37 CELL CYCLE ANALYSIS IN IN VITRO-CULTURED ADULT SKIN FIBROBLASTS OF THE GORAL (NAEMORHEDUS CAUDATUS)

Abstract

The present study was undertaken to examine the cell cycle characteristics of goral adult skin fibroblasts under a variety of cell cycle-arresting treatments. Gorals are listed as an endangered animal in CITES Appendix I. Experimental findings suggest that G0/G1 stages of the cell cycle give better results in somatic cell nuclear transfer (SCNT) in terms of normal ploidy and development of reconstructed embryos. In this regard, synchronization of the cell cycle stages in G0/G1 phase is suggested to be one of the key factors for determining the success of nuclear transplantation. Serum deprivation, contact inhibition, chemical inhibitors such as protease, and antioxidant inhibitors are widely used for cell cycle synchronization and inhibition of apoptosis. Four experiments were performed, and each with one-way completely randomized design involving three replicates of all treatments using the general linear models procedure in the statistical analysis system (SAS Institute, Inc., Cary, SC, USA). Least significant difference was used to determine statistical significance (P < 0.05) among treatment groups. In Exp. 1, effects of cyclic confluency, serum starvation, or full confluency on goral cells were studied. In Exp. 2, treatments with different antioxidants such as 2 mM beta-mercaptoethanol (B-ME), cysteine, or glutathione for 24 h were examined in fully confluent cells without serum starvation. In Exp. 3, three protein inhibitors, 2 mM 6-dimethylaminopurine (6-DMAP), 7.5 µg/mL cycloheximide, or 7.5 µg/mL cytochalasin B, were treated as in Exp. 2. In Exp. 4, different concentrations (0, 0.5, 1.0, or 2.5%) of dimethyl sulfoxide (DMSO) were treated as in Exp. 2. In all experiments, cell cycle stages were analyzed by fluorescence-activated cell sorting (FACS) analysis. In Exp. 1, more cells (70.4%) were found in the G0/G1 stage in the serum starved medium compared to cells in the cyclic (66.1%) and fully confluent growth phase (65.5%). In Exp. 2, supplementing with B-ME (61.8%) or cysteine (62.0%) in the culture medium synchronized cell cycle to the G0/G1 stage better than glutathione (53.1%). In Exp. 3, more cells were synchronized to the G0/G1 stage in media supplemented with cyclohexamide (62.3%) than with 6-DMAP (5.7%) or cytochalasin B (13.3%). Incidence of apoptosis was higher in media containing 6-DMAP (91.8%) or cytochalasin B (76.6%). In Exp. 4, supplementing the medium with 0.5% (76.0%) or 1.0% (75.9%) DMSO synchronized the cells effectively to the G0/G1 stage compared to the untreated control medium (69.3%) or medium supplemented with 2.5% of DMSO (8.0%). In conclusion, cyclohexamide, B-ME, cysteine, or DMSO at the optimum concentration can synchronize the cell cycle effectively, which may have a positive impact on the outcome of SCNT in the goral.

This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-04-00).