title: 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 pronuclear-stage 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< or =RM<0.6.
authors.affiliation: Interdisciplinary Graduate School of Science and Technology, Shinshu University, Nagano, Japan.
authors.forename: Hany
authors.initials: H
authors.lastname: Abdalla
authors.forename: Masumi
authors.initials: M
authors.lastname: Hirabayashi
authors.forename: Shinichi
authors.initials: S
authors.lastname: Hochi
mesh_terms: D044503:5-Methylcytosine / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D005612:Freeze Drying; D016678:Genome; D008297:Male; D008745:Methylation; D008954:Models, Biological; D015233:Models, Statistical; D009865:Oocytes / Q000378:metabolism*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013094:Spermatozoa / Q000378:metabolism*; D013997:Time Factors; D015053:Zygote / Q000378:metabolism*
chemical_list: D044503:5-Methylcytosine