Preparations of Luminal Fluids from the Rat Initial Segment, Caput, and Cauda

Luminal fluid from the initial segment, caput, and cauda of adult rats was collected as described previously . This luminal fluid was centrifuged at 3000 rpm in an Eppendorf microcentrifuge to remove the spermatozoa. The supernatant was collected and stored at — 80°C before use. buy antibiotics online

Preparations of Samples

Sham-operated and EDL-operated rats were killed with carbon dioxide. The initial segment, caput, corpus, and cauda regions of the epididymis and the testis were collected, minced with scissors, and washed twice with ice-cold PBS (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM sodium chloride, pH 7.4). For the comparative study of GGT catalytic activity among various tissues and of the effects of EDL on GGT catalytic activity in these tissues, washed tissues were subjected to homogenization with a pestle homogenizer in 0.3 M sucrose homogenization buffer (0.3 M sucrose, 0.05 M Hepes, pH 7.45, 0.05 M magnesium chloride, 0.2 mM PMSF, 0.5 mM dithiothreitol [DTT]). For the observation of whether testicular factors and/or bFGF could recover the GGT catalytic activity and GGT protein level, the washed initial segments from the sham-operated and EDL-operated rats were then incubated with Dulbecco’s Modified Eagle’s medium (DMEM) containing 10-40% RTF preparations or various concentrations of bovine bFGF (Sigma), recombinant human epidermal growth factor (EGF; Sigma), phor-bol 12-O-tetradecanoylphorbol 13-acetate (TPA; Sigma), or fetal calf serum (FCS; Sigma) in a 35°C water bath with gentle shaking. After 6-h incubation, the tissues were collected and homogenized as described above. The homog-enates were extracted with a Triton solution (0.5% Triton X-100, 0.5 M Tris, pH 8.0) at 4°C overnight and then centrifuged at 5000 X g for 15 min. The supernatants were then harvested and stored at -20°C until analyzed for GGT catalytic activity and Western blot analysis.