DNA methylation status in DMRs of imprinted genes in spermatozoa of OHFD analyzed by COBRA. DNA methylation patterns in DMRs of paternally imprinted genes H19 (423 bp), Gtl2 (425 bp), Rasgrf1 (284 bp) and maternally imprinted genes Peg3 (444 bp) and Snrpn (420 bp) in spermatozoa of OHFD (n = 9-10) and OCD mice (n = 10) were evaluated by COBRA. Oocytes were utilized as a control. Enzymes used are shown in the right column; Oo, oocyte. (A) H19; (B) Gtl2; (C) Rasgrf1; (D) Peg3, red arrowheads showed the digested bands; (E) Snrpn; (F) a shorter region located in DMR of H19 was amplified with Peg3 in the first-round PCR and the methylation pattern was analyzed by COBRA.