Effects Of MICU1-mediated Mitochondrial Calcium Intake On Mouse Vitrified MⅡ Oocytes

Oocyte vitrification has been widely used in curing human infertility and preserving animal germplasm resources.However,vitrification-induced mitochondrial damage adversely affects oocyte development.Several studies have reported that mitochondrial calcium uptake protein 1（MICU1）regulates the uptake of mitochondrial calcium and subsequently controls aerobic metabolism and oxidative stress in mitochondria,but research considering oocytes remains unreported.Retrieved MⅡ oocytes of mice were classified as vitrified or control groups.After thawing,oocytes of vitrified group were cultured with or without DS16570511（MICU1 inhibitor）and MCU-i4（MICU1 activator）for 2 h.We evaluated whether the addition of MICU1 regulator can affect mitochondrial calcium ion concentration,mitochondrial active oxygen species,mitochondrial membrane potential,pyruvate dehydrogenase activity,ATP content,chromosome spindle morphology and developmental potential after vitrificated MⅡ oocytes resuscitated.1.The results of the study on effect of MICU1 expression on mitochondrial calcium intake in vitrified oocytes showed that MICU1 protein expression in vitrified oocytes increased abnormally.When vitrified oocytes were supplemented with MCU-i4,MICU1activator,the level of MICU1 protein was significantly increased（p<0.01）.In contrast,the level of MICU1 protein was significantly decreased in the presence of MICU1 inhibitor DS16570511（p<0.01）.MICU1 inhibition resulted in a decrease in mitochondrial Ca²⁺uptake（p<0.01）,whereas MICU1 activation resulted in an increase in mitochondrial Ca²⁺uptake（p<0.01）.It illuminates that elevated MICU1 in vitrified oocytes promotes mitochondrial Ca²⁺uptake.2.The results of the study on effect of vitrification procedure on pyruvate metabolism of oocytes revealed that the oocyte vitrification increased the uptake of cytoplasmic Ca²⁺by mitochondria（p<0.01）,although the level of PDH phosphorylation in mitochondria decreased significantly（p<0.05）,pyruvate oxidation activity increased,but ATP level still decreased（p<0.01）,indicating that vitrification can activate mitochondrial calcium uptake,accompanied by increased pyruvate oxidation and a significant decrease in ATP.3.The results of the study on effects of MICU1-mediated mitochondrial Ca²⁺Uptake on pyruvate oxidation and energy metabolism in vitrified mouse oocytes showed that MICU1 activator MCU-i4 reduced the phosphorylation level of PDH in vitrified oocytes（p<0.05）,pyruvate oxidation activity and ATP level increased（p<0.05）;MICU1 inhibitor DS16570511 increased the phosphorylation level of PDH in vitrified oocytes（p<0.05）,pyruvate oxidation activity decreased,ATP level decreased（p<0.01）.Revealing that MICU1-mediated mitochondrial Ca²⁺intake promotes pyruvate oxidation by activating PDH,which can compensate for energy consumption during oocyte vitrification to a certain extent.4.The results of the study on effects of MICU1-mediated mitochondrial Ca²⁺uptake on embryonic developmental potential in vitrificated oocytes showed that MICU1 activator MCU-i4 did not affect the 2-cell and blastocyst rates of vitrificated oocytes（p<0.01）,and MICU1 inhibitor DS16570511 significantly decreased the 2-cell and blastocyst rates of vitrificated oocytes（p<0.01）.Indicating that MICU1-mediated mitochondrial Ca²⁺uptake in vitrificated oocytes is beneficial to maintaining subsequent developmental potential.5.The results of the study on effects of MICU1-mediated mitochondrial Ca²⁺uptake on the mitochondria of vitrified oocytes showed that MICU1 inhibitor or activator had no significant effect on ROS production in vitrified oocytes（p>0.05）,mitochondrial membrane potential（MMP）was significantly decreased after vitrificated oocytes were treated with MICU1 inhibitor（p<0.01）,MMP was significantly increased after vitrificated oocytes treated with MICU1 activator（p<0.01）,indicating that MICU1-mediated mitochondrial Ca²⁺uptake has a positive effect on MMP of vitrificated oocytes.6.The results of the study on effect of MICU1-mediated mitochondrial Ca²⁺uptake on chromosome-spindle morphology of the vitrified oocytes showed that the proportion of abnormal spindles and misplaced chromosomes in vitrified oocytes was significantly higher than that in fresh group（p<0.05）,and the spindle-chromosome dislocation of vitrified oocytes treated with MICU1 inhibitor was more obvious（p<0.01）,indicating that MICU1 had a positive effect on the recovery of microtubule distribution and chromosome arrangement of oocytes after vitrification.Conclusions:The increase of MICU1-mediated mitochondrial calcium uptake in vitrificated MⅡ oocytes promoted pyruvate oxidation and compensated for ATP consumption,which was beneficial to the recovery of mitochondrial activity,chromosome-spindle morphology and maintenance of developmental potential of oocytes.