Dynamic Expression Of AID, ELP3and TET3in Mouse Preimplantaion Embryos

DNA methylation as an epigenetic marker refers to a methyl group added to the cytosine bases on the fifth carbon atoms. It plays important roles in gene transcriptional regulation. Aberrant DNA methylation may lead to cancer and stamp-related diseases.During embryonic development, gametogenesis and cell reprogramming, DNA methylation patterns exhibit dynamic changes, including erasing and reestablishing of DNA methylation modifications. There are two obvious DNA demethylation during mammalian early embryonic development.So far, the active DNA demethylation in mammalian are mainly through three ways. The first way is the hydroxylation of5-methylcytosines (5mC) to5hydroxymethyl cytosines (5hmC). This reaction is catalyzed by the proteins of ten-eleven translocation (TET) family, which consists three members, TET1, TET2and TET3. The second way is the DNA repair-associated active demethylation pathways. The third way is the direct removal of cytidines by other proteins such as the extension of complex3protein (elongator complex3, ELP3).The first active DNA demethylation in mammalian life course occurs at pronuclear stage embryos after fertilization, in which the active demethylation factors in oocyte cytoplasm conduct rapid and extensive demethylation to male pronuclear. This process is important for the activation of paternal genome and early embryonic development.In this study, we selected three demethylation protein AID, ELP3, TET3, which is implied to participate in catalytizing active demethylation. We used real-time quantitative PCR and immunofluorescence staining method to analyze the mRNA and protein expression of these genes in oocyte, pronuclear stage embryos and2-cell stage embryos. The main results are as follows:1. The Aid mRNA began to be expressed in GV stage oocytes, further accumulated in MⅡ oocytes, consumed after fertilization, and synthesized after zygotic genome activation. In the parthenogenetic embryos, the transcription of Aid is not restarted in the two-cell stage, indicating the presence of paternal genome is required for the transcription of Aid gene. Immunofluorescence staining found that AID protein could be detected in the cytoplasm of oocytes, zygotes and2-cell embryos, and there is no obvious nucleoplasm position. The fluorescence intensity of AID increased in PN4embryos, and decreased in2-cell embryos. In parthenogenetic embryos, the fluorescence signal was barely detectable in2cells. Considering the AID expression in oocytes and oocytes’ characteristics, namely oocytes accumulate a large number of mature mRNA and protein during maturation, we speculate that abundant expression of AID in MⅡ stage may have a effect in the process of fertilization.2. The Elp3mRNA began to be accumulated in oocytes, significantly increased in pronuclear embryos after fertilization, and reduced after zygotic genome activation. Immunofluorescence staining showed that ELP3signal was were detected in GV and MⅡ oocytes, as well as in PN2, PN4and two-cell embryos. The fluorescent signal was distributed in both the cytoplasm and the nucleus, suggesting ELP3may be involved in the active demethylation of male pronucleus. Moreover, the fluorescence signal of ELP3also co-localized with spindles in MⅡ stage oocytes, suggesting that it may play a regulatory role in the dynamic changes of spindle structure in meiosis by acting as a microtubule binding protein.3. The Tet3mRNA began to be expressed in oocytes, significantly elevated in PN2embryos after fertilization, and significantly reduced in two-cell embryos. The fluorescent signal of TET3protein was detected and dispersed in cytoplasm. Moreover, the signal showed colocalization with the spindles in MⅡ oocytes and two-cell embryos derived from parthenogenesis. It can be speculated that TET3may participate in meiotic division regulation by interacting with relevant regulatory factors, or in the process of meiotic spindle structure dynamics as a microtubule-binding proteins in specific period.4. Expression of Aid, Elp3and Tet3in different period of early embryonic development was compared and found that all they have a certain amount of mRNA reserves in oocytes. The expression levels of Aid and Elp3were significantly lower than that of Tet3. The transcription of Aid disappeared after fertilization in prokaryotic stage, while Tet3gene was still significantly higher than Elp3. It is speculated that Tet3gene may be the main acting factor for active demethylation of male pronuclear after fertilization, while Aid and Elp3may be more involved as a cofactor. Comparison of the expression of these three genes in different stages showed that the expression of Aid was detected in oocytes and2-cell embryos, but not in the other periods.it is speculated that its role for actively demethylation was not strong in male pronuclear. The expression levels of Elp3and Tet3significantly decreased in the various phases after2cells stages, and kept at a low level until the blastocyst stage. It is speculated that these three genes acted additional funcitons other than demethylation in the preimplantation embryos after active demethylation of male pronucleus.This study provides theoretical basis for the in-depth study of active demethylation mechanism during early preimplantation embryo development.