The Effects Of CNP On Oocyte Maturation And Embryo Development Of The Yak

Yak(Bos grunniens)is an important livestock species in Qinghai-Tibet Plateau and its adjacent areas by providing means of production and livelihood to the local herders.Embryo transfer is an important means to improve the genetic performance and increase the production efficiency of livestock.However,rates of in vitro maturation(IVM)of oocytes and blastocyst are low,which largely determines the development of yak embryo transfer.C-type natriuretic peptide(CNP)is a natural meiotic arrest factor of oocytes.Addition of CNP to IVM medium can synchronize nuclear and cytoplasmic maturation of oocytes,in hence improves the quality of mature oocytes.Therefore,this study intends to investigate the effects of CNP on oocyte maturation,early embryonic development and molecular regulation mechanisms of yaks.In the experiment,the biphasic IVM system(Biphasic IVM)was used to culture the cumulus-oocyte complexes(COCs),including a pre-IVM in IVM media supplemented with different concentrations of CNP(0,50,100,200 nM)for a period(0,4,6,8,12,16,and 24 h),followed by a conventional IVM phase.The meiotic arrest effects of concentrations of CNP and culture phase were assessed by germinal vesicle(GV)ratio of oocytes after pre-IVM.COCs were IVM for 24 h,26 h and 28 h after Pre-IVM,respectively.The maturation rate,cleavage rate and blastocyst rate were compared between different group after in vitro fertilization(IVF)and in vitro culture(IVC)to evaluate the effect of CNP in pre-IVM on oocyte maturation and embryo development of yaks.The effects of CNP on oocyte cytoplasmic maturation were evaluated by the levels of transzonal projections(TZPs),glutathione(GSH)and reactive oxygen species(ROS)in oocytes.The relative expression levels of CNP receptor(NPR2),epidermal growth factor(EGF)and its receptor(EGFR),apoptosis related genes(BCL-2 and BAX),DNA methylation transfer synthase(DNMT1),bone morphogenetic protein(BMP15)and growth differentiation factor(GDF9)in yak IVM-oocytes and early embryos were evaluated by q PCR.The results showed that(1)the GV percentage of COCs cultured in 100 nM CNP for 6 h pre-IVM was greater than other treatments(78.15 ± 5.66%,P<0.05).(2)IVM system including pre-IVM for 6 h in IVM mefium containing 100 nM CNP,followed by an extended IVM phase for 28 h resulted in greater maturation rate(79.42 ±0.58%),cleavage rate(63.76 ± 0.36%)and blastocyst rate(32.97 ± 0.36%)than those in other groups(P<0.05).(3)TZPs level and GSH level of oocytes in pre-IVM(100 nM CNP)for 6 h were significantly higher than that in the conventional IVM group(P<0.05),but ROS level was significantly lower than that of conventional IVM group(P<0.05).(4)Pre-IVM in 100 nM CNP increased the expression of NPR2,BCL-2,GDF9 and BMP15 genes in mature oocytes,but inhibited the expression of EGF and BAX genes,and had no significant effect on DNMT1.2-,4-,8-cell embryo,morula and blastocyst derived from Biphasic IVM containing 100 nM CNP had greater expression levels of BCL-2,EGF,BMP15,GDF9 and NPR2 genes(P<0.05)compared with embryos derived from conventional IVM/IVF.GDF9 and BCL-2 expression levels were significantly up-regulated from 2-cell to 8-cell stage(P<0.05).NPR2 expression was significantly up-regulated in morula and blastocyst stage(P<0.05),and DNMT1 was significantly up-regulated in blastocyst stage(P<0.05).The expression of BAX gene was inhibited in 4-cell and blastocyst stage.In summary,the optimum concentration of CNP in biphasic in vitro culture system of yak oocytes is 100 nM,and the optimum culture time is pre-IVM 6 h +IVM 28 h,The addition concentration and culture time can significantly improve the quality,maturation rate and blastocyst rate of yak oocytes.CNP may play its regulatory role through regulating the expression of BAX,BCL-2,EGF,EGFR,BMP15,GDF9,NPR2 and DNMT1 genes of oocytes and early embryos of yaks.