Using the Cre/loxP system, we studied the roles of SF-1 in ovarian physiology in vivo, focusing specifically on its functions in granulosa cells. Consistent with our initial description [21
], the granulosa cell-specific inactivation of SF-1 caused marked postnatal gonadal defects, including ovarian hypoplasia, altered uterine differentiation, and defects in both estrogen biosynthesis and fertility. Although these findings do not exclude important roles of LRH-1 in ovarian function, they define essential roles of SF-1 in granulosa cell function that cannot be replaced by LRH-1. In most respects, SF-1 Het mice exhibited no reproductive phenotype; they had mild ovarian hypoplasia with normal ovarian function and approximately 65% of the normal number of oocytes harvested after superovulation. Moreover, the gene expression profiles in SF-1 Het mice generally paralleled those in WT controls both basally and after eCG treatment, indicating that the presence of one SF-1 allele suffices for largely normal ovarian function.
SF-1 KO mice exhibited markedly reduced ovarian size, with a significantly decreased number of growing follicles and the absence of CLs (), suggesting that they are impaired in terminal stages of follicle differentiation and/or ovulation. A careful inspection of ovarian histology in young adult SF-1 KO mice showed a paucity of primary and secondary follicles, which led us to evaluate the number of follicles just before the initiation of puberty. At age 21 days, SF-1 KO mice already had a decreased number of follicles (). These data again suggest that SF-1 has important roles in ovarian development and follicular maturation. This defect was not apparent during fetal development of SF-1 KO ovaries up to Embryonic Stage 16.5 [21
We also evaluated ovarian function by determining the plasma levels of ovarian steroid hormones both basally and after stimulation with gonadotropins (). The basal E2 level in SF-1 KO mice did not differ significantly from the levels in WT and SF-1 Het mice, but marked impairment was noted in the response to eCG. Moreover, basal plasma FSH revealed a significantly increased concentration in SF-1 KO mice compared with mice of other genotypes. These data together indicate that ovarian estrogen production is defective in mice with granulosa cell-specific KO of SF-1. Although developing ovarian follicles are the main source of E2, other tissues (e.g., adipose tissue) can produce estrogen. The normal basal E2 level in SF-1 KO mice may derive from these other sources.
] have demonstrated that AMH—which is produced by granulosa cells of the growing follicles—suppresses development of the primordial follicles. Thus, the FSH-dependent recruitment of small antral follicles in Amh
KO mice was increased, and premature exhaustion of the primordial follicle reserve occurred [31
]. SF-1 has a key role in Amh
]; consistent with this, Amh
transcripts were decreased in our qPCR analyses, and only a few follicles contained immunoreactive AMH (). Therefore, the decreased levels of AMH in the granulosa cell-specific SF-1 KO mice may contribute to the diminished follicle number observed.
Similarly, the reduced induction of Cyp19a1 expression in ovarian granulosa cells seen in the qPCR and immunohistochemical analyses likely contributes to the deficient E2 production. The hypoplastic uteri with impaired glandular differentiation were consistent with estrogen deficiency in the granulosa cell-specific SF-1 KO mice. We cannot exclude the possibility that these ovaries had few follicles and therefore had lower capability to produce E2 after gonadotropin induction.
] in female aromatase KO mice showed severely impaired E2 synthesis and follicle development, resulting in defective ovulation. The phenotype described herein resembles several aspects of aromatase KO mice but apparently was less pronounced in terms of hormone deficiencies and gonadal morphology. The attenuated phenotype in mice with granulosa cell-specific KO of SF-1 likely arises because they lack SF-1 and CYP19A1 only in ovarian granulosa cells, whereas aromatase KO mice lack CYP19A1 expression in all tissues. By avoiding any exogenous source of estrogen with a soy-free diet [34
], we showed that our mice can still produce E2 but not at levels sufficient to induce ovulation. Moreover, we found that the ovarian hemorrhagic cysts previously described [21
] occurred in only a few of these mice fed a phytoestrogen-free diet.
As a proposed target gene of SF-1 in ovarian cells, we also examined inhibin-alpha expression in the granulosa cell-specific SF-1 KO mice. This gene is transcriptionally activated by FSH in ovarian granulosa cells during follicular growth [24
]. Transient transfection assays using the inhibin-alpha promoter showed that both SF-1 and LRH-1 can increase inhibin-alpha promoter activity [20
]. A dynamic association/dissociation model has been proposed to explain the interactions of NR5A members and the inhibin-alpha promoter: at baseline, SF-1 is bound to the inhibin-alpha promoter, but FSH induces LRH-1 to replace SF-1 [20
]. In our investigations, increased basal expression of Inha
was detected in SF-1 KO mice, localizing to granulosa and theca/interstitial cells. Therefore, we hypothesize that LRH-1 can replace SF-1 to induce basal Inha
expression in granulosa cells. However, LRH-1 alone was unable to increase Inha
expression after gonadotropin stimulation, as revealed by the reduced Inha
transcript levels in SF-1 KO mice. These findings, together with the elevated FSH concentration, suggest that adult ovarian granulosa cells of SF-1 KO mice are unable to respond appropriately in terms of gene expression and steroid biosynthesis. A similar defect is seen in aging mice where the negative regulation of FSH by ovarian inhibin is lost, supporting an accelerated ovarian senescence phenotype in these mice. Chronically elevated expression of inhibin-alpha was reported in ovaries of acyclic aging female rats, and increased expression of INHA has been associated with lack of ovulation and loss of estrous cyclicity due to loss of estrogen secretion [36
]. The extent to which altered expression of Inha
contributes to the phenotype seen in the granulosa cell-specific SF-1 KO mice remains to be determined.
The impaired induction of cyclin D2 transcripts by eCG, the lower immunoreactivity for cyclin D2 and MKI67, and the intense signal of CDKN1B suggest that SF-1 KO mice have an underlying defect in granulosa cell proliferation and follicle growth [27
]. Thus, changes in proliferation may be a major factor in their phenotype. We previously showed that male mice with Leydig cell-specific SF-1 KO had impaired somatic cell proliferation during fetal development [21
], and evidence from other studies [38
] strongly implicates SF-1 as a key regulator of adrenocortical proliferation. By analogy, it is likely that SF-1 regulates other genes involved in granulosa cell proliferation; the identification of these genes will be an important target for future studies.