Cross Talk Between Estradiol and mTOR Kinase in the Regulation of Ovarian Granulosa Proliferation

Treatment of ovarian granulosa cells and follicles with the mammalian target of rapamycin (mTOR) kinase inhibitor results in biphasic effects where nanomolar rapamycin (RAP) results in reduced proliferation, mitotic anomalies, and attenuated follicle growth, while the picomolar RAP results in accelerated follicle growth. Here, we tested whether such effects are specific to RAP or could be mimicked by 2 alternative mTOR inhibitors, everolimus (EV) and temsirolimus (TEM), and whether these effects were dependent on the presence of estradiol (E2). Spontaneously immortalized rat granulosa cells (SIGCs) were cultured in dose curves of RAP, EV, TEM, or vehicle with or without E2. Proliferation and phosphorylation of mTOR targets p70S6 kinase and 4E-binding protein (BP) were determined. Cell cycle gene array analysis and confirmatory quantitative reverse transcriptase polymerase chain reaction were performed upon cells treated with picomolar RAP versus controls. Nanomolar RAP, EV, and TEM reduced SIGC proliferation and decreased phospho-p70 and 4E-BP. Picomolar concentrations accelerated proliferation without affecting mTOR substrate phosphorylation. Acceleration of growth by picomolar inhibitor required E2. Picomolar drug treatment altered the transcription of cell cycle regulators, increasing Integrin beta 1 and calcineurin expression, and decreasing inhibin alpha, Chek1, p16ARF, p27/Kip1, and Sestrin2 expression. At nanomolar concentrations, mTOR inhibitors attenuated granulosa proliferation. Accelerated growth and alterations in cell cycle gene transcription found with picomolar concentrations required E2 within the intrafollicular concentration range. The low concentrations of inhibitors required to increase granulosa proliferation suggest a novel use to support the growth of ovarian follicles.

用雷帕霉素（mTOR）激酶抑制剂处理卵巢颗粒细胞和卵泡会产生双相效应，纳摩尔雷帕霉素（RAP）会导致增殖减少、有丝分裂异常以及卵泡生长减缓，而皮摩尔RAP则会导致卵泡生长加速。在此，我们测试了这种效应是否为RAP所特有，或者是否可被另外两种mTOR抑制剂依维莫司（EV）和替西罗莫司（TEM）模拟，以及这些效应是否依赖于雌二醇（E2）的存在。将自发永生化大鼠颗粒细胞（SIGCs）在含或不含E2的RAP、EV、TEM或溶媒的剂量曲线中培养。测定mTOR靶点p70S6激酶和4E - 结合蛋白（BP）的增殖和磷酸化情况。对用皮摩尔RAP处理的细胞与对照细胞进行细胞周期基因芯片分析以及验证性定量逆转录聚合酶链反应。纳摩尔RAP、EV和TEM降低了SIGC的增殖，并降低了磷酸化 - p70和4E - BP。皮摩尔浓度加速增殖但不影响mTOR底物磷酸化。皮摩尔抑制剂促进生长需要E2。皮摩尔药物处理改变了细胞周期调节因子的转录，增加了整合素β1和钙调神经磷酸酶的表达，降低了抑制素α、Chk1、p16ARF、p27/Kip1和Sestrin2的表达。在纳摩尔浓度下，mTOR抑制剂减弱颗粒细胞增殖。在皮摩尔浓度下发现的加速生长和细胞周期基因转录的改变在卵泡内浓度范围内需要E2。增加颗粒细胞增殖所需的低浓度抑制剂提示了一种支持卵巢卵泡生长的新用途。