蛋白质异戊二烯化调控初级卵泡/次级卵泡转换的作用与机制研究

During the follicle growth, oocyte and surrounding granulosa cells develop together as a unit through information communication and material exchange. We found that when we deleted the gene of geranylgeranyl diphosphate synthase (GGPPS) in mouse oocyte, a key enzyme controlling the balance of protein geranylgeranylation and farnesylation, the developmental transition of primary follicle to secondary follicle was prohibited because of the defect of granulosa proliferation and mitophage of oocyte mitochondria. In this project, we will spatiotemporally delete GGPPS in oocyte and granulosa cell through crossing GGPPS-LoxP and different Cre mice to study how the balance of geranylgeranylation and farnesylation regulates the communication between oocyte and granulosa, and the function of mitochondria in oocyte during the follicle development. We will focus the mediation of Stat3 phosphorylation, which has been proved to regulated by geranylgeranylated Rac1, that controls the nucleus localization that activate gene transcription like GDF9, and mitochondrion localization that regulate oxidative phosphorylation. We will also identify the mitochondria proteins that might be geranylgeranylated and farnesylated and study their function on oxidative phosphorylation, mitophage and primary to secondary follicle transition. We further propose the concept of "mitochondria checkpoint" during follicle development that would examine the quality and quantity of mitochondria. To fulfill the condition of mitochondria checkpoint is critical for the transition of follicle to next stage. This concept would be able to apply to other physiological and pathological processes.

卵泡发育过程中，卵母细胞/颗粒细胞相互作用共同生长。在卵母细胞特异敲除蛋白质异戊二烯化修饰的关键基因香叶基香叶基焦磷酸合成酶(GGPPS)后，小鼠出现初级卵泡/次级卵泡发育阻滞的现象，其原因可能是颗粒细胞增殖受到抑制，卵母细胞线粒体自噬增加导致的能量代谢低下。本课题进一步构建GGPPS卵母细胞和颗粒细胞的时空特异敲除小鼠，以初级卵泡/次级卵泡发育转换为研究对象，从卵母细胞/颗粒细胞物质和信号交流以及线粒体功能的全新角度阐明卵泡发育的新机制。研究GGPPS控制的蛋白质异戊二烯化修饰如何影响Stat3的磷酸化，调控其进入细胞核和线粒体行使功能；鉴定线粒体的异戊二烯化修饰蛋白质，研究其在氧化磷酸化、线粒体自噬和初级卵泡/次级卵泡转换过程中的作用。我们提出卵泡发育存在“线粒体检验点”的概念：即线粒体的质量和数量对卵泡进入下一个发育阶段至关重要，这一概念对细胞的其他生理和病理过程具有广泛的指导意义。

卵泡发育过程中，卵母细胞/颗粒细胞相互作用，共同生长。围绕蛋白质异戊二烯化调控初级卵泡/次级卵泡转换的作用与机制研究，本课题通过构建Ggps1卵母细胞和颗粒细胞的时空特异敲除小鼠，发现甲羟戊酸途径代谢小分子GGPP在调控卵母细胞-颗粒细胞交流过程中扮演了重要的作用。一方面，早期卵母细胞中的GGPP通过调控小G蛋白Rac1的香叶基化，调控Stat3的入核活性进而调控卵源性分泌因子GDF9的分泌，参与了卵母细胞对颗粒细胞的调控；另一方面，卵母细胞中GGPP也可能通过调控卵母细胞线粒体的质量从而影响初级卵泡的发育。此外，我们发现Ggps1调控的线粒体质量尤其在完全生长期卵母细胞的成熟过程中有显著的影响，完全生长期卵母细胞中缺失Ggps1不影响颗粒细胞的额增殖，但是影响卵母细胞自身的质量和胚胎发育潜能的下降，并且线粒体质量的下降引起了后代胚胎质量的问题，提示GGPP调控线粒体的质量在卵母细胞的质量维持中起到至关重要的作用。另外，我们发现颗粒细胞中Ggps1调控的蛋白质异戊二烯化并非卵母细胞-颗粒细胞交流所必须，提示Ggps1调控的蛋白质异戊二烯化在卵巢中的发挥了阶段特异性和细胞特异性的作用。发现GGPP可能作为一种调控生育力的小分子，在卵母细胞早期和成熟过程中都发挥了重要的作用，早期补给GGPP可能为改善卵母细胞与颗粒细胞的交流提供新的方法；生长期卵母细胞GGPP的补给可能在临床改善卵母细胞质量，提高临床IVF结局中发挥有效作用。