Homozygous inactivation of Sox9 causes complete XY sex reversal in mice

In the presence of the Y-chromosomal gene Sry, the bipotential mouse gonads develop as testes rather than as ovaries. The autosomal gene Sox9, a likely and possibly direct Sry target, can induce testis development in the absence of Sry. Sox9 is thus sufficient but not necessarily essential for testis induction. Mutational inactivation of one allele of SOX9/Sox9 causes sex reversal in humans but not in mice. Because Sox9(-/-) embryos die around Embryonic Day 11.5 (E11.5) at the onset of testicular morphogenesis, differentiation of the mutant XY gonad can be analyzed only ex vivo in organ culture. We have therefore conditionally inactivated both Sox9 alleles in the gonadal anlagen using the CRE/loxP recombination system, whereby CRE recombinase is under control of the cytokeratin 19 promoter. Analysis of resulting Sox9(-/-) XY gonads up to E15.5 reveals immediate, complete sex reversal, as shown by expression of the early ovary-specific markers Wnt4 and Foxl2 and by lack of testis cord and Leydig cell formation. Sry expression in mutant XY gonads indicates that downregulation of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our results provide in vivo proof that, in contrast to the situation in humans, complete XY sex reversal in mice requires inactivation of both Sox9 alleles and that Sox9 is essential for testogenesis in mice.     

SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development

In mammals, male sex is determined by the Y-chromosomal gene Sry (sex-determining region of Y chromosome). The expression of Sry and subsequently Sox9 (SRY box containing gene 9) in precursors of the supporting cell lineage results in the differentiation of these cells into Sertoli cells. Sertoli cells in turn orchestrate the development of all other male-specific cell types. To ensure that Sertoli cells differentiate in sufficient numbers to induce normal testis development, the early testis produces prostaglandin D(2) (PGD(2)), which recruits cells of the supporting cell lineage to a Sertoli cell fate. Here we show that the gene encoding prostaglandin D synthase (Pgds), the enzyme that produces PGD(2), is expressed in Sertoli cells immediately after the onset of Sox9 expression. Promoter analysis in silico and in vitro identified a paired SOX/SRY binding site. Interestingly, only SOX9, and not SRY, was able to bind as a dimer to this site and transactivate the Pgds promoter. In line with this, a transgenic mouse model showed that Pgds expression is not affected by ectopic Sry expression. Finally, chromatin immunoprecipitation proved that SOX9 but not SRY binds to the Pgds promoter in vivo.     

Functional analysis of Sox8 and Sox9 during sex determination in the mouse

Sex determination in mammals directs an initially bipotential gonad to differentiate into either a testis or an ovary. This decision is triggered by the expression of the sex-determining gene Sry, which leads to the activation of male-specific genes including the HMG-box containing gene Sox9. From transgenic studies in mice it is clear that Sox9 is sufficient to induce testis formation. However, there is no direct confirmation for an essential role for Sox9 in testis determination. The studies presented here are the first experimental proof for an essential role for Sox9 in mediating a switch from the ovarian pathway to the testicular pathway. Using conditional gene targeting, we show that homozygous deletion of Sox9 in XY gonads interferes with sex cord development and the activation of the male-specific markers Mis and P450scc, and leads to the expression of the female-specific markers Bmp2 and follistatin. Moreover, using a tissue specific knock-out approach, we show that Sox9 is involved in Sertoli cell differentiation, the activation of Mis and Sox8, and the inactivation of Sry. Finally, double knock-out analyses suggest that Sox8 reinforces Sox9 function in testis differentiation of mice.     

From SRY to SOX9: mammalian testis differentiation

Sry (sex-determining region on the Y chromosome) is a master gene that initiates testis differentiation of the bipotential indifferent gonad in mammals. In mice, Sry expression is transiently activated in a center-to-pole wave along the anteroposterior (AP) axis of developing XY gonads. Shortly after the onset of Sry activation, Sox9 (Sry-related HMG box-9), a fundamental testis-differentiation gene common to all vertebrates, is also activated in a center-to-pole pattern similar to the initial Sry expression profile. Several male-specific cellular events, such as glycogenesis, coelomic epithelium proliferation, mesonephric migration and vasculogenesis, are induced in XY gonads following the onset of Sry and Sox9 expression. This paper mainly focuses on recent advances in elucidating the regulatory mechanisms of Sry and Sox9 expression and male-specific cellular events immediately downstream of SRY action during the initial phases of testis differentiation.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

Regulation of male sexual development by Sry and Sox9

Sry, a gene from the Y chromosome, is known to initiate testis formation and subsequent male differentiation in mammals. A related gene, Sox9, also plays a critical role in testis determination, possibly in all vertebrates. A number of models have been presented regarding the molecular modes of action of these two genes. However, details regarding their regulation, regulatory target genes, and interacting protein factors and co-factors have not been established with any certainty. In this review, we examine new evidence and re-examine existing evidence bearing on these issues, in an effort to build up an integrative model of the network of gene activity centred around Sry and Sox9. J. Exp. Zool. 290:463-474, 2001.     

Large-scale screen for genes involved in gonad development

The vertebrate gonad develops from the intermediate mesoderm as an initially bipotential organ anlage, the genital ridge. In mammals, Sry acts as a genetic switch towards testis development. Sox9 has been shown to act downstream of Sry in testis development, while Dax1 appears to counteract Sry. Few more genes have been implicated in early gonad development. However, the genetic networks controlling early differentiation events in testis and ovary are still far from being understood. In order to provide a broader basis for the molecular analysis of gonad development, high-throughput gene expression analysis was utilized to identify genes specifically expressed in the gonad. In total, among 138 genes isolated which showed tissue specific expression in the embryo, 79 were detected in the developing gonad or sex ducts. Twenty-seven have not been functionally described before, while 40 represent known genes and 12 are putative mouse orthologues. Forty-five of the latter two groups (86%) have not been described previously in the fetal gonad. In addition, 21 of the gonad specific genes showed sex-dimorphic expression suggesting a role in sex determination and/or gonad differentiation. Eighteen of the latter (86%) have not been described previously in the fetal gonad. In total we provide new data on 72 genes which may play a role in gonad or sex duct development and/or sex determination. Thus we have generated a large gene resource for the investigation of these processes, and demonstrate the suitability of high-throughput gene expression screening for the genetic analysis of organogenesis.     

siRNA介导Sry基因沉默对小鼠胚胎性别决定基因表达的影响

为研究Sry基因的调控网络, 采用siRNA表达载体介导的RNAi技术, 特异性地抑制睾丸决定因子Sry在小鼠胚胎中的表达, 并观察Sry基因沉默后对在两性性腺分化中起重要作用的Wt1, Sf1, Dax1, Gata4, Sox9及Amh基因表达的影响。利用本课题组先前构建的siRNA重组表达载体(pSilencer4.1/Sry217及pSilencer4.1/Sry565), 通过尾静脉注射法导入妊娠9.5天(9.5 dpc)的母鼠体内, 在11.5 dpc时取胚胎, 对性别鉴定为雄性的胚胎以RT-PCR法和Western-blot检测Sry基因的表达抑制效果, 并同时用定量PCR法检测Wt1等上述性别决定相关基因表达变化情况。结果表明, 注射干扰质粒后48 h Sry基因的mRNA和蛋白表达水平均降低, 其中siRNA表达质粒pSilencer 4.1/Sry 565的抑制效果显著, 可达到80%的抑制率。Sry基因沉默后, Wt1基因表达量显著升高; Sf1, Dax1, Gata4, Sox9基因表达水平没有明显变化; Amh基因无表达。试验结果表明, Sry基因表达抑制会导致Wt1基因表达升高; 另外, Sry基因激活Sox9基因的表达可能需要其他的辅助因子协同作用。