雌激素膜受体GPR30与PI3K/AKT/mTOR介导的雌激素对山羊卵泡体细胞与卵母细胞互作的调控机制研究

雌激素对卵泡发育具有重要调节作用，但其通过雌激素膜受体GPR30发挥作用的分子机制尚不明确。申请人前期研究发现，雌二醇（E2）通过激活GPR30调控山羊卵丘细胞与卵母细胞间通讯和卵母细胞成熟进程，并有研究证明E2/GPR30通路可激活PI3K及其下游通路且二者存在协同调控关系。本研究以山羊卵泡为主要材料，应用卵泡培养、卵泡荧光探针注射与活细胞成像、RNA干扰、qRT-PCR、WB、免疫荧光等技术，系统研究E2/GPR30对卵母细胞生长与成熟、卵泡发育或闭锁相关基因表达调控，E2/GPR30对PI3K/AKT/mTOR通路激活及其关键分子表达与活化状态，以及颗粒细胞凋亡与自噬，卵母细胞核成熟与纺锤体组装、线粒体分布及其活性、RNA转录和蛋白合成效率，颗粒细胞与卵母细胞间通讯及相关基因表达，解释E2/GPR30与PI3K/AKT/mTOR通路在卵泡体细胞与卵母细胞互作中的分子调控机制。

Estrogen plays an important role in the regulation of follicular development, however, its molecular mechanism through the estrogen membrane receptor GPR30 is unclear. We previous found that estradiol (E2) regulated the communication between goat cumulus cells and oocytes and the maturation process of oocytes by activating GPR30. It has been proved that E2/GPR30 pathway could activate PI3K and its downstream pathways through a cooperative interaction with each other. In this study, goat follicles will be used as the main materials. Take advantage of follicle culture, intra-follicle fluorescent probe injection and live cell imaging, RNA interference, quantitative PCR, Western blot, immunofluorescence and other techniques, the genes expression related to oocyte growth, maturation and follicle development or atresia, the expression and activities of key molecules in PI3K/AKT/mTOR signaling pathway, granulosa cell apoptosis and autophagy, oocyte nuclear maturation and spindle assembly, mitochondrial distribution and activity, RNA transcription and protein synthesis efficiency in oocytes, and intercellular communication between granulosa cells and oocytes and related gene expression, will be systematically investigated during follicular development, in order to elucidate the regulation mechanism of E2/GPR30 and PI3K/AKT/mTOR pathway in the interaction between follicle somatic cells and oocytes in goats.