Study Of DNA Methylation And During Early Development Of Bovine Embryos

During preimplantation development of embryos, the chromosomes undergo extensive changes of epigenetic modifications, such as DNA demethylation and methylation, histone demethylation and methylation, histone deacetylation and acetylation etc. Abnormal epigenetic modifications of embryos would lead to abnormal development after birth or abortion. The terminal differentiated somatic cell could be reprogrammed to a totipotent state when transfer into matured enucleated oocytes by somatic cell nuclear transfer(SCNT). SCNT allows the generation of a new whole organism from a signal differentiated somatic cell. This technique has great potential for biomedical industry, agriculture, and endangered species conservation. But the incomplete reprogramming of somatic cell lead to lower cloning efficiency, which restricts the development of this technology. Therefore, study of the epigenetic modifications during the implantation embryonic development is important. In our study, changes of DNA methylation(5-mC), DNA hydroxyl methylation(5-hmC), and histone methylation(H3K9me2, H3K9me3) during IVF and SCNT embryos pronucleus stage and preimplantation embryo development were detected and analyzed systematically. The epigenetic modifications in bovine SCNT and IVF embryos were compared. A simple and efficient transfer siRNA into SCNT embryos method was established. The effect of DNMT1 on the development and epigenetic reprogramming of bovine SCNT embryos were investigated. And the effect of the O-GlcNAc glycosylation on the epigenetic reprogramming and in vitro development of bovine SCNT embryos were also investigated. Following results have been obtained:1. Immunofluorescence was used to detect the changes of DNA methylation(5-mC), DNA hydroxyl methylation(5-hmC), histone methylation(H3K9me2, H3K9me3) during pronucleus stages and preimplantation development of IVF and SCNT embryos. The results showed that during pronucleus stage of bovine IVF embryos, before the formation of the zygote(2hpa-10hpa), on female pronucleus, the level of 5-mC had no significant change, the level of 5-hmC increased slowly, and the level of H3K9me2 and H3K9me3 gradually decreased; while the 5-mC level of male pronucleus gradually decreased and to the lowest at 10 hpa, and the level of 5-hmC steady increase, and H3K9me2 and H3K9me3 decreased. After zygote formation(16hpa-22hpa), the levels of 5-mC, 5-hm C, H3K9me2 and H3K9me3 were steadily increased. During SCNT embryos development, the levels of 5-mC, H3K9me2 and H3K9me3 of the pseudo pronucleus were gradually reduced from 2hpa to 10 hpa and then steadily increase form 16 hpa to 22 hpa. The 5-hmC level of pseudo pronucleus was increased from 2hpa to 22 hpa. From 2-cell to blastocyst stage, the levels of 5-mC, 5-hmC and H3K9me3 of IVF and SCNT embryos were slowly decreased from 2-cell to 8-cell stage, after zygotic gene activation(8-cell stage), the signals increase gradually. The signal of H3K9me2 of IVF embryos was weak at 2-cell to 8-cell stage, and then increased gradually to blastocyst stage; the signal of H3K9me2 of SCNT embryos was weak at 2-cell stage and gradually increased from 4-cell to blastocyst stage. These results suggested that there were general epigenetic reprogramming during pronucleus stages and preimplantation development of IVF and SCNT embryos.2. The epigenetic modifications during pronucleus stage and early development of bovine IVF and SCNT embryos were compared. The results showed that, at 22 hpa of pronucleus stages, the levels of 5-mC and H3K9me3 in SCNT embryos were significantly higher, and the level of 5-hmC and H3K9me2 in SCNT embryos was significantly lower than in IVF embryos. And the levels of 5-mC, 5-hmC and H3K9me2 in SCNT embryo was significantly higher than in IVF embryo, the 5-mC level was significantly at 2-cell to morula stage, the 5-hmC level was significantly at 2-cell to 8-cell and blastocyst, and the H3K9me2 level was significantly at 4-cell to blastocyst. The signal of H3K9me3 in SCNT embryos were significantly higher than in IVF embryos at 2-cell to 8-cell stage, but significantly lower from morula to blastocyst stage. These results suggest that, after the somatic cells enter oocytes, abnormal epigenetic modifications were with embryo development, extremely higher 5-mC, 5-hmC, H3K9me2 and abnormal expression of H3K9me3 might be the reasons that lead abnormal SCNT embryos development.3. Establish a simple and effective method to transfer siRNA into SCNT embryos.4. The effect of DNMT1 on the in vitro development and epigenetic reprogramming of SCNT embryos was evaluated. The results showed that, after successful transferring siRNA into SCNT embryos, the expression of DNMT1 significantly decreased, mRNA level was significantly at 2-cell stage to 8-cell stage, protein level was significantly at 4-cell and 8-cell stages. After transfecting with DNMT1 siRNA, the 5-mC signal of SCNT embryos were significantly decreased, but no significantly difference were observed on the in vitro development. These results suggested that interference of DNMT1 could effectively decrease the extremely high 5-mC level of SCNT embryos, repair the abnormal epigenetic modifications, But had no effect on the in vitro development of embryos.5. The effect of the O-GlcNAc glycosylation on the in vitro development and epigenetic reprogramming of SCNT embryos by treated with DON or PUGNAc. The results showed that, treatment with DON or PUGNAc in high concentrations or for a long time could inhibit embryo development, reduced blastocyst rate. SCNT embryos treated with 25 nM DON for 24 h could significantly improve 5-hmC level and decrease 5-mC level of pseudo pronucleus; and the 5-mC signal was significantly lower at 2-cell to morula stage, in contrast, the 5-hmC signal of DON treated SCNT embryos had no significantly change from 2-cell to blastocyst stage. SCNT embryos treated with DON for 24 h had a significant higher cleavage rate and blastocyst rate, lower apoptosis rate of blastocyst, and higher number of inner cell mass of blastocyst; the in vitro development capabilities of embryos were also improved. SCNT embryos treated with 100 nM PUGNAc for 24 h had no significantly effect on the 5-mC and 5-hmC signal, and had no obvious improvement on the developmental ability. These results suggested that dynamic equilibrium of glycosylation played an important role during embryo development, treatment with DON or PUGNAc in high concentrations or for a long time would break this balance and inhibit the embryo development. Treatment with 25 nM DON for 24 h could repair the abnormal epigenetic modifications during early development of SCNT embryos, regulate gene expression and enhance the nuclear reprogramming.