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, a nd granulosa cell apoptosis. For this study, individual large granulos a cells were isolated from immature rat ovaries after sequential incub ation with EGTA and EGTA-sucrose solutions. Granulosa cells were then cultured for 24 h in RPMI-I640 (control) supplemented with progesteron e and (or) the progesterone antagonist RU 486. The cells were then fix ed 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 co mpared with single granulosa cells (p < 0.05). Aggregated granulosa ce lls were observed to be connected by gap junctions. Compared with cont rols, progesterone reduced and RU 486 increased the percentage of sing le and aggregated apoptotic granulosa cells present after culture. In the presence of RU 486, progesterone reduced the percentage of apoptot ic single granulosa cells from 84 +/- 4% (RU 486 alone) to 66 +/- 8%. In granulosa cell aggregates, progesterone reduced the incidence of ap optosis from 86 +/- 3% to 44 +/- 7% (p < 0.05). Progesterone in the pr esence of RU 486 was more effective in inhibiting apoptosis of aggrega ted granulosa cells than in single granulosa cells (p < 0.05). Taken t ogether, these data indicate that (i) progesterone acts through the pr ogesterone receptor to inhibit granulosa cell apoptosis and (ii) cell- cell adhesion enhances progesterone's anti-apoptotic actions.