Effects Of Antifreeze Protein Ⅲ On The Development And Mitochondria Of Vitrified Mouse Oocytes

In order to explore the protective effect of antifreeze protein Ⅲ(AFPⅢ)on vitrified mouse oocytes,and to find the most appropriate way and concentration of AFPⅢ.In this study,AFPⅢ was added in three different ways(vitrification solution,equilibration solution,equilibration solution and vitrification solution),and parthenogenetic activation and in vitro fertilization experiments were performed on MⅡ oocytes in AFPⅢ groups with different concentrations(250,500,750 and 1000 ng/m L).The content of mitochondria,ROS,ATP and mitochondrial membrane potential of oocytes in each group were detected,the mitochondrial distribution and the co-localization of mitochondrial and lysosomal were observed.It is expected to provide a theoretical basis for improving the formula of vitrification solution and reducing the freezing damage of vitrification to mouse and other mammalian oocytes.Results found:1.The survival rate,parthenogenetic cleavage rate and 8-cell rate of vitrified oocytes were improved by adding 250,500,750 and 1000 ng/m L AFPⅢ in three different ways,respectively.Among the three different addition methods,the survival rate and parthenogenetic cleavage rate of 500 ng/m L AFPⅢ groups were significantly higher than those in other AFPⅢ groups(P<0.05).When 500ng/m L AFPⅢ was added to both equilibrium solution and vitrification solution,the parthenogenetic 8-cell rate of vitrified oocytes was significantly higher than that of the vitrification control group(P<0.05).2.The survival rate,IVF cleavage rate and 8-cell rate of vitrified oocytes were improved by adding 250,500,750 and 1000 ng/m L AFPⅢ in three different ways,respectively.Among the three different addition methods,the survival rate and IVF cleavage rate of 500 ng/m L AFPⅢ groups were significantly higher than those in other AFPⅢ groups(P<0.05).When 500ng/m L AFPⅢ was added to both equilibrium solution and vitrification solution,the IVF 8-cell rate of vitrified oocytes was significantly higher than that of the vitrification control group(P<0.05).In conclusion,adding 500 ng/m L AFPⅢ to both equilibrium solution and vitrification solution has the best protective effect on the development of vitrified mouse oocytes.Therefore,this addition method and concentration were selected as the vitrification scheme for the following experiments.3.Compared with the fresh control group,after vitrification,the mitochondrial content of oocytes was significantly decreased and the ROS content was significantly increased(P<0.05),some mitochondria were abnormal distributed,and the co-localization of mitochondria-lysosome were increased.Compared with the vitrification control group,the mitochondrial content of oocytes in the AFPⅢ group was significantly increased and the ROS content was significantly decreased(P<0.05).The co-localization of mitochondria and lysosome was decreased,and the distribution of mitochondria was not significantly different from that of the fresh control group.Therefore,AFPⅢ can significantly reduce the content of ROS in vitrified mouse oocytes,and reduce the decrease of mitochondrial content,the abnormal distribution of mitochondria and the co-localization of mitochondria-lysosome caused by vitrification.4.Compared with the fresh control group,the mitochondrial membrane potential and ATP content of oocytes after vitrification were significantly decreased(P<0.05),while the mitochondrial membrane potential and ATP content in AFPⅢ groups were significantly higher than those in the vitrification control group(P<0.05).Therefore,AFPⅢ has a protective effect on the mitochondrial function of vitrified mouse oocytes.In conclusion,the supplementation of 500 ng/m L AFPⅢ to both equilibration solution and vitrification solution is helpful to improve the survival and development of mouse oocytes after vitrification,reduce oxidative stress and protect mitochondria and their functions.