Vitamin C Promotes In Vitro Development Of Porcine Early Embryos By Regulating Mitochondrial Function

The partial pressure of oxygen in the oviduct of developing embryos in vivo is 5%,whereas in vitro culture usually employs normoxia(21%).The hyperoxic environment of in vitro culture causes the embryo produce excessive reactive oxygen species(ROS),triggering oxidative stress,which in turn damage the mitochondria of embryo and subsequently affect embryonic development in several ways.Vitamin C(Vc)is a potent antioxidant that can protect the embryo from oxidative stress through removing free radicals,and also is an important component of the mitochondrial endogenous antioxidant defense.Previous studies have shown that the addition of Vc in culture medium can promote the developmental potential of embryos during in vitro cultures,but the underlying mechanism still unclear.Therefore,this study was aim to systematically explore the effects of Vc on the in vitro development of porcine early embryos as well as on the mitochondrial function of embryos.The main results of this study are as follows:1.To analyze differences of gene expression profile between in vivo and in vitro developing embryos.Comparative analysis of gene expression profiles of morulae produced in vitro and in vivo was performed by reanalysis the published transcriptome data from the geo database.The results revealed a total of 1276 differentially expressed genes(DEGs),including 507 upregulated and 769 downregulated genes.Go functional enrichment analysis of DEGs showed that genes were enriched in pathways such as mitochondria,inner mitochondrial membrane,mitochondrial respiratory chain complex I,and ATP synthesis proton transport;KEGG pathway enrichment analysis of DEGs showed that genes were enriched in the oxidative phosphorylation signaling pathway.These pathways are all associated with mitochondrial function,suggesting that mitochondrial function in early embryos closely relative to the developmental potential of embryos cultured in vitro.2.The effect of Vc on the in vitro development of porcine early embryos was explored.The results showed that the addition of 12.5 μg/m L Vc in culture medium can significantly increase the blastocyst rate of porcine early embryos w(P < 0.05),and the culture efficacy was dependent on the duration of Vc addition;Compared with embryos cultured under low partial pressure(5% O2),early embryos cultured under normoxia(21% O2)had significantly higher ROS levels(P < 0.05)and lower blastocyst rates(P < 0.05);However,the addition of Vc caused a slight decrease in ROS levels(P > 0.05),a significant increase in the blastocyst rate(P < 0.05),and increase the expression levels of embryonic anti-apoptotic genes(P < 0.05).3.The effect of Vc on mitochondrial function in in vitro culture porcine early embryos was explored.Adding Vc(12.5 μg/m L)to culture increased the mitochondrial membrane potential(P < 0.05),upregulated expression levels of mitochondrial function related genes MFN1 and OPA1(P < 0.05),and increased expression levels of TCA cycle key enzyme genes PDHA1 and OGDH(P <0.05).The above results indicate that the mitochondrial function of early embryos is closely related to their in vitro developmental potential,and Vc may promote early pig embryo development in vitro by alleviating oxidative stress and improving mitochondrial function.