Demethylation dynamics of the paternal genome in pronuclear-stage bovine zygotes produced by in vitro fertilization and ooplasmic injection of freeze-thawed or freeze-dried spermatozoa

Abstract

This study was designed to investigate the dynamics of the paternal genome demethylation in pronuclearstage bovine zygotes produced either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using freeze-thawed (FT) as well as freeze-dried (FD) bull sperm stored at +4 or -196 C for one year. Zygotes were fixed and immunostained using anti-5-methyl-cytosin at 8, 10, 14 and 18 h post IVF (hpi) and at 6 and 12 h post ICSI (hpic). In conventional IVF-derived zygotes, the overall average of the relative methylation (RM; male/female) decreased from 0.92 at 8 hpi to 0.69 at 10 hpi (P<0.05) without any additional decrease at 14 and 18 hpi (0.67 and 0.64, respectively; P>0.05). This was accompanied by higher proportions of zygotes showing RM<0.6 (45.5, 37.5 and 38.2% at 10, 14 and 18 hpi, respectively; P<0.05) compared with 3.7% at 8 hpi. The overall averages of the RM in the FT-ICSI derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in the corresponding IVF-derived zygotes (8 and 14 hpi), but a higher proportion of the 6 hpic zygotes (37.8%; P<0.05) showed an RM<0.6 compared with the 8 hpi zygotes (3.7%). The proportions of FD-ICSI derived zygotes at 12 hpic showing an RM<0.6 (60.6 and 62.4% for +4 and -196 C storage, respectively) were higher than that of the FT-ICSI derived zygotes (39.4%; P<0.05). Thus, the bovine paternal genome rapidly demethylated within 10 h after IVF and 6 h after ICSI, and the freeze-drying and/or the storage process had no adverse effect on demethylation of the paternal genome. The extent of demethylation in the pronuclear-stage bovine zygotes was moderate, with 0.4≤RM<0.6.