FangXiong Shi

Regulation of FoxO1 Transcription Factor by Nitric Oxide and Cyclic GMP in Cultured Rat Granulosa Cells

Abstract FoxO1 is a transcription factor implicated in a multitude of physiological processes including cell cycle progression, apoptosis and insulin signaling. Recent findings indicate that FoxO1 is a key regulator during the proliferation and maturation of granulosa cells. Over the past several years, it has become evident that nitric oxide (NO) and cGMP modulate ovarian function. There has been no information, however, about whether NO-cGMP affects FoxO1 expression or about the relationship between NO-cGMP and FoxO1. In the present study, we used immunoblot analysis to determine whether NO and cGMP affect FoxO1 expression in cultured granulosa cells. Our results clearly showed that FSH suppressed FoxO1 expression in a time-dependent manner, and that NO-cGMP stimulated FoxO1 expression in cultured granulosa cells. In addition, this stimulatory effects of NO and cGMP can be blocked by FSH in cultured granulosa cells. These findings demonstrate that NO and cGMP influence FoxO1 expression possibly through antagonizing the action of FSH in cultured granulosa cells. Results of both immunoblot analysis and immunohistochemistry also show that estradiol implantation do not affect the expression of FoxO1 in rat granulosa cells as gonadotrophins do, indicating that mechanism of estradiol on granulosa cells is different from gonadotrophins. Together, our experiments suggest that expression of FoxO1 in rat granulosa cells can be regulated by gonadotrophins and the NO/cGMP signaling pathway.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum inhibin concentrations and inhibin immunostaining during follicular development in female Sprague-Dawley rats

Intact and hypophysectomized immature rats were pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0 or 32 microg/kg p.o.) and sacrificed throughout synchronized follicular development (0, 12, 24, 48, and 72 h after equine chorionic gonadotropin, eCG). TCDD administration to intact rats resulted in a premature elevation of serum FSH and LH by 12 h post-eCG. In intact rats pretreated with TCDD, the intensity of ovarian immunoreactivity for inhibin and the number of ovarian follicles staining for inhibin in midsaggital ovarian sections were decreased at the time of eCG administration (24 h post-TCDD) in comparison to controls. However, this decreased ovarian staining for inhibin was not associated with alterations in serum inhibin concentrations. Serum inhibin was suppressed in TCDD-treated rats when compared to intact controls only at 24 h post-eCG. Hypophysectomized animals exhibited no effect of TCDD on serum inhibin at any timepoint but did have decreased estradiol concentrations during follicular development. In summary, TCDD reduced serum concentrations of inhibin after the premature increases in FSH and LH suggesting that inhibin is not important in the initial elevation of FSH following exposure to TCDD.

Localization of Ovarian Inhibin/Activin Subunits in Follicular Dominance during the Estrous Cycle of Guinea Pigs

Abstract The cellular localization of inhibin α, βA and βB subunits in cyclic ovaries of the guinea pig was investigated. The immunoreactivity of inhibin α, βA and βB subunits was localized to the granulosa cells of some large healthy follicles in each ovary throughout the estrous cycle. The number of follicles that stained was in accordance with the number of offspring typical in guinea pigs. Inhibin βB was also localized to the granulosa cells of small antral follicles on Day 4. There were two kinds of staining patterns for inhibin α, βA and βB subunits on Day 12: strongly stained follicles identical to those observed on Days 8 and 16, and weakly stained follicles that showed atresia in hematoxylin and eosin (HE) stained sections. Two types of ovarian cysts were found throughout the estrous cycle in this experiment: serous cysts and follicular cysts. The incidence of serous cysts and follicular cysts were 64% and 24% of animals, respectively. There was no positive reaction for inhibin α, βA and βB subunits in the corpora lutea, other follicles or any kind of ovarian cyst during the estrous cycle. These results comfirm that only dominant follicles stain positively for inhibin α and βA subunits and are in agreement with the phenomenon that the follicular development of guinea pigs shows two waves of growth. This study is also the first to describe the ovarian cysts during the estrous cycle in guinea pigs systematically.

Follicle selection in cyclic guinea pigs with active immunization against inhibin α-subunit.

Abstract Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125 I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine α-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3±0.3 to 7.0±1.6 per each ovary), and preovulatory sized follicles (from 1.8±0.6 to 7.0±1.6 per each ovary), and follicles stained positively for inhibin α-subunit (from 2.3±0.5 to 6.3±1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin α-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.