Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells

Abstract

The nuclear receptor steroidogenic factor 1 (SF-1 [officially designated NR5A1]) is essential for fetal gonadal development, but its roles in postnatal ovarian function are less well defined. Herein, we have extended our analyses of knockout (KO) mice with markedly decreased SF-1 expression in granulosa cells. As described, these SF-1 KO mice had hypoplastic ovaries that contained a decreased number of follicles and lacked corpora lutea. In the present study, we showed that SF-1 KO mice exhibited abnormal estrous cycles, were infertile, and released significantly fewer oocytes in response to a standard superovulation regimen. Moreover, they had blunted induction of plasma estradiol in response to gonadotropins. The granulosa cell-specific SF-1 KO also significantly affected ovarian expression of putative SF-1 target genes. Consistent with their decreased follicle number, these mice had reduced ovarian expression of anti-müllerian hormone (Amh), which correlates with the reserve pool of ovarian follicles, as well as decreased gonadotropin-induced ovarian expression of aromatase (Cyp19a1) and cyclin D2 (Ccnd2). In contrast, perhaps because of their abnormal cyclicity, SF-1 KO ovaries had higher basal expression of inhibin-alpha. They also had decreased immunoreactivity for genes related to proliferation (Ccnd2 and Mki67 [also known as Ki67]) and increased expression of Cdkn1b, also known as p27, which inhibits cyclin-dependent kinases, arguing for a role of SF-1 in granulosa cell proliferation. These findings demonstrate that SF-1 has a key role in female reproduction via essential actions in granulosa cells.

FIG. 1.

Oocyte yields after gonadotropin-induced ovulation in 8- to 12-wk-old WT, SF-1 Het, and SF-1 KO mice. All mice were treated with eCG (5 IU), followed 48 h later with hCG (5 IU). Oocytes were harvested from the oviducts and counted at approximately 16 h after hCG treatment. Each data point represents the oocyte yield of an individual mouse of the respective genotypes. The horizontal line represents the mean value of each group. The numbers of mice studied for each genotype were WT (n = 8), SF-1 Het (n = 6), and SF-1 KO (n = 7).

FIG. 2.

Hematoxylin-eosin staining of ovarian histology of adult WT and granulosa cell-specific SF-1 KO mice reveals the decreased size of the KO ovary and the absence of CLs. For SF-1 immunoreactivity, lower-magnification (middle panels) and higher-magnification (bottom panels) views are shown of sections of WT and granulosa cell-specific SF-1 KO mice that were analyzed for SF-1 expression by immunohistochemistry as described in Materials and Methods. The asterisk indicates a follicle in the KO section in which granulosa cells still retain some SF-1 immunoreactivity. Bar = 200 μm.

FIG. 3.

Gross structure and histology of genital tracts in adult females. A) Genital tracts of WT, SF-1 Het, and SF-1 KO mice. Ruler scale is mm. B) Uterine histology at lower magnification. Bar = 100 μm. C) Uterine histology at higher magnification. Sections from all mice have three uterine layers: myometrium (my), endometrial stroma with endometrial gland (eg), and epithelium (ep). The WT and SF-1 Het sections reveal a thick stromal layer and a considerable number of endometrial glands, whereas the SF-1 KO section shows a less differentiated glandular structure. Bar = 200 μm.

FIG. 4.

Analysis of ovarian gene expression both basally and at 48 h after eCG treatment. The WT, SF-1 Het, and SF-1 KO mice were killed at the indicated times, whole ovaries were collected, tRNA was extracted, and transcript levels were determined by qPCR as described in Materials and Methods. Gene expression levels in samples are shown relative to basal expression in WT ovaries, which is set to 1. * P < 0.05. ** P ≤ 0.01 for comparison between SF-1 KO and WT at the same time point. ^# P < 0.05 for comparison between SF-1 KO and SF-1 Het at the same time point. ^§ P < 0.05 for comparison between SF-1 Het and WT at the same time point.

FIG. 5.

Immunohistochemical detection of SF-1 target genes in ovarian serial sections. Ovarian sections from adult female WT and granulosa cell-specific SF-1 KO mice were prepared and analyzed by immunohistochemical analysis as described in Materials and Methods using the indicated specific antibodies. The bottom panel shows the expression of CYP11A1 after eCG treatment. The asterisk indicates the position of the oocyte within the same large follicle in serial sections of the SF-1 KO ovary. Bar = 200 μm.

FIG. 6.

Immunohistochemical detection of cyclin D2, MKI67 (Ki67), and CDKN1B (p27) in ovarian sections. Ovarian sections from adult female WT and granulosa cell-specific SF-1 KO mice were prepared and analyzed by immunohistochemistry as described in Materials and Methods using the indicated specific antibodies. The asterisk indicates the position of the oocyte within the same large follicle in serial sections of the SF-1 KO ovary. Bar = 200 μm.