FSH对体外培养绵羊卵巢原始卵泡激活过程中PI3K-AKT通路的影响

Primordial follicles activation(PFA) is a major biological checkpoint in the follicles development of female mammal. The results of the in vivo study showed that, the PFA process is independent of pituitary gonadotropins, such as FSH and LH. However, recent studies have provided strong evidence that FSH might be involved in early development of follicle in indirect or direct pathway, in which PI3K-Akt signaling pathway might be an important way in the regulation of PFA process, however, at present, the effect of FSH on the PFA process and PI3K-Akt signaling pathway is not clear. This object will focus on the primordial follicles of newborn female Chinese Merino Sheep (Xinjiang Junken Type) , by addition of FSH in vitro culture process in different ways, analysis of the effects of FSH on the PFA process and the proteins worked in PI3K-Akt signaling pathway, from three aspects of cell, protein and gene levels, meanwhile, the method of histology, immunohistochemistry, RNA-Seq, QT-PCR, RT-PCR, protein and phosphorylation chip will be used, and finally to explore the effect mechanism of FSH on the PFA process and PI3K-Akt signaling pathway. This research will provide a new vision in PFA study, deepen our understanding of the law of sheep follicle development, and provide theoretical basis for in vitro culture of sheep primordial follicle. Thus, it is of great significance to improve the reproductive potential of animals, and solve insufficient of oocytes for gene cloning and embryo production in vitro.

原始卵泡激活（PFA）是雌性哺乳动物卵泡发育过程中一个重要的生物学控制节点；体内研究结果表明PFA不依赖于促卵泡素（FSH）等促性腺激素，但近年来体外实验表明FSH可能通过直接或间接的方式参与早期卵泡的发生,其中PI3K-Akt通路可能是其调控PFA的一条重要途径，目前对FSH在这一通路中的具体作用还不清楚。本项目以新生雌性新疆军垦型细毛羊原始卵泡为研究对象，在体外培养的条件下，采用不同的FSH添加方式，以组织学，免疫组化，RNA-Seq，QT-PCR，RT-PCR、蛋白磷酸化芯片等方法，从细胞、蛋白、基因三个层面分析FSH对PFA以及PI3K-Akt信号通路中各效应蛋白的影响，探讨其通过PI3K-Akt通路对PFA的作用机制。项目的实施可加深对绵羊卵泡发育规律的了解，为开发原始卵泡体外培养技术提供理论依据，对提高动物繁殖潜力，解决基因克隆、胚胎体外生产中卵母细胞来源不足等具有重要意义。

原始卵泡激活（PFA）是卵泡发育命运中一个重要的生物学控制节点；体内研究表明PFA是 促卵泡素（FSH）非依赖性的，但近年来体外实验表明FSH可能通过直接或间接的方式参与早期 卵泡的发生,其中PI3K-Akt通路可能是其调控PFA的一条重要途径，目前对FSH在这一通路中的具体作用还不清楚。本项目以新生雌性新疆军垦型细毛羊原始卵泡为研究对象，在体外培养的条件下，采用不同的FSH添加方式，以组织学，免疫组化，RNA-Seq、蛋白磷酸化组学分析等方法，从细胞、蛋白、基因三个层面分析FSH对PFA以及PI3K-Akt信号通路中各效应蛋白的影响，探讨其通过PI3K-Akt通路对PFA的作用机制。研究结果表明添加FSH可显著促进体外培养卵巢皮质中原始卵泡激活发育和维持存活，转录组学和蛋白磷酸化组学分析结果均表明FSH添加可以显著促进该通路中相关蛋白如RTK，AKT1/2/3，FOX03/4, S6K,TSC1/2，PI3K，SMG1等的基因表达和蛋白活化，其可能通过RTK-PI3K-AKT通路参与绵羊原始卵泡的体外激活。本研究的实施可加深对绵羊卵泡发育规律的了解，为开发原始卵泡体外培养技术提供理论依据，对提高动物繁殖潜力，解决基因克隆、胚胎体外生产中卵母细胞来源不足及女性生殖保健等具有重要意义。