Study On Antioxidant Maslinic Acid Regulating Oxidative Stress In Porcine Early Embryos In Vitro

Early embryo in vitro culture,a crucial part of the in vitro production procedure in mammalian embryos,is a highly essential technique for biotechnology applications such as cloning,in vitro fertilization and transgenic animal.However,early embryos are susceptible to external environmental factors during in vitro culture,resulting in low developmental potential.In particular,excessive accumulation of ROS for a long time may cause oxidative stress in early embryos,resulting in blocked early embryonic development.Therefore,relieving oxidative stress of early embryos in vitro is of great significance for reducing embryo loss and promoting the development of embryo engineering.Maslinic acid（MA）,a natural remarkable antioxidant,can scavenge free radicals in cells or tissues,but its role in mammalian embryos has been rarely reported.Therefore,this study aims to investigate the role of MA in regulating oxidative stress in porcine early embryos in vitro.The results were as follows:In this study,we first assessed the in vitro developmental capacity of embryos by adding different concentrations of MA during in vitro culture of porcine parthenogenetic embryos.The results showed that 1μM MA significantly increased blastocyst formation rate（p<0.01）,hatching rate（p<0.05）and total cell numbers（p<0.05）and was better than other concentration groups,while the 5μM MA group significantly decreased blastocyst hatching rate（p<0.01）and diameter（p<0.05）compared to the 1μM MA group;however,there was no significant difference in the cleavage rate（p>0.05）of embryos cultured in vitro in all groups.Cell proliferation and apoptosis assays showed that MA significantly promoted blastocyst cell proliferation（p<0.05）and inhibited blastocyst cell apoptosis（p<0.01）,while MA up-regulated the m RNA expression levels of the cell proliferation gene DHODH（p<0.05）and down-regulated the apoptosis gene BAX/Bcl-2（p<0.05）.Further,MA was also found to promote the developmental potential of somatic cell nuclear transfer and in vitro fertilized embryos（p<0.05）.Thus,MA can significantly ameliorate the developmental potential of early-stage embryos.Next,RNA-seq was performed on porcine in vitro blastocysts from the MA and control groups to analyze the differentially expressed genes between the two groups,and the resulting differentially expressed genes were analyzed for GO and KEGG pathway enrichment.The sequencing results showed that a total of 946 differentially expressed genes were detected in the two groups;373 genes were up-regulated（p<0.05）and 573 genes were down-regulated（p<0.05）in the MA group compared with the control group.Among them,DHODH,LRPAP1,CYP2G2,FEZ1,AKAP12,RPL31,CPLANE1,POLA1 and CLDN1 genes were significantly differentially expressed in both groups（p<0.05）and were verified by RT-q PCR in early pig embryos and found to be consistent with the results of RNA-seq.GO analysis showed that differentially expressed genes were mainly enriched in functions such as negative regulation of TOR signaling,redox process,mitochondria,and ATP binding activity（p<0.05）;KEGG enrichment showed that MA was mainly closely associated with PI3K-AKT signaling,m TOR signaling,and peroxisome pathways in porcine blastocysts（p<0.05）.These functions or pathways are directly or indirectly involved in the regulation of intracellular oxidative stress.Therefore,MA promotion of early porcine embryonic development in vitro may be related to oxidative stress regulation.To verify whether MA promotes the developmental potential of porcine early embryos in vitro by regulating oxidative stress pathways,the antioxidant capacity of porcine early embryos in vitro was first examined.The results showed that MA not only significantly reduced intracellular ROS levels at the 4-cell stage and blastocyst stage（p<0.01）,but also reduced ROS levels in the presence of H₂O₂-induced oxidative damage（p<0.01）.It also significantly increased intracellular GSH levels at the 4-cell stage and blastocyst stage（p<0.01）.In addition,MA also improved mitochondrial function in porcine blastocysts by increasingΔΨm（p<0.01）as well as intracellular ATP levels（p<0.0001）.Not only that MA supplementation promoted the relative protein expression levels of pluripotency marker OCT4（p<0.01）,antioxidant enzyme SOD1（p<0.0001）and CAT（p<0.05）,but also upregulated the m RNA expression level of antioxidant gene HO-1 in porcine blastocysts.In summary,the present context suggests that MA supplementation effectively improves the in vitro developmental potential of porcine parthenogenetic activation,somatic cell nuclear transfer and in vitro fertilization embryos.and RNA-seq results reveal a correlation between MA promotion of porcine early embryos into blastocysts and oxidative stress regulation.and RNA-seq reveal a correlation between MA promotion of porcine early embryos into blastocysts and oxidative stress regulation,as well as MA can effectively alleviate the challenge of oxidative stress injury in early embryos by up-regulating the expression of SOD1,CAT and HO-1 and improving mitochondrial function.Therefore,MA is a potential candidate antioxidant for improving the in vitro production of porcine early embryos.