Research On The Mechanism Of AMPK-MCU Affecting The Maturation Of Mouse Oocytes

The maintenance of mouse oocytes arrest in pre-meiosis I depends on the high concentration of 3’-5’-cyclic adenosine phosphate（c AMP）.In vivo respond to the proliferation of luteinizing hormone（LH）and the mechanical release of mature follicles in vitro,which are both lead to the degradation of c AMP in oocytes and induce AMP-activated Protein Kinase（AMPK）activation.AMPK is the key signal for mouse oocytes to enter the germinal vesicle breakdown（GVBD）-phase,which can regulate various biological events in the cytoplasm and provide energy/material supply for the maturation of oocytes.In addition,AMPK can induce mitochondrial ATP production by directly acting on the Mitochondrial Calcium Uniporter（MCU）,which is mediated by mitochondrial Ca²⁺influx.In a variety of organelles,mitochondria act as both a regulator of Ca²⁺input and a decoder,with responding quickly to intracellular Ca²⁺demand by virtue of its dynamics.The increase of cytoplasmic Ca²⁺will cause the mitochondria to quickly absorb Ca²⁺,and the subsequent enhancement of Ca²⁺in the mitochondrial matrix can induce the efficiency of the mitochondrial electron transport chain and tricarboxylic acid（TCA）cycle,thereby increasing the efficiency of mitochondrial respiratory metabolism and ATP production capacity.In addition,AMPK can directly act on the mitochondrial Ca2+uniporter（MCU）to couple the energy demand in the cytoplasm with the production of mitochondrial ATP,and this process is mediated by mitochondrial Ca²⁺influx.The increase of cytoplasmic Ca²⁺level will cause the mitochondria to quickly absorb Ca²⁺,and the subsequent increase of Ca²⁺in the mitochondrial matrix can increase the efficiency of the mitochondrial electron transport chain and tricarboxylic acid（TCA）cycle,thereby increasing the efficiency of mitochondrial respiratory metabolism and ATP production capacity.The unique role of MCU and its complex in the regulation of Ca²⁺flow into mitochondria determines that it participates in a variety of cellular metabolic reactions and is coupled with biological processes such as energy supply,lipid metabolism and cell death.In the first part of the study,based on the important roles of different functional sites of AMPKα1 catalytic subunit in cell energy supply,lipid,protein and glucose metabolisms,mutations of AMPK were respectively made at its D157,T172,S485,and S173 sites.And then,they were overexpressed in mouse oocytes.The results showed that AMPK showed important energy regulation in the GVBD-phase of mouse oocytes.Overexpression and activation of AMPKα1 will advance the development of oocytes,but it does not significantly affect the first polar body（PBI）rate of oocytes,indicating that AMPK does not show a more important regulatory role in the late stage of oocyte maturation.In the second part,by inhibiting and overexpressing the Mcu gene,we explored its role as the direct downstream of AMPK in the maturation of oocytes.Simultaneously,the mutant gene Mcu-D261Q-E264Q of its Ca²⁺uptake sites was made to reveal that roles in oocytes relating with mitochondrial Ca²⁺influx.The results showed that the inhibition of MCU significantly affected the maturation of mouse oocytes,accompanied by a decrease in the ability of mitochondrial oxidative phosphorylation（OXPHOS）;Simultaneously,The expression of Mcu gene in mouse oocytes also increased the expression of apoptosis-related genes,indicating that MCU-mediated mitochondrial Ca²⁺overload also affected the maturation process of oocytes.The final results revealed that AMPK plays diverse roles as a key energy and stress effector,and participates in a variety of biological processes in the recovery and regulation of oocytes premeiosis arrest.However,in the later development process,the MCU responds to AMPK induced energy deficiency and mediates rapid energy supply response,which benefits the MI-phase chromosomes separating and eventually expel the first polar body.Besides,MCU may play important roles in additional oocyte regulatory events which need further investigation.