To characterize the hormonal regulation of cyclin D2, cyclin E, and p27 in the ovary, we used the hypophysec-tomized rat, in which the effects of estradiol, FSH, and LH on granulosa cell proliferation and differentiation have been well characterized. In situ hybridization of ovarian sections from hormone-treated hypophysecto-mized (H) rats showed that cyclin D2 and p27 exhibit distinctly regulated patterns of expression in granulosa cells (Fig. 4). The results were verified by Western analysis of protein obtained from granulosa cells or luteal cells of these same animals. Specifically, cyclin D2 mRNA and protein are expressed at high levels in granulosa cells of preovulatory follicles of H rats treated with estradiol and FSH (HeF). When HEF rats are injected with hCG, cyclin D2 is down-regulated within 4 h and remains low throughout luteinization. As shown by in situ localization, the down-regulation of cyclin D2 mRNA occurs specifically in granulosa cells of preovulatory follicles that are destined to ovulate and undergo terminal differentiation or luteinization. Cyclin D2 mRNA continues to be expressed in smaller growing follicles that lack LH receptors.
The p27 cdk inhibitor is expressed in the preovulatory granulosa cells of HEF rats, and like cyclin D2, is reduced to low levels after 4 h of hCG treatment (Fig. 4). However, after this time its expression pattern differs dramatically from that of cyclin D2. By 24 h after hCG, p27 mRNA and protein are expressed at very high levels and, as shown by the in situ hybridization, are localized to the luteinizing granulosa cells of the corpus luteum. buy asthma inhaler
On the basis of the observations that both FSH and estradiol induce cyclin D2 mRNA and protein expression in granulosa cells, we sought to determine whether these hormones also affect the expression of cyclin E, a downstream mediator critical for cell cycle progression through the G1 checkpoint. Western analysis (Fig. 5) of whole cell extracts showed that levels of cyclin E were low in granulosa cells isolated from ovaries of H rats. Treatment in vivo with FSH increased cyclin E levels within 2 h, after which cyclin E remained elevated at this level. Interestingly, treatment with estradiol caused a greater increase in cy-clin E within 2 h than did FSH. Cyclin E levels then continued to increase progressively in the estradiol-treated rats for 48 h. Additional experiments showed that cyclin E expression is also regulated as cells luteinize in response to an ovulatory stimulus of hCG. As in the previous experiment, granulosa cells from untreated H rats contained low levels of cyclin E that were increased in response to estradiol (HE) or estradiol followed by FSH (HEF).