Testicular membrane preparations were obtained as previously described by Le Gac et al. . Briefly, testes were homogenized as described above in homogenization buffer containing 0.3 M sucrose and inhibitors of proteolytic enzymes and were centrifuged at 600 X g for 30 min in order to eliminate undisrupted tissue and cell nuclei. The supernatant was recentrifuged at 45 000 X g for 45 min, and the resulting pellet was resuspended as described above. Crude hepatic preparations were also obtained according to the method of Yao et al. All preparations were used immediately in the binding assay. Buy Asthma Inhalers Online
Testicular cells were isolated and cultured as previously described by Loir and Loir and Le Gac in order to identify GH target cells in testis. In brief, gonads in stages III and VI of spermatogenesis were dissociated by perfusion with collagenase (0.8 mg/ml) in culture medium without Ca2+ for 4-5 h. The dispersed tissues were incubated overnight in 100 ml culture medium containing 1% BSA before final mechanical dispersion of the cells. Most of the spermatozoa were removed by centrifugation into an isotonic Percoll solution. The total cells were directly used for binding studies or were separated to obtain a population of purified germ cells, a population of isolated spermatozoa, a population enriched in Sertoli cells, and a pellet of remaining cells containing mixed germ cells and somatic cells. Total cells and isolated germ cells were tested for binding immediately after separation or after 4 days of culture, while the Sertoli cell population was used after 4 days of culture.