Figure 5. The effect of melatonin on DNA methylation on the NNAT promoter of prolonged-culture oocytes. Porcine oocytes matured in vitro were continuously cultured in medium supplemented with or without 10−3 M melatonin for 24 h. (A) Primer and CpG loci examined at the NNAT differentially methylated region (DMR) are shown. DNA methylation was examined by bisulfite sequencing in fresh (B), prolonged-culture (C) and prolonged-culture + Mel oocytes (D). Each line presents an individually cloned allele, with an open circle for a non-methylated CpG site and filled circles for methylated CpG. Ten clones successfully sequenced for each group are presented. Methylation level (%) = (Methylated CpG/Total CpG) × 100. PC, prolonged-culture; Mel, melatonin.