298 EFFECTS OF TRICHOSTATIN A DURING IN VITRO FERTILIZATION OF BOVINE OOCYTES ON SUBSEQUENT DEVELOPMENT, CELL NUMBER, AND ALLOCATION OF RESULTING EMBRYOS

Abstract

Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization or of transferred cell genomes after nuclear transfer to establish a totipotent state for normal development. In the fertilization of bovine oocytes, asynchronous histone acetylation occurs during pronuclear formation in the manner that modification of the paternal genome precedes that of the maternal genome (Wee et al. 2006 J. Biol. Chem. 281, 6048–6057). In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse ovaries were in vitro-matured (IVM) for 21 h in TCM-199 supplemented with 5% v/v FCS, 0.5 mM sodium pyruvate, 0.02 AU mL^-1 FSH, and 1 µg mL^-1 estradiol-17β at 39°C under 5% CO₂ in air. After IVM, the oocytes were subjected to IVF with 3 × 10⁶ mL^-1 of Percoll gradient-selected sperm in a defined medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were freed from cumulus cells and cultured in mSOF medium until 168 h post-insemination (hpi) at 39°C under 5% CO₂, 5% O₂, and 90% N₂ with high humidity. Cleavage and blastocyst development were assessed at 48 and 168 hpi, respectively. Inner cell mass (ICM) and trophectoderm (TE) of blastocysts were differentially stained by the method of Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29) to assess cell number and ICM/TE ratio. Experiments were replicated 3 times. Data are presented as means ± SEM and statistically analyzed by multiple comparison with the Holm method. Rates of cleavage (0 nM: 71.0 ± 7%, n = 102; 5 nM: 75.5 ± 5%, n = 106; 50 nM: 68.8 ± 6%, n = 105; and 500 nM: 71.7 ± 4%, n = 98) and blastocyst formation (21.4 ± 5%, 22.3 ± 6%, 17.8 ± 2%, and 18.2 ± 2%, respectively) were similar among the groups. However, 500 nM TSA significantly (P < 0.05) increased ICM and total cell numbers (59.8 ± 4 and 143.5 ± 7, respectively, n = 31) compared with the control (43.1 ± 3 and 120.9 ± 7, n = 31). In addition, ICM/TE ratios were higher in the 50 nM (0.81 ± 0.08, n = 29) and 500 nM (0.92 ± 0.2, n = 31) groups than in the control (0.59 ± 0.04, P < 0.05). These results suggest that TSA treatment during IVF of bovine oocytes does not affect the blastocyst rate but alters the cell numbers and their allocation to ICM and TE. Overriding epigenetic modification of the genome during fertilization may have a carryover effect on cell proliferation and differentiation in pre-implantation embryos.

This study was supported by a grant from Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST.