Androgen metabolism by and binding to mature rabbit epididymal tissue: studies on cytosol

The rabbit epididymis is capable of metabolizing [³H]-testosterone to [³H]-5α-androstan-17β-hyroxy-3-one (5αDHT) and to [³H]-5α-androstan-3α, 17β-diol (3α-diol) under in vitro conditions. These reactions occurred more rapidly at 23°C than 0°C and were dependent upon time and substrate concentration in the linear manner characteristic of enzymatic reactions. Androgens were bound to two or more components in cytosol preared from epididymal tissue. Androgens dissociated slowly from one of the components (the epididymal androgen receptor) and rapidly from the other component(s) (androgen binding protein (ABP) and/or testosterone binding globulin (TeBG). Differences in dissociation rates among these molecules were utilized to determine which androgens were bound to the androgen receptor. This was accomplished by adding unlabeled testosterone to pre-labeled cytosol to dissociate label that was bound to the rapidly dissociating moieties and separating the bound and free radioactivity by gel filtration chromatography. Androgens were extracted from the column eluates with diethyl ether, separated by thin layer chromatography, and identified by recrystallization to constant specific activity. The androgen receptor strongly favors the binding of 5αDHT since although 5αDHT constituted only 10–20% of the total pool of radioactive androgens, it comprised 50% of the androgens bound to the receptor. The remaining receptor-bound androgen consisted of testosterone and 3α-diol. The androgen receptor could be demonstrated in cytosol from both intact and castrated rabbits using these methods. However, the concentration of available receptor sites was more than twice as great in castrated animals than in intact ones. Total bound radioactivity in cytosol was examined by gel chromatography of labeled cytosol to which no unlabeled hormone had been added. Epididymal tisue from 3-day castrated rabbits produced significantly less 5αDHT (P = 0.005) than did epididymal tissue from intact rabbits.