TET去甲基化酶对猪卵母细胞发育的影响  

Effects of TET Demethylase on Porcine Oocyte Development

作　　者：黎明国 李清春 陈鑫 卢世豪 何凡 祁梦凡 张化鹏 任玉军 张庆泽 符彬彬 徐梦思 艾子凯 闫坤 冯赟 华再东[2] 黄涛[1] 毕延震[2] LI Mingguo;LI Qingchun;CHEN Xin;LU Shihao;HE Fan;QI Mengfan;ZHANG Huapeng;REN Yujun;ZHANG Qingze;FU Binbin;XU Mengsi;AI Zikai;YAN Kun;FENG Yun;HUA Zaidong;HUANG Tao;BI Yanzhen(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding,Institute of Animal Science and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan 430064,China)

机构地区：[1]石河子大学动物科技学院,石河子832003 [2]湖北省农业科学院畜牧兽医研究所,动物胚胎工程及分子育种湖北省重点实验室,武汉430064

出　　处：《中国畜牧兽医》2023年第5期1907-1917,共11页China Animal Husbandry & Veterinary Medicine

基　　金：新疆生产建设兵团科技创新人才计划项目中青年领军人才(2020CB018);湖北省重点研发计划(青年科学家项目)(2021BBA221);湖北省农业科技成果转化资金项目(2021ABC022);湖北省农业科技创新中心项目(2023-620-000-001-20)。

摘　　要：【目的】研究TET(ten-eleven translocation)去甲基化酶对猪卵母细胞发育的影响。【方法】将收集的猪卵丘-卵母细胞复合体(COCs)置于不同浓度(0(对照组)、100、200和400μmol/L)Bobcat339抑制剂的体外成熟培养液中培养42 h,测量卵丘扩展直径,统计成熟率,确定最小有效浓度。在体外成熟培养液中培养27 h后,利用免疫荧光(IF)检测5-甲基胞嘧啶(5mC)与5-羟甲基胞嘧啶(5hmC)信号强度以及纺锤体组装情况;利用DCFH-DA染料检测卵母细胞活性氧(ROS)水平;利用JC-1染色法检测线粒体膜电位水平(MMP);利用ATP检测试剂检测卵母细胞ATP水平;利用实时荧光定量PCR检测42 h时COCs卵丘扩展相关基因表达水平和27 h时卵母细胞抗氧化相关基因表达水平。【结果】与对照组相比,100、200和400μmol/L组卵丘细胞扩展水平均极显著降低(P<0.01),100、200和400μmol/L组成熟率显著或极显著降低(P<0.05;P<0.01),确定最小有效浓度为100μmol/L,后续试验均用该浓度。IF检测结果显示,与对照组相比,27 h时100μmol/L组卵母细胞中5mC信号强度显著升高(P<0.05),而5hmC信号强度没有显著降低(P>0.05)。与对照组相比,27 h时100μmol/L组卵母细胞纺锤体排列紊乱,组装异常。与对照组相比,ROS水平极显著升高(P<0.01),MMP和ATP水平极显著降低(P<0.01)。与对照组相比,42 h时100μmol/L组COCs中卵丘扩展相关基因透明质酸合成酶2(Has2)表达水平显著降低(P<0.05),穿透素3(Ptx3)基因表达量极显著降低(P<0.01),27 h时100μmol/L组卵母细胞中抗氧化相关基因超氧化物歧化酶1(Sod1)与Sod 2表达水平显著降低(P<0.05)。【结论】TET去甲基化酶通过清除猪卵母细胞中的5mC,降低ROS水平,提高MMP水平来指导纺锤体正常组装排列,维持COCs的正常扩展,保证猪卵母细胞正常成熟。【Objective】The purpose of this study was to investigate the effects of ten-eleven translocation(TET)demethylase on the development of porcine oocytes.【Method】The collected porcine cumulus-oocyte complexes(COCs)were cultured in vitro maturation medium with different concentrations(0(control group),100,200 and 400μmol/L)Bobcat339 inhibitors for 42 h.Cumulus expansion diameter was measured,maturity rate was calculated,and the minimum effective concentration was determined.After 27 h culture in in vitro mature medium,the signal strength of 5mC and 5hmC and the spindle assembly were detected by immunofluorescence(IF).Reactive oxygen species(ROS)were detected by DCFH-DA dye.Mitochondrial membrane potential(MMP)was detected by JC-1 staining.ATP level of oocyte was detected by ATP detection reagent.The expression levels of cumulus expansion related genes at 42 h and antioxidation-related genes in oocytes at 27 h were detected by Real-time quantitative PCR.【Result】Compared with control group,the cumulus expansion level of 100,200 and 400μmol/L groups were extremely significantly decreased(P<0.01),the maturity rate of 100,200 and 400μmol/L groups were significantly or extremely significantly decreased(P<0.05 or P<0.01).The minimum effective concentration was determined to be 100μmol/L,which was used in subsequent tests.IF test results showed that compared with the control group,5mC signal intensity in 100μmol/L group was significantly increased at 27 h(P<0.05),while 5hmC signal intensity was not significantly decreased(P>0.05).Compared with control group,ROS levels were extremely significantly increased(P<0.01),while MMP and ATP levels were extremely significantly decreased(P<0.01).Compared with control group,the expression level of mound expansion related gene hyaluronic acid synthetase 2(Has2)in the COCs of 100μmol/L group was significantly decreased at 42 h(P<0.05),and the expression level of penetrin 3(Ptx3)gene was extremely significantly decreased(P<0.01).The expression of antioxidation-related gene

关 键 词：猪卵母细胞 TET去甲基化酶 甲基化 纺锤体 活性氧 线粒体膜电位 

分 类 号：S814.6[农业科学—畜牧学]