AMPKα1不同功能位点的突变对小鼠卵母细胞成熟能力的影响  

Effects of Mutations at Different Functional Sites of the AMPKα1 on the Maturation Ability of Mouse(Mus musculus)Oocytes

作　　者：石丽君 任雪华 虞莲 王丽丽 雷安民[1] SHI Li-Jun;REN Xue-Hua;YU Lian;WANG Li-Li;LEI An-Min(College of Veterinary Medicine/Shaanxi Stem Cell Engineering and Technology Research Center,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物医学院/陕西省干细胞工程技术研究中心,杨凌712100

出　　处：《农业生物技术学报》2022年第3期413-424,共12页Journal of Agricultural Biotechnology

基　　金：陕西省重点研发项目(2020NY-011);西藏昌都市横向课题(K4040121029)。

摘　　要：单磷酸腺苷活化蛋白激酶(adenosine 5'-monophosphate-activated protein kinase,AMPK)作为哺乳细胞内调控代谢的关键酶,在卵母细胞成熟过程中发挥重要作用,但目前AMPK的催化亚基AMPKα1在小鼠(Mus musculus)卵母细胞成熟过程中发挥的作用尚不清楚。本研究利用重叠延伸PCR(splicing by overlap extension PCR,SOE-PCR)技术对小鼠AMPKα1基因的多个位点进行了突变,分别获得AMPKα1-D157A、AMPKα1-T172D、AMPKα1-S485A以及AMPKα1-S485A-S173A突变体,在Hela细胞系中验证其功能后体外转录得到cRNAs,并通过显微注射技术在小鼠卵母细胞中超表达含有各突变位点的AMPKα1基因。结果显示,AMPKα1及其突变体在Hela细胞的胞质内广泛分布,且超表达AMPKα1-S485A突变体的细胞增殖数量和活性均增加;同时,AMPKα1及其突变体在小鼠卵母细胞中的超表达呈聚集分布,并与减数分裂过程中的染色质分离相关联。与对照组相比,除超表达AMPKα1-S485A组之外,其余各组的超表达都加速了小鼠卵母细胞的成熟进程,而超表达AMPKα1-S485A突变体的卵母细胞生发泡破裂(germinal vesicle breakdown,GVBD)率与对照组相比无显著差异,且增加了卵母细胞的极体排出率以及降低了卵母细胞的死亡率。以上结果表明,AMPKα1在小鼠卵母细胞成熟过程发挥着重要作用,为其后续在卵母细胞中的作用研究提供了参考。Adenosine 5'-monophosphate-activated protein kinase(AMPK),as a key enzyme in mammalian cell metabolism,plays an important role in the maturation of oocytes.However,the role of AMPKα1,the catalytic subunit of AMPK,remains unclear in the maturation of mouse(Mus musculus)oocytes.In this study,the different functional sites of AMPKα1 were mutated by splicing by overlap extension PCR(SOE-PCR)to obtain AMPKα1-D157A,AMPKα1-T172D,AMPKα1-S485A and AMPKα1-S485A-S173A mutants,respectively.After verifying their functions in Hela cell lines,cRNAs were transcribed in vitro,and the AMPKα1 genes containing various mutation sites were overexpressed in mouse oocytes by microinjection technology.The results showed that AMPKα1 and its mutants were widely distributed in the cytoplasm of Hela cells,and cells overexpressing AMPKα1-S485A mutants showed increased proliferation and activity.Meanwhile,overexpression of AMPKα1 and its mutants in mouse oocytes showed aggregative distribution,and it was seemed to be associated with chromatin separation during meiosis.Compared with the control group,the overexpression of each group except the AMPKα1-S485A overexpression group accelerated the maturation process of mouse oocytes,and the germinal vesicle breakdown(GVBD)rate of the oocytes overexpressing AMPKα1-S485A mutant was the same as that of the control group.In addition,the overexpression of AMPKα1-S485A could increase the polar body excretion rate of oocytes and reduce the mortality rate of oocytes.These results showed that AMPKα1 played an important role in the maturation of mouse oocytes,and this study provides a reference for the subsequent study of AMPKα1 in mouse oocytes.

关 键 词：单磷酸腺苷活化蛋白激酶(AMPK) 功能位点突变 显微注射 小鼠卵母细胞成熟 

分 类 号：S857.2[农业科学—临床兽医学]