LPA对牦牛卵丘细胞主要卵源因子表达的影响  

Effect of LPA on the Expression of Main Oocytesreted Factors in Cumulus Cells of Yak(Bos grunniens)

作　　者：刘斌[1] 王靖雷 高丽青 许瑞华 张倩[1] 王萌[1] 王立斌[1] 余四九[1] 徐庚全[1] 潘阳阳[1] LIU Bin;WANG Jing-Lei;GAO Li-Qing;XU Rui-Hua;ZHANG Qian;WANG Meng;WANG Li-Bin;YU Si-Jiu;XU Geng-Quan;PAN Yang-Yang(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《农业生物技术学报》2023年第5期989-1000,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32160859);甘肃省科技重大专项(21ZD10NA001);甘肃省重点人才项目(2022-0623-RCC-0307)。

摘　　要：一定浓度的溶血磷脂酸(lysobisphosphatidic acid,LPA)可以促进卵母细胞成熟,而卵丘细胞以及卵源因子(oocyte-secreted factors,OSFs)能够保障卵母细胞生长发育及功能的正常发挥。本研究旨在对卵丘细胞所分泌的卵源因子受到外源性溶血磷脂酸的调控后,其表达量的变化进行研究。以体外培养的牦牛(Bos grunniens)卵丘细胞为研究对象;添加外源性LPA(0,5,15,30和50μmol/L)进行调控,采用PCR、qPCR、Western blot及间接免疫荧光等方法,检测经LPA调控后卵丘细胞分泌的OSFs的表达变化。qPCR结果显示:当添加的LPA浓度为15μmol/L时,生长分化因子9(growth differentiation factor 9,GDF9)和成纤维细胞生长因子(fibroblast growth factors,FGF10)mRNA的相对表达水平达到最高,浓度大于15μmol/L时,GDF9和FGF10 mRNA的表达水平降低。LPA浓度为30μmol/L时,骨形态发生蛋白1(bone morphogenetic proteins 1,BMP1)和BMP15 mRNA的相对表达水平达到最高,浓度大于30μmol/L时,BMP15 mRNA的表达水平降低,BMP1 mRNA的相对表达水平变化不显著。Western blot结果显示:当LPA浓度为30μmol/L时,GDF9、BMP1、BMP15蛋白的相对表达水平最高,浓度大于30μmol/L时,BMP1、BMP15蛋白的表达水平降低,而GDF9蛋白的表达受到抑制。相比较其他OSFs蛋白对LPA的应答,在一定LPA浓度范围内,FGF10蛋白相对表达水平与LPA的浓度之间呈现剂量依赖性。免疫荧光结果显示:0μmol/L处理组OSFs在卵丘细胞的荧光标记主要集中在细胞质和细胞核,而LPA处理后的样本,OSFs在卵丘细胞细胞质中的荧光标记明显增强。本研究证实,在牦牛卵丘细胞体外培养过程中,外源性LPA能够在基因与蛋白水平上调节OSFs在卵丘细胞中的表达,添加适当浓度的LPA能够增加卵丘细胞相关细胞因子的分泌,进而对卵母细胞的发育进行调节。本研究对于构建牦牛以及其他哺乳动物卵母细胞成熟的体外培养模式具有借鉴意义。A certain concentration of lysobisphosphatidic acid(LPA)can promote oocyte maturation,while cumulus cells and oocyte-secreted factors(OSFs)can guarantee the normal development and function of oocytes.The purpose of this study was to investigate the expression of oocyte-secreted factors(OSFs)regulated by exogenous lysophosphatidic acid(LPA)in cumulus cells.The cumulus cells of Bos grunniens cultured in vitro were used as the research object,and exogenous LPA(0,5,15,30 and 50μmol/L)was supplemented into the cultured cumulus cells.PCR,qPCR,immunofluorescence tech and Western blot(WB)were used to evaluate the expression status of OSFs after exogenous LPA regulation.qPCR results showed that when the concentration of exogenous LPA was 15μmol/L,the relative expression levels of growth differentiation factor 9(GDF9)and fibroblast growth factors(FGF10)mRNA were the highest.And when the concentration was greater than 15μmol/L,the expression levels of GDF9 and FGF10 mRNA decreased.The relative expression levels of bone morphogenetic proteins 1(BMP1)and BMP15 mRNA were the highest when the concentration of LPA was 30μmol/L.However,when the concentration was greater than 30μmol/L,the expression of BMP15 mRNA decreased,and the relative expression level of BMP1 mRNA did not change significantly.Western blot results showed that when the concentration of LPA was 30μmol/L,the relative expression level of GDF9,BMP1 and BMP15 protein was the highest.However,when the concentration of LPA was more than 30μmol/L,the expression levels of BMP1 and BMP15 proteins were decreased,while the expression of GDF9 protein was inhibited.Compared with the response of other OSFs proteins to LPA,the relative expression level of FGF10 protein and the concentration of added LPA were dose-dependent within a certain range of LPA concentrations.Immunofluorescence results showed that the fluorescence labeling of OSFs in the cumulus cells of 0μmol/L treatment group was mainly concentrated in the cytoplasm and nucleus,while the fluorescence labeling

关 键 词：牦牛 卵丘细胞 卵源因子(OSFs) 溶血磷脂酸(LPA) 

分 类 号：S857.2[农业科学—临床兽医学]