| Throughout ovarian development and function in mammals, a highly orchestrated, periodic process known as follicular atresia occurs that destroys and eliminates follicles and oocytes from the ovary. Follicular atresia is pervasive.In humans, it is estimated to account for99.9%of the loss of oocytes from development of the fetal ovary until reproductive senescence. Growth of follicles is an ongoing process, beginning with the growth of primordial follicles. When these follicles develop into mature, preovulatory size follicles, they will be atresia without ovulation. It is generally believed that granulosa cell apoptosis is considered the underlying mechanism of follicular atresia. Mechanism of atresia in preovulatory follicles is still unclear now. In this study, we make the experimental mice exposed to FSH treatment. So the majority of ovarian follicles in the mouse can develop into the stage of preovulatory size at the same time. As we know, preovulatory follicles will undergo atresia without the peak of LH. We successfully constructed a model of large follicular atresia with the mice exposed to FSH treatment but not to LH. This study investigated the effect of FSH on the preovulatory follicular atresia and the regulation of FSH on some important regulatory factors in the granulosa cells. Our results showed that:1) By the TUNEL assay, we show that as long as the follicles became atresia, the apoptosis of granulosa cells in the follicles became more and more serious. In this process of follicular atresia, FSH can inhibit granulosa cell apoptosis. From the result we can also indicate that the morphological form of preovulatory follicular atresia is antral atresia which is characterized by the initial elimination of granulosa cells proximal to the antrum.2) In order to examine the relationship between Caspases family members and granulosa apoptosis, we performed RT-PCR comparisons of the level of Caspases in healthy preovulatory follicles versus atresic follicles. We found that the expression of Caspase-8did not change significantly in the atresic follicles. And FSH has no effect on Caspase-8in this process. In the contrary, Caspase-9and Caspase-3have a different expression level in the different state of the follicle. Based on this result we speculate that apoptosis of granulosa cells in this process may be media by the mitochondrial apoptotic pathway.3) We also have performed RT-PCR to compare the level of FoxO1in healthy preovulatory follicles versus atresic follicles. We found that FoxO1level was low in granulosa cells of healthy preovulatory follicles. And FSH can inhibit FoxO1expression in atresic follicles. The amount of FoxO1protein exhibits the same regularity with the mRNA.4) In the cultured granulosa cells, the stimulation of FSH results in ERalpha protein translocation from the cell nucleus into the cytoplasm. Chromatin immunoprecipitation (ChIP) and real time quantitative PCR were used to examine the interaction between ERalpha protein and FoxO1gene promoter. The result showed that in the atresic follicles there is a stronger interaction than that in the healthy follicles, and FSH can inhibit this interaction in the atresic follicles.Thus, through this study, we can indicate that FSH can inhibit the expression of ERalpha and the localization of ERalpha in the nucleus in the atresic preovulatory follicles. We also found that ERalpha protein can interact with the FoxO1promoter. Based on these results, we can speculate that the inhibition of FSH on follicles atresia may be through this way. |
