| The incomplete nuclear reprogramming of donor cells cause the low rate of somatic cell nuclear transfer(SCNT),incliuding DNA methylation,genomic imprinting,X chromosome inactivation,histone variants,and so on.Therefore,we investigated the effects of donor cell sex difference on the development of buffalo SCNT embryos,and analyzed DNA methylation and the relevant genes,Histone 3 lysine 9 di-and tri-methylation(H3K9me2,H3K9me3),the methylation level and expression of Xist,H19,IGF2 in SCNT-♀ and SCNT-♂early embryos.And then,we reduced the expression levels of H3K9me3 and Xist to improve the development of SCNT embryos.The results of all the works were summarized in the following:1.Effects of different sexual buffalo fetal fibroblasts on the development of SCNT embryosEffects of different sexual fibroblasts on the development and speed of buffalo SCNT embryos were compared.The results showed:the blastocyst rate of SCNT-♀ was significantly higher than SCNT-♂ group(P<0.05),however,it was no significant difference among of the SCNT-♀、ICSI-X.ICSI-Y and IVF groups(P>0.05).In addition,the growth speed of SCNT-♀ and SCNT-♂embryos was all faster than the IVF embryos:the developmental rate of 8-cell and blastocysts of SCNT-♀ embryos and SCNT-♂ embryos were significantly higher than that of IVF embryos(P<0.05).Using Q-RT-PCR to test the expression level of OCT4,SOX2,NANOG in various types of embryos,the results showed:the expression of OCT4 and NANOG at morula stage,and the expression of NANOG at blastocyst stage that in SCNT-♀ group were significantly higher than those of SCNT-♂ group(P<0.05),but the expression of OCT4 at blastocyst.stage in SCNT-♂ group was higher than those of SCNT-♀ group(P<0.05).There was no significant difference in the SOX2 between SCNT-♀ and SCNT-♂ groups at morula and blastocyst stage(P>0.05).2.The methylation of SCNT-♀ and SCNT-♂ embryosTo explore the molecular mechanism of methylation in cloned.embryos,the expression of DNA methylation level,DNA methyltransferases,H3K9me2 and H3K9me3 in SCNT-♀ and SCNT-♂ embryos were analysed.Immunostaining was used to examine the expression of 5mC,5hmC,H3K9me2 and H3K9me3.The expression of Dnmts and TETs was analysed by Q-RT-PCR.The results showed:5mC and 5hmC were mainly expressed in the SCNT-♀ and SCNT-♂ embryos,respectively.The DNA methylation levels of SCNT-♀ embryos were significantly lower than those of SCNT-♂ embryos.The expression patterns of Dnmt1、Dnmt3a and Dnmt3b in SCNT-♀ embryos were similar to the ICSI-X embryos,but it was significantly different between SCNT-♂ embryos and ICSI-Y embryos.TET1 mRNA was expressed mainly in the SCNT-♂ embryos.TET2 and TET3 were inactive in the early embryos.H3K9me2 was expressed majorly in the IVF embryos.H3K9me3 was significantly expressed SCNT-♂embryos at the 8-ell stage(P<0.05).3.Methylation patterns and regulation of imprinted genes in SCNT-♀and SCNT-♂ embryosBisulfite Sequencing PCR was used to examine the methylation of Xist,H19 and IGF2 in SCNT-♀ and SCNT-♂embryos.We found that the methylation levels of Xist in IVF-X and IVF-Y embryos were respectively 44.8%and 72.3%.The methylation of Xist in SCNT-♀ embryos was similar to that of IVF-X embryos,but in SCNT-♂ embryos were 0%.The methylation level of H19 and IGF2 genes in IVF-X,IVF-Y,SCNT-♀ and SCNT-♂ embryos were in the range from 80%to 94%.KDM4B ectopic expression reduced the level of H3K9me3 and improved the development of SCNT embryos(P<0.05).4.The expression and regulation of imprinted genes in SCNT-♀ and SCNT-♂ embryosQRT-PCR method was employed to examine the expression of Xist,H19 and IGF2 in ICSI-X、ICSI-Y、SCNT-♀、SCNT-♂ embryos.Furthermore,Xist-shRNA was injected to reduce the level of Xist in SCNT-♀ embryos.The results showed:Xist gene expressed highly in the 8cell stage of various types of embryos(P<0.05).Moreover,the expression level of Xist was higher in SCNT-♀ embryos than that of SCNT-♂ embryos at various developmental stages.H19 and IGF2 were highly expressed in 2cell and 8cell stage of SCNT-♀and SCNT-♂ embryos(P<0.05).Injecting of the Xist-shRNA injecting could reduce the expression level of Xist and significantly improve the developmental rate of blastocysts in SCNT-♀ embryos(P<0.05).5.Methylation and expression of imprinted genes in different sexual cloned BuffaloesWe respectively analysed the methylation level and expression of Xist,H19 and IGF2 in different sexual cloned buffalos by BSP and QRT-PCR,including the liveborn cloned buffalos(♀ L1-3,♂L 1-3),stillborn cloned buffalos(♀ S1-3,♂S1-3)and natural reproduction buffalos(♀Nl-3,♂N1-3).The results showed:(1)The methylation level of Xist in ♀L group was similar to that of ♀N group(P>0.05),whereas ♀S group was significantly lower than ♀N group(P<0.05),there was no significant difference among ♂L,♂S and ♂N groups(P>0.05).(2)The methylation level of H19 was no significant difference among ♀L,♀S,♀N,♂L,♂S and ♂N groups(P>0.05).Formore,The methylation level of IGF2 was not significantly different among ♀$L,♀S,♀N,♂L and ♂N groups(P>0.05),but it was significantly lower in ♂S group(P<0.05).(3)The expression of Xist in ♀S group was significantly higher than ♀L and ♀N groups(P<0.05),no significant difference among the ♂L,♂S and ♂N groups(P>0.05).The expression of IGF2,H19 were significantly expressed in ♀S and ♂S groups,(P<0.05),but no difference between ♀L and ♀N,♂L and ♂SN groups(P>0.05).In conclusion,(1)the developmental potential of SCNT-♀ preimplantation embryos is greater than that of SCNT-♂ embryos.(2)The global DNA methylation in SCNT-♀ embryos is lower than that of SCNT-♂ embryos.Moreover,the expression patterns of Dnmts and the methylation patterns of Xist in SCNT-♀ embryos show a more normal trend compared with SCNT-♂embryos.(3)Reducing the level of H3K9me3 can improve the development of SCNT-♀ embryos.(4)Impeding the expression of Xist can facilitate the development of SCNT-♀ embryos.(5)The methylation and expression of Xist、H19 and IGF2 show normal levels in the living cloned buffalos,while there are hypomethylated and highly expressed in the dead cloned buffalos. |
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