Blood samples were collected from the jugular vein at Day 3 and Day 7, and daily from Day 10 to Day 20 postestrus. In addition, blood was collected every two days from ewes that maintained at least one CL at the time of the second surgery until they were determined to be in estrus. Serum was stored at -20°C until analyzed for progesterone. Ewes that did not return to estrus by Day 45 postestrus (n = 5) were killed, and their ovaries were examined for the presence of marked CLs. The University of Missouri Animal Care and Use Committee (protocol #2745) approved procedures used in this study.
Concentrations of progesterone in serum were determined by RIA. Assay sensitivity for progesterone was 0.2 ng/ml, and intra- and interassay coefficients of variation were 8.2% and 14.4%, respectively. buy antibiotics online
Isolation of Total RNA and Northern Blot Analysis
Uterine endometrium was collected from ewes that did not return to estrus by Day 45 postestrus (n = 5) and from single ewes at Days 10 and 13 postestrus (nonpregnant) and Days 16, 25, and 45 of pregnancy. Tissue was snap-frozen in liquid nitrogen and stored at — 80°C. Total cellular RNA was extracted, denatured, and separated by electrophoresis in a 1.2% (w:v) agarose gel. The RNA was transferred to a nylon membrane and was hybridized first to a 32P-labeled cDNA probe for ovine uterine serpin, a marker of prolonged exposure of uterine tissue to progesterone, and subsequently to a 32P-labeled cDNA for ovine actin.