| With the in-depth study of the genetics and development of mammals,the rapid development of in vitro fertilization,somatic cell nuclear transfer,induced pluripotent stem cells and transgenic biotechnology,the large number of animal eggs and embryos due to in vitro research requirements,the long-term of eggs and embryos,Balanced supply is very important,and cryopreservation technology facilitates the solution of this problem.In particular,cryopreservation of gametes not only can provide sufficient gametes and embryo sources across time and space,but also facilitate the exchange of excellent germplasm resources and the maintenance of biodiversity.However,recent studies have shown that vitrification not only damages the ultrastructure of cell membranes,mitochondria,but also the DNA methylation level of oocytes or embryos,the modification of histones,and the expression of imprinted genes.Affects and hinders the normal development of the embryo.Resveratrol is a polyphenolic substance with a variety of biological activities,such as anti-oxidation,anti-tumor and anti-aging effects.Recent studies have shown that resveratrol can regulate the expression of genes on epigenetic modifications and participate in genomic DNA methylation and histone modification.In this study,Cryotop vitrification method was used to increase the refrigerating efficiency and embryonic development potential of oocytes by simultaneously adding resveratrol to the vitrification and thawing solution of mouse MII oocytes.Abnormal epigenetic modifications are improved.In this study,TUNEL staining,immunofluorescence staining and q RT-PCR were used to analyze the level of DNA methylation,the histone H3K9 acetylation level,and the deacetylase SIRT1 of resveratrol in frozen MII oocytes.The expression level and the effect of blastocyst quality and the use of bisulphite sequencing method to detect the effect of resveratrol on the differential methylation status of the imprinted genes PEG3 and GTL2 in 2-cell and blastocyst.The following research results have been obtained:1.In the oocyte vitrification freeze-thaw process,the addition of resveratrol can significantly improve the developmental potential of oocytes.In this study,2 μM resveratrol was added to the oocytes frozen and thawed solution.The study found that the fertility rate of frozen resveratrol supplemented group was higher(63.8%)than that of frozen non-resveratrol group(42.9%).(P<0.05),but still significantly lower than the unfrozen group(85.8%)(P<0.05).There was no significant difference in the cleavage rate between the three groups(P>0.05).The embryo rate(68.3%)of frozen resveratrol supplemented group was significantly higher than the frozen non-resveratrol group(50.2%),and it was not significantly different from the unfrozen group(74.1%)(P>0.05);apoptosis staining of blastocysts revealed that the apoptotic rate of the frozen resveratrol supplemented group was significantly lower than that of the frozen non-resveratrol group(P<0.05),but there was no significant difference with unfrozen groups(P>0.05).;The number of blastocyst cells and the number of cells in the inner cell mass of the frozen resveratrol supplemented group was significantly higher than that of the frozen non-resveratrol group(P<0.05),and there was no significant difference with the unfrozen group(P>0.05).2.Adding resveratrol frozen oocytes can improve the apparent state of oocytes.This study showed that the expression level of 5m C and the deacetylase SIRT1 in oocytes of frozen non-resveratrol group were significantly decreased(P<0.05),and the H3K9 acetylation level was significantly increased(P<0.05).The 5m C level,histone H3K9 acetylation level,and SIRT1 expression level of the frozen resveratrol supplemented group were significantly improved,and there was no significant difference from the unfrozen group(P>0.05).The expression levels of the imprinted genes PEG3 and GTL2 in the frozen non-resveratrol group were significantly lower than those in the unfrozen group(P<0.05),and there was no significant difference between the non-frozen group and the unfrozen group after the addition of resveratrol(P>0.05).After in vitro fertilization of oocytes,the methylation status of the differential methylated region(DMR)of the imprinted genes PEG3 and GTL2 in 2-cell and blastocysts was examined and found that the methylation level of GTL2 differentially methylated region of blastocysts in frozen resveratrol supplemented group was significantly lower than that in the frozen non-resveratrol group(P<0.05).There was no significant difference between the frozen and added resveratrol group(P<0.05);the methylation level of PEG3 differentially methylated region of blastocysts in frozen resveratrol supplemented group was significantly higher than that in the frozen non-resveratrol group(P<0.05).There was no significant difference between the frozen and added resveratrol group(P<0.05)In summary,after adding resveratrol in the vitrification process,the fertilization rate and blastocyst rate of oocytes can be significantly increased,and the blastocyst apoptosis rate can be reduced;the addition of resveratrol can restore the level of 5m C,histone H3K9 acetylation and SIRT1 of MII phase oocyte in mice,and can improve the methylation status of the differentially methylated regions of the imprinted genes PEG3 and GTL2 at the time of blastocyst. |
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