细胞周期检查点激酶1的表达对猪卵母细胞体外成熟的影响  

Effect of Chk1 expression on in vitro maturation of porcine oocytes

作　　者：韩国波 李英花[1,2] 叶孟傲然 许永男 HAN Guobo;LI Yinghua;YEMENG Aoran;XU Yongnan(College of Agriculture,Yanbian University,Yanji 133000,China;Yanbian Yellow Cattle Germplasm Resources Conservation Engineering Research Center,Yanji 133000,China)

机构地区：[1]延边大学农学院,吉林延吉133000 [2]吉林省延边黄牛种质资源保护工程研究中心,吉林延吉133000

出　　处：《畜牧与兽医》2020年第11期7-13,共7页Animal Husbandry & Veterinary Medicine

基　　金：吉林省教育厅“十三五”科学技术项目(JJKH20190032KJ);延大科合字(2018)第09号。

摘　　要：细胞周期检查点激酶1(Chk1)作为DNA复制检查点和DNA损伤反应的重要成员,在有丝分裂和减数分裂中都具有重要作用。为探究Chk1对猪卵母细胞减数分裂的影响,本研究首先采用免疫荧光染色和实时荧光定量PCR技术检测了Chk1在猪卵母细胞不同时期的表达和定位,随后利用chir-124抑制Chk1的表达,研究其对猪卵母细胞减数分裂的影响,以及相关基因的表达和纺锤体的变化。结果表明,Chk1在4个时期(GV、GVBD、MI、MII)均有表达,GV期主要定位在生发泡内,GVBD期主要定位在核周围,MI期和MII期主要定位在纺锤体上;用不同浓度的chir-124处理卵母细胞来抑制Chk1,发现0.5μmol/L的chir-124对卵母细胞生发泡破裂(GVBD)的影响最大,可显著提高猪卵母细胞处于GV-IV期的比例(P<0.05),并且可显著提高卵母细胞达到GVBD期的比率(P<0.05),但对后续的成熟没有显著影响;随后对MII期卵母细胞的基因表达进行检测发现,Chk1的抑制可调节cdk1、cdc25C、cyclin b1基因的表达,并使gwl基因的表达显著下降(P<0.05),从而调节卵母细胞恢复减数分裂的进程;Chk1的表达会激活纺锤体组装检查点(SAC),阻止同源染色体分离,将猪卵母细胞定位在MI期,而减少Chk1的表达后,猪卵母细胞可渡过MI期,进入MII期。综上,在GV期使用chir-124抑制Chk1的表达可促进猪卵母细胞恢复减数分裂,并促进后续的成熟。Cell cycle checkpoint kinase 1(Chk1),as an important member of DNA replication checkpoints and DNA damage responses,plays an important role in mitosis and meiosis.In order to investigate the effect of Chk1 on the meiosis of porcine oocytes,in this study,immunofluorescence staining and real-time quantitative PCR were used to detect the expression and localization of Chk1 in porcine oocytes at different periods,and then Chk1 expression was inhibited by chir-124 to study its influence on meiosis of pig oocytes,as well as the expression of related genes and changes in spindles.The results showed that Chk1 was expressed in 4 periods(GV,GVBD,MI,MII),the GV phase was mainly located in the raw foam,the GVBD phase was mainly located around the nucleus,the MI and MII phases were mainly located on the spindle;treatment of oocytes with different concentrations of chir-124 to restrain Chk1,found that 0.5μmol/L chir-124 had the greatest effect on oocyte GVBD,which could increase the proportion of porcine oocytes in GV-IV phase(P<0.05),and could increase the rate at which oocytes reach GVBD phase(P<0.05),but had no effect on subsequent maturation;subsequently,the gene expression of MII oocytes was detected,and it was found that the consumption of Chk1 could regulate the expression of cdk1,cdc25 C,cyclin b1 genes and make gwl Gene expression was significantly decreased(P<0.05),thereby regulating the progression of oocyte recovery to meiosis;and Chk1 expression activates the spindle assembly checkpoint(SAC),prevents homologous chromosome segregation,and localizes porcine oocytes in the MI phase,and reducing the expression of Chk1,the porcine oocytes could cross the MI phase and enter the MII phase.In conclusion,the use of chir-124 to restrain the expression of Chk1 can promote the recovery of meiosis in porcine oocytes and promote subsequent maturation.

关 键 词：猪 卵母细胞 体外成熟 CHK1 chir-124 

分 类 号：S828.9+9[农业科学—畜牧学]