p66Shc在绵羊卵母细胞中的表达及其与胞质氧化还原稳态的关系  

Expression of p66Shc and Its Relationship with Cytoplasmic Redox Homeostasis in Sheep Oocytes

作　　者：张通[1,2] 栗瑞兰 范晓梅[1,3] 刘春洁 海日汗[1] 霍敏 张家新 ZHANG Tong;LI RuiLan;FAN XiaoMei;LIU ChunJie;HAI RiHan;HUO Min;ZHANG JiaXin(Inner Mongolia Autonomous Region Key Laboratory of Animal Genetics,Breeding and Reproduction/College of Animal Science,Inner Mongolia Agricultural University,Hohhot 010018;College of Medicine,Shanxi Datong University,Datong,037009 Shanxi;Basic Medical College,Inner Mongolia Medical University,Hohhot 010110)

机构地区：[1]内蒙古农业大学动物科学学院/内蒙古自治区动物遗传育种与繁殖重点实验室,呼和浩特010018 [2]山西大同大学医学院,山西大同037009 [3]内蒙古医科大学基础医学院,呼和浩特010110

出　　处：《中国农业科学》2019年第12期2183-2192,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31460598)

摘　　要：[目的]研究p66Shc在绵羊卵母细胞中的表达特征及其与卵母细胞胞质氧化还原稳态的关系,为揭示p66Shc参与调控卵母细胞胞质氧化还原稳态分子机制提供理论依据。[方法]试验所用卵丘-卵母细胞复合体(cumulus-oocyte complexes, COCs)来源于屠宰场的绵羊卵巢。分别收集成熟前优质和劣质、常规成熟24 h及成熟30 h老化的卵母细胞,采用实时荧光定量PCR对成熟前后不同质量绵羊卵母细胞中p66Shc mRNA的表达量进行分析,利用细胞免疫荧光结合MitoTracker Red探针对p66Shc蛋白和线粒体进行共定位,同时利用荧光探针DCFH-DA和卵母细胞自发荧光分别对成熟前后不同质量卵母细胞内ROS水平和氧化还原稳态进行检测。此外,采用外源H2O2诱导的氧化应激处理成熟前优质卵母细胞,并对p66Shc蛋白的表达及定位进行了检测分析。[结果]实时荧光定量PCR结果显示,成熟前劣质和成熟后老化卵母细胞中p66Shc mRNA表达量分别显著(P<0.05)高于成熟前优质和常规24 h成熟的卵母细胞。然而优质卵母细胞成熟前后p66Shc mRNA表达量没有显著差异(P>0.05)。共定位结果表明,p66蛋白主要分布在线粒体分布活跃的区域。细胞免疫荧光结果显示,成熟前劣质和成熟后老化卵母细胞中p66Shc蛋白表达量也分别显著(P<0.05)高于成熟前优质和常规24 h成熟的卵母细胞。与成熟前优质和常规24 h成熟的卵母细胞相比,成熟前劣质和成熟后老化卵母细胞表现出线粒体分布紊乱和活性下降、ROS水平升高以及氧化还原稳态的失衡。此外,与未添加外源H2O2的对照组相比,外源H2O2处理成熟前优质卵母细胞诱导的氧化应激显著(P<0.05)上调p66Shc蛋白的表达并促使p66Shc由胞质向细胞核定位。[结论]p66Shc基因在劣质和老化的绵羊卵母细胞中呈现高水平的表达,H2O2诱导的氧化应激显著上调p66Shc蛋白的表达并影响其亚细胞定位。总之,绵羊卵母细胞中p【Objective】The aim of this study was to investigate the expression characteristics of p66Shc and its relationship with cytoplasm redox homeostasis in different quality sheep oocytes before and after maturation, which could provide a theoretical basis for revealing the molecular mechanism of p66Shc involved in the regulation of cytoplasmic redox in oocytes.【Method】The sheep cumulus-oocyte complexes (COCs) of ovaries used in this experiment were derived from the slaughterhouse. High quality and poor quality immature oocytes, conventional 24 h matured oocytes and aged oocytes (matured 30 h) were collected, respectively. The expression of p66Shc in different quality sheep oocytes before and after maturation was analyzed by quantitative real-time PCR. The p66Shc protein and mitochondria were co-located by using cellular immunofluorescence combined with MitoTracker Red probe. Simultaneously, the ROS level and the redox homeostasis of different quality oocytes were detected by fluorescence probe DCFH-DA and oocyte spontaneous fluorescence, respectively. In addition, exogenous H2O2-induced oxidative stress was used to treat high quality immature oocytes, and the expression and localization of p66Shc protein were detected and analyzed.【Result】The real-time fluorescent quantitative results showed that the expression of p66Shc mRNA in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes (P<0.05). However, there was no significant difference in the expression of p66Shc mRNA in high-quality oocytes before and after maturation (P>0.05). The co-localization results showed that the regional distribution of p66 protein was consistent with active mitochondrial distribution. The cellular immunofluorescence results showed that the expression of p66Shc protein in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes (P< 0.05

关 键 词：绵羊卵母细胞 P66SHC 线粒体 ROS 氧化还原稳态 

分 类 号：S826[农业科学—畜牧学]