Testis-Like Steroidogenesis: MATERIALS AND METHODS(2)

18 Apr

Homogenates were brought to a final volume of 3 ml with 0.05 M Tris. The following cofactors were added: NADP at 1 mM, glucose-6-phosphate at 10 mM, and glu-cose-6-phosphate dehydrogenase at 5 U/ml (Sigma Chemical Co., St. Louis, MO). [4-14C]Progesterone (0.1 ^Ci; NEN, Boston, MA) or 1 ^Ci of [1,2,6,7-3H]androstenedione was added to the homogenates. Samples were incubated at 37°C with continuous agitation for 30 min with [14C]progesterone or for 15 min with [3H]androstenedione: these incubation times ensured that the entire radiolabeled steroid was not metabolized by the end of the incubation period. Steroids were extracted twice with ethyl acetate (Fisher, Santa Clara, CA). The remaining aqueous phase was counted in a scintillation counter to determine the percentage recovery of labeled steroids, which was greater than 95%. Ethyl acetate was evaporated from the extracted steroids with nitrogen at 45°C before resuspension in 50 ^l of ethyl acetate. Steroid metabolites in each sample were identified by thin-layer chromatography. buy flovent inhaler

Thin-Layer Chromatography

Samples were loaded onto Whatman (Clifton, NJ) 20cm X 20-cm Linear K TLC plates with silica gel as absorbent (Fisher). Each sample was loaded onto two plates; one was developed in chloroform:methanol (98:2), the other in benzene:ethyl acetate (3:1). Standard steroids were [3H]progesterone, androstenedione, testosterone, dihydrotestosterone, and estradiol (NEN). Radioactive metabolites were detected by a Berthold (Wildbad, Germany) TLC scanner. To confirm the identity of the metabolites, regions corresponding to the standards were scraped from the plate, reextracted with ethyl acetate, and run on a new plate in the solvent that was alternative to the original.