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FoxO1 is a transcription factor implicated in a growing number of physiological processes, including apoptosis, cell cycle progression, and insulin signaling. Recent findings indicate that FSH and growth factors influence ovarian functions in part through regulation of FoxO1. The present study utilized immunohistochemical analysis to determine the ovarian localization and regulation of FoxO1 protein levels in neonatal rats, immature rats during gonadotropin-induced follicular development, ovulation, and luteinization, and in spontaneously developing ovarian cysts of aging rats. In postnatal rats, FoxO1 immunoreactivity was very faint in ovaries of 5- and 10-day-old females. In contrast, strong immunoreactivity was observed in granulosa cells of larger developing follicles at 25 days of age. To stimulate follicle development, immature female rats received equine chorionic gonadotropin (eCG) followed 52 h later by an ovulatory dose of human chorionic gonadotropin (hCG). Prior to gonadotropin treatment, moderate FoxO1 immunoreactivity was observed in granulosa cells of small follicles. Subsequently, treatment with eCG markedly decreased FoxO1 protein levels in granulosa cells of healthy antral and preovulatory follicles. Interestingly, FoxO1 staining was observed in cumulus and antral, but not mural granulosa cells of preovulatory follicles. Induction of ovulation and luteinization with hCG further decreased ovarian FoxO1 levels, with no staining evident in corpora lutea. At all time points, the most intensive FoxO1 staining was observed in granulosa cells of atretic follicles, with predominantly nuclear localization. Similarly, while FoxO1 levels were low in granulosa cells of preovulatory follicles in proestrous rats, FoxO1 staining was intense in granulosa cells of spontaneously developing cystic follicles in aged, acyclic females. Together, these findings indicate that FoxO1 is expressed in a regulated, cell-specific manner during ovarian follicular development, atresia and luteinization, suggesting roles in these physiological processes.

Previous studies demonstrated inhibitory effects of nitric oxide (NO) and cGMP on ovarian steroidogenesis. This study examined the effects of NO on estrogen levels and cAMP accumulation from immature cultured rat granulosa cells. Granulosa cells were incubated with media alone (control), FSH or FSH plus increasing concentrations of the NO generator, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO). While FSH increased estrogen levels 15-fold compared with controls, DETA/NO inhibited FSH-stimulated aromatase activity in a dose-dependent manner. Time-course studies revealed that the inhibitory effects of DETA/NO on aromatase activity persisted throughout the 72 h culture period. Treatment with DETA/NO also inhibited the stimulatory effects of forskolin on estrogen production, indicating that NO can influence steroidogenesis by actions downstream of the FSH receptor. Incubation of cells with FSH plus DETA/NO increased cGMP accumulation over 100-fold, compared with cells treated with media or FSH alone. In this regard, a cGMP analog mimicked the inhibitory effects of NO on FSH- and forskolin-stimulated estrogen production, indicating a potential mechanism of NO action. NO also decreased FSH-stimulated (cAMP) accumulation from cultured cells, indicating an antagonistic effect of NO on the second messenger mediating FSH actions. These findings demonstrate that NO inhibits estrogen production from rat granulosa cells, potentially reflecting actions on the second messengers cGMP and cAMP.