| DNA demethylation is involved in many processes of mammalian life, and plays an important role in early embryonic development and primordial germ cells formation. Over the past few decades, the mechanisms underlying active DNA demethylation have triggered a lot of discussion and attention, but many of them remain controversial. Tet proteins and their oxidation product 5hmC, have opened up a new field for the study of mechanisms of DNA active demethylation. As an intermediate product,5hmC is an important epigenetic marker involved in the process of DNA demethylation. This study investigated the roles of Tet proteins on DNA demethylation during early embryonic development in mice, thus providing new insights for further understanding the mechanisms of active DNA demethylation in early mouse embryos.The localization and dynamic changes of 5mC and 5hmC in fertilized embryos and parthenogenetic embryos were examined during mouse early embryonic development. Results showed that:the distribution of 5mC and 5hmC in fertilized eggs is asymmetrical; during pronuclear stage from PN1 to PN5, the level of 5mC was moderately decreased and the level of 5hmC was slightly increased in the female pronucleus, while in the male pronucleus, the 5mC level was quickly decreased and 5hmC level was dramatically increased. In parthenogenetic embryos, only a small amount of DNA was oxidized to 5hmC at the pronuclear stage, but a bulk of 5hmC reprogramming was seen at the 2-cell stage. This result indicated that although massive generation of 5hmC in paternal genome mainly occurred at 1-cell stage, the 5hmC generation in maternal genome happened at 2-cell stage. Further examination showed that the level of Stella was decreased significantly in 2-cell embryos, implying that the maternal genome at this stage may be less protected by Stella, thus favoring the Tet protein-mediated hydroxymethylation. During the development from 2-cell stage to blastocyst stage, the level of 5mC and 5hmC and the expression of Tet genes exhibited a similar dynamic pattern between parthenogenetic and fertilized embryos. However, the 5hmC level and Tet gene expression in parthenogenetic embryos were generally lower than that in fertilized embryos, suggesting some differences between the two types of embryos in DNA methylation reprogramming.Detailed study of parthenogenetic and fertilized 2-cell embryos showed that the level of 5hmC in the late stage was significantly higher than that in the early stage. This indicated that there was a large amount of 5hmC generated in 2-cell embryos. Down-regulation of Tet gene expression by RNAi suggested that, the generation of 5hmC, in either parthenogenetic or fertilized 2-cell embryos, was regulated by all three Tet proteins. It seemed that Tetl mainly acts on parthenogenetic embryos, while both Tetl and Tet3 have a role on fertilized embryos. This indicated that Tet proteins could not only induce the generation of 5hmC in 1-cell embryos, but also mediate the 5hmC generation in 2-cell embryos. RNAi experiments also showed that down-regulation of Tet gene expression significantly decreased the blastocyst development. This suggested that Tet protein family played an important role in the embryonic development.After the nuclei of mouse fetal fibroblast cells were transferred into the cytoplasm of a blastomere of 2-cell embryos, the level of 5hmC level was significantly increased, indicating that the 2-cell embryos still have the ability to regulate 5hmC reprogramming.In summary, the massive generation of 5hmC in paternal genome predominantly occurred in 1-cell embryos, while the 5hmC generation in maternal genome mainly occurred in 2-cell embryos. Tet genes could not only induce the generation of 5hmC in 1-cell embryos, but also mediate the 5hmC generation in 2-cell embryos. Mouse 2-cell embryos have the ability to regulate 5hmC reprogramming. These results would provide new information for understanding the function of Tet protein family and the mechanism of DNA demethylation in early embryonic development. |
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