Animals, Treatments, Sample Collection, and Processing
Wistar rats were bred and maintained in the Medical Research Council animal facility (Edinburgh). All animal studies were performed under license from and according to the legal requirements of the United Kingdom Home Office. Rats were administered one of the following treatments postnatally. In our studies the day of birth was assigned as Day 1. Based on the nomenclature below, the rats treated in a preliminary study (study 1) received either treatment 1, 2, 3, or 5. In a follow-up study (study 2) a cohort of rats was treated with one of the six regimens listed below.
1. Subcutaneous injection of 20 pl of corn oil administered on alternate days from Day 2 through Day 12 (control);
2. Subcutaneous injection of ethinyl estradiol (Sigma Chemical Company, Dorset, U.K.) at a dose of 10 pg/20 pl of corn oil administered on alternate days from Day 2 through Day 12;
3. Subcutaneous injection of DES (Sigma) at a dose of 10 pg/20 pl of corn oil administered on alternate days from Day 2 through Day 12;
4. Subcutaneous injection of 10 pg of DES coadministered with 200 pg of testosterone esters (DES + TE; Sustanon, Organon Labs, Cambridge, U.K.) administered in 20 pl of corn oil on alternate days from Day 2 through Day 12. The rationale for the TE dose has been fully described elsewhere;