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| Effects of Mutations at Different Functional Sites of the AMPKα1 on the Maturation Ability of Mouse (Mus musculus) Oocytes |
| 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 |
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Abstract 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.
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Received: 24 September 2021
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Corresponding Authors:
**anminleiryan@nwsuaf.edu.cn
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| About author:: *These authors contributed equally to this work |
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