MATERIALS AND METHODS(1)

Animals

Normal adult male retired breeder Sprague-Dawley rats (450-550 g; Hilltop Laboratories, Philadelphia, PA) were maintained on a 12L:12D cycle, with free access to food and water, in the University of Virginia vivarium. All experiments in this study were approved by the University of Virginia Animal Welfare Committee.

Efferent Duct Ligation (EDL)

To determine whether one or more testicular factors can affect GGT catalytic activity and GGT protein level in the rat epididymis, unilateral EDL surgeries were performed as described previously . The controls were sham operated on the side contralateral to the EDL in the same animal. The sham-operated and EDL-operated rats were killed with carbon dioxide at 1 day, 3 days, 5 days, or 14 days after EDL. ventolin inhaler

RTF Preparations

Ovine RTF and androgen-binding protein (ABP)-deplet-ed oRTF (no-ABP oRTF) were kindly provided by Dr. Rupert Amann (Colorado State University, Fort Collins, CO). Charcoal oRTF was prepared using dextran-coated charcoal (Sigma Chemical Co., St. Louis, MO) to extract the steroids from the oRTF without ABP . No-ABP oRTFs > 10 kDa and < 10 kDa were prepared using an ultrafiltration centrifuge filter with a cutoff membrane of Mr 10 000 (MSI, Westborough, MA) according to the manufacturer’s instructions. Boiled charcoal oRTF was obtained by boiling the charcoal oRTF at 100°C for 5 min. Rat RTF (rRTF) was collected from the rete testis of 16-20h-EDL rats by mi-cropuncturing the efferent ducts as described previously . The supernatant (rRTF) was obtained after centrifugation at 3000 rpm for 10 min in an Eppendorf (Hamburg, Germany) microcentrifuge. Synthetic RTF (sRTF) was prepared that contained concentrations of inorganic electrolytes similar to those of ram RTF . This sRTF was also used as artificial rRTF since it contained inorganic components similar to those of rRTF . All RTF preparations were stored at — 80°C before use.