5. Subcutaneous injection of 10 mg/kg of a long-acting gonadotropin-releasing hormone antagonist (GnRHa; Antarelix, Europeptides, Ar-genteuil, France) in 5% mannitol administered on Postnatal Days 2
5. This regimen has been shown to abolish gonadotropin secretion until Postnatal Days 15-20 and to lower testosterone levels;
6. Subcutaneous injection of the androgen-receptor antagonist, flutamide (Sigma), administered at a dose of 50 mg/kg in 20 pl of corn oil on alternate days from Day 2 through Day 12. These animals were compared with an additional group of control rats.
For all treatments, pups were weaned at 22 days and fed standard rat and mouse breeding diet no. 3 (SDS, Dundee, Scotland). Animals were killed on Day 25 by inhalation of CO2, followed by cervical dislocation. The right epididymis from each animal was fixed for ~5 h in periodate-lysine paraformaldehyde (PLP), which contains 2% paraformaldehyde, and the whole left epididymis was snap-frozen and stored at —70°C prior to protein extraction. In study 2, the right testis was weighed after necropsy and the other reproductive organs were snap-frozen for use in other experiments.