Adult female Sprague-Dawley rats were obtained from Charles River Breeding Labs. (Portage, MI) at 8 wk of age. Animals were housed under a controlled lighting schedule of 12L:12D (lights-on 0600-1800 h, experiment 1; lights-on 0600-1800 h or 0500-1700 h, experiment 2; lights-on 0500-1700 h, experiment 3). Examination of vaginal epithelium was carried out daily, and only those rats showing at least four consecutive 4-day cycles were used. Monitoring of vaginal epithelium began at 9-11 wk of age, and animals were killed between 11 and 17 wk of age. For experiment 3, rats were obtained from Charles River at 10 wk of age, after the animals had shown 2 consecutive 4-day estrous cycles. These rats were killed after a minimum of 2 consecutive 4-day cycles, at 12-14 wk of age. Animal procedures were approved by the University Committee on the Use and Care of Animals at the University of Michigan. antibiotic levaquin
In experiment 1, rats were killed either on the morning (0830-1100 h) of proestrus (n = 8) or estrus (n = 5), or on the afternoon (1700-1800 h) of estrus (n = 5). Rats were killed by decapitation following ether anesthesia, and the ovaries were immediately removed and placed on ice. One ovary was frozen in OCT compound (Miles Laboratories, Inc., Elkhart, IN) for immunohistochemistry for monocytes/macrophages (ED1-positive), differentiated macrophages (ED2-positive), and MCP-1 protein and for in situ detection of apoptosis in the corpora lutea of different ages.