Vitamin C Regulates Bovine Cumulus Cell Proliferation And Apoptosis Through CPEB3

Cumulus cells are a specific type of granular cells that surround oocytes and have special physiological functions.The proliferation,apoptosis,and spread of cumulus cells are extremely important for oocyte maturation,fertilization,and early embryonic development.Studies have shown that vitamin C can regulate the apoptosis of bovine granular cells by affecting the related DNA methylation.In this process,cytoplasmic polyadenylation element binding protein 3(CPEB3)may play a potential role.CPEB3 is a member of the cytoplasmic polyadenylation element binding family,which has been found to regulate dormancy and mask mRNA translation in oocytes,but whether and how it affects the physiological state of cumulus cells is unclear.Therefore,in this study,primary bovine cumulus cells were first isolated,and 100 ?M of vitamin C was added to the culture medium.CCK-8 and flow cytometry were used to determine the effect of vitamin C addition on cumulus cell proliferation,cell cycle,apoptosis,and Effect of ROS level.The results show that vitamin C can promote the progression of the S phase of the cell cycle,thereby promoting the proliferation of bovine cumulus cells and increasing intracellular ATP levels.Vitamin C can also inhibit bovine cumulus cell apoptosis by reducing intracellular ROS levels.During this process,thbs4,IRX1,COL1A2,GSN,Khdrbs2 and CPEB3 were differentially expressed.Subsequently,this study suppressed the expression of CPEB3 in bovine cumulus cells by siRNA,and detected cell cycle progression,proliferation,and apoptosis by cell cycle,CCK-8,EdU,apoptosis,JC-1,ROS,qRT-PCR Death,reactive oxygen content,mitochondrial membrane potential,and cell proliferation related gene expression changes.The results showed that after CPEB3 inhibition,cumulus cells had an abnormal cell cycle,and the ratio of G0 / G1 phase cells decreased from 75.99 ± 1.25% in the control group to 68.10 ± 0.76%(P <0.05).In contrast,the proportion of S-phase cells increased from 8.39 ± 1.49% of the control group to 14.20 ± 0.68%(P <0.05),and the proportion of G2 / M phase cells increased from 9.92 ± 1.12% to 13.1 ± 0.32%(P <0.05).The results of the CCK-8 test showed that the cell proliferation in the CPEB3 inhibition group at 24 h(P <0.05),48 h(P <0.01)and 72 h(P <0.01)was significantly higher than that in the control group.EdU test results showed that the proportion of EdU-positive cumulus cells in the CPEB3 inhibition group was 1.43 ± 0.36 times higher than that in the control group.The apoptosis rate of cumulus cells decreased from 14.04 ± 1.70% of the control group to 10.75 ± 0.60%(P <0.05).We also tested the change in mitochondrial membrane potential in bovine cumulus cells after CPEB3 inhibition.The CPEB3 inhibition group had a red / green fluorescence intensity ratio of 1.69 ± 0.16 times that of the control group.The relative ROS level in the CPEB3 inhibition group was reduced by 10.46 ± 4.31% compared with the control group(P <0.05).The DCFH-DA fluorescence intensity of the CPEB3 inhibition group also decreased to 96.34% ± 0.51% of the control group level(P <0.05).CCNA,CCND,CCNE,CDK2,CDK4,CDK6,p21 and P27 mRNA expression were down-regulated,and CCNB,CDK1,HAS2,PTGS2,PTX3 and CEBPB mRNA expression were up-regulated.Therefore,CPEB3 plays a potential role in regulating the biological and physiological functions of bovine cumulus cells.This study deepens the research on the biological function of vitamin C,provides a theoretical basis for further exploring the genes,physical and chemical changes and response mechanisms involved in the regulation of vitamin C on bovine cumulus cells,and is effective for the development of modern reproduction technology and detection technology Using vitamin C provides new ideas.