Effects Of Mouse Oocyte Vitrification On DNA Damage And Expression And DNA Methylation Of H19 And Gtl2

As an important part of assisted reproductive technology,vitrification technology has been widely used in the field of breeding of livestock,rare animals and endangered animals and treatment of human infertility.With the continuous efforts made by researchers,vitrification technology is of increase popularity and beco ming incresingly advanced.However,recent studies have found that vitrification is not only adversely affecting cell ultrastructure and cell membrane,but also cause aberrant DNA methylation status,gene expression,DNA damage and imprinting genes in oocyte or embryo,resulting in potential risks for embryonic development and offspring.In this study,the effects of oocyte vitrification on DNA damage in oocytes and embryos were analyzed by oocyte single cell gel electrophoresis,immunofluorescence staining,qRT-PCR and TUNEL staining.QRT-PCR and the bisulfite sequencing method were used to detect the effects of oocyte vitrification on the DNA methylation status and expression of H19 and Gt12 differentially methylated regions(DMRs)in M?oocytes and offspring.The following results were obtained:1.In the single cell gel electrophoresis,there was no significant difference between control group and vitrified group in tail DNA% and OTM.There was no significant difference in the level of ?H2AX between the two groups in,and the 5hmC level increased significantly(P<0.05).The number of ?H2AX foci in vitrified group was significantly higher than that in control group(P<0.05),M?oocytes with the 5hmC level significantly increase(P<0.05).In M?oocytes,the expression of 53BP1,Casp3,Fas,Gadd45 a and Ddit3 were not significantly different in two groups(P<0.05),while the expression of 53BP1,Gadd45 a and Ddit3 in 2-cell significantly increased after vitrification(P<0.05).The cleavage rate and blastocyst rate in vitrified group significantly decreased after parthenogenetic activation(P<0.05)and the number of apoptotic cells in blastocyst significantly increased(P<0.05).2.There was no significant difference in H19 methylation status between control group and vitrified group(P> 0.05)in M?oocytes,while the average methylation level of the Gtl2 differentially methylate region in vitrified group was significantly lower than that in control group(P<0.05).There was no significant difference in the expression level of H19 in M?oocytes after vitrification(P>0.05),while the expression level of GT12 significantly decreased.In the heart and spleen,there was no significant difference found in the DNA methylation level of Gt12 DMR between two groups(P>0.05).In the liver,the DNA methylation level of Gt12 DMR in vitrified group was significantly higher than that in control group(P<0.05).Real-time quantitative PCR results showed that the expression of Gtl2 in the spleen did reduce significantly(P> 0.05),while the expression of Gtl2 in the liver and heart was significantly increased(P<0.05).Conclusions: Oocyte vitrification does not affect the DNA damage of M?oocytes,but it can cause 2-cell DNA damage,leading to upregulation of 53BP1,Gadd45 a and Ddit3.It can also affect the development of early embryos,with the cleavage rate and blastocyst rate being decrease after vitrification.Also the blastocyst apoptosis was affected,with the increase in the number of apoptotic cells in blastocyst.Oocyte vitrification can affect the expression and DNA methylation of imprinted genes in M?oocytes and offspring.