| Somatic cell nuclear transfer(SCNT)can be applied in research,animal breeding,transgenic animal production,medical treatment and many other fields.However,low efficiency seriously restricts the application of this technology.Because of the fact that serial SCNT(SSCNT)can reuse cytoplasm to reprogram donor cells,so theoretically,reprogramming efficiency,and subsequent cloned embryo development could be enhanced.Hence,in this study buffalo SSCNT and its underlying mechanism were systematically detected.The results of these investigations are summarized in the following1.The developmental efficiency and epigenetic modifications of buffalo SSCNT embryos were detected in this section.SSCNT was operated through using three types of donor cells(BFF,BFF-PRL and 8811).The results revealed that the clone blastocyst rate of reconstructed embryos derived from 8811(SSCNT blastocysts)was significantly promoted compared to the blastocyst derived from BFF and BF-PRL,meanwhile,the expression level of 5mC and H3K9me3 were also down-regulated in SSCNT blastocysts(P<0.05).Moreover,the expression pattern of Dnmtl,HDAC1,HDAC2 were decreased while the expression level of Oct4,Sox2 and Nanog were increased significantly(P<0.05)in SSCNT blastocyst.These results revealed that the developmental potential of buffalo SSCNT embryos was higher than normal SCNT embryos.2.The underlying mechanism of buffalo SSCNT was investigated from the perspective of donor cells.The difference in physiological characteristics and epigenetic modifications among BFF,BFF-PRL and 8811 were detected.The results revealed that no significant difference was found in cell morphology and growth ability among three types of donor cells.Meanwhile,no significant difference was existed in 5mC,AcH3K18,histone H3K9me2/me3,methylation status of imprinted genes H19 and IGF2,as well as the expression of related genes(P>0.05)among the three types of donor cells.Here the results we got suggested that the promotion of SSCNT embryo developmental potential may be not caused by the initial epigenetic modifications of donor cells.It was speculated that the improvement of SSCNT embryonic development efficiency was related to the incubation between donor cells and oocyte cytoplasm.3.The underlying mechanism of buffalo SSCNT was checked from the perspective of recipient oocytes.The effects of oocyte extracts incubation on BFFs and subsequent SCNT embryos were detected.The results revealed that the expression level of 5mC and histone H3K9me3 in BFFs were decreased significantly(P<0.05)after BFF was incubated with GV oocyte extracts.Subsequent experiments showed that the rate and blastocyst cell number of SCNT blastocyst were also significantly promoted(P<0.05).Moreover,the expression level of 5mC and histone H3K9me3 in blastocyst were decreased after donor cells were treated with GV oocyte extracts(P<0.05).At the same time,the expression level of Oct4,Sox2,and Nanog in blastocyst were all increased after donor cells were incubated with GV oocyte extracts(P<0.05).The results we got in this section revealed that the incubation between oocyte cytoplasm and donor cells could alert the epigenetic modifications of donor cells,and then improve the developmental ability of cloned embryos.It was concluded that some maternal factors exist in oocytes cytoplasm played a key role in this process.4.As one key maternal factor,buffalo KDM1A gene plays important roles during histone methylation regulation.Therefore,the expression pattern of KDMIA in GV/MII oocytes were detected and the results showed KDMIA was highly expressed in buffalo GV and MⅡ oocytes,especially in GV oocytes(P<0.05).Then the CDS of buffalo KDMIA gene was cloned and analyzed.At last,the eukaryotic expression vector pcDNA3.1(+)-KDM1A was constructed and the expression level of KDMIA can be significantly promoted in BFF after transfection.5.In this section,the effects of KDMIA on buffalo SCNT embryos were explored by overexpressing KDMIA in buffalo SCNT embryos.Through using microinjection technology,it was found that the expression level of KDM1A could be up-regulated significantly {P<0.05),overexpressing of KDM1A led to a significant increase in 8cell rate,morula rate and blastocyst rate of SCNT embryos and a significant decrease in H3K9me3.Moreover,ZGA related genes Ssuch as elF-3a,TRC,SUPT4H1,SNAI1,ZSC.AN5B,and histone demethylase related genes KDM4B,KDM4D were all up-regulated significantly(P<0.05)after KDMIA was overexpressed.This results revealed that overexpression of maternal factor KDM1A in buffalo cloned embryos could down-regulate the level of H3K9me3,enhance the ZGA and so improve the developmental efficiency of cloned embryos.In summary,all those results above reveal that:(1)The developmental potential of buffalo SSCNT embryos is higher than that of SCNT embryo,this phenomenon may be due to the fact that donor cells are easier to be reprogrammed,but not the initial status of donor cell epigenetic modifications.(2)Incubating donor cells with oocyte extracts can promote the reprogramming efficiency of donor cells and subsequent developmental ability of SCNT embryos.(3)Maternal factor KDMIA is highly expressed in GV oocytes,by acting its role in demethylation and ZGA,the methylation level of histone H3K9me3 is decreased and the expression level of ZGA related genes are increased,and then improve the developmental ability of SSCNT embryos. |
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