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.

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Background: Kisspeptins are important regulators of the development and function of the hypothalamic-pituitary-gonadal axis. However, the importance of kisspeptin at the pituitary level is unclear. Methods: We examined the expression profile of kisspeptin in the mouse pituitary during development and in adulthood using RT-PCR, quantitative PCR and immunohistochemistry. Results: Kiss1 mRNA was detected in both embryonic and postnatal pituitaries. Kisspeptin-immunoreactive (⁺) cells were detected from embryonic day (E) 13.5 throughout adulthood, being localized to the rostroventral portion in the anterior pituitary (AP) in embryos, and also to the dorsocaudal AP postnatally. A large proportion of kisspeptin⁺ cells were double-labeled with gonadotrope markers including Foxl2, SF-1, and LHβ, and the percentage of LHβ⁺ cells in kisspeptin⁺ cells increased during development. No kisspeptin⁺ cells were positive for the proliferating cell marker MCM7 (minichromosome maintenance protein 7), but a few kisspeptin⁺ cells co-expressed the stem/progenitor cell marker Sox2. Kisspeptin expression was similar between sexes and between agonadal SF-1 knockout embryos and wild-type littermates. Kiss1 mRNA levels were not significantly different between sexes or during early postnatal development, but levels in females increased when puberty began and were significantly higher than in males at postpubertal ages. Conclusions: These results suggest that kisspeptin is expressed in gonadotrope precursors during gonadotrope differentiation, and that kisspeptin expression begins soon after the initiation of αGSU production and is extinguished soon after the initiation of LH production. Furthermore, pituitary kisspeptin expression may be regulated in a gonad-independent manner during development, but may be associated with gonadotrope function in adulthood.

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