Molecular basis of apoptosis in human granulosa cells

卵泡闭锁和黄体消退过程中颗粒细胞的凋亡是卵巢生理的一个重要方面，其异常调控可能导致卵巢功能障碍。作为阐明细胞凋亡的分子基础的第一步，我们之前在体外受精患者的颗粒细胞中证实了一组凋亡机制的表达。然而，原代培养体系在进一步分析细胞凋亡机制方面存在不足。我们用SV-40大T抗原转染正常卵巢颗粒细胞，建立了永生化细胞系。使用其中一种细胞系作为模型系统，我们证实了凋亡机制的表达，这已经在体外受精患者的颗粒细胞中得到证实。该细胞系组成性地表达Apaf-1、Bcl-X _L、Bax、Caspase-3、Caspase-9及其剪接变体以及DFF-45、DFF-40和AIF的转录本。此外，还证实了磷脂酰丝氨酸（PS）受体的表达，该受体在凋亡细胞的吞噬中起作用。用staurosporine处理这些细胞以caspase依赖和不依赖的方式诱导凋亡。我们研究了几种内源性因子作为潜在的细胞凋亡诱导剂。在研究的这些因素中，睾酮（Test）以剂量依赖的方式有效地诱导细胞凋亡。3 μM Test处理后6小时内，细胞表面有PS表达，细胞核和DNA出现断裂。caspase抑制剂Z-VAD- fmk部分抑制了细胞死亡过程。同时给予17β-雌二醇治疗无明显效果。总之，我们首次证明了雄激素依赖性诱导人颗粒细胞凋亡。

Apoptosis of granulosa cells during follicular atresia and corpus luteum regression is a crucial aspect of ovarian physiology, and the abnormal regulation may lead to ovarian dysfuncti on. As an initial step to clarify the molecular basis of apoptosis , we previously demonstrated the expression of a set of apo- ptotic machinery in granulosa cells from patients undergoing IVF.However, the primary culture system had disadvantages for further analysis of the apoptotic machinery. We established immortalized cell line s from granulosa cells of normal ovaries by transfection with SV-40 large T antigen. Using one of these cell lines as a model system, we demonstrated the expression of apoptotic machinery, which had been demonstrated in granulosa cell s from IVF pa-tients. The cell line constitutively expresses transcripts for Apaf-1, Bcl-X _L, Bax, Caspase-3, Caspase-9 and its splicing variants as well as DFF-45, DFF-40, and AIF.In addition, expression of phosphatidylserine (PS) receptor, which plays a role for phagocytosis of apoptotic cells, was also demonstrated. Treatment with staurosporine of these cells induced apoptosis in a caspase-dependent and -independent manner. We examined several endogenous factors as potential apoptosis inducers. Among those factors examined, testosterone (Test) efficiently induced apoptosis in a dose-dependent manner. Within 6 hours after treatment with 3 μM Test, PS was expressed on the cell surface accompanying with nuclear and DNA fragmentation . The cell death process was partially suppressed by a caspase inhibitor Z-VAD- fmk. Concomitant treatment with 17β- estradiol exhibited no effect. In conclusion, we have demonstrated for the first time the androgen-dependent induction of apoptosis in human granulosa cells.