Mechanisms of luteinization in the primate ovary

描述(申请人提供)：最近在灵长类动物中的证据表明，颗粒细胞的增殖在排卵间期的第一个季度内停止。然而，细胞周期进程的特定标记物，如细胞周期蛋白D2，在细胞周期停滞的同时，细胞周期抑制因子如P53的水平降低。这一应用将检验这样一种假设，即排卵刺激诱导颗粒细胞增殖的瞬时爆发，这是灵长类动物终末分化和黄体形成的基本特征。实验计划在体外黄体化剂量的促性腺激素对颗粒细胞和在体内对成年雌性恒河猴排卵刺激后的早期时间点检测颗粒细胞的细胞周期特征(目标1)。增殖爆发对颗粒细胞终末分化和黄体化的功能后果将使用体外方案来确定(目标2)。这一应用将进一步检验雌激素通过调节关键的细胞周期成分来调节增殖爆发，以及早期排卵期雌激素作用对于功能性黄体的形成至关重要的假说(目标3)。拟议的研究将利用体内控制的卵巢刺激模型，在该模型中可以确定促性腺激素和类固醇的不同作用。颗粒细胞将从接受控制性卵巢刺激的恒河猴身上获得，然后再进行排卵刺激，用于体外实验。用实时定量RT-PCR检测mRNA的表达水平，用蛋白质印迹、凝胶漂移和激活法测定蛋白质的表达水平和活性。类固醇激素的浓度将通过放射免疫法进行测量。拟议的研究有望证明灵长类颗粒细胞在排卵刺激下经历增殖爆发，而排卵刺激是黄体化的一个重要组成部分。到目前为止，人们预计雌激素通过调节关键的细胞周期成分，在推动增殖爆发方面发挥着迄今意想不到的作用。这些研究将为卵巢癌和某些类型的不孕症的病因提供洞察力，并为避孕药提供潜在的新途径。

DESCRIPTION (provided by applicant): Recent evidence in primates demonstrates that proliferation of granulosa cells is arrested within the first quarter of the periovulatory interval. However, specific markers of cell cycle progression, e.g., cyclin D2, are increased while levels of cell cycle suppressors such as p53 are reduced in concurrence with cell cycle arrest. This application will test the hypothesis that an ovulatory stimulus induces a transient burst of granulosa cell proliferation that is an essential feature of terminal differentiation and luteal formation in the primate. Experiments are planned to examine cell cycle characteristics of granulosa cells at early time points following a luteinizing dose of gonadotropin to granulosa cells in vitro and after an ovulatory stimulus to adult, female rhesus monkeys in vivo (aim 1). The functional consequences of the proliferative burst on terminal differentiation and luteinization of granulosa cells will be determined using in vitro protocols (aim 2). This application will further test the hypothesis that estrogen mediates the proliferative burst via regulation of key cell cycle components, and that early periovulatory estrogen action is essential to the formation of a functional corpus luteum (aim 3). The proposed studies will utilize an in vivo controlled ovarian stimulation model in which the differential effects of gonadotropins and steroids can be determined. Granulosa cells will be obtained from rhesus monkeys undergoing controlled ovarian stimulation prior to an ovulatory stimulus for in vitro experimentation. Levels of mRNA will be determined with real time RT-PCR, and protein levels and activity by western blot, gel shift, and kinase assays. Concentrations of steroid hormones will be measured by RIA. The proposed studies are expected to demonstrate that primate granulosa cells undergo a proliferative burst in response to the ovulatory stimulus that is an essential component of luteinization. It is expected that estrogen plays a heretofore unexpected role in driving the proliferative burst through the regulation of key cell cycle components. These studies will provide insight into the etiologies of ovarian cancer and certain kinds of infertility, as well as provide potential novel avenues for contraceptive agents.