Melatonin Affects The Development Of Parthenogenetic Embryos From Vitrified-warning Mouse Oocytes,Potentially By Modulating SIRT1

Our previous studies have shown that oxidative stress increased,and the developmental potential of parthenogenetic embryos decreased after cryopreservation of mouse oocytes at the MII stage.Melatonin effectively reduces the ROS level in vitrified-warmed oocytes and promotes subsequent embryonic development,but the underlying molecular mechanism remains unclear.SIRT1 is a NAD⁺dependent deacetylase,which plays a role in maintaining oocyte mitochondrial function and participating in oxidative stress regulation.Therefore,we hypothesized that melatonin regulates oxidative stress through SIRT1 and promotes the parthenogenetic development of vitrified-warmed mouse oocytes.The results of this study can enrich and improve the theory of oocyte cryopreservation and provide a reference for the conservation,development and utilization of animal germplasm resources.Firstly,mouse MII oocytes were randomly divided into three groups:Fresh group（F）,Vitrification group（V）,and Vitrification+Melatonin group（VM）.SIRT1expression level,oxidative stress level（Glutathione,GSH;Reactive oxygen species,ROS）and Lipid Peroxidation level（Recombinant glutathione peroxidase 4,GPX4;lipid ROS）were detected in parthenogenetic 2-cell embryos.The embryo development rate after parthenogenesis activation were calculated.Secondly,MII oocytes were randomly divided into three groups:Vitrification+MT group（VM）,Vitrification+MT+EX527 group（VME）,Vitrification+SRT-1720 group（VS）.Melatonin(10^-9mol/L),EX527(2×10^-5mol/L,SIRT1 protein inhibitor)and SRT-1720(10^-6mol/L,SIRT1protein agonist)were added into recovery,parthenogenesis activation and embryo culture solution.The parthenogenesis rate of oocytes in VM,VME and VS groups was analyzed.At last,the protein expressions of SIRT1,NRF2,the levels of reactive oxygen species（ROS）and glutathione（GSH）of parthenogenesis 2-cell embryos were detected.The results are presented as follows:（1）Antioxidant effects of melatonin on the parthenogenetic embryo development of vitrified mouse oocytes.Detected the parthenogenesis 2-cell embryos from vitrified MII mouse oocytes,the ROS level was significantly increased and the GSH level was significantly decreased in the vitrified group compared with the fresh group（P<0.05）.When 10^-9mol/L melatonin was added,the ROS level was significantly decreased and the GSH level was significantly increased in the vitrification group,with no significant difference from the fresh group（P>0.05）.Further findings showed no significant difference in lipid ROS in parthenogenesis 2 cells among all groups（P>0.05）.However,the expression level of GPX4 in the vitrification group was significantly lower than that in fresh group（P<0.05）.After melatonin was added,the GPX4 level was significantly increased（P<0.05）,and had no significant difference with the fresh group（P>0.05）.As for the oxidative stress regulator SIRT1,the expression level of parthenogenetic 2-cell embryos in the vitrified group was significantly lower than that in the fresh group（P<0.05）.The SIRT1 expression level was significantly higher than that of the vitrified group after melatonin was added（P<0.05）,but had no significant difference with the fresh group（P>0.05）.In addition,parthenogenetic development was statistically analyzed in each group.Parthenogenesis activation cleavage rate（75.68±2.38%vs.93.02±1.71%）,4-cell rate（72.97±2.92%vs.88.37±3.34%）,morula rate（62.16±2.67%vs.87.21±3.09%）and blastocyst rate（56.76±2.84%vs.77.91±3.08%）of vitrification group were significantly decreased（P<0.05）compared with fresh group.The parthenogenetic activation cleavage rate（94.74±2.70%）,4-cell rate（89.47±3.73%）,morula rate（82.89±3.61%）and blastocyst rate（80.26±3.81%）were significantly increased in the vitrification group when 10^-9mol/L melatonin was added（P<0.05）.Moreover,there was no significant difference between the fresh and vitrification+MT groups（P>0.05）.In conclusion,melatonin alleviates oxidative stress and SIRT1 injury induced by cryopreservation.After,the development of mouse oocyte parthenogenesis was significantly improved during melatonin treatment.（2）Effects of melatonin on the parthenogenetic embryo development of vitrified mouse oocytes were mediated by SIRT1.The parthenogenetic activation cleavage rate（94.67±1.63%）,4-cell rate（86.67±4.72%）,morula rate（81.33±3.27%）and blastocyst rate（76.00±2.36%）of VM group were significantly higher（P<0.05）than that in VME group（81.11±3.07%,68.89±4.51%,62.07±4.01%,52.22±1.68%）,and had no significant difference（P>0.05）with VS group:（93.33±2.19%,86.67±2.19%,77.33±1.44%,70.67±3.13%）.These findings suggest that melatonin improves the development potential of vitrified-warmed oocytes by modulating SIRT1.（3）Effects of melatonin on oxidative stress of parthenogenetic embryo development was mediated by SIRT1.When 10^-9mol/L melatonin was added into the recovery solution,parthenogenesis activation solution and embryo culture solution after oocyte cryopreservation,the SIRT1 level in parthenogenesis activated 2-cell was significantly higher than that in the VME group（P<0.05）,but had no significant difference compared with VS group（P>0.05）.Meanwhile,NRF2 level were detected,and the results showed no significant differences in NRF2 level among the three groups（P>0.05）.Then,the oxidative stress level of 2-cell parthenogenetic embryos was detected:ROS level in the VM group was significantly higher than that in the VME group（P<0.05）but was not significantly different from VS group（P>0.05）.These results suggested that melatonin regulates intracellular ROS levels through SIRT1.Similarly,there was no significant difference in the GSH level of 2-cell embryos after the supplementation of melatonin or SRT-1720（P>0.05）while the GSH level was significantly decreased in the VME group（P<0.05）.It follows that melatonin reduces oxidative stress of parthenogenetic 2-cell embryos from vitrified-warmed oocytes by up-regulating the SIRT1 level.In summary,cryopreservation of mouse MII oocytes caused abnormal SIRT1levels,increased oxidative stress levels in parthenogenetic 2-cell embryos.The addition of 10^-9mol/L melatonin reduced oxidative stress by regulating the expression level of SIRT1 protein in 2-cell embryos,thus potentially improving the development of parthenogenetic embryos from the vitrified-warmed oocytes.