Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004

Sex determination: a ‘window’ of DAX1 activity

Louisa M. Ludbrook and Vincent R. Harley

Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, VIC 3168, Australia

Traditionally, DAX1 was considered an ‘anti-testis’ that are probably important for male sex determination because DAX1 duplications in XY individuals cause have yet to be identified, because some 75% of sex reversal male-to-female sex reversal: dosage-sensitive sex rever- cases remain unexplained genetically [15]. Some progress sal (DSS). In DSS, two active DAX1 on one has been made in deciphering the roles and complex X can abrogate testis formation. By con- relationships of the known sex-determining genes during trast, mutations and deletions of DAX1 cause adrenal gonadogenesis. Here, we describe the emerging role of hypoplasia congenita (AHC). Although AHC patients DAX1 in male testis formation and discuss the possible develop testes, gonadal defects include disorganized molecular mechanisms through which DAX1 regulates testis cords and hypogonadotropic hypogonadism, this pathway. which is not completely restored with gonadotropin or androgen therapy. Recent evidence of XY sex reversal Expression of DAX1 in Dax1-deficient mice strongly supports a role for Dax1 DAX1 RNA expression is restricted to certain tissue types as a ‘pro-testis’ gene. Therefore, perhaps DAX1/Dax1 and is largely coexpressed with SF1, also crucial for both acts within a ‘window’ of activity, outside of which tes- adrenal and gonadal development [16–18]. Based on in tis formation does not occur. Here, we discuss the func- situ hybridization analyses, Sf1 and Dax1 are expressed in tion and possible mechanisms of DAX1 action in male both developing and adult adrenal, gonadal, hypothalamic gonadogenesis. and pituitary tissues [10]. During human and murine gonadal development, both DAX1 and SF1 are coexpressed Sex determination in most mammals (Box 1) is controlled in the gonads of both sexes before genital ridge differen- by the presence or absence of the sex-determining region tiation, with Sf1 expression slightly preceding that of Dax1 Y chromosome gene (SRY in humans, Sry in mice) [1]. SRY [10]. In humans, DAX1 expression levels are the same in deletions or mutations give rise to females with XY the embryonic testis and [19]. In XY mice, Dax1 is gonadal dysgenesis [2]. Sry is expressed in the bipotential upregulated strongly in Sertoli cells at 12.5 days post gonad just before overt differentiation into a testis [3] and, coitum (dpc), just after Sry. Sertoli Dax1 expression drops when Sry is transgenically introduced into XX mice, they soon after, but increases in the interstitial cells between develop as males [4]. SRY is thought to initiate a genetic 13.5 and 17.5 dpc [20]. In the XX mouse gonadal cascade resulting in testis differentiation [5]. Other sex- determining genes act downstream of SRY within this pathway (Figure 1). Box 1. Sex determination in mammals Through studies of the genomes of sex-reversed The human embryo develops a sexual identity six to seven weeks patients (XY females, XX males), several sex-determining into gestation. Sex determination involves the differentiation of the genes have been identified. SOX9 mutations cause XY sex bipotential embryonic gonads into the male testes or the female . The presence of a testis enables further development of reversal in 75% of patients with campomelic dysplasia [6]. male-specific structures and external genitalia. Up to this point, the Sox9 is upregulated directly after Sry in the XYembryonic sex of an embryo appears ambiguous; a common gonadal ridge is mouse gonad [7] and XX mice transgenic for Sox9 [under present in both males and females, which contains bipotential the control of Wilms’ tumor-1 (encoded by WT1) regulatory supporting and steroidogenic cell lineages, in addition to the regions] develop as males [8]. Steroidogenic factor-1 (SF1 primordial germ cells destined to form spermatogonia or oogonia. As differentiation ensues, the supporting cell lineage gives rise to in humans; Sf1 in mice; official gene symbol NR5A1) and male Sertoli cells or female follicle cells, whereas the steroidogenic WT1 are both initially expressed before Sry in the gonad cells form testicular Leydig cells or ovarian thecal cells responsible [9,10]. In humans, SF1 mutations are responsible for XY for production of steroid sex hormones. sex reversal [11,12] and WT1 mutations cause severe Also present in the sexually indifferent embryo are the cardinal structures of the Wolffian (male) and Mullerian (female) accessory kidney and gonadal defects, including XY gonadal dysgen- reproductive ducts, the future genital tracts. Depending on gonadal esis [13]. Gene duplication of DAX1 (Dax1 in mice; encodes sex, the development of one ductal system will be promoted in the DSS–AHC on gene 1; official gene symbol embryo and the alternate accessory structures will degenerate. Sex- NR0B1) is associated with XY sex reversal in patients with specific development of external genitalia follows during week 8. The dosage-sensitive sex reversal (DSS) [14]. Many more genes onset of sex determination is genetically controlled, and is initiated by the Y chromosome gene SRY and its expression in the supporting cells of the gonadal ridge. Corresponding author: V.R. Harley ([email protected]). www.sciencedirect.com 1043-2760/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tem.2004.02.002 Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004 117

DAX1

MIS

WT1/SF1 SRY SOX9 SF1 Testis NTF3, FGF9 Other gonad-forming No SRY Ovary genes

TRENDS in Endocrinology & Metabolism

Figure 1. Molecular genetic events surrounding the initiation of sex determination. Solid lines represent interactions for which experimental evidence exists; dashed lines represent suggested, but as yet undetermined interactions. In the week 7 urogenital ridge, WT1 and SF1 are expressed in both sexes. In the XY gonad, SRY is activated fol- lowed by upregulation of SOX9 by processes possibly involving DAX1, NTF3 and FGF9. SOX9 in turn activates SF1, which is proposed to activate unknown testis-forming genes. SF1 and SOX9 also activate AMH. DAX1 could act in multiple modes and at multiple steps. In the XX gonad, SRY is absent and an ovary develops independent of DAX1 expression. Abbreviations: AMH, anti-Mullerian hormone gene; DAX1, DSS–AHC on X chromosome gene 1; FGF9, fibroblast growth factor-9 gene; NTF3, neurotro- pin 3 gene; SF1, steroidogenic factor-1 gene; SOX9, SRY box-9 gene; SRY, sex-determining region Y chromosome gene; WT1, Wilms’ tumor gene-1. primordium, Dax1 is expressed between 12.5 and 14.5 dpc, DAX1 C-terminus [32,33], one of which (Alien) is after which expression decreases [20]. expressed in the testis [34]. Investigation into DAX1 function in steroidogenesis has DAX1 structure and function revealed several possible mechanisms for DAX1 action DAX1 encodes a member of the orphan nuclear hormone during gonadal development. In cultured cells, DAX1 (NHR) family of transcriptional regulators. DAX1 represses Sf1-mediated transcriptional activation of ster- is 470 amino acids in length and has two distinct protein oidogenic genes, such as Cyp11A, Cyp17 and Cyp19 [35]. domains (Figure 2). The C-terminal domain shows Co-transfection of SF1 with DAX1 DNA into choriocarci- homology to the ligand-binding domain (LBD) of related noma cells showed a 75% repression of an SF1 artificial NHRs, although a ligand remains elusive. The N-terminal response element/luciferase reporter compared with domain represents a novel domain comprising 3.5 repeats transfection with SF1 alone [36]. This repressive effect of a 65–67-amino acid motif containing two putative zinc was dose dependent and relatively specific for SF1- fingers, and possibly defining a nucleic acid-binding mediated activity, because inhibition of the related thyroid domain [21,22]. DAX1 is present in mammalian species was not apparent [36]. Adrenal hypo- (mice [21], birds [23], humans and other primates [14,24]) plasia congenita (AHC) missense mutations in DAX1 (all of and non-mammalian vertebrates (reptiles, amphibians which localize to the C-terminal LBD) significantly reduce and fish [25–27]). Human and mouse DAX1 show 75% its repressive effects in cell culture assays [36,37], similarity in amino acid sequence [21]. The N-terminal implicating the LBD as a mediator of repression. Recent repeats contain conserved leucine LXXLL motifs resem- evidence indicates that many AHC mutant DAX1 bling the box protein interaction motifs show defective nuclear localization, accounting for the lack [28]. The closest relative of DAX1 is small heterodimer of repression observed in cell culture assays [38,39]. partner, SHP, which also lacks a traditional N-terminal Although correct protein folding and stability of the DNA-binding domain, and is able to interact with protein C-terminal LBD are crucial for DAX1 nuclear import [39], targets through LXXLL-related motifs [29,30]. DAX1 has a evidence is emerging of a direct role for the N-terminal bipartite transcriptional silencing domain (Figure 2), LXXLL motifs in mediating repression. Zhang and which has been proposed to interact directly with colleagues showed that the DAX1 LXXLL motifs interact corepressors to mediate repression [31]. At least two with the AF2 domain of ligand-bound estrogen receptors, corepressors, Alien and NCoR, interact in vitro with the ERa and ERb? and repress ER-mediated activation of target reporter genes [28]. Transient transfection assays showed Dax1-mediated transcriptional repression of an Putative DNA binding SF1 target, CYP11A1, was reduced when each of the three or protein interaction Putative ligand binding DAX1 LXXLL motifs was separately mutated, and domain domain completely removed when all three motifs were disrupted, N C presumably because the SF1–DAX1 interaction is lost LYNLL LYSNL LYSLL Silencing domains [40]. A direct interaction between SF1 and DAX1 (via its LXXLL motifs) was demonstrated [36]. Together, these TRENDS in Endocrinology & Metabolism studies suggest that direct protein interaction between the DAX1 LXXLL motifs and target nuclear receptors, such as Figure 2. Features of the DAX1 protein. DAX1 has two distinct domains; the C-ter- SF1, occurs during DAX1-mediated transcriptional minus is homologous to the ligand-binding domain of the nuclear hormone recep- tor family. The unique N-terminus contains 3.5 repeats of a 65–67-amino acid repression. motif. Within these repeats are three LXXLL motifs, involved in mediating protein It is probable that DAX1 and SF1 interact during interactions [38,40]. Integrity of a bipartite transcriptional silencing domain is required for DAX1 repression [31]. Corepressors, such as NcoR and Alien, can gonadogenesis in a similar manner to that seen during interact directly with the DAX1 C-terminus [32,33]. steroidogenesis. Their transcripts are coexpressed early in www.sciencedirect.com 118 Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004 the urogenital ridge and are directly implicated in male strain) were used, complete male-to-female sex reversal sex determination by clinical cases of XY sex reversal. was seen. Although dosage sensitivity during gonadal DAX1 duplication causes DSS, and SF1 mutations are differentiation appears to vary between humans and mice, described in individuals with XY sex reversal [12,14].In dosage experiments in mice suggest that Dax1 and Sry act cell-based assays, DAX1 indeed represses SF1-mediated antagonistically [47]. Thus DAX1, rather than the other upregulation of the gene encoding anti-Mullerian hormone genes contained within the 160 kb duplicated region of (Amh, also known as Mullerian inhibiting substance; Mis) Xp21, is significantly, if not solely, responsible for the DSS during male sexual differentiation [41]. In transfected phenotype. immature rat Sertoli cells, direct DAX1–SF1 interaction causes repression of GATA4–SF1 synergy of an AMH– Possible mechanisms of DAX1 action in DSS reporter promoter [41]. This repression did not occur when Several explanations for the DSS phenotype can be the study was repeated in a DAX1 mutant that removes considered, based upon known mechanism(s) of DAX1 the N-terminal (SF1 interaction) domain, suggesting that action. DAX1 might repress SF1 target genes during sex direct protein interaction is required for repression [41].In determination, in a manner similar to that seen during a related study, DAX1 repressed synergy between SF1 and steroidogenesis. One such candidate target for DAX1 WT1 during activation of AMH–reporter constructs [42]. repression is SRY (Figure 1). SF1 upregulated human SRY Repression of SF1–WT1 synergy probably involves the 2–2.5-fold in reporter gene studies in the NT2/D1 direct interaction of DAX1 with SF1, as with repression of teratocarcinoma cell line, by binding to a consensus GATA4–SF1 synergy. Together, these studies suggest a NHR binding site at –327 to –298 in the SRY promoter common function for DAX1 as a repressor of SF1 trans- [48]. Mutation of the SF1-binding site within the SRY activation in both steroidal and gonadal tissues. promoter reduced its activity to basal levels [48]. Although DAX1 might also regulate through activation of SRY by SF1 is relatively subtle, events direct binding to DNA and/or RNA. One early study occurring in the embryonic gonad are highly sensitive to showed that DAX1 could bind directly in vitro to the dosage changes. Indeed, several familial SRY mutations retinoic acid-responsive element, RARE [14]. Current causing XY gonadal dysgenesis encode mutant proteins evidence supports a role for DAX1 in the recognition of with ‘wild-type’ biochemical activities, suggesting that nucleic acid structure rather than sequence. DAX1 binds SRY acts close to a threshold level [49]. Furthermore, the to DNA loops, mediating repression during adrenal porcine SRY promoter is trans-activated by SF1 in development, via direct DNA binding to the promoter of cultured cells, probably through binding to the SF1- target genes [43]. In DNA-binding assays, DAX1 interacts binding site identified –1369 bp from the SRY transla- with DNA hairpin sequences present in both steroidogenic tional initiation site [50]. DAX1 attenuation of SF1 acute regulatory protein (encoded by Star) and Dax1 activation of SRY could explain the complete XY gonadal promoter regions. In cultured cells, transfected DAX1 dysgenesis phenotype in patients with DSS who have significantly repressed the activation of both Star and excess DAX1 activity. However, no measurements of Sry in Dax1 [43]. However, in DAX1-deficient male mice, the Dax1 transgenic gonads have been reported to confirm levels of Star gene expression were not significantly this. Downstream of SRY action, SOX9 acts to upregulate altered [44]. DAX1 might also act at a post-transcriptional level, SF1 [51], which probably contributes to upregulation of directly binding to RNA and polyribosomes and acting as a other later-acting sex-determining genes. Once identified, shuttling protein for export of RNA species into the these genes might also prove to be potential candidates for nucleus [22]. Further evidence supporting an RNA- DAX1 over-repression leading to DSS. binding mode of action of DAX1 is eagerly awaited. Gonadal DAX1 plays a role during sexual differen- tiation to repress SF1 upregulation of AMH. Mutation of Dosage-sensitive sex reversal: an ‘anti-testis’ role for AMH causes persistent Mullerian duct syndrome (PMDS) DAX1 in humans and internal pseudohermaphroditism in knock- Patients with DSS have a 46, XY karyotype and no testes. out mice [52,53]. It has been suggested that an ‘over- The region duplicated in such patients spans 160 kb at repression’ of AMH by DAX1 could partly explain the DSS Xp21 [45]. In addition to DAX1, this region contains the sex-reversed phenotype [35]. However, this is unlikely for DAM (DSS–AHC critical interval MAGE) family genes several reasons. First, the complete lack of testicular related to the melanoma antigen gene family, which tissue in some patients with DSS suggests the disruption encode tumor-associated antigens [45]. Dax1 was not of early testis-forming genes. Second, AMH is not a testis- seen to have a role in ovarian development and was forming gene because, in both XY PMDS humans and therefore termed an ‘anti-testis’ gene [46]. In 1998, Swain Amh-knockout mice, testis development appears to be and colleagues generated a mouse model to determine normal and male infertility results from associated whether Dax1 was responsible for DSS. Transgenic mice secondary and structural problems [52–54]. Third, (Mus musculus musculus origin) carrying extra copies of although Dax1 represses Amh through disruption of SF1 Dax1 were bred. Sex reversal was not observed in function in cell-based assays [41,42], mutating the offspring. However, mice with the highest Dax1 expression proximal SF1-binding site within the Amh promoter in (almost five times that of normal levels) exhibited retarded vivo does not cause pseudohermaphroditism in XY mice testis formation [47]. When alternate ‘late acting’ Sry [54]. However, a distal SF1-binding site [55] was not tested alleles (SryPOS from the Mus domesticus poschiavianus [54]. Although SF1 contributes to AMH upregulation, it is www.sciencedirect.com Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004 119 unclear whether ‘over-repression’ of this effect by DAX1 Dax1-knockout mice were XY sex reversed when crossed will significantly reduce AMH expression in vivo. with the M. d. poschiavianus strain containing the Y chromosome SryPOS allele [64]. These Dax12/YPOS mice DAX1 mutations in AHC have neither testicular cords nor Sertoli and Leydig cell Humans with loss-of-function mutations in DAX1 causing markers, and show complete sex reversal with fully AHC have abnormal testes but do not exhibit sex reversal penetrant stages of follicular development [64]. Expression [56]. Most AHC males suffer pubertal onset hypogonado- of Sox9 was significantly reduced in the Dax12/YPOS gonad tropic hypogonadism (HHG) and show impaired sperma- at 12.5 dpc, when Sox9 is usually upregulated in XY togenesis (both attributed to the underlying gonadotropin gonads following Sry expression (which is reportedly at deficiency). Administration of exogenous gonadotropins or normal levels in Dax12/YPOS mice) [64]. This implicates androgens does not correct spermatogenesis or improve Dax1 as an important player in male sex determination fertility in AHC individuals [57,58], suggesting an and suggests a position in the pathway after Sry induction, underlying testicular defect, although a more suitable yet before Sox9 upregulation at 12.5 dpc (Figure 1). comparison would be with gonads of hormone-treated non- In Dax1-knockout mice, the second exon of the Dax1 AHC patients with HHG. gene is effectively excised, removing the C-terminal domain, as in human AHC. As reported, low levels of an Dax1-knockout mice: a ‘pro-testis’ role for DAX1 abnormal Dax1 transcript were still detected in the The initial description of the phenotype of Dax1-knockout knockout mice, originating from the remaining gene, and male mice (Dax12/Y) suggested that Dax1 was essential for probably comprising a partial N-terminal protein product maintenance of the testicular germinal epithelium and a [46]. It remains possible that this partial product retains necessary factor for spermatogenesis and gametogenesis in some function or might have a disruptive effect, perhaps a adult mice [46]. Although this correlates with the infertility dominant negative one, within the developing testis, given seen in XYAHC adults, Dax12/Y mice showed normal serum recent insight into the importance of protein interactions testosterone, gonadotropin and adrenal steroid hormone of the DAX1 N-terminal domain LXXLL motifs [40]. levels (unlike patients with AHC), supporting a role for Dax1 outside the hypothalamic–pituitary axis [46,59].Further Possible mechanisms of a pro-testis action for DAX1 analysis of the phenotype of Dax12/Y mice performed on The mechanism of DAX1 action in promoting testis 11.5–17.5 dpc embryonic gonads revealed a decrease in determination is unclear. The possible targets for DAX1 proliferation of peritubular myoid cells, and early defects in regulation affecting cellular proliferation and migration testicularcord developmentand organization [60].Although are the genes encoding fibroblast growth factor-9 (Fgf9) or Sertoli and germ cells are present in the Dax12/Y gonad, neurotropin 3 (NTF3 in human, Ntf3 in mice), based upon albeit disorganized, the migration and/or development of knockout phenotypes resembling that of Dax12/Y. Fgf9- fetal Leydig cells is disrupted [60]. This implicates Dax1 as a knockout mice phenotypes range from testicular hypo- crucial factor early in sex determination and suggests a role plasia to XY sex reversal [65]. Where testicular tissue was for it in regulating cell migration and/or proliferation in the detected, seminiferous cord formation was disorganized fetal testis. and peritubular myoid cells were depleted. NTF3 directly The spermatogenesis defects seen in the Dax12/Y testis promotes mesonephric cell (pre-peritubular cell) migration can be partly explained by the dysregulation of steroid into the gonad in rat testis organ cultures [66]. Inhibition hormone production. Through repression of Sf1 trans- of the NTF3 receptor, trkC, inhibited both cell migration activation, DAX1 regulates the expression of Cyp19, which and seminiferous cord formation [66]. In both Fgf9- encodes aromatase, the key enzyme responsible for knockout mice and NTF3-blocked rat gonads, Sox9 conversion of testosterone to estrogen [61]. Overexpres- expression was also reduced. Further investigation sion of Cyp19 in the transgenic AROM þ mouse gonad might identify a role for DAX1 in regulating testis-forming results in Leydig cell hyperplasia and abnormal semini- target genes, such as those outlined above. ferous tubules [62]. Given that Cyp19 expression is upregulated in Leydig cells of Dax12/Y mice [44], it was A ‘window’ of Dax1 activity in male sexual development important to determine whether the Dax12/Y testis The phenotypic similarities between low Dax1 (the phenotype could be attributed to excess Cyp19 production. Dax12/YPOS-knockout and testis phenotype in AHC) and A Dax1 transgene under control of the luteinizing hormone high Dax1 (the Dax1 transgenic mouse and patients with receptor promoter (LHR–DAX1) was specifically intro- DSS) activity suggest that Dax1 might function within an duced into the Leydig cells of the knockout mouse [63].In activity ‘window’ (Figure 3). In a certain dose/activity this rescue, aromatase expression was comparable to that range, DAX1 can play a regulatory role in testis formation, in wild-type mice, and sperm count and fertility were yet, should this be reduced below a dosage threshold or partially restored. However, the testis pathology remained exceed that of an upper level threshold, testis development unchanged [63]. It appears that additional functions of becomes misregulated. The molecular action of DAX1 in DAX1 (to those in developing Leydig cells) are responsible sex determination remains to be defined. Although for the defects in gonadogenesis, although the dysregula- evidence clearly demonstrates the capacity of DAX1 to tion of Cyp19 might contribute to the infertility of the repress target gene expression (directly or indirectly via Dax12/Y mice, SF1 inhibition) and protein function (such as ER), it is More definitive evidence that Dax1 acts in a ‘pro-testis’ difficult to reconcile both the knockout and transgenic manner arose from the recent observation that Dax1 mouse phenotypes if DAX1 acts solely as a negative www.sciencedirect.com 120 Review TRENDS in Endocrinology and Metabolism Vol.15 No.3 April 2004

presence of corepressors, such as Alien, DAX1 exerts a repressive effect, whereas in the presence of coactivators or an elusive ligand, it functions as a positive regulator. DAX1-mediated repression might involve binding through the LXXLL motif to the LBD of target NHRs such as SF1, or might involve direct binding of DAX1 to DNA. Chromatin immunoprecipitation assays should be used DAX-1activity to identify potential direct DNA targets of DAX1 during gonadal differentiation. The ‘testis-promoting’ function Male might stem from its proposed role as a post-transcriptional window transport protein (e.g. through binding to SRY mRNA). Co- immunoprecipitation together with proteomics techniques Phenotypic sex might reveal components of the multiprotein regulatory complexes in which DAX1 is presumed to act, providing XY mice * important insights into this mysterious molecule.

XY humans Acknowledgements We thank Anne Reutens, Eric Vilain, Michael Clarkson and Peter Fuller for critical reading of the manuscript. V.R.H. is supported by the NHMRC TRENDS in Endocrinology & Metabolism (Australia). We thank Sue Pankridge for artwork.

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