The Study On The Repair Effects Of Resveratrol On Mitochondrial Injury Caused By 2-Cell Embryo Vitrification In Mouse

In the past few decades,although vitrification biology has made tremendous progress and the vitrification preservation efficiency of mammalian embryos is more and more high,the embryo after cryopreservation will inevitably damaged to a certain extent,especially mitochondrion.Mitochondria are the energy factories of embryo,through oxidative phosphorylation providing energy to embryo.Mitochondrial distribution and stage matching the different energy requirement in cells of different area are important to embryo.Mitochondrion is closely related to the generation and scavenging of reactive oxygen species(ROS)and the oxidative stress of embryo.Therefore,the abnormal distribution and damage of mitochondrial may seriously affect the embryo development.Resveratrol is a kind of natural antioxidant,which can trigger the mitochondria biosynthesis and protect mitochondria.So,we hypothesized that resveratrol was beneficial to the mitochondria injury caused by vitrification.We examined the mitochondrial damage caused by the vitrification of mouse 2-cell embryo using nylon net.Then we researched whether resveratrol can repair the mitochondrial damage caused by vitrification to provide reference for further research on human oocyte and embryo vitrification technology.The contents and results of this research are as follows:(1)Fresh and vitrified/thawed 2-cell embryos were detected the mitochondrial distribution,mitochondrial membrane potential(??m)and intracellular ROS level.The results showed that mitochondria in fresh embryo was evenly distributed in the cytoplasm;and were scattered in irregular aggregation in most of vitrified embryos;??m of fresh embryo was significantly higher than vitrification embryo(P<0.05);The ROS level of fresh embryos was significantly lower than vitrification embryo(P<0.05).These results showed that vitrification induced the abnormal distribution and functional damage of mitochondrial in mouse 2-cell embryos.(2)Fresh 2-cell embryos were cultured respectively in CZB supplemented with different concentrations(0.01 mol/L,0.1 mol/L,1 mol/L,10 mol/L)of resveratrol for 72 h;vitrified/thawed 2-cell embryos were cultured respectively in CZB supplemented with different concentrations(0.01 mol/L,0.1 mol/L,1 mol/L)of resveratrol for 72 h.Screen the suitable concentration of resveratrol.Mouse 2-cell embryos were cultured 72 h in CZB added with different concentrations of resveratrol,and then calculated blastocyst rate,and blastocyst cell.The results showed that the hightest blastocyst rate was in 0.1 ?mol/L resveratrol so,we choose 0.1 ?mol/L resveratrol for further research.(3)Fresh embryo,fresh embryo with resveratrol(0.1 ?mol/L),vitrified embryo group and vitrified embryo group with resveratrol(0.1 ?mol/L)were cultured for 72 h.The results showed that after 72 h,the mRNA levels of mitochondrial function related genes-SIRT1 and PGC-1? of the embryos with resveratrol treatment were significantly lower than the embryos with no resveratrol treatment(P<0.05).Therefore,we speculated that vitrified/thawed 2-cell embryos were repaired by resveratrol at the beginning of culture,and then high expression of SIRT1 and PGC-1? to improve the mitochondrial function were no longer needed.(4)According to above results and speculation,we examined SIRT1 and PGC-1? mRNA levels of the embryos with resveratrol treatment for 8 h.The results showed that there was no significant difference between the embryos with resveratrol treatment and the embryos with no treatment,which indicated that after vitrification,during the cleavage period of 2-cell embryos to 4-cell embryos,resveratrol may had completed its basic role of repairing mitochondrial injury.Therefore,we cultured the vitrified/ thawed embryos with resveratrol treatment for 8 h in CZB medium.Results showed that the blastocyst rate of resveratrol treatment group was significantly higher than that untreated group(P<0.05),and there was no significant difference between resveratrol treatment group and fresh embryos,which meaned that co-cultured with resveratrol,embryos developed to 4-cell stage and resveratrol completed basically its role in the protection and restoration of mitochondria to ensure the subsequent development of vitrified/thawed embryos.(5)On the basis of above research results,and considering that long-time treatment with resveratrol may have toxic effects on embryo development,we explored that wherther shortening the time of resveratrol treatment can repair the distribution and function of mitochondria or not.Four groups of 2-cell embryos culturing for 2 h,were detected the SIRT1 and PGC-1? mRNA expression levels,the blastocyst rate of culturing to 72 h subsequently in the conventional culture medium,and mitochondrial distribution,ROS level and mitochondrial membrane potential of 2-cell embryos.Results showed that the SIRT1 and PGC-1? mRNA levels of embryos with resveratrol co-culture for 2 h were significantly higher than that with no resveratrol treatment(P<0.05),which indicated that in 2 h incubation,resveratrol activated embryos' mitochondrial biogenesis and improved mitochondrion function;the blastocyst rate of embryos with resveratrol treatment was significantly higher than that with no resveratrol treatment(P<0.05),and there was no significant difference between embryos with resveratrol treatment and fresh embryos,which indicated that 2 h resveratrol co-culture repaired the mitochondrial damage caused by vitrification and may reach the similar level of development potential to fresh embryo.After resveratrol co-culture for 2h,the model of the mitochondrial distribution in most vitrified/thawed embryo was similar to fresh embryos;the intracellular ROS level of embryos with resveratrol treatment was significantly lower than that with no resveratrol treatment(P<0.05);??m of embryos was significantly higher than that with no resveratrol treatment(P<0.05).These results demonstrated that co-culturing with resveratrol for 2h can repair the mitochondrial damage of embryo caused by vitrification.Above results showed that vitrification induced the injury of abnormal mitochondrial distribution and mitochondrial function damage of mouse 2-cell embryos,however,in vitrified/thawed embryos with resveratrol co-culture for 2 h,the abnormal mitochondrial distribution and mitochondrial dysfunction were significantly improved;and the subsequent development ability of vitrified/thawed 2-cell embryos was significantly higher than vitrified/thawed 2-cell embryos with no resveratrol treatment.