SETD2在猪卵母细胞体外成熟中的作用  

Effects of SETD2 on in vitro Maturation of Porcine Oocytes

作　　者：高乐鹏 余思聪 刘双 马馨[1] 李所[1] GAO Lepeng;YU Sicong;LIU Shuang;MA Xin;LI Suo(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林农业大学动物科学技术学院,长春130118

出　　处：《吉林农业大学学报》2023年第3期365-371,共7页Journal of Jilin Agricultural University

基　　金：吉林省科技发展计划项目(20190103149JH);吉林省教育厅项目(JJKH20220361KJ);国家自然科学基金项目(31902162)。

摘　　要：SETD2是H3K36me3特异性甲基转移酶,参与组蛋白甲基化修饰,并通过调节多种基因的表达影响细胞功能。为探究SETD2在猪卵母细胞体外成熟(in vitro maturation,IVM)过程中的表达规律及作用,通过免疫荧光染色检测SETD2及H3K36me3在猪卵母细胞IVM过程中各个时期(GV、MⅠ、MⅡ期)的表达定位情况;在GV期显微注射SETD2干扰RNA(SETD2-siRNA)构建干扰模型,检测干扰后对SETD2、H3K36me3、DNA双链断裂标志物(γH2AX)以及第一极体(PBI)排出率的影响;通过qPCR技术检测干扰后对DNA甲基化转移酶DNMT3B、凋亡和抗凋亡相关基因BAX、BCL2表达的影响。结果表明:SETD2信号GV期聚集在DNA周围,MⅠ和MⅡ期均在细胞质中表达,而H3K36me3在整个减数分裂过程中均与核共定位;进一步研究发现,SETD2-siRNA干扰后显著降低了SETD2和H3K36me3表达水平,增加了γH2AX表达(P<0.05),同时显著降低PBI排出率(P<0.05);qPCR结果显示,SETD2-siRNA干扰后DNMT3B表达差异不显著,但BCL2表达水平显著下降,凋亡相关基因BAX显著升高(P<0.05)。说明SETD2在卵母细胞体外成熟中起着重要作用,SETD2-siRNA干扰后降低H3K36me3甲基化水平,导致DNA断裂增加,引起细胞凋亡,影响猪卵母细胞IVM成熟效率及质量。SETD2 is H3K36Me3-specific methyltransferase,which participates in histone methylation modification and affects cell function by regulating the expression of multiple genes.The purpose of this experiment is to explore the expression rule and function of SETD2 in vitro maturation of porcine oocyte(IVM).The expression and localization of SETD2 and H3K36me3 in porcine oocyte IVM at different stages(GV,MⅠ,MⅡ)were detected by immunofluorescence staining.The interference model was constructed by microinjection of SETD2 interfering RNA(SETD2-siRNA)at GV stage,and the effects of interference on SETD2,H3K36me3,DNA double strand break marker(γH2AX)and the first polar body(PBI)excretion rate were detected.qPCR was used to detect the effects of interference on DNA methyltransferase DNMT3B,apoptosis and anti-apoptosis related genes BAX and BCL2 expression.The results showed that the GV phase of SETD2 was clustered around DNA,MⅠand MⅡwere expressed in cytoplasm,and H3K36me3 was colocalized with nuclei throughout meiosis.Further study showed that SETD2 and H3K36me3 expression levels were significantly decreased after SETD2-siRNA interference,whileγH2AX expression was increased(P<0.05),and PBI excretion rate was significantly decreased(P<0.05).qPCR results showed that the expression of DNMT3B was not significantly different after SETD2-siRNA interference,but the expression level of BCL2 was significantly decreased,and the apoptosis-related gene BAX was significantly increased(P<0.05).In conclusion,SETD2 plays an important role in in vitro maturation of oocytes.Interference by SETD2-siRNA reduces the methylation level of H3K36me3,leads to increased DNA breakage,induces cell apoptosis,and affects the maturation efficiency and quality of porcine oocytes IVM.

关 键 词：SETD2 猪卵母细胞 H3K36me DNA损伤 

分 类 号：S862[农业科学—野生动物驯养]