Preliminary Study On Mitochondrial DNA Damage And Its Mechanism Of Mice Oocytes By Vitrification

Mitochondria are the organelles that produce the most ATP in cells,and are also important production sites of ROS in vivo.Therefore,mitochondria and mitochondrial DNA(mtDNA)may be in a high oxidative stress environment,and are prone to oxidative damage.The singularity of mammalian mitochondria is derived from oocytes.The damage of mitochondria or mtDNA in oocytes will inevitably affect the maturation of oocytes and the development of whole fertilized eggs.In order to explore the effect of vitrification freezing on mtDNA damage in mice and the further mechanism,this study used mice oocytes as materials and divided them into three groups.There are fresh control group,cryopreservation group and cryo-resuscitation group.The effects of vitrification on mtDNA copy number,3867 BP(9089 locus to 12956 locus)deletion,.8-OHdG content and beta-galactosidase activity in oocytes were investigated by PCR,qPCR,Elisa and other experimental methods.The results is as follows:1.The CQ value of oocyte mtDNA in the cryo-resuscitation group(33.52±0.4419)was significantly higher than that in fresh control group(22.63±0.3404)and the cryopreservation group(24.35±0.1015)(P<0.05).The higher the fluorescence quantitative CQ value,the lower the mtDNA copy number of oocyte.Results indicated that copy number of mtDNA in oocytes of freeze-recovery group was significantly lower than that of fresh control group and cryopreservation group.It could be seen that vitrification significantly reduces the copy number of mtDNA in oocytes.2.The deletion of mtDNA 3867 bp fragment in oocytes of each group was detected by semi-quantitative PCR.It was found that after PCR amplification,the mtDNA of oocytes produced a large number of 3867 bp fragments,and there were related bands in the electropherogram.Appeared,but no relevant bands appeared in the fresh control and cryopreservation groups.3.Detection of the activity of ?-galactosidase in oocyte of each group by Elisa method.The absorbance of ?-galactosidase in oocyte of cryo-resuscitation group(0.0529±0.0002)was significantly lower than that of fresh control group(0.0734±0.0006)and freeze solution treatment group(0.0756±0.0007)(P<0.05).The lower absorbance of ?-galactosidase,the higher activity.The results showed that freezing-resuscitation resulted in a significant change in activity of?-galactosidase in oocyte,which was significantly higher than that in fresh control group and freezing solution treatment group.4.The 8-OHdG concentration of oocyte mtDNA which used Elisa method in the cryo-resuscitation group(0.1538 ±0.0987)was significantly higher than that in the fresh control group(0.0339±0.0007)and the toxicity exposure group(0.0642±0.0018)(P<0.05),and the 8-OHdG concentration of oocyte mtDNA in the cryopreservation group was also significantly higher than that in the fresh control group(P<0.05).It can be seen that the freeze-thaw solution and vitrification process used in this study will significantly increase the concentration of 8-OHdG in the oocyte.Conclusion:The results showed that the vitrification process will reduced the copy number of mitochondrial DNA in mouse oocytes,increased the deletion of 3867 bp,increased the concentration of 8-OHdG on mtDNA,and increased the activity of ?-galactosidase.