Research Of The Role And Mechanism Of Gadd45a On DNA Damage Repair Of Vitrification Mouse Oocytes

Vitrification technology plays an important role in animal germplasm conservation,embryo engineering and reproductive medicine.However,a series of physical and chemical changes such as sudden drop in temperature,osmotic stress and toxicity of cryoprotectants during vitrification and thawing have adverse effects on oocytes,leading to DNA damage of oocytes.DNA damage repair of oocytes is important for maintaining the stability of oocyte genome,ensuring genetic fidelity and restoring oocyte developmental potential.It was found that that the DNA of mouse oocytes was damaged after vitrification and freezing,and the expression of DNA damage repair factor Gadd45 a increased,but its role and regulatory mechanism were not clear.In this study,real-time PCR,immunofluorescence and Western blot were used to detect the DNA damage of mouse oocytes and early embryos after vitrification.And the expression changes of DNA damage repair gene Gadd45 a,Gadd45a regulatory gene(ATM and p53),Gadd45 a interaction protein(TDG and PCNA)during the frozen-thaw process also been detected.To investigate the role and mechanism of Gadd45 a in DNA damage response induced by oocyte vitrification,and improve the vitrification technology of oocytes from the point of view of improving the DNA damage repair ability of oocytes.The results of the study are as follows:1.After vitrification of oocytes,the average fluorescence intensity of DNA damage marker γH2AX in MⅡ oocytes was significantly higher than that in control group(p<0.001).The mean fluorescence intensity of γH2AX in 2-cell embryos active by parthenogenesis was higher than that in the control group(p<0.05).The results indicated that the DNA of oocyte was damaged after vitrification and freezing,and the damage lasted until the 2-cell stage.2.The expression level of DNA damage repair gene Gadd45 a in oocytes after vitrification showed that the m RNA level of Gadd45 a increased after vitrification of MⅡ oocytes(p<0.05),and the expression of Gadd45 a protein detected by immunofluorescence significantly increased(p<0.01).In the 2-cell embryos,the m RNA level of Gadd45 a significantly increased after vitrification(p<0.01),and the expression of Gadd45 a protein detected by immunofluorescence was extremely increased(p<0.001).3.The expression levels of Gadd45 a function-related genes showed that the m RNA level of ATM,the upstream gene of Gadd45 a,increased significantly in MⅡ oocytes after vitrification(p<0.01),and the m RNA and protein levels of TDG increased significantly(p<0.05).The m RNA level of ATM was significantly decreased(p<0.01)in the 2-cell embryos,and the protein level of p-ATM was extremely significantly increased(p<0.001).The m RNA and protein levels of p53 and PCNA in MⅡ oocytes and parthenogenetic 2-cells in the vitrified group were not significantly different from those in the control group(p>0.05).These results indicate that Gadd45 a may be expressed through ATM regulation.CO-IP results showed that TDG protein interacted with PCNA protein in MⅡ oocytes,which was involved in DNA damage repair after vitrification.4.KU55933 treatment for 2 hours significantly increased the average fluorescence levels of p-ATM,Gadd45 a and γH2AX in MⅡ oocytes(p<0.001),and significantly decreased pATM(p<0.01)and γH2AX fluorescence level(p<0.05)after parthenogenetic activation to 2-cell embryos.The average fluorescence level of γH2AX in the KU55933-treated group was lower than that in the vitrified without addition group(p<0.05).In conclusion,KU55933 can promote DNA damage repair of oocyte.According to Gardner scoring system,blastocysts of the frozen group without addition and the KU55933 treatment group were graded.The development degree of blastocysts in the vitrified without addition group was concentrated in grade 2(41%)and grade 3(30.19%),and the development degree of blastocysts in the KU55933-treated group was concentrated in grade 3(38.84%)and grade 4(38.90%).The proportion of grade 2 blastocysts in the vitrified without addition group was significantly higher than the KU55933-treated group(p<0.01),and the proportion of grade 3 and 4blastocysts in the KU55933-treated group was both significantly higher than the vitrified without addition group(p<0.05).Conclusions: Vitrification of MⅡ oocytes can cause DNA damage,which can last until the parthenogenetic activated 2-cell stage.During this process,the increase of DNA damage repair gene Gadd45 a m RNA and protein levels can be detected.Gadd45 a is involved in ATMmediated DNA damage repair during vitrification of oocytes.The high expression of Gadd45 a and ATM can reduce the degree of DNA damage in early embryos and improve the developmental potential of vitrified oocytes.The expression of TDG protein in MⅡ stage oocytes increased after vitrification.TDG protein and PCNA protein are involved in DNA damage repair of oocytes through protein interaction.