Effects Of AZD5438 On The In Vitro Development Of Porcine Parthenogenetic Activation And Somatic Cell Nuclear Transfer Embryos

Nowadays,the study of parthenogenetic activation method and the activation parameters have matured,but the efficiency of the activation is still quite low.In addition,since oocytes activation is a key part of cell nuclear transfer,improved efficiency of parthenogenetic activation also promotes the success rate of somatic cell nuclear transfer.AZD5438 works as a cyclin-dependent kinase inhibitor and AZD5438 can regulate the cell cycle by inhibiting the phosphorylation of CDK substrates in vitro.This study was carried out to evaluate the potential effects of AZD5438 on the early development of porcine parthenogenetic activated and somatic cell nuclear transfer embryos,optimize efficiency of porcine oocytes activation and provide a basis for new MPF inhibitors.Main contents and results of this study are as follows:1.Experiment was designed to reveal the optimum treatment condition.Porcine oocytes activated by electric pulse were treated with different concentration of AZD5438 for 4 h.According to the blastocyst rate,the experimental group treated with 10 ?M AZD5438 could significantly increase the blastocyst rate,the difference was significant(46.4%vs.34.5%,P<0.05).Then porcine oocytes activated by electric pulse were treated with 10 ?M AZD5438 for different durations.The results showed that supplemented 10 ?M AZD5438 for 4 h after activation can improve the blastocyst rate,the difference was not significant(42.8%vs.38.6%,37.2%),but there was no significant difference.2.Experiment was designed to compare the effect of AZD5438 and 6-dimethylaminopurine(6-DMAP)on the early development of porcine parthenogenetic activated and somatic cell nuclear transfer embryos.Porcine parthenogenetic activated and somatic cell nuclear transfer embryos activated by electric pulse were separately treated with 10?M AZD5438 and 6-DMAP for 4 h.According to the blastocyst rate of porcine parthenogenetic activation(42.4%vs.34.3%)and somatic cell nuclear transfer(13.40%vs.11.11%),the results showed that the blastocyst rate were similar between 10?M AZD5438 and 6-DMAP,the difference was not significant.3.Experiment was designed to investigate the karyotype of porcine parthenogenet ic activated embryos treated by 10 ?M AZD5438 for 4 h.Blastocysts induced from 10 ?M AZD5438 group or 5 ?g/ml CB group were subjected to karyotype analysis.The results showed that 60 percent of porcine parthenogenetic activated embryos treated by 10 ?M AZD5438 was diploid,10 percent was haploid,10 percent was tetraploid and 20 percent was hybrid.4.Experiment was designed to investigate the MPF activity of porcine parthenogenetic activated embryos treated by 10 ?M AZD5438 for 4 h.The results showed that supplemented 10 ?M AZD5438 for 4 h after activation significantly decrease the level of MPF(125.8 vs.288.7,P<0.05).5.Experiment was designed to investigate the effect of AZD5438 on the relative gene expression level of porcine parthenogenetic activated embryos by RT-PCR.By observing the expression of pluripotency-related genes(Sox2?Oct4?Nanog)and apoptosis-related genes(Bcl-2?Bax)of porcine parthenogenetic activated embryos treated by 10 ?M AZD5438 for 4 h.The results showed that there were no significant difference between 10 ?M AZD5438 group or 5 ?g/ml CB group on the relative gene expression level of porcine parthenogenetic activated embryos treated with 10 ?M AZD5438 group or 5 ?g/ml CB(P>0.05).Conclusion can be made by the study as followings:AZD5438 can enhance the development of porcine parthenogenetic activated and somatic cell nuclear transfer embryos by reducing the level of MPF,and the optimal treating condition of AZD5438 is that supplemented 10 ?M AZD5438 for 4 h after activation.In addition,there were no significant difference in the relative gene expression level of pluripotency-related genes(Sox2?Oct4?Nanog)and apoptosis-related genes(Bcl-2?Bax)between two groups.The study showed that AZD5438 treatment can improve the early development of porcine parthenogenetic activated and somatic cell nuclear transfer embryos.And AZD5438 can be used as a new MPF inhibitor in the porcine oocytes activation.