Decreased progesterone levels and progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells

We tested the hypothesis that progesterone (P-4) acts at a local level to i nhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesi s. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 m M) +/- P-4 (500 ng/ml) for 48 h. Luteal cells were recovered and snap froze n for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P-4 level s by RIA. Aminoglutethimide inhibited P-4 synthesis by > 95% and increased the level of apoptosis as evidenced by P-32-labeled oligonucleosomal DNA fr agmentation (> 40%). P-4 supplementation inhibited the onset of apoptosis t hat was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining te chnique and together strongly suggest that P-4 has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to iso late a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine ovi ductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was presen t in both large and small luteal cells. To determine whether the anti-apopt otic effect of P-4 was regulated at the receptor level, luteal cells were c ultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h . Both antagonists caused approximately a 40% increase in P-32-labeled olig onucleosomal DNA fragmentation. Interestingly, there was no difference (P g reater than or equal to 0.05) in P-4 levels after treatment with PR antagon ists. These observations support the concept that P-4 represses the onset o f apoptosis in the CL by a PR-dependent mechanism.