We elected to use the 64 mg/ kg MXC dose in the transgenic animal experiments because our experiments in CD-1 mice showed that this dose was the most effective in inducing atresia without overt toxicity. Transgenics and their wild-type counterparts were aged between 30 and 66 days at the start of the dosing experiments.
All animals were dosed for 20 continuous days via intraperitoneal injection and were killed during estrus in the early part of the day (between 0800 and 1300 h). Vaginal cytology was used to determine the day of estrus. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, killing, and tissue collection.
Histological Evaluation of the Number of Follicles and Corpora Lutea
After dosing, ovaries were collected and fixed in Dietrick solution for 24 h. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific, West Chester, PA), serially sectioned (8 |xm), mounted on glass slides, and stained with Weigert hematoxylin-picric acid methylene blue.