| The process of embryonic development is the process of reprogramming of sperm-egg combination. DNA methylation, one of the major epigenetic reprogramming modifications, participates in a variety of physiological activitives of cells, DNA methylation allows the orderly expression of genes involved in embryonic development, maintaining the normal development of embryos. Cattle are singleton animals, in order to increase the breed of cattle, so we obtain more embryos by in vitro culture way and transplantation, but due to various reasons, the survival rate of embryo in vitro is lower than in vivo, causing the early death of embryos and certain loss for the livestock production.Now, we have confirmed that the abnormal DNA methylation lead to the embryo to fail. The article from the perspective of bovine early embryonic death, using bisulfite sequencing, studied on related genes OCT4and Nanog promoter methylation pattern of bovine early embryonic development, revealing the methylation patterns of the related genes of bovine early embryonic development, speculating the signal of the early embryonic death, enhancing the survival rate of embryos in vitro, guiding the laboratory production. The results were shown as follows:1. In our test, we used bovine parthenogentic embryos to explore methods for higher concertration of genomic DNA. The results indicated that first removing transparent with proteinase K, we could obtain the relatively high concentration of genomic DNA by this method.2. We investigated the methylation pattern of OCT4gene promoter in the early embryo of death at different stages. The results were shown that the methylation levels of OCT4gene promoter at2-cell,4-cell,8-16cell and morula stage were90.50%,90.00%,92.30%,77.70%in cloned bovine embryos, while the methylation levels at different stages were87.80%、81.10%、88.50%、70.60%in bovine IVF embryos of early death. Taken together, the methylation level of OCT4promoter was high in early dead bovine cloned embryos, and the methylation level of OCT4promoter was higher in cloned embryos of early death than IVF embryos of early death, but there had no significant difference (P>0.05) at different stages between cloned embryos and in vitro-fertilized embryos; while between cloned embryos and in vitro-fertilized bovine embryos, the methylation level of OCT4decreased from8-16cell to morula stage.3. We investigated the methylation pattern of Nanog gene promoter in the early embryos of death at different stages. The results were shown that the methylation levels of Nanog gene promoter at2-cell,4-cell,8-16cell and morula stage were46.15%、42.311%、48.46%、53.85%in cloned bovine embryos, while the methylation levels at different stages were63.85%、56.92%、69.23%、53.07%in bovine IVF embryos of early death. Taken together, the methylation level of Nanog promoter was higher in in vitro fertilized embryos. In cloned bovine embryos, from8-16cell to morula stage, the methylation level of Nanog promoter increased, but in in vitro-fertilized embryos decreased. The methylation value of Nanog promoter was higher at each stage in IVF embryos compared to those in cloned embryos.4. Taken all together, in bovine embryos of early death, the methylation level of OCT4promoter was higher compared to Nanog, we induce that the abnormal methylation of OCT4leads to the abnormal methylation of Nanog, and the abnormal methylation of OCT、Nanog cause the death of early embryos.5. For the stages of significant differences, we made quantitative analysis. The results were shown:no matter what the way we got the embryos of early death, the expression of OCT4was higher than Nanog, and only at morula stage we deceted little Nanog expression. We induce that OCT4may limit the expression of Nanog.6. Combining with the above, we induce that2-cell stage may be the signal of early embryonic death, thereby we cosider add the concentration of the inhibitors to improve the survival rate in vitro. |
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