| Melatonin?MT?has an important function in anti-oxidative stress,however,the mechanism of action of melatonin in relieving heat stress?HS?injury of porcine oocytes is still lacking in-depth research.In this study,porcine crossbreds ovary was used as the test object and oocytes were collected.In the first experiment,the mechanism of melatonin alleviating the damage of heat stress on porcine oocytes in vitro was studied.Experiment 2 investigated the effect of melatonin on cumulus cell expansion and gene expression after heat stress.Experiment 3 analyzed the influence of melatonin on the development ability and expression of apoptosis genes of parthenogenetic activated and cloned embryos after heat stress.This has great theoretical and practical significance for improving oocytes utilization efficiency and increasing animal fertility in high-temperature seasons.Experiment 1 studied the mechanism of melatonin in alleviating the damage of heat stress on mature oocytes of porcine in vitro.This study was divided into 3 groups:the control group was treated with porcine oocytes matured in vitro for 42 h in maturation medium at 38.5?,the heat stress group was treated with porcine oocytes matured in vitro for 4 h in maturation medium at 41.5?,and then transferred back to38.5?for 38 h,the heat stress and melatonin group was treated with porcine oocytes matured in vitro for 4 h in maturation medium with 10-9 M melatonin at 41.5?,and then transferred back to 38.5?for 38 h.The results showed that melatonin significantly reduces ROS levels,the abnormal distribution rate of oocyte mitochondria?59.6%±3.06%vs.68.3%±1.95%;P<0.05?,and improves the survival rate and maturation rate of oocytes.The mechanism study through three time points of 4 h,18 h and 42 h found that HS significantly increased the morphological abnormality rate of?-tubulin and F-actin,significantly reduced the ratio of Bcl-2/Bax mRNA,and significantly increased 4 h and 18 h HSP70 expression,the expression of Nrf2 mRNA at 18h and42h.Melatonin significantly increased the ratio of Bcl-2/Bax mRNA at 4 h,18 h and42 h;significantly reduced the morphological abnormalities of?-tubulin and F-actin at18 h and 42 h,Significantly improve the activity of oocytes at 4 h and 42 h,significantly promote the mRNA expression of HSP70 at three time points of 4 h,18 h and 42 h,and significantly reduce the expression of Nrf2 mRNA at 18 h and 42 h.Based on the above results,under the conditions of this study,melatonin cooperates with intracellular HSP70 and Nrf2 factors to jointly inhibit HS-induced oxidative damage to porcine oocytes.Experiment 2 investigated the effect of melatonin on cumulus cell expansion and gene expression after heat stress.The experiment was continued on the basis of the grouping of experiment 1.After 42 hours of oocyte culture,the cumulus expansion index of each group was counted.After the cumulus cells were isolated,the expressions of cumulus expansion,apoptosis,and antioxidant-related genes were detected.The results showed that melatonin treatment enhance the expression of cumulus expansion index?1.61±0.024 vs.2.09±0.065;P<0.05?,Ptgs2,Ptx3,Tnfaip6,and CAT mRNA were significantly increased.The mRNA expression of CAT was still significantly lower than that of the control group,and the ER mRNA expression was not significantly different.The results show that melatonin can reduce the damage of porcine cumulus cells by promoting the expansion of cumulus cells and the expression of related anti-apoptosis,antioxidant-related genes,and inhibiting the expression of pro-apoptotic genes.Experiment 3 analyzed the influence of melatonin on the development ability and expression of apoptosis genes of parthenogenetic activated and cloned embryos after heat stress.Continue the experiment based on the grouping of Experiment 1,detect the development of parthenogenetic activated embryos and cloned embryos in each group,count the cleavage rate and blastocyst rate of each group,and detect the expression of apoptosis genes.The results showed that melatonin increase the total cell numbers of blastocysts of parthenogenetic embryos and cloned embryos significantly.Among them,the cleavage rate?77.67%±3.18%vs.84.67%±1.33%;P<0.05?and blastocyst rate?26.59%±1.45%vs.33.42%±1.67%;P<0.05?of parthenogenetic embryos increased significantly,while the cleavage rate and blastocyst rate of cloned embryos did not change significantly.The ratio of Bcl-2/Bax mRNA was significantly increased.The overall results of this study indicate that melatonin alleviates heat stress damage to cumulus cells and oocytes during the in vitro maturation of porcine oocytes,inhibits apoptosis of cells and embryos,and can significantly enhance the development potential of parthenogenetic embryos.The results of this study indicate that melatonin reduces intracellular ROS content,improves mitochondrial distribution,repairs?-tubulin and F-actin,inhibits oocyte apoptosis,promotes HSP70 gene expression,mediates Nrf2 signaling pathway,and alleviates HS induction of porcine oocytes are oxidatively injury,thereby promoting the development of oocytes and parthenogenetic embryos.And melatonin reduces the damage caused by heat stress to cumulus cells by promoting cumulus expansion,inhibiting cumulus cell apoptosis,and improving antioxidant capacity. |
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