiation of the male pathway is an active process diverting the gonadal primordia towards testis fates before ovarian commitment. Although the male and female gonads are morphologically identical until around E12, the two differentiation pathways have previously started to branch out at the molecular level. In mice, Sry is switched on in the XY gonad a few hours after E10.5. Its expression peaks around E11.5 and is turned off shortly before E12.5. Little is known about the regulation of SRY transcription, although three factors, Gata4, Fog2 and Wt1(+KTS) have been implicated [1,2,3]. Sertoli cells, the supporting cell lineage of the testis, are responsible for AMH production and for supporting the proliferation and differentiation of germ cells. They are the first somatic cells of the gonads to be committed to a testicular fate and they play a fundamental role in the first stages of testis differentiation. Sry is expressed in Sertoli cell precursors, and — at least in mouse — must act during a very crucial time-window in order to properly activate male testis determination [6,7]. One of the first cellular events triggered by Sry expression is cellular proliferation in the XY coelomic epithelium, possibly to produce sufficient Sertoli cell precursors to initiate testis development [8]. Fgf9 and possibly IGF have been shown to play a role in this process [9,10]. At the molecular level, the molecular target(s) of SRY are still elusive, although several indirect observations suggest that the transcription factor Sox9 is one of these [6,11]. Low levels of Sox9 are expressed in XX and XY gonadal primordia. Once the SRY protein product reaches a threshold concentration level, Sox9 is up-regulated in XY gonads and, at around the same period, is downregulated in XX gonads [12]. Moreover, the Sox9 protein, which is cytoplasmic in both sexes prior to the onset of Sry expression, is translocated to the nucleus of XY preSertoli cells [13]. Sry, a protein involved in chromatin remodelling and transcription regulation, might act cellautonomously to trigger differentiation of Sertoli cells and expression of Sox9. However, historical XX $ XY chimera studies and more recent data revealed that not all Sox9-expressing Sertoli cell have experienced Sry expression, suggesting the existence of a paracrine signal — possibly prostaglandin D2 (PGD2) — driving the Sox9 expression in these cells [14,15]. This mode of Sertoli cells’ recruitment might serve to increase the number of supporting cells during testis development. PGD2 has also been shown to induce SOX9 nuclear translocation in NT2–D1 Sertoli-like cells [16]. Current Opinion in Genetics & Development 2006, 16:289–292 Sex determination in XX gonads In contrast to the situation in the male gonad, where dramatic morphological changes follow Sry expression, in the female gonad the first cellular event occurs at E13.5, when germ cells enter into meiosis. However, the femalespecific molecular program is activated as early as it is in the male gonad [17–19]. In a recent large-scale transcriptional analysis, 2306 genes were found to be expressed in a sex-specific manner in the somatic compartment of the gonads between E10.5 and E13.5: 1223 were overexpressed in XX embryos, and 1083 in XY embryos [17]. Mechanisms for ovarian commitment and early development remain elusive. Three genes, upregulated in XY gonads during the period of sex determination, have been proposed as candidate ovary-determining. Overexpression of DAX1 in the male gonads results in XY sex-reversal in both humans and mice. However, knockout and transgenic studies in mice indicate that Dax1 is not required for normal ovarian development but is crucial for testicular development [4]. Wnt4-null XX mice are masculinized as a result of the ectopic presence of androgen-producing cells and the lack of a Müllerian duct. Nevertheless, Wnt4 has been shown to be directly required for Müllerian duct development, and the androgen-producing cells express markers specific to adrenal steroidogenic cells. However, Wnt4 possesses some ’anti-male’ properties, because its expression appears to inhibit the migration of endothelial and steroidogenic cells into the developing ovary (see below) [4]. Finally, Pailhoux et al. [20] demonstrated that the deletion of an 11.7-kb DNA element is responsible for PIS syndrome, a XX sex-reversal condition in the goat. The deletion was shown to affect the transcription of at least 2 genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frames; and FOXL2. Mutations in FOXL2 in humans cause BPES syndrome (blepharophimosis ptosis epicanthus inversus syndrome), which is associated with premature ovarian failure [21]. Again, null mutations of Foxl2 in mice do not affect initial ovary formation, but lead to failure in primary follicle formation [22,23]. Sex determination and sex reversal The gonadal primordium is unique among all organs because it can develop in two different organs, a testis or an ovary. Defects in the establishment of the gonadal primordia or in the differentiation of the gonads after sex determination generally result in defective organ formation. By contrast, mutations affecting the sex determination process can result in real sex reversal (i.e. in redirecting a genetically female gonad to a testicular fate or vice versa). In theory, XX sex-reversal can be produced by the expression of testis-determining genes in XX gonads (i.e. gain-of-function mutations) or by loss-of-function mutations in ovary-determining genes. Approximately 85% of the cases of XX sex-reversal in man are due to www.sciencedirect.com Sex determination and sex reversal Camerino, Parma, Radi and Valentini 291 the translocation of the SRY gene to the X chromosome or to an autosome. SRY-independent XX sex-reversal is extremely rare and its molecular basis in man unknown. In the mouse model, XX sex-reversal has been artificially produced only by the ectopic expression of the testisdetermining genes Sry and Sox9 in the developing XX gonads [24,25,26,27]. As previously discussed, loss-offunction mutations in the three candidate ovary-determining genes, Dax1, Foxl2 and Wnt4, do not result in complete XX sex-reversal in mouse [22,23,28–33]. A heterozygous mutation in WNT4 has been associated with Müllerian duct regression and virilization in an XX patient [34]. Loss-of-function mutations in human SRY are characterized by gonadal dysgenesis after birth, and only account for approximately 10–15% of XY sex-reversal in man. Haploinsufficiency for SOX9 causes campomelic dysplasia associated with XY sex-reversal. To our knowledge, no gain-of-function mutations have been associated with XY sex-reversal. ambiguous genitalia. Conversely, mutations in genes required for ovarian development might cause ovarian dysgenesis [4]. Conclusions More than fifteen years after the SRY gene was identified, a large proportion of the genes involved in the genetic pathway for gonadal sex determination and differentiation still remain unidentified. Accordingly, mutational screenings in sex-reversed patients have demonstrated that only a small subset of patients carry mutations in known genes. The integration of new and old approaches is likely to reveal many new players in the field. Positional cloning efforts and large-scale mutagenesis screens in families and in sporadic cases with subtle genomic rearrangements are in progress. Large-scale transcriptional analyses for genes expressed in a sex-specific manner have identified genes that are dimorphically expressed in the first stages of gonadal differentiation. Functional analysis of many of these genes is underway. Acknowledgements Gonadal differentiation Following sex determination and Sertoli cell differentiation, several cellular events take place in the XY gonad. Sertoli cells polarize and aggregate around germ cells, thereby causing the reorganization of the gonad into two compartments: the testis cords, composed of Sertoli and germ cells; and the interstitial space between the cords. Migration of mesonephric cells into the XY gonad, mainly consisting of endothelial, perivascular and peritubular myoid cells, is also a male-specific event. Peritubular myoid cells surround Sertoli cells and cooperate to deposit the basal lamina at the periphery of testis cords. Migrating endothelial cells associate to establish the coelomic vessel that promotes the efficient export of testosterone from the early testis. Finally, fetal Leydig cells, the androgen-producing cells, differentiate between E12.5 and E13.5 in the interstitial space between cords (reviewed in [1,2]). In the female gonad, the first cellular event occurs at around E13.5, when germ cells enter into meiosis and will arrest at prophase 1 at birth. Germ cells play an essential role in the development of the ovary, because XX gonads depleted of germ cells fail to form ovarian follicles and degenerate [5,35]. In the presence of meiotic germ cells, the somatic cells of the gonad differentiate into follicles that surround the oocytes. Several of the genes required for follicle formation have been identified, including the two transcription factor genes Figa and Foxl2, whereas Wnt4 and follistatin are required during early gonad development to repress aspects of testis differentiation in XX gonads [4]. Loss-of-function mutations in several of the genes involved in the differentiation of the testis (e.g. Dhh, Arx and Pod1 [1,2,3]), cause XY sex-reversal or www.sciencedirect.com Work in our laboratory was supported by grants from Coperativa Est Ticino, Telethon, European Community, MIUR and CNR. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Brennan J, Capel B: One tissue, two fates: molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 2004, 5:509-521. This comprehensive and intelligent review summarizes a wealth of information on the genetic cascade and cellular events involved in gonadal sex determination and differentiation. 2. Ross AJ, Capel B: Signaling at the crossroads of gonad development. Trends Endocrinol Metab 2005, 16:19-25. 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