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Treatment In Vivo1
a The Women's Research Institute, Wichita, Kansas 67214-3199
b Department of Obstetrics and Gynecology, University of Kansas School of Medicine-Wichita, Wichita, Kansas 67214-3199
c Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114
d Department of Physiology, University of Arizona, Tucson, Arizona 85724-5051
Caspase-3, a vertebrate homologue of the protein encoded by the Caenorhabditis elegans cell death gene, ced-3, induces apoptosis when overexpressed in eukaryotic cells. Since apoptosis occurs during corpus luteum (CL) regression in many species, including the ewe, these studies were conducted to 1) isolate a cDNA encoding ovine caspase-3, 2) measure steady state amounts of caspase-3 mRNA in the CL during luteolysis induced by prostaglandin F2
(PGF2) and during the time of maternal recognition of pregnancy, and 3) measure changes in caspase activity during PGF2-initiated luteal regression. Oligonucleotide primers corresponding to a human caspase-3 cDNA sequence were combined with total RNA from ovine CL in a reverse transcription-polymerase chain reaction-based procedure to amplify a 640-base pair partial cDNA with a nucleotide sequence 86% and 81% identical to the human and rat caspase-3 cDNAs, respectively. CL were collected from ewes at 0, 12, or 24 h after treatment with PGF2 on Day 10 of the estrous cycle and from nonpregnant and pregnant ewes on Day 12 or Day 14 of the cycle. Northern blot analysis of total cellular RNA from ovine CL and a radiolabeled ovine caspase-3 cRNA probe indicated the presence of a single mRNA transcript of approximately 2.5 kilobases. Levels of caspase-3 mRNA were approximately 3-fold higher (p < 0.05) in CL at 12 h and 24 h after PGF2 in comparison to those levels measured in matched CL from untreated ewes. There were no differences (p > 0.05) in amounts of caspase-3 mRNA in CL on Day 12 or Day 14 of the estrous cycle compared to Day 12 or Day 14 of pregnancy, respectively. Caspase activity in CL (measured by the ability of CL lysates to cleave an artificial caspase substrate) was also significantly (p < 0.05) increased in CL collected after treatment with PGF2 compared to CL collected from nontreated ewes. We conclude that physiological cell death during PGF2-induced luteal regression in the ewe is mediated, at least in part, via increased expression and activity of the caspase family of pro-apoptotic proteases.
2 Correspondence: Bo R. Rueda, The Women's Research Institute, 1010 N. Kansas, Wichita, KS 67214-3199. FAX: 316 293 1881; brueda{at}kumc.edu
3 Current address: USDA-CSREES-NRI, 1400 Independence Ave., SW, Stop 2241, Washington, DC 20250-2241.
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