Hugh S. Keeping

Assay of primate seminiferous tubule androgen receptors using [3H]mibolerone

The synthetic radiolabelled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) was used to characterize androgen receptor binding in the seminiferous tubules from Cynomolgus monkey testis. Mibolerone binding was of high affinity (Kd = 0.6-5.4 nM) and limited capacity (37-50 fmol/mg protein), and was androgen specific. Sucrose density gradient centrifugation using a vertical tube rotor permitted the identification of a 9S molybdate-stabilized receptor under low salt conditions. The receptor bound to DEAE-cellulose. Methyltrienolone, but not mibolerone, also bound to a low affinity high capacity binding site in tubule cytosol, which probably represents glucocorticoid receptor binding, since it could be displaced by excess dexamethasone. However, occupancy of this low-affinity binding site by dexamethasone in an androgen receptor assay with [3H]methyltrienolone lead to a 33% underestimation of receptor binding, which appeared to relate to radioactive decomposition. Mibolerone, as well as methyltrienolone, bound to a progestin-binding protein in seminiferous tubule cytosol. These studies provide methods for the study of seminiferous tubule androgen receptors in subhuman primates and indicate that, due to its greater stability and lack of binding to glucocorticoid receptor, mibolerone is a useful new ligand in the study of androgen receptors in testis and its constituent cells.