FSH对兰州大尾羊卵泡细胞凋亡与FSH受体表达调控机制研究

A large number of follicles happen the apoptosis and atresia during the follicular development. The follicular atresia are caused mainly by cell apoptosis. The apoptosis is an important factor affecting the female reproductive functions. Follicle-stimulating hormone (FSH) inhibits or promotes the follicular apoptosis or atresia by binding to its receptor subtypes (FSHRα/FSHRβ). However, this actions of FSH has not been reported in Lanzhou fat-tailed sheep, especially in aspects of the ovaries FSH receptors (FSHR) expression and the mechanism of follicular cell apoptosis.The aims of the present project are as followings. (1) to explore the inhibition or promotion effects of FSH on the follicle apoptosis in the different follicle stages by combining FSHR; (2) to investigate the regulatory effects of FSH on the FSHR gene and FSHR protein expressions in the ovaries of Lanzhou fat-tailed sheep in vivo and in vitro conditions, as well as the correlations between the follicle apoptosis and apoptosis-related gene (Bcl-2/Bax, Fas/FasL, PTK-STAT, Caspase-3, Caspase-9) using immunohistochemistry, TUNEL assay,hybridization in situ , RT-PCR, Western blot, flow cytometry, Annexin-V assay and microscopy histology methods; (3) to demonstrate the FSH modulation mechanisms on follicle apoptosis during the follicular development in Lanzhou fat-tailed sheep. So as to enhance ovarian reserve capacity and reproductive performance for Lanzhou fat-tailed sheep, also to provide new ideas.

细胞凋亡是影响生殖的重要因素，卵泡刺激素（FSH）对卵泡细胞凋亡或闭锁有明显调节作用,但 FSH对卵泡细胞凋亡和凋亡相关基因及FSHR表达的作用和调控机理在兰州大尾羊至今无研究报道。本项目以兰州大尾羊为研究对象，利用免疫组织化学、TUNEL分析、原位杂交、RT-PCR、Western blot、流式细胞仪、Annexin-V分析、显微和超微组织学等方法，通过在体和离体研究，（1）探讨不同发育阶段卵泡中FSH与FSHR结合对细胞凋亡的促进或抑制作用；（2）研究卵泡细胞凋亡和凋亡相关基因(Bcl-2/Bax, Fas/FasL, PTK-STAT, Caspase-3,Caspase-9等)表达及相互关系, 分析不同浓度FSH对卵巢FSHR基因和FSHR蛋白表达的调控作用；（3）阐明FSH对兰州大尾羊卵泡发育过程中细胞凋亡的调控机制和通路，为提高兰州大尾羊卵巢储备能力和繁殖性能提供技术依据。

目的：全面探讨FSH、马绒毛膜促性腺激素（ECG）、FHS受体结合抑制剂对绵羊卵母细胞体外成熟的影响和作用机制以及信号通路。.方法: 在培养液中分别加入5、10、20、30IU/mL的促卵泡激素（FSH）和ECG,并设10IU/mL的FSH为阳性对照组，分别分为FSH-1 (对照组), FSH-2, FSH-3, FSH-4 and FSH-5五组。添加FSH受体结合抑制剂（FRBI）(0, 10, 20, 30 and 40μg/mL)进行卵丘卵母细胞复合体（COCs）体外培养24h，计算成熟率；实时荧光定量PCR（qPCR）和WB方法检测FSHR和LHR 基因和蛋白表达水平；ELISA法测定培养液中FSH、LH 和caspase-3浓度。.结果：FSH对绵羊卵母细胞体外成熟培养有显著的影响并且对绵羊卵母细胞的第一极体排出有显著的促进作用；FSH-3的成熟率最高，最适宜浓度是10IU/mL。FSH 处理能够增强卵母细胞的成熟率，减低凋亡率。FSH-3和FSH-4 组FSHR蛋白的表达水平高于FSH-1组。绵羊卵母细胞的成熟率随着ECG浓度升高而增加，eCG-4组成熟率高于其他组（P＜0.05）。FRBI-3和FRBI-4 组的IP3含量低于FSH组。FRBI添加剂量的增加，COCs成熟率逐渐减低，FRBI-4组凋亡率显著低于对照组和FSH组（P<0.05）。FRBI-3组合FRBI-4组COCs中FSHR蛋白与LHR蛋白表达水平低于FSH组。表明FRBI能够明显影响绵羊卵母细胞的成熟与凋亡。FRBI处理组小鼠的FSHR mRNAs表达水平逐渐降低，FRBI-3 和FRBI-4组分别减少29.37% 和43.65%。较高剂量的FRBI能够抑制小鼠卵巢中FSHR mRNAs的含量和水平。.结论：结果表明FSH对绵羊卵母细胞体外成熟培养有显著的影响，最适宜卵母细胞体外成熟培养的浓度是10IU/mL。eCG 能提高绵羊卵母细胞的成熟率，增加FSHR、LHR和GnRHR 基因和蛋白表达。卵母细胞的IVM成熟率与FSHR、LHR和 GnRHR mRNA和蛋白表达水平有显著的正相关性。表明FRBI能够明显影响绵羊卵母细胞的成熟与凋亡，主要是降低了Caspase-3浓度及FSHR蛋白和LHR蛋白表达水平。较高剂量的FRBI能够抑制小鼠卵巢中FSHR mRNAs和蛋白及雌激素受体（