Effects of Progesterone and RU 486 on Ovine Endometrial Oxytocin Receptors
Presented by: Kathrin Dunlap
Mentor: Dr. Stormshak
Animal Science, Oregon State University
Two experiments are being conducted to investigate the effects of the steroid hormone, progesterone (P4), and the progesterone antagonist RU 486, on the ability of the peptide hormone oxytocin to bind to its receptor. To determine whether treatment with either P4 or RU 486 had an effect upon the binding of oxytocin, 11 mature, ovariectomized ewes were assigned randomly to three groups (n= 3 or 4). Each animal then received two days of sub-cutaneous injections of 25 µg estradiol-170 (E2), followed by five days of s.c. injections of 10 mg P4 (all dissolved in a vehicle - com oil) to simulate a normal estrous cycle. Each ewe's specific hormone treatment began 24 hours after her last P4 injection and covered a time period of three days. The first group, serving as a control, received daily s.c. injections 25 µg E2, the second group received daily s.c. injections of 25 µg E2 plus 10 mg P4, while the third group received daily s.c. injections of 25 µg E2 plus 10 mg RU 486. Laparotomies, to remove endometrial tissue, were performed approximately 14 to 17 hours after the animals' last injection of hormone. In the first experiment, endometrial tissue was quick frozen in liquid N2 for future analysis of oxytocin receptor (OTR). The membrane fraction will be isolated and analyzed for OTR. Eventually the mRNA of the endometrial tissue will be isolated to determine if OTR is being synthesized during treatments of P4 or RU 486.
The second experiment utilizes tissue from control animals, which upon removal from the uterus, was immediately transported to the laboratory where the membrane fraction was isolated and incubated. The membranes were incubated with either: 1) vehicle (ethanol), 2) 5 ng/ml P4, or 3) 5 ng/ml P4 plus 10 ng/ml RU 486 for 60 min at room temperature. The membranes were washed, centfifuged at 100,000 x g and then frozen for future OTR analysis. The objectives of this study are to gain insights regarding the effects that P4 and RU 486 may have upon the binding of oxytocin to its receptor and whether these effects are genomic or non-genomic.