Studies On The Relationship Between5-Hydroxymethylcytosine And Active DNA Demethylation During Early Development Of Mouse Embryos

The life cycle of mammals begins after a sperm enters an egg. Immediately afterfertilization, both the maternal and paternal genomes undergo dramatic changes toprepare for reprogramming. One of the molecular events that take place during thistransition is the active demethylation of the paternal genome. DNA methylation playsan important role in the regulation of gene expression and is essential for normalmammalian development. Genome-wide DNA methylation changes have beenobserved during at least two stages of mouse embryogenesis. The first event occursduring early development of mouse embryos after a sperm enters an egg while theother occurrence is in primordial germ cells (PGCs). In zygotes, the paternal genomeand the maternal genome undergo different reprogramming processes. Afterfertilization, the paternal genome rapidly demethylates, independent of genomic DNAreplication, which is called active demethylation. In contrast, the maternal genomeresists demethylation. During embryo cleavage, the maternal genome undergoesdemethylation during DNA replication in the absence of DNA methyltransferase1(DNMT1). Due to the fact that the process of active demethylation is independent ofDNA replication, factors must exist in zygotes that possess the ability to activelydemethylate. Over the past three decades, many enzymes have been implicated in theprocess of active DNA demethylation. Despite extensive efforts, the factorsresponsible for active DNA demethylation have not been identified, and many ofthose that have been implicated remain controversial.5hmC, which is generated byoxidation of5mC by the TET family of enzymes, is thought to be potentially involvedin active DNA demethylation. Further researchs suggested that TET family ofenzymes had three members: Tet1, Tet2and Tet3. Therefore, whether the5hmC isinvolved in active DNA demethylation caused the wide attention. Genomic methylation and hydroxymethylation were visualized withimmunofluoresence (IF) staining of5-methylcytosine and5-hydroxylmethylcytosinein our study. IF results showed that during early pronuclear embryonic stages, thefluorescence intensity of5-hydroxymethylcytosine (5hmC) in the paternal pronucleusincreased from PN1to PN5, while5hmC staining of maternal pronucleus exhibited noobvious differences. By the BSP and GluMS-PCR we found the ratio of5hmC/5mCin LINE1increased from PN2to PN5in fertilized zygotes. At50h, we observedexpression of the EGFP protein in embryos after methylated plasmid injection.Cytosine (C) methylation of gene promoter regions usually inhibits gene expression,and the plasmid is incapable of replication in cells. Thus, expression of EGFP isindicative of an active demethylation process. To further investigate the involvementof5hmC in this process, we searched for the existence of5hmC in promoter region ofmethylated plasmid with qGluMS-PCR. The qGluMS-PCR showed that5hmCappeared in the promoter region of methylated plasmid, while unmodified plasmidhad no5hmC. The qRT-PCR results showed that there was moderate expression ofTet1and Tet2in the eight-cell stage and morula stage embryos, but trace levels ofTet1and Tet2expression were present in zygotes and two-cell stage embryos.However in fertilized embryos, Tet3showed high expression levels in MII oocytesand zygotes, but its expression was reduced during the two-cell stage. It indicated thatthe conversion between5mc to5hmc is mainly catalyzed by Tet3in zygotes. By theBSP and GluMS-PCR we found that after the4-cell stage, a demethylation processwhich had roles in LINE1potentially occurred. In blastocysts, de novo5mC and5hmC staining was clearly visible in the ICM. These results indicated that themethylation and demethylation were existed in cleavage stage embryos, and they werea dynamic process. Our results will lay the foundation to elucidate the mechanisms ofactive DNA demethylation.