Abstract
Steroidogenic factor 1 (NR5A1) is essential for gonadal development. To study the importance of NR5A1 during early gonadal sex differentiation, we generated Sox9-Cre-Nr5a1 conditional knockout (cKO) mice: Sox9-Cre;Nr5a1flox/flox and Sox9-Cre;Nr5a1flox/− mice. Double-immunostaining for NR5A1 and AMH revealed silenced NR5A1 in Sertoli cells and reduced AMH+ cells in the gonads of XY Sox9-Cre-Nr5a1 cKO mice between embryonic days 12.5 (E12.5) and E14.5. Double-immunostaining for SOX9 and FOXL2 further indicated an early block in Sertoli cells and ectopic granulosa cell differentiation. The number of cells expressing the Leydig cell marker 3βHSD obviously reduced in the gonads of XY Sox9-Cre;Nr5a1flox/− but not Sox9-Cre;Nr5a1flox/flox mice at E15.5. The presence of STRA8+ cells indicated that germ cells entered meiosis in the gonads of XY Sox9-Cre-Nr5a1 cKO mice. The results of qRT-PCR revealed remarkably reduced and elevated levels of testis and ovary markers, respectively, in the gonads of XY Sox9-Cre-Nr5a1 cKO mice at E12.5‒E13.5. These data suggested that the loss of Nr5a1 abrogates the testicular pathway and induces the ectopic ovarian pathway, resulting in postnatal partial/complete male-to-female gonadal sex reversal. Our findings provide evidence for the critical role of NR5A1 in murine gonadal sex determination in vivo.
Highlights
In mammals, bi-potential undifferentiated gonads, which develop from the gonadal primordium in the urogenital ridge, differentiate into either testes or ovaries[1,2]
Immunohistochemistry (IHC) findings confirmed that Cre was expressed by Sertoli cells in XY sex-determining region-box 9 (Sox9)-Cre, but not in XY Nr5a1f./− mice at E12.5–E15.5 (Supplementary Fig. 1)
Because the IHC findings of NR5A1 and markers of Sertoli, Leydig, and germ cells did not differ among the three control groups (Supplementary Fig. 2), these groups were combined in the subsequent analyses
Summary
Bi-potential undifferentiated gonads, which develop from the gonadal primordium in the urogenital ridge, differentiate into either testes or ovaries[1,2]. The gonads of XY Amh-Cre-Nr5a1 cKO mice are strikingly hypoplastic, and the loss of Sertoli and germ cells along with dysgenic testis cords from E15.5 suggested a role for NR5A1 in Sertoli cell survival and proliferation during embryonic testis differentiation after sex determination. To study the role of NR5A1 during early testis differentiation, we generated a different Nr5a1 cKO using the Sox9-Cre transgene, which is active in the gonads, osteo-chondrogenic tissues, intestine, spinal cord, and pancreas, during mouse embryogenesis[18]. We analysed the expression of cell type-specific gonadal markers between E12.5 and E15.5 to determine the onset and progression of sex reversal in XY Sox9-Cre-Nr5a1 cKO mice
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