EPIDERMAL GROWTH-FACTOR INHIBITS LARGE GRANULOSA-CELL APOPTOSIS BY STIMULATING PROGESTERONE SYNTHESIS AND REGULATING THE DISTRIBUTION OF INTRACELLULAR FREE CALCIUM

The initial study was designed to determine whether all granulosa cells (GCs) undergo apoptosis in vitro. GCs were isolated from immature rat ovaries and separated on a 15-45% Percoll gradient. Twelve fractions were collected, and GCs were pooled according to size: small GCs (similar to 50 mu(2); fractions 2-5) and large GCs (greater than or equal to 75 mu(2); fractions 6-8). GCs were cultured in serum-free medium for 24 h. After 24 h of culture, fragmented DNA, detected by in situ end labeling of the 3'OH ends of DNA fragments, was observed within 70-80% of large GCs. Similarly, in situ DNA staining demonstrated that at lease 50% oflarge GCs possessed apoptotic nuclei. These degenerative changes in DNA were observed within less than or equal to 5% of small GCs. These studies demonstrate that in serum-free medium, most large GCs die via an apoptotic mechanism within 24 h. Subsequent studies focused on the mechanism by which epidermal growth factor (EGF) inhibits large GC apoptosis. EGF reduced the percentage oflarge GCs with apoptotic nuclei from 47 +/- 1% for controls to 18 +/- 2% (P < 0.05). EGF also increased progesterone (P-4) secretion from large GCs (6.3 +/- 0.7 for controls vs. 18.7 +/- 1.0 ng/ml for EGF treatment; P < 0.05). The effect of EGF on apoptosis was mimicked by P-4 and attenuated by the P-4 antagonist, RU 486, and aminoglutethimide (AG), an inhibitor of P-4 synthesis. The effect of AG was overridden by P-4. Therefore, EGF reduces large GC apoptosis by stimulating P-4 synthesis, with P-4 mediating its action through its receptor. Intracellular free calcium ([Ca2+](i)), as assessed by fluo-3 fluorescence, was localized to cytoplasmic foci prior to culture and in the presence of P-4. In the absence of P-4 or in the presence of RU 486, calcium became dispersed throughout the cytoplasm and ultimately resulted in an apparent increase in [Ca2+](i) within 28% of the GCs. This suggests that P-4 acts through its receptor to prevent a redistribution and increase in [Ca2+]i that may subsequently result in GC apoptosis.