本项目将探索FSH通过BimEL变化调控猪卵泡自噬的分子机制。首先，以猪体外培养卵泡为对象，通过电镜观察、免疫共沉淀、蛋白检测和磷酸化抑制剂，明晰FSH引起的卵泡自噬活性改变与BimEL及磷酸化BimEL的关系。其次，以颗粒细胞为模型，运用超表达和RNAi敲降技术研究BimEL调控自噬活性的机制，同时用基因定点突变技术探索BimEL磷酸化调控自噬活性的路径，再结合细胞通路抑制剂明晰FSH通过BimEL变化调控细胞自噬活性的分子通路。然后，用RNAi、通路抑制剂结合特异磷酸化抗体检测对以上路径在卵泡体外发育中的功能进行验证。最后，以猪卵巢为材料，通过电镜观察、免疫组化、蛋白检测和免疫共沉淀等技术比较不同阶段卵泡中BimEL、磷酸化BimEL和自噬的水平变化规律，确认目标通路是否调控体内卵泡的发育。研究结果将从自噬角度重新诠释卵泡发育调控机制，还可能为卵泡发育控制技术或药物研发提供新理论依据。

This project is to investigate the molecular pathway of autophagy regulated by FSH via BimEL in porcine follicular cell. First, The electronic microscope observation, co-immunoprecipitation, western blot assay and protein phosphate inhibitors will be employed to confirm the relationships between follicle autophagy flux and BimEL or phosphorylated BimEL in the follicle treated with FSH. Then, BimEL overexpression and knockdown experiments will be performed in primary culture porcine granulosa cells to find the pathway of autophagy regulated by BimEL, and the pathway regulated by phosphorylated BimEL will be also studied by the single nucleotide mutant in bim gene. Based on the above results, specific phosphorylation inhibitor will be used for determining the exact pathway of autophagy in granulosa cell controlled by FSH via BimEL. Next, siRNA targeting bim gene and signaling pathway inhibitors experiments will be carried out in vitro culture porcine follicles to investigate the physiological function of above pathway in follicle development in vitro. Finally, the immunohistochemistry technique, electronic microscope observation and western blot assay will be used to detect the changes of BimEL, specific phosphorylated BimEL, LC3-II, Beclin-1 and autophagesome in different stage follicles to assure whether the regulating pathway regulates the follicle development in normal ovary. The results are anticipated to bring forward a new theory of follicle development from the aspect of autophagy, and will provide new targets for novel technologies and new medicine research to control follicle development in domestic animals.