Genome-wide and gene specific paternal demethylation in androgenetic embryos

In the present study, a genome-wide measure of demethylation employing the 5 methylcytidine antibody as well as a gene specific approach by bisulphite sequencing of a house keeping and imprinted genes was investigated to confirm if the active paternal demethylation was occurred correctly in mouse haploid, diploid androgenetic and triploid polyspermic embryos. The results indicated that the active demethylation of paternal genome in haploid, diploid, and triploid embryos occurred as similar as the normal ICSI embryos and completed the full demethylation within 6, 8, and 10 h after fertilization, respectively. The methylated CpG dinucleotides sites of alpha-actin, paternally expressed Igf2, paternal methylated Gtl2 and H19 gene loci were hypermethylated in mature sperm. After fertilization, methylated sites of two paternal methylated genes retained their methylation status whereas the paternal alleles of alpha-actin and Igf2 rapidly underwent active demethylation in the diploid androgenetic embryos produced by two sperm injection into enucleated oocytes or pronuclear transplantation. These results indicated that no specificity of paternal specific active demethylation was found in androgenetic or triploid polyspermic embryos.