The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. However, only partial effects have been reported for candidate genes experimentally tested so far. Here we demonstrate that inactivation of two ovarian somatic factors, Wnt4 and Foxl2, produces testis differentiation in XX mice, resulting in the formation of testis tubules and spermatogonia. These genes are thus required to initiate or maintain all major aspects of female sex determination in mammals. The two genes are independently expressed and show complementary roles in ovary morphogenesis. In addition, forced expression of Foxl2 impairs testis tubule differentiation in XY transgenic mice, and germ cell-depleted XX mice lacking Foxl2 and harboring a Kit mutation undergo partial female-to-male sex reversal. The results are all consistent with an anti-testis role for Foxl2. The data suggest that the relative autonomy of the action of Foxl2, Wnt4 and additional ovarian factor(s) in the mouse should facilitate the dissection of their respective contributions to female sex determination.

Disorders of sex development (DSDs) are conditions affecting development of the gonads or genitalia. Variants in two key genes, SRY and its target SOX9, are an established cause of 46,XY DSD, but the genetic basis of many DSDs remains unknown. SRY-mediated SOX9 upregulation in the early gonad is crucial for testis development, yet the regulatory elements underlying this have not been identified in humans. Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9. Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testis. This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.

During female reproductive life, ovarian follicle reserve is reduced by maturation and atresia until menopause ensues. Foxo3 is required to maintain the ovarian reserve in mice. Here we show that overexpression of constitutively active FOXO3 can increase ovarian reproductive capacity in mice. We find increased follicle numbers and decreased gonadotropin levels in aging FOXO3-transgenic mice compared with wild-type littermates, suggesting maintenance of a greater ovarian reserve. Based on cumulative progeny in aging animals, we find 31–49% increased fertility in transgenic females. The gene expression profile of Foxo3−/− knockout ovaries appears older than that of wild-type littermates, and the transgene induces a younger-looking profile, restoring much of the wild-type transcriptome. This is the first gain-of-function model of augmented reproductive reserve in mice, thus emphasizing the role of Foxo3 as a guardian of the ovarian follicle pool in mammals and a potential determinant of the onset of menopause.

Background: Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit. Results: Following Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers. Conclusion: The results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility.

Sex determination in fetal germ cells depends on a balance between exposure to retinoic acid (RA) and the degradation of RA achieved by the testis‐specific expression of the catabolic cytochrome P450 enzyme, CYP26B1. Therefore, identification of factors regulating the expression of the Cyp26b1 gene is an important goal in reproductive biology. We used in situ hybridization to demonstrate that Cyp26b1 and transcription factor genes steroidogenic factor‐1 ( Sf1 ) and Sry ‐related HMG box 9 ( Sox9 ) are coexpressed in Sertoli cells, whereas Cyp26b1 and Sf1 are coexpressed in Leydig cells in mouse fetal testes. In the mouse gonadal somatic cell line TM3, transfection of constructs expressing SOX9 and SF1 activated Cyp26b1 expression, independently of the positive regulator RA. In embryonic gonads deficient in SOX9 or SF1, Cyp26b1 expression was decreased relative to wild‐type (WT) controls, as measured by quantitative RT‐PCR (qRT‐PCR). Furthermore, qRT‐PCR showed that Cyp26b1 up‐regulation by SOX9/SF1 was attenuated by the ovarian transcription factor Forkhead box L2 (FOXL2) in TM3 cells, whereas in Foxl2 ‐null mice, Cyp26b1 expression in XX gonads was increased ~20‐fold relative to WT controls. These data support the hypothesis that SOX9 and SF1 ensure the male fate of germ cells by up‐regulating Cyp26b1 and that FOXL2 acts to antagonize Cyp26b1 expression in ovaries.—Kashimada, K., Svingen, T., Feng, C.‐W., Pelosi, E., Bagheri‐Fam, S., Harley, V. R., Schlessinger, D., Bowles, J., Koopman, P. Antagonistic regulation of Cyp26b1 by transcription factors SOX9/SF1 and FOXL2 during gonadal development in mice. FASEB J. 25, 3561–3569 (2011). www.fasebj.org

Follistatin is a secreted glycoprotein required for female sex determination and early ovarian development, but the precise mechanisms regulating follistatin (Fst) gene expression are not known. Here, we investigate the roles of bone morphogenetic protein 2 (BMP2) and forkhead-domain transcription factor L2 (FOXL2) in the regulation of Fst expression in the developing mouse ovary. Bmp2 and Fst showed similar temporal profiles of mRNA expression, whereas FOXL2 protein and Fst mRNA were coexpressed in the same ovarian cells. In a cell culture model, both FOXL2 and BMP2 up-regulated Fst expression. In ex vivo mouse fetal gonad culture, exogenous BMP2 increased Fst expression, but this effect was counteracted by the BMP antagonist Noggin. Moreover, in Foxl2-null mice, Fst expression was reduced throughout fetal ovarian development, and Bmp2 expression was also reduced. Our data support a model in which FOXL2 and BMP2 cooperate to ensure correct expression of Fst in the developing ovary. Further, Wnt4-knockout mice showed reduced expression of Fst limited to early ovarian development, suggesting a role for WNT4 in the initiation, but not the maintenance, of Fst expression.