In Vitro Cell Cycle Synchronization of Sheep Granulosa Cells

Purpose: To investigate the effect of different methods of synchronization on sheep granulosa cell cycle. Materials and Methods: Granulosa cells were aspirated from ovarian follicles and plated in a DMEM medium containing 15% FBS. Upon 70-80% confluency, the medium of the primarycultured as well as the passaged-5 cells were replaced with the medium containing either 0.5% FBS for 24, 48 and 72 h or 0.5 mg mimosine for 24 h. In the last group the cells were cultured in a base medium for further 4 days. In the present investigation, for each culture system, the cells were examined in terms of their cell cycle stage using flow cytometry. Moreover, the cultures were investigated with respect to their apoptotic as well as the proliferating cell contents by using Brdu labeling and TUNEL staining. Results: At primary as well as passaged-5 cultures subjected to serum starvation for 24 h, the frequency of G0/G1, proliferating as well as apoptotic cells were similar to those of control group. At culture with 48 and 72h serum starvation, the percentage of G0/G1 cells tended to increase significantly to 83% and 85% at primary culture and 89% and 90% at passage-5 culture respectively. Moreover, treating the cultures with mimosine caused the G0/G1 cell to increase. The percentages of apoptotic cells in cultures with either serum starvation (for 24 and 48 h) or with mimosine did not increase compared to those of control cultures.Conclusion: According to our results, 72 h after serum starvation, frequency of the apoptotic cells appeared to increase significantly.