| Although many mammals have been successfully cloned by somatic cell nuclear transfer technology,the low cloning efficiency still restricts the application of this technology.Regulating cell cycle of the donor is one of the core steps in improving nuclear transfer technology,because the cell cycle coordination of donor and recipient cells is closely related to cloning efficiency.Rapamycin is a protein-specific inhibitor of mTOR(mammalian target of rapamycin,mTOR),which can blocks the cell progression from G1 to S phase,the cell cycle is stopped in the G1 phase.Therefore,this study explored the effects of Rapamycin and serum starvation on the BEF cell cycle and embryonic developmental competence of somatic cell nuclear transfer,in order to find a suitable method of donor cell synchronization and provide theoretical and technical basis for improving cloning efficiency of buffalo.First,the effects of different concentrations of Rapamycin(0,0.5,1,2,4μM)on cell cycle and viability of BEF were compared.The results showed that the viability of the cells treated with 1 μM Rapamycin and the proportion of G0/G1 phase were significantly higher than control group and other experimental groups(P<0.05).When the effects of Rapamycin on BEF at different times(0,48,72,96 h)were compared,the results showed that the proportion of G0/G1 phase in cells treated with 1 μM Rapamycin for 72 h and 96 h was significantly higher than that of other groups(P<0.05).Secondly,the effects of Rapamycin and serum starvation on the cell cycle of BEF were compared.The results found that the Rapamycin synchronization efficiency was significantly higher than the serum starvation(P<0.05).qPCR results showed that Rapamycin and serum starvation treatment significantly down-regulated the expression of CyclinD1,CyclinD3,CDK4(P<0.05)and up-regulated the expression of p27(P<0.05),but the expression level of p27 in Rapamycin group was significantly higher than serum starvation Group(P<0.05).Rapamycin also significantly down-regulated the expression of CDK2(P<0.05).Western blot analysis revealed that both Rapamycin and serum starvation increased the expression of p27 protein(P<0.05)and decreased the expression of CyclinD1 protein(P<0.05),but Rapamycin also significantly inhibited the phosphorylation level of mTOR protein(P<0.05).Next,the effects of Rapamycin and serum starvation on the physiological indexes of BEF were explored.Apoptosis was detected by Annexin V-FITC double staining.The results showed that the level of apoptosis in serum starvation group was significantly higher than Rapamycin group(P<0.05).qPCR results showed that Rapamycin significantly up-regulated the expression level of apoptosis-inhibiting gene Bcl-2(P<0.05),and significantly down-regulated the expression of the pro-apoptotic genes Casepase3 and Casepase9(P<0.05),while the serum-starved group was significantly up-regulated the expression level of the apoptotic gene Bad(P<0.05).Cell ROS assay showed that serum starvation significantly increased cell ROS levels compared with Rapamycin-treated group(P<0.05).The detection of BEF cell metabolism-related gene expression revealed that the Rapamycin-treated group significantly up-regulated the expression levels of the metabolic related genes ACADVL,Ass,and HMGCL(P<0.05).In addition,the proportion of BEF cells with normal chromosome multiple treated with Rapamycin was significantly higher than serum-starved groups(P<0.05).Finally,this study compared the effects of Rapamycin and serum starvation on the embryonic developmental competence of somatic cell nuclear transfer.The results showed that the 8-cell rate and blastocyst rate of nuclear transfer embryos treated with Rapamycin were significantly higher than control group and serum-starved group(P<0.05).The above results show that:(1)Rapamycin at an appropriate concentration(l1μM)could improve the cell viability and proportion of G0/G1 phase in BFF cells treated for a certain time(72 h).(2)After BFF cells treated Rapamycin,the percentage of G0/G1 phase was significantly higher than serum starvation.which may be due to the fact that Rapamycin can affect the expression of cell cycle-related genes by inhibiting phosphorylation of mTOR.(3)After BFF cells treated Rapamycin,the level of apoptosis was significantly lower than serum-starved group,which may be related to Rapamycin reducing cell ROS levels and regulating metabolism in vivo.(4)Rapamycin significantly improves the embryonic developmental competence of somatic cell nuclear transfer.This effect may be related to that Rapamycin can increase percentage of G0/G1 phase and cell viability in vivo. |
PDF Full Text Request