高雄激素通过核受体NR4A1调节卵泡发育的机制研究

NR4A1 is a differentially expressed gene between normal and PCOS ovaries, which is down-regulated in PCOS ovary. Previous study suggests that over-dose androgen decreased the expression of NR4A1 in mouse follicles and in ovarian granulosa cells. Moreover, hyperandrogen can promote granulosa cells proliferation and inhibit its apoptosis. Therefore, we put forward the hypothesis that the down-expression of NR4A1 by hyperandrogen may regulate the proliferation or apoptosis of ovarian granulosa cells, which may further influence early follicular development. By mouse follicles and granulosa cells cultured in vitro, ①To explore the effects of NR4A1 over-expression and down-expression on the growth of mouse follicles and on proliferation and apoptosis of granulosa cells. ②After ovarian granulosa cells were treated with over-dose androgen, the effects of NR4A1 over-expression on granulosa cell proliferation and apoptosis were subsequently analyzed. Thus conforming hyperandrogen might be involved in the process of proliferation and apoptosis of follicular granulosa cells by regulating the expression of NR4A1. ③To examine the expression patterns of Ki-67、XIAP、cyclinD、Bcl-2、Bax、FasL、TRAIL and Caspase3，8，9 by qPCR and Western Blot after NR4A1 over-expressed or down-expressed in follicular granulosa cells. The pathway and the possible mechanisms involved in NR4A1 regulating granulosa cells proliferation and apoptosis were subsequently analysised. To date, there is no report about the effect of NR4A1 on proliferation or apoptosis of granulosa cells. This study illustrated the effect of NR4A1 on the the regulation of follicular development, and partially explained the pathophysiology of PCOS follicular development disorders.

核受体NR4A1为多囊卵巢综合征（PCOS）与正常人卵巢的差异基因，在PCOS卵巢低表达。前期研究表明，高雄激素降低NR4A1在小鼠卵泡及颗粒细胞的表达；且高雄激素促进颗粒细胞增殖、抑制细胞凋亡。由此我们提出科研假说，高雄激素可能通过NR4A1调节颗粒细胞的增殖、凋亡，进而影响卵泡发育，部分解释PCOS卵泡发育异常。本研究拟：①观察NR4A1超表达和表达沉默对小鼠卵泡生长及颗粒细胞增殖、凋亡的影响；②研究高雄激素作用下，超表达NR4A1对颗粒细胞增殖、凋亡的影响，证实高雄激素通过NR4A1调节卵泡发育；③检测NR4A1超表达和表达沉默后Ki-67、XIAP、cyclinD、Bcl-2、Bax、FasL、TRAIL及 Caspase3，8，9等因子的变化，探讨NR4A1调节颗粒细胞增殖、凋亡的分子信号通路。本研究阐明NR4A1在介导卵泡发育调节中的作用，部分解释了PCOS卵泡发育障碍的机制。

PCOS患者体内高水平的雄激素是导致卵泡发育异常的重要的病理机制环节，但卵巢内高雄激素影响卵泡发育的具体机制至今仍不清楚。核受体NR4A1在高雄激素的多囊卵巢综合征患者的卵巢中表达降低。NR4A1在高雄激素和卵泡发育异常之间是一个“间桥”，NR4A1表达下调既是高雄激素的“果”，又成为卵泡发育异常的“因”。本课题研究核受体NR4A1参与高雄激素对小鼠未成熟卵泡颗粒细胞的增殖、凋亡调控，从而对窦前卵泡的发育调节作用进行了系统研究。主要研究结果：（1）NR4A1在小鼠卵巢窦前及窦卵泡的颗粒细胞及卵泡膜细胞内均有表达，并主要定位于细胞核内，提示NR4A1作用于卵巢颗粒细胞进而调节卵泡的发育。（2）NR4A1的表达沉默明显抑制了小鼠窦前卵泡中颗粒细胞的凋亡，促进了其生存与增值。此结果与高雄激素水平下的卵泡表现相一致。提示高雄激表象素可能通过NR4A1抑制窦前卵泡的凋亡。（3）NR4A1的过表达显著增加了小鼠窦前卵泡中颗粒细胞的凋亡。一定程度上抵消了高雄激素所引起的对窦前卵泡凋亡的抑制作用。改善了高雄激素环境下的卵泡凋亡障碍，提示NR4A1在高雄激素引起的卵泡凋亡障碍中起的重要作用，可通过增加其表达水平修正窦前卵泡的凋亡障碍。（4）在高雄激素的影响下，NR4A1的表达降低，并进一步降低了线粒体相关凋亡因子的表达。其现象与NR4A1表达沉默后的凋亡改变相一致，提示高雄激素通过NR4A1调节线粒体凋亡通路，并最终达到抑制窦前卵泡凋亡的作用。结论：NR4A1通过线粒体凋亡通路影响窦前卵泡的发育和凋亡。NR4A1的表达沉默导致窦前卵泡中颗粒细胞的凋亡受到抑制，这与PCOS中的高雄激素环境下窦前卵泡凋亡抑制现象一致。通过过表达NR4A1，成功逆转了高雄激素对窦前卵泡凋亡的抑制作用。这一研究有助于进一步阐明NR4A1在介导卵泡发育调节中的作用，部分解释了PCOS卵泡发育障碍的机制，也为探寻PCOS新的靶向治疗方法积累了实验资料。