Effects Of Melatonin And Cumulus Cells On In Vitro Maturation And Embryo Development Of Porcine Oocytes

Melatonin as a scavenger of free radicals can eliminate the adverse effects of oxidative stress on oocyte by scavenging reactive oxygen species in oocyte,and cumulus cells are closely associated with oocyte maturation,and the cumulus cells play an important role in oocyte maturation and fertilization.This paper mainly studies the effects of melatonin and cumulus cells on in vitro maturation and embryo development of oocytes.Thereby further optimizing the culture system of oocyte maturation in vitro.The first experiment was the control group,the melatonin group,the melatonin receptor inhibitor group and the melatonin + melatonin receptor inhibitor group,which mainly detected the embryo development index,reactive oxygen species,mitochondria distribution,Glutathione content and related gene expression.The results showed that the cumulus expansion index,polar body excretion rate,embryo cleavage rate and blastocyst rate of melatonin were significantly higher than those of the other three groups(P<0.05).The content of glutathione in melatonin group was not significantly different from that in melatonin + inhibitor group,but significantly higher than that in control group and inhibitor group(P<0.05).The proportion of oocytes in the melatonin group was significantly higher than that in the control group(P<0.05),and the level of reactive oxygen species in the oocytes was significantly lower than that in the control group(P<0.05).The melatonin group had no significant effect on the expression of GDF9 gene related to oocyte maturation(P>0.05),but significantly decreased the expression of proapoptotic genes P53 and BAX(P<0.05),and increased the expression of antioxidant gene CAT and mature related gene BMP15.The results of this experiment indicate that melatonin acts on its receptor to enhance the in vitro maturation and development of porcine oocytes and affect the expansion of oocyte cumulus,which can promote the production of glutathione and mitochondrial space in porcine oocytes.Distribution,and can reduce intracellular reactive oxygen species;adding melatonin significantly reduced the expression of proapoptotic genes P53 and BAX,and increased the expression of antioxidant gene CAT and mature related gene BMP15.The second experiment was mainly in the nude egg group,the co-culture groupand the cumulus-oocyte complexes(COCs)group.The main indicators of embryo development and related gene expression were detected.The survival rate of COCs group was significantly higher than that of nude mice(P<0.05).There was no significant difference in survival rate between COCs group and co-culture group(P>0.05).The first polar body rate of COCs group was significantly higher than that of COCs group.In the co-culture group and the nude egg group(P<0.05),the cleavage rate and blastocyst rate of the COCs group were significantly higher than those of the co-culture group and the nude egg group(P<0.05).The expression of Gpx3,Gpx4,Gclm,Gsto1 and Gclc was significantly down-regulated in the naked egg group(P<0.05),but the expression of glutathione thiotransferase Gstz1 ??was not significantly different from that in the COCs group(P>0.05).The results of this experiment indicated that co-culture of naked eggs with cumulus cells significantly increased the maturation rate of naked eggs and their late embryo development ability,but their maturation and developmental ability were still lower than that of cumulus-oocyte complexes.The cumulus cells can promote the expression of glutathione anabolism-related genes Gclc,Gclm,Gpx3,Gpx4 and Gtso1 in oocytes.