Cultures of Dispersed Leydig Cells
The medium containing fetal testes was replaced by 15 ml of culture medium containing 0.4% collagenase type II (Sigma) and DNAse I (105 Kunitz U/L; Sigma), and the testes were incubated for 30 min at 37°C in a shaking water bath. Thereafter, the testes were dispersed mechanically by aspirating the tissue suspension through a pipette at 5-min intervals, continuing the incubation until the tissue was completely disintegrated. The cells then were centrifuged at 200 X g for 5 min at 4°C, and the supernatant was discarded. The cells finally were washed twice with 50 ml of medium, resuspended in fresh medium, divided equally into the 24 wells of a culture plate (Nunc, Roskilde, Denmark), and cultured for 24 h at 37°C in an atmosphere of 5% C02 in air to recover and stabilize. buy diabetes drugs
The purity of Leydig cells was assessed by a cytochemical 3fJ-hydroxysteroid dehydrogenase (Зр-HSD) reaction, and 30-40% of 3(3-HSD-positive cells were found in the fetal testicular cell suspensions. Thereafter, the medium was removed, and 1.0 ml fresh medium containing 0.2 mM l-methyl-3-isobutylxan-thine (MIX; Aldrich Chemie, Steinheim, Germany) and 10-6 M VIP (Peninsula Labs., Inc., Belmont, CA), 10 6 M PACAP-27 (Peninsula), or 30 |xg/L highly purified hCG (CR-121, 11500 IU/mg; NIH, Bethesda, MD) was added, and the cells were incubated for 4 h. Thereafter, the medium was collected (diluted 1:1 with 2 mM theophylline [Sigma] in the case of с AMP measurement), heated at 100°C for 5 min, and stored at -20°C until analyzed (see below). All the experiments were run in triplicate or quadruplicate and repeated at least twice.