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a The Women's Research Institute, Wichita, Kansas 67214
b Department of Obstetrics and Gynecology, University of Kansas School of Medicine-Wichita, Wichita, Kansas 67214
c Veterans Affairs Medical Center, Wichita, Kansas 67218
d Department of Biological Sciences, Wichita State University, Wichita, Kansas 67260
e Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331
f Oregon Regional Primate Research Center, Beaverton, Oregon 97006
We tested the hypothesis that progesterone (P4) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) ± P4 (500 ng/ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P4 levels by RIA. Aminoglutethimide inhibited P4 synthesis by > 95% and increased the level of apoptosis as evidenced by 32P-labeled oligonucleosomal DNA fragmentation (> 40%). P4 supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P4 has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P4 was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in 32P-labeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P 
0.05) in P4 levels after treatment with PR antagonists. These observations support the concept that P4 represses the onset of apoptosis in the CL by a PR-dependent mechanism.
1 Supported in part by NIH RO1-HD35934, Wesley Medical Research Institute, KURI, and the Department of Veterans Affairs.
2 Correspondence: Bo R. Rueda, The Women's Research Institute, Department of Ob/Gyn, The University of Kansas School of Medicine-Wichita, 1010 N. Kansas, Wichita, KS 67214-3199. Fax: 316 293 1881; brueda{at}kumc.edu
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