H3K9me3对猪卵母细胞体外成熟和早期胚胎发育的影响  

Effects of H3K9me3 in vitro on Maturation of Porcine Oocytes and Early Embryo Development

作　　者：田钗 张彩玉 杨婷[1] 王婷[1] 黄时海[1] 石德顺[1] 李湘萍[1] TIAN Chai;ZHANG Caiyu;YANG Ting;WANG Ting;HUANG Shihai;SHI Deshun;LI Xiangping(State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,Guangxi University,Nanning 530004,China)

机构地区：[1]广西大学亚热带农业生物资源保护与利用国家重点实验室,南宁530004

出　　处：《西南大学学报（自然科学版）》2022年第3期68-74,共7页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金项目(31560632,32060754);广西自然科学基金项目(2020GXNSFAA238039)。

摘　　要：H3K9的异常甲基化被认为是影响胚胎发育障碍的主要表观遗传修饰之一.毛壳素是H3K9me3的特异性抑制剂,能抑制其甲基转移酶SUV39H1/2和G9A的活性,下调H3K9me3水平.本实验通过在猪卵母细胞体外成熟过程中添加毛壳素,探究调控H3K9me3对猪卵母细胞体外成熟和早期胚胎发育的影响.在0~22 h用不同浓度毛壳素(0 nmol/L,2 nmol/L,5 nmol/L,10 nmol/L)处理猪卵母细胞,与未处理组相比,发现2 nmol/L毛壳素可显著提高卵母细胞的第一极体排出率(81.5%VS 72.62%),孤雌胚胎4-细胞率(74.43%VS 68.69%)和囊胚率(40.43%VS 27.48%)(p<0.05).qRT-PCR结果表明,与未处理组相比,2 nmol/L毛壳素显著降低卵母细胞中SUV39H1/2和G9A的表达,显著提高囊胚中Nanog,Oct4,CDX2的表达(p<0.05).免疫荧光分析发现,与未处理组相比,2 nmol/L毛壳素显著降低囊胚中H3K9me3的表达(p<0.05),H3K9me3在GV期卵母细胞中有表达,在MI,MII期未检测到表达.在0~44 h用不同浓度毛壳素(0 nmol/L,2 nmol/L,5 nmol/L,10 nmol/L)处理猪卵母细胞,未处理组与各处理组的卵裂率、4-细胞率、囊胚率及囊胚细胞总数均无差异不具有统计学意义(p>0.05).5 nmol/L处理组的第一极体排出率显著提高(87.39%VS 71.95%,p<0.05).以上结果表明,毛壳素可通过降低卵母细胞中SUV39H1/2和G9A的表达,上调囊胚中多能性基因Nanog,Oct4,CDX2的表达,调控H3K9me3的水平,从而促进猪卵母细胞体外成熟和早期胚胎发育.Abnormal methylation of H3 K9 is considered to be one of the main epigenetic modifications affecting embryonic developmental disorders.Chaetocin is a specific inhibitor of H3 K9 me3 that suppresses the activity of its methyltransferases,SUV39H1/2 and G9A,and down-regulate thelevel of H3 K9 me3.This experiment explored the effects of regulating H3 K9 me3 on in vitro maturation and early embryonic development by adding chaetocin during the in vitro maturation of porcine oocytes.Firstly,porcine oocytes were treated with different concentrations of chaetocin(0 nmol/L,2 nmol/L,5 nmol/L,10 nmol/L)from 0-22 h.2 nmol/L of chaetocin was found to significantly increase the discharge rate of first polar(81.5%VS 72.62%),4-cell rate(74.43%VS 68.69%)and blastocyst rate(40.43%VS 27.48%)(p<0.05)compared to untreated group.qRT-PCR results showed that 2 nmol/L chaetocin significantly reduced expressionof SUV39H1/2 and G9A in oocytes and significantly improvedexpressionof Nanog,Oct4,CDX2 compared to the blastocyst(p<0.05).Immunofluorescence assays found that 2 nmol/L chaetocin significantly reduced the expression of H3 K9 me3 in the blastocyst compared to the untreated group(p<0.05).H3 K9 me3 was expressed in GV oocytes with no significant differences(p>0.05)that of control,but it was not detected in MI and MII phases.Porcine oocytes were treated with different concentrations of chaetocin(0 nmol/L,2 nmol/L,5 nmol/L,10 nmol/L)from 0-44 h,but no significant differences were found in cleavage rate,4-cell rate,blastocyst rate and total cystocytescompared to the untreated group(p>0.05).The first pole discharge rate increased significantly in the 5 nmol/L treatment group(87.39%VS 71.95%,p<0.05).The above results show that chaetocin can regulate the level of H3 K9 me3 by reducing the expression of SUV39H1/2 and G9A in oocytes,upregulating the expression of the multipotent gene Nanog,Oct4,CDX2 in blastocyst to regulate the level of H3 K9 me3,thereby promoting in vitro maturation and early embryo development of porcine.

关 键 词：SUV39H1/2 H3K9me3 胚胎发育 卵母细胞成熟 

分 类 号：S814.8[农业科学—畜牧学]