269 DEMETHYLATION DYNAMICS OF PATERNAL GENOMES IN PRONUCLEAR BOVINE ZYGOTES PRODUCED BY IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION WITH NONDRIED OR FREEZE-DRIED SPERMATOZOA

Abstract

Epigenetic reprogramming in early preimplantation embryos, which refers to erasing and remodeling epigenetic marks, including DNA methylation, is essential for differentiation and development. In many species, the paternal genome is subjected to genome-wide active demethylation before DNA replication commences, whereas the maternal genome maintains its methylation status until being demethylated passively during the subsequent cleavage divisions. This study was designed to investigate the dynamics of paternal genome demethylation in pronuclear-stage bovine zygotes produced by conventional IVF or intracytoplasmic sperm injection (ICSI) using conventionally frozen, nondried and freeze-dried (FD) bull sperm stored at either +4 or –196°C for 1 year. Zygotes were fixed and immunostained using anti-5 methyl cytosin (Calbiochem, San Diego, CA, USA) at 8, 10, 14 and 18 h post IVF (hpi) and at 6 and 12 h post ICSI (hpic). After digital optical sections of the zygotes had been collected by a Bio-Rad confocal laser scanning microscope equipped with a krypton/argon ion laser, the fluorescence emitted by each pronucleus was quantitatively analyzed by Image-J software. The relative methylation of the male pronucleus to the female pronucleus in each zygote (RM) was calculated. Differences among groups regarding proportions of zygotes showing different levels of RM were analyzed using Fisher’s exact probability test, whereas differences among groups regarding the overall average of RM were analyzed using Student’s t-test. In conventional IVF, the overall average RM significantly decreased (P < 0.05) from 0.92 at 8 hpi to 0.69 at 10 hpi with no additional significant decrease at 14 and 18 hpi (0.67 and 0.64, respectively). This decrease was accompanied by significantly higher proportions of zygotes showing RM < 0.60 (45.5, 37.5, and 38.2% at 10, 14, and 18 hpi, respectively) compared with 3.7% at 8 hpi. Because spermatozoa take about 2 h to penetrate into oocytes in a bovine IVF system, zygotes at 6 and 12 hpic may be developmentally comparable with those at 8 and 14 hpi. The overall averages of RM in ICSI-derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in corresponding IVF-derived zygotes (8 and 14 hpi), but a significantly higher proportion of 6 hpic zygotes (37.8%) than 8 hpi zygotes (3.7%) showed RM < 0.60. Surprisingly, the proportions of 12 hpic zygotes produced with FD sperm showing RM < 0.60 (60.6 and 62.4% for +4 and –196°C storage, respectively) were significantly higher than the proportions of those produced with nondried sperm (39.4%). These findings indicate that demethylation of the bovine paternal genome occurred early after IVF and ICSI, and that the freeze-drying of the paternal genome resulted in a higher demethylation level.