Wnt4 overexpression disrupts normal testicular vasculature and inhibits testosterone synthesis by repressing steroidogenic factor 1/beta-catenin synergy.

Genetic studies in mice suggest that Wnt4 signaling antagonizes expression of male hormones and effectively blocks male development in the female embryo. We recently identified an XY intersex patient carrying a chromosomal duplication of the WNT4 locus and proposed that this patient's feminization arises from an increased dosage of WNT4. To test this hypothesis, a transgenic mouse was generated with a large genomic P1 containing the human WNT4. Although a complete male to female intersex phenotype was not observed in WNT4 transgenic male mice, a dramatic reduction in steroidogenic acute regulatory protein was detected consistent with the marked reduction in serum and testicular androgen levels. Furthermore, a mild reduction of germ cells and a disorganized vascular system were observed in testes of WNT4 transgenic males. Consistent with these in vivo data, Wnt4 repressed steroidogenesis in adrenocortical and Leydig cell lines, as evidenced by reduced progesterone secretion and 3beta-hydroxysteroid dehydrogenase activity. In vitro studies showed that Wnt4 antagonizes the functional synergy observed between the major effector of the Wnt signaling pathway, beta-catenin and steroidogenic factor 1, and chromatin immunoprecipitation showed that Wnt4 attenuates recruitment of beta-catenin to the steroidogenic acute regulatory protein promoter. Our findings suggest a model in which Wnt4 acts as an anti-male factor by disrupting recruitment of beta-catenin at or near steroidogenic factor 1 binding sites present in multiple steroidogenic genes.

Regulation of the orphan nuclear receptor steroidogenic factor 1 by Sox proteins.

Steroidogenic factor 1 (SF-1) is an essential factor in endocrine proliferation and gene expression. Despite the fact that SF-1 expression is restricted to specialized cells within the endocrine system, the only identified regulatory factors of SF-1 are the ubiquitously expressed E-box proteins (upstream stimulatory factors 1 and 2). Sequence examination of the SF-1 proximal promoter revealed a conserved site of AACAAAG (Sox-BS1), which matches exactly the defined consensus Sox protein binding element. Among the approximately 20 known members of the Sox gene family, we focused on Sox3, Sox8, and Sox9, based on their coexpression with SF-1 in the embryonic testis. Indeed, all three of these Sox proteins were capable of binding the proximal Sox-BS1 within the SF-1 promoter (-110 to -104), albeit with differing affinities. Of the three Sox proteins, Sox9 exhibited high-affinity binding to the Sox-BS1 element and consistently activated SF-1 promoter-reporter constructs. Mutating the Sox-BS1 attenuated SF-1 promoter activity in both embryonic and postnatal Sertoli cells, as well as in the adrenocortical cell line, Y1. Our findings, taken together with the overlapping expression profiles of Sox9 and SF-1, and the similar intersex phenotypes associated with both SOX9 and SF-1 human mutations, suggest that Sox9 up-regulates SF-1 and accounts partially for the sexually dimorphic expression pattern of SF-1 observed during male gonadal differentiation.