Flow cytometric cell cycle analysis of two subpopulations of porcine granulosa cells

Two types of granulosa cells from 120 individual follicles (forty follicles in particular stages of development) were analysed by DNA flow cytometry to determine the percentage of cells with degraded DNA (apoptosis) and the cell cycle analysis. The distribution of cells in the cell cycle (G(1), S, G(2)/M) was studied to show relation to location within a follicle and follicular development. Isolated granulosa cells were scraped from the vesicle walls with rounded-tip ophtalmological tweezers and separated into weakly-associated (AGc) and tightly-bound (MGc) according to Ford (1991) and Duda (1997). Granulosa cells after fixation in 70% ethanol and staining with propidium iodide (PI) were analysed. At least 20,000 events were collected in each specimen. The S-phase fraction (SPF) and apoptosis were calculated using the ModFit LT programme for the cell cycle analysis (Verity Software House Inc., USA). In AGc a population with degraded DNA was found, containing less fluorescence than the G(1)/G(0) peak as shown in the DNA histograms. The percentage of apoptotic AGc ranged from 39.29 in small, to 58.9 in medium and was significantly higher than in large follicles (26.13%; p < 0.05). The percentage of apoptotic MGc was significantly lower than in the AGc (p < 0.05) and was equal to 3.78 in small, 0.10 in medium and 0.08% in large follicles. There are no significant statistical differences between the mean percentage of SPF in MGc of small and medium follicles (4.94, 7.25%). However, SPF was significantly lower in large follicles (1.31%). The number of SPF in AGc decreased during follicular development (35.92, 26.98, and 19.62%). Our data indicate lack of apoptotic cell death in MGc which seem to be more differentiated, and lose their mitotic potential. In AGc however, which are undifferentiated and undergo numerous mitosis, apoptosis was observed.