Abstract

The kodkod is the smallest wild cat in South America and its population is in constant decrease. The SCNT might be a valuable tool for preserving the genetic pool of the kodkod. The cell cycle synchronization of donor cells plays a crucial role in the SCNT. The objective of this research was to evaluate the effect of two different treatments, serum starvation (SS) and contact inhibition (CI), for induction of cell quiescence, on skin fibroblast derived from domestic cat and kodkod. All experiments were performed with fibroblasts in the passes 5 to 6. SS was achieved by replacing culture medium with 10% FBS by medium with 0.5% FBS. CI was performed by allowing the cells to reach 100% confluence. Both treatments were evaluated at day 1, 3 and 5, and growing cells (60-80% confluence; GC) were used as control. Three replicates of each treatment were performed. For cell cycle analysis, fixed cells were incubated with 50 μg/ml of propidium iodide and 100 μg/ml of RNase for 30 min at 37ºC. Flow cytometry was conducted on the Attune® NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific). At least 10,000 events were scored using the BL2-A channel (filter 574/26 nm). Results were analysed using the Attune® NxT SW v1.1 software. Apoptosis was evaluated by gene expression analysis of BAX and BCL2 by RT-qPCR, SDHA was used as internal control. Flow cytometry results were analyzed by ANOVA using the GLM procedure in the SAS software, means comparison was made by Tukey’s test. RT-qPCR results were analyzed by the Kruskal-Wallis test using the Infostat software. In all experiments, differences were considered at P < 0.05. Flow cytometry analysis revealed that in fibroblasts of domestic cat, SS and CI, both at 3 and 5 days of treatment, increased the percentage of cells in G0/G1 and reduced the fraction of cells in S and G2/M stages compare to GC (P < 0.05). On the other hand, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of cells in G0/G1 in fibroblasts of kodkod (P < 0.05). Furthermore, only SS for 5 days reduced the percentage of cells in G2/M stage compare to GC, in kodkod fibroblasts (P < 0.05). Regarding to gene expression analysis, in fibroblasts of domestic cat, no statistical differences were found in the BAX/BCL2 ratio between both SS and CI (at 1, 3 and 5 days) compared to GC. However, in kodkod cells BAX/BCL2 ratio was high in CI at 3 and 5 days of treatment compared to SS at 3 and 5 days (P < 0.05). In SS the relative expression of BCL2 increases with the time of treatment, while in CI the relative expression of BCL2 is not enough to reduce the levels of BAX. In conclusion, fibroblasts of domestic cat and kodkod respond differently to different methods of cycle synchronization. In kodkod fibroblasts CI had a negative effect after 3 days, which was related to a high apoptosis incidence. We suggest SS for 3 or 5 days for cell cycle synchronization in kodkod fibroblasts. These results are relevant for production of kodkod embryos by SCNT.