25 CRYOPRESERVATION AND IN VITRO CULTURE AFFECT HISTONE ACETYLATION LEVELS OF BOVINE FIBROBLASTS

Abstract

Long-term culture of bovine fibroblasts increases histone acetylation levels (Enright et al. 2003 Biol. Reprod. 69, 1525–1530), and blastocyst development and pregnancy rates are enhanced by using donor cells having high levels of histone acetylation (Yang et al. 2007 Reproduction 133, 219–230; Yang et al. 2006 Biol. Reprod. 76, 36–42). When histone acetylation levels are increased in amino acid residues, the nucleosome is less tightly bound to DNA and, as a consequence, relaxation of the chromatin structure occurs (Zlatanova et al. 2000 FASEB J. 14, 1697–1704). Exposure of the donor cell with relaxed chromatin structure to oocyte cytoplasm factors after nuclear transfer (NT) may be effective for nuclear reprogramming (Tóth et al. 2004 J. Cell Sci. 117, 4277–4287). Bovine embryos can be reconstructed with donor cells thawed immediately before NT, but it is not known if epigenetic changes arise during cell cryopreservation, and if NT efficiency is affected. The acetylated form of histone H3-lysine 9-lysine 14 (acH3K9/14) is associated with active chromatin configuration (Rice and Allis 2001 Curr. Opin. Cell Biol. 13, 263–273) and active transcription (Fuks 2005 Curr. Opin. Genet. Dev. 15, 490–495) and, thus, can be used as a marker to determine epigenetic changes in somatic cells. The purpose of our study was to evaluate histone acetylation levels of cultured and cryopreserved bovine fibroblasts. Cells were derived from skin of three adult cows and cultured in GMEM for 15 days. Fibroblasts from each of the three cell lines were analyzed at passage 1 (P1), 2 (P2), and 10 (P10). At each of the three passages, cells were cultured until reaching 100% confluence, followed by an additional 3 days in culture during which time acetylation levels were measured in fresh and frozen cells. For cryopreservation, cells at P1, P2, and P10 were disaggregated with accutase, resuspended in CryoStor™ (CS10; BioLife Solutions, Bothell, WA, USA), and cooled at 1.0°C min–1 to –80°C prior to storage in liquid nitrogen. Cells were fixed with ethanol for 12 h and incubated for 30 min with antibody directed against acetylated lysine 9 on histone 3 (H3K9). Then, cells were incubated with a fluorescein isothiocyanate (FITC) conjugated secondary antibody and DNA stain and evaluated by flow cytometry. Overall, histone acetylation levels in frozen cells (70%) were lower than levels in fresh cells (86%; P < 0.05), and all cell lines, whether fresh or frozen, showed lower acetylation levels at P1 (61%) than at P2 and P10 (88% and 85%, respectively; P < 0.001). No significant differences were observed between individual cell lines for fresh and frozen treatments. However, frozen cells from cell line 3 showed lower histone acetylation levels than fresh cells from cell line 3 and cell lines 1 and 2 at each passage. In summary, histone acetylation levels were lower in cryopreserved bovine fibroblasts and were higher in cell lines cultured for longer times. Table 1. Flow cytometrically detected acetylation levels of lysine 9 on histone 3 in cultured or cryopreserved bovine fibroblast cell lines passed for 1, 2, or 10 times