PROGESTERONE AND CELL-CELL ADHESION INTERACT TO REGULATE RAT GRANULOSA-CELL APOPTOSIS

Ovarian follicles are composed of both small and large granulosa cells, but only the large granulosa cells undergo apoptosis within 24 h of culture in serum-free medium. The present study was designed to assess the relationship between cell-cell contact, progesterone treatment, and granulosa cell apoptosis. For this study, individual large granulosa cells were isolated from immature rat ovaries after sequential incubation with EGTA and EGTA-sucrose solutions. Granulosa cells were then cultured for 24 h in RPMI-I640 (control) supplemented with progesterone and (or) the progesterone antagonist RU 486. The cells were then fixed and assessed for apoptosis by either electron microscopy or in situ end labeling of DNA fragments. After 24 h of culture, the proportion of apoptotic granulosa cells was twofold lower for aggregated cells compared with single granulosa cells (p < 0.05). Aggregated granulosa cells were observed to be connected by gap junctions. Compared with controls, progesterone reduced and RU 486 increased the percentage of single and aggregated apoptotic granulosa cells present after culture. In the presence of RU 486, progesterone reduced the percentage of apoptotic single granulosa cells from 84 +/- 4% (RU 486 alone) to 66 +/- 8%. In granulosa cell aggregates, progesterone reduced the incidence of apoptosis from 86 +/- 3% to 44 +/- 7% (p < 0.05). Progesterone in the presence of RU 486 was more effective in inhibiting apoptosis of aggregated granulosa cells than in single granulosa cells (p < 0.05). Taken together, these data indicate that (i) progesterone acts through the progesterone receptor to inhibit granulosa cell apoptosis and (ii) cell-cell adhesion enhances progesterone's anti-apoptotic actions.