Effect Of Histone Deacetylase Inhibitors On Nuclear Reprogramming Of Cloned Pig Embryos

Somatic cell nuclear transfer(SCNT)is a valuable tool for basic mammalian biomedical research.However,the efficiency of animal cloning remains unsatisfactorily low,limiting the application of cloned pigs.At present,this low cloning efficiency is due to abnormal epigenetic modifications.Histone deacetylase inhibitors(HDACi)can repair the aberrant epigenetic modifications and subsequently improve the in vitro developmental capacity of porcine SCNT embryos.MGCD0103,PCI-24781,M344,Quisinostat,and CI994 are five HDACi.They can elevate the level histone acetylation,but yet never applied in pig SCNT embryos.In this study,for the first time,we explored the effects of these five HDACi on the developmental competence of cloned pig embryos.Methods:To selected optimal treatment concentration,porcine SCNT embryos were treated with different concentrations of five HDACi(MGCD0103,PCI-24781,M344,Quisinostat,and CI994)for 24 h,and pig SCNT embryos were treated with optimal concentration for different treatment time.After cloned embryos treated optimal condition,investigating the level of histone acetylation,DNA methylation,apoptosis,and pluripotency-and apoptosis-related gene expressions by immunostaining and PCR.In addition,each HDACi-treated porcine SCNT embryos were transferred into the oviducts of surrogates and investigated the in vivo developmental capacity.Results:(1)Our results showed that treating with 0.2 ?M MGCD0103 for 24 h effectively improved the development of SCNT embryos,in comparison to the control group(blastocyst formation rate,25.5 vs.10.7%,P<0.05).Furthermore,MGCD0103 supplementation significantly(P<0.05)increase the acetylation level of histone H3 at lysine 9(AcH3K9)and histone H4 at lysine 12(AcH4K12).To examine the in vivo development,MGCD0103-treated SCNT embryos were transferred into two surrogate sows,and three fetuses developed.These results suggest that MGCD0103 can enhance the nuclear reprogramming and improve in vitro developmental potential of cloned pig embryos.(2)Our results showed that treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos,in comparison to the control group(25.3%vs 10.5%,P<0.05).Furthermore,PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12.TUNEL assay revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos.Also,PCI-24781-treated embryos were transferred into three surrogate sows,and two fetuses developed.These results suggest that PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.(3)Our results indicated that treatment with 5 ?M M344 for 6 h improved the development of porcine embryos,in comparison with the untreated group(25.1%vs.10.9%;P<0.05).Moreover,M344-treated embryos had increased average fluorescence intensity of AcH4K12 at the pseudo-pronuclear stage(P<0.05).However,no differences exist in Oct4,NANOG,and SOX2 expression in M344-treated and untreated SCNT blastocysts.In evaluating the effect of M344 on in vivo development,845 M344-treated embryos were transferred into 3 surrogates,and developed 3 fetuses.These findings suggested that M344 elevated the level of histone acetylation,facilitated the nuclear programming,and subsequently improved the developmental competence of pig SCNT embryos.(4)Our results showed that treatment with 10 nM Quisinostat for 24 h significantly improved the development of reconstructed embryos,in comparison to the untreated group(19.0%vs.10.2,P<0.05).Furthermore,Quisinostat-treated SCNT embryos significantly increased the average fluorescence intensity of AcH3K9 at the pseudo-pronuclear stage(P<0.05)and led to a great improvement of the immunostaining intensity of POU5F1 at blastocyst stage(P<0.05).While there was no statistical difference in BAX expression,the transcript level of BCL2 was significantly different in quisinostat treatment group compared to control group.Finally,Quisinostat-treated cloned embryos were transferred into three surrogates,and six fetuses developed.These findings suggested that Quisinostat regulated the gene expression and epigenetic modification,facilitated the nuclear reprogramming,and subsequently improved the developmental competence of pig SCNT embryos and blastocyst quality.(5)Treatment with 10 ?M CI994 for 24 h significantly improved the blastocyst formation rate of SCNT embryos in comparison with the untreated group(21.9%vs.11.4%,P<0.05).Moreover,the average fluorescence intensities of AcH3K9 and AcH4K12 in CI994-treated embryos were remarkably increased at the pseudo-pronuclear stage,but not at the blastocyst stage.The intensity of POU5F1 was higher in CI994-treated blastocysts than in control blastocysts,whereas that of 5mC did not differ between the two groups.The percentage of apoptotic cells in blastocysts was significantly higher in the untreated group than in the CI994-treated group.mRNA levels of POU5F1 and SOX2 were significantly increased in the CI994-treated group.Finally,CI994-treated cloned embryos were transferred into three surrogates,and three fetuses developed.These observations suggest that optimum exposure(10 ?M for 24 h)to CI994 after activation elevates the level of histone acetylation and subsequently improves the in vitro development of pig SCNT embryos.Conclusions:Optimum exposure to MGCD0103(0.2 pM,24 h),PCI-24781(0.5 nM,24 h),M344(5 ?M,24 h),Quisinostat(10 nM,24 h)post-activation improved the in vitro developmental competence of cloned pig embryos by enhancing histone acetylation,inhibiting apoptosis,regulating pluripotency related gene expression,and improving somatic nuclear reprogramming.