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 t o determine the percentage of cells with degraded DNA (apoptosis) and the c ell 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 folli cular development. Isolated granulosa cells were scraped from the vesicle w alls with rounded-tip ophtalmological tweezers and separated into weakly-as sociated (AGc) and tightly-bound (MGc) according to Ford (1991) and Duda (1 997). Granulosa cells after fixation in 70% ethanol and staining with propidium i odide (PI) were analysed. At least 20,000 events were collected in each spe cimen. The S-phase fraction (SPF) and apoptosis were calculated using the M odFit LT programme for the cell cycle analysis (Verity Software House Inc., USA). In AGc a population with degraded DNA was found, containing less flu orescence than the G(1)/G(0) peak as shown in the DNA histograms. The perce ntage of apoptotic AGc ranged from 39.29 in small, to 58.9 in medium and wa s significantly higher than in large follicles (26.13%; p < 0.05). The perc entage 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 percent age 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 A Gc 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 dif ferentiated, and lose their mitotic potential. In AGc however, which are un differentiated and undergo numerous mitosis, apoptosis was observed.