Effects Of Oocytes Vitrification On DNA Methylation And The Investigation Of Mechanisms In Mouse

Cryopreservation of oocytes is of great value for human assisted reproduction and protection of animal genetic resources.In recent years,the development of vitrification technology has significantly improved the success rate of cryopreservation of human and animal oocytes,and promoted the establishment of human oocyte bank.However,studies have shown that vitrification also reduces the developmental potential of oocytes to a certain extent,especially leading to abnormal changes in some epigenetic modifications.DNA methylation reprogramming is critical during oocyte and early embryo development.The purpose of this study is to deeply analyze the effects of vitrification of mouse oocytes on DNA methylation,explore related mechanisms,and take corresponding measures to improve the cryopreservation efficiency of oocytes.The specific research content and results obtained are as follows:(1)Effects of oocyte vitrification on DNA methylation and imprinted genes.By immunostaining,it was found that the level of DNA methylation in oocytes and 2-cell stage embryos was significantly reduced after vitrification.qRT-PCR was used to detect the effect of vitrification on the expression of imprinted genes in oocytes and early embryos.It was found that the expression of maternal imprinted genes Peg3,Peg10 and Igf2 r was abnormal in vitrified oocytes.In 2-cell stage embryos,Peg3 and Peg10 and the parental imprinted gene Gtl2 was abnormal;the expression of the imprint protective gene Zfp57 was significantly increased.Further,the effects of oocyte vitrification on the expression of imprinted genes in tissues and organs of offspring were detected.It was found that the expression of the parental imprinted genes Gtl2 and H19 in the liver was abnormally increased in the vitrified individuals,but H19 was significantly decreased in the spleen.The expression of the imprinted genes Igf2,Igf2 r and Snrpn was significantly increased in the spleen,and the expression of Peg3 in the heart was significantly decreased.Bisulfite sequencing(BSP)was used to detect the effect of oocyte vitrification on DNA methylation in the imprinted regulatory regions of tissues and organs from the offspring.It was found that the level of Peg3 methylation in the heart increased(21.2% vs 9.1%);H19 methylation level increased in the spleen(78.5% vs 60.0%),while decreased in the liver(15.2% vs43.6%);methylation level of Gtl2 increased in the liver(94.2% vs 81.0)%).(2)Effects of oocyte vitrification on DNA methylation at a single base resolution.The whole genome methylation sequencing(WGBS)method was used to detect the changes of DNA methylation site distribution and methylation level after oocyte vitrification.It was found that the proportion of C-site methylation in the vitrified group was decreased(2.69).%vs 3.63%),and there was also a decrease under the CG sequence environment(13.28% vs17.98%).The average DNA methylation level in the vitrified group was lower than that in the control group.The decreased methylation mainly occurred in the promoter region,exon region and up/downstream regions of the gene.The differential methylation area(DMR)between the control group and the vitrified group was detected by DSS software.The average methylation level of DMRs in the vitrified group was lower than that of the control group,and the methylation level of the vitrified group was decreased in each functional area of the genome.The number of declined DMRs is more than the number of elevated DMRs.Finally,the genes anchored by DMRs were enriched and analyzed.The GO analysis enriched items were mainly concentrated in biological processes.The KEGG analysis was mainly enriched in the c GMP-PKG pathway.(3)The effect of oocyte vitrification on the expression changes of DNA methylation-related genes.The effect of oocyte vitrification on DNA methylation-related gene expression was examined by qRT-PCR.The results showed that the expression of DNA methylase Dnmt1 was decreased in the vitrified oocytes,but the difference was not significant.The cofactor Dnmt3 l of de novo DNA methyltransferase was significantly increased,and the expression of demethylase Tet3 and TDG was significantly increased.In the 2-cell stage embryos,the expression of Dnmt1 was significantly decreased in the vitrified group,and the expression of Tet3 and TDG was still significantly higher than that of the control group.(4)Investigation of the relationship between DNA methylation changes and DNA damage caused by oocyte vitrification.The effect of oocyte vitrification on the expression of DNA damage and repair-related genes was detected by qRT-PCR.It was found that the expression of Fas was significantly decreased in the oocytes of the vitrified group,and the expression of 53BP1,Ddit3 and Gadd45 a was significantly increased in the 2-cell stage embryos.Immunofluorescence staining was used to detect the effect of oocyte vitrification on DNA damage marker γH2AX in preimplantation embryos.It was found that γH2AX levels were significantly increased in fertilized oocytes and 2-cell embryos in the vitrified group.Immunofluorescence co-staining was used to detect the effect of oocyte vitrification on γH2AX and 5hm C in 2-cell stage embryos.It was found that the number of γH2AX positive sites in the vitrified group increased,while the 5hm C level decreased significantly.(5)The effect of resveratrol addition on the epigenetic modification and embryo development potential after oocyte vitrification.qRT-PCR and Western blot analysis showed that resveratrol supplementation restored the abnormally low expression of deacetylase Sirt1 in the vitrified group.Immunofluorescence staining showed that the addition of resveratrol corrected the abnormal H3K9 ac and DNA methylation in the vitrified oocytes and 2-cell embryos.qRT-PCR analysis showed that the expression of the imprinted genes Peg3 and Gtl2 returned to normal after resveratrol addition.Reactive oxygen species(ROS)assay showed significantly increased ROS levels in vitrified oocytes and early embryos,and they returned to normal after resveratrol addition.The abnormally increased γH2AX content in the fertilized and 2-cell embryos of the vitrified group returned to normal,and the abnormal 5hm C level in the 2-cell stage embryo was restored.The fertilization and blastocyst rates were significantly decreased after oocyte vitrification,while there was no significant difference between the resveratrol and the control group.The quality of the blastocysts was also improved.The total cell numbers and inner cell numbers increased significantly.Embryo transfer experiments showed that the birth rate of the vitrified group was significantly lower than that of the control group,and was significantly increased after the resveratrol addition.In conclusion,this study found that oocyte vitrification caused abnormalities in DNA methylation at the overall level and at the single base level,affecting the imprinting of imprinted genes.The main reasons may be due to abnormal expression of DNA methylation-related genes and DNA damage caused by cryopreservation.Resveratrol was added during the vitrification process,which reduced DNA damage,improved DNA methylation and partial imprinted gene expression,and significantly improved embryo development potential before and after implantation.This study provides a theoretical and practical reference for improving the cryopreservation efficiency of oocytes from the perspective of DNA methylation.