| Epigenetic modification patterns are obviously reprogrammed during mammalian preimplantation embryo development, such as DNA methylation and histone modifications reprogramming. However, comparison with in vivo fertilization preimplantation embryos, the epigenetic modification reprogramming of mice SCNT preimplantation embryo were aberrant. The abnormal development of SCNT embryos may be caused by the incomplete epigenetic modification reprogramming of the somatic cell nucleus in an enucleated oocyte. Cattle as a domestic animal have an important economic value in our country. Bovine nuclear transfer technology has been established for many years, but the efficiency is still very low. The epigenetic modification reprogramming during embryonic development vary among species. So, in this study, we analyzed the DNA methylation reprogramming during bovine preimplantation embryo development. The main results are as the follow;1. The dynamic changes of DNA methylation and H3K9me3 during bovine IVF preimplantation embryo development.(1) IF staining indicated that the 2-cell embryos showed strong IF signals for 5 m C, and the signal gradually decreased until 8-cell embryos were formed and then increased until the blastocyst stage during bovine IVF preimplantation embryo development. In IVF blastocysts, both inner cell mass(ICM) and trophectoderm cells were methylated. IF staining for 5 hm C showed the existence of 5 hm C in all bovine IVF preimplantation embryos, and 5 hm C existed in both ICM and trophectoderm cells, as did 5 m C in bovine IVF blastocysts.(2) High expression of TET3 was observed in oocytes and IVF embryos at the 2-cell and 4-cell stages, but the TET3 level was significantly decreased in IVF blastocysts. TET1 and TET2 expression remained low both in IVF and SCNT preimplantation embryos.(3) satellite I, α-satellite, POU5F1 and NANOG showed obviously DNA demethylation progress; imprint gene H19 showed resistance to DNA demethylation and maintained the moderate methylation situation all time; SOX2 and CDX2 were always hypomethylation or not methylation.(4) The results showed that POU5F1 and NANOG were highly expressed in IVF blastocyst stage embryos; however, the expression levels in oocyte and early IVF stage embryos were significantly lower than those in IVF blastocyst stage embryos. SOX2 and CDX2 m RNA expression levels were always kept high in oocytes, bovine IVF preimplantation embryos.(5) Immunofluorescence staining results showed that strong H3K9me3 signals were observed in each stage of embryos and no obvious change of H3K9me3 signals was found during bovine in vitro fertilization pre-implantation embryo development. Immunoflurescence staining for H3K9me3 in bovine in vitro fertilization blastocysts results also indicated that there was no difference in H3K9me3 signals between inner cell mass(ICM) and trophectoderm(TE) cells.2. The dynamic changes of DNA methylation and Vitamin C influence during bovine SCNT preimplantation embryo development.(1) Each bovine SCNT preimplantation embryo showed stronger IF signals for 5 m C than that in the corresponding IVF preimplantation embryo, and the signal of 5 m C did not show obvious changes in the bovine SCNT preimplantation embryos. More interestingly, no 5 hm C signal was observed in each bovine SCNT preimplantation embryo developmental stage.(2) TET3 showed significantly lower expression in SCNT embryos when compared with IVF embryos, and the expression level was further reduced following the development of SCNT preimplantation embryos, while no TET3 expression was observed in blastocysts of SCNT embryos. No expression of TET1 was observed in the 4-cell stage of IVF embryos; while, in SCNT preimplantation embryos, the expression of TET1 was observed at the 4-cell stage, and the level increased until the blastocyst stage.(3) Compared to the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes(POU5F1 and NANOG) and repeated elements(satellite I and α-satellite) showed insufficient demethylation and hypermethylation states in the SCNT group. Second, a differentially methylated region(DMR) of an imprint control region(ICR) in H19 exhibited excessive demethylation and hypomethylation states. Third, some pluripotency genes(CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups.(4) Vitamin C could improve satellite I, α-satellite and H19 DNA methylation reprogramming and made the methylation states of those gene closer to IVF blastocyst; however, Vitamin C showed adverse influence on POU5F1 and NANOG methylation reprogramming and no influence on SOX2 and CDX2 methylation reprogramming in SCNT preimplantation embryos.3. Effect of DNMT inhibitor(RG108) on bovine parthenogenetic embryo development.(1) The supplement of RG108(100 μM, 200 μM and 500 μM) significantly decreased the blastocyst rate of bovine parthenogenetic embryo development; however, did not show any influence on embryo cleavage rate and 8-cell formation rate.(2) DNMT1 and DNMT3 b showed high expression in the parthenogenetic 2-cell stage embryo, then, the expression levels of DNMT1 and DNMT3 b decreased during the following developmental stages. The DNMT3 a showed lower expression compared to DNMT1 and DNMT3 b during bovine parthenogenetic preimplantation embryo development.(3) The results showed that there was no NANOG transcription until the blastocyst stage in the control group; however, there were NANOG gene transcripts at 8-cell stage in the RG108 group. Based on the RT-PCR results, we found that the NANOG transcription level significantly increased at the blastocyst stage for the RG108 group compared to the control group. Then, we studied the methylation states at the NANOG gene promoter region. The NANOG promoter methylation levels in the 8-cell and blastocyst stage embryos were significantly lower in the RG108 group than control group.(4) satellite I and a-satellite regions had lower methylation levels in the 8-cell and blastocyst stage embryos in the RG108 group than the control group.(5) There was no difference in the numbers of apoptotic cells in the 8-cell stage embryos in both the control and RG108 groups; however, the number of apoptotic cells in the RG108 group blastocyst stage embryos was higher than the control group. There was no difference in the BAX transcription levels for 8-cell stage embryos of the two groups. However, the BAX transcription level was significantly higher in the blastocyst stage embryos in the RG108 group compared with control group. These results indicate that the correct DNA methylation reprogramming was indispensable for bovine preimplantation embryo development. |
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