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 cell s (GCs) undergo apoptosis in vitro. GCs were isolated from immature ra t ovaries and separated on a 15-45% Percoll gradient. Twelve fractions were collected, and GCs were pooled according to size: small GCs (sim ilar 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 fo r 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 l ease 50% oflarge GCs possessed apoptotic nuclei. These degenerative ch anges in DNA were observed within less than or equal to 5% of small GC s. These studies demonstrate that in serum-free medium, most large GCs die via an apoptotic mechanism within 24 h. Subsequent studies focuse d on the mechanism by which epidermal growth factor (EGF) inhibits lar ge 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 eff ect 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 sy nthesis. The effect of AG was overridden by P-4. Therefore, EGF reduce s 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 fo ci 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 recept or to prevent a redistribution and increase in [Ca2+]i that may subseq uently result in GC apoptosis.