Nuclear androgen binding sites in the male rat. II. Seminiferous tubules

A nuclear exchange for androgens in the seminiferous tubules of the mature male rat has been established. The exchange, utilizing technology established on the ventral prostate gland described in the previous paper [7], is based upon LH-20 chromatography of salt extract of purified nuclei. The extract is incubated with [3H]methyltrienolone (R 1881) or [3H]dihydrotestosterone (DHT) at 4 degrees C for 21 h. Bound is separated from free on a hydroxylapatite microcolumn. Nonspecific binding is determined by use of 100-fold molar excess of the unlabeled ligand. Specific binding in an incubation volume of 2 ml containing 1.4 nM [3H]R 1881 was 5.78 +/- 0.57 fmol/mg DNA (mean +/- SE). Specific binding was linearly related to protein concentration. Of the steroids studied, only testosterone competed with DHT in suppression of [3H]DHT (one-sixth the affinity). In 4 experiments by saturation analysis, specific binding was saturable and of high affinity: Kd = 6.1 +/- 0.93 X 10(-10), n = 2.2 +/- 0.41 pM (3.75 +/- 0.77 fmol/mg DNA). A second receptor of low affinity and high capacity was also observed: Kd = 3.28 X 10(-7); n = 161 pM (2.46 pmol/mg DNA). In an analysis of specific binding by ultracentrifugation on an 8-34% glycerol density gradient, 2 radioactively labeled peaks were observed, one at 5.6 S, completely suppressed by 100-fold molar excess of unlabeled steroid, and a second larger peak at 3 S, only partially suppressed. The possibility that marginal binding was due to proteolysis could not be confirmed: (1) Addition of nuclear extract from seminiferous tubules to nuclear extract from the ventral prostate enhanced specific binding in the latter; (2) A 4-h delay in processing of seminiferous tubules did not decrease specific binding; (3) A protease inhibitor (PMSF) did not enhance binding. However, sonication of the testes, retaining only late spermatids and spermatozoa, triple specific binding (16.2 +/- 0.8 fmol/mg DNA). These results confirm our previous reports in which we observed by radioimmunologic analysis the presence of androgen receptors in seminiferous tubules, and in late spermatids and spermatozoa. It is suggested that the sonicated testis is an ideal model in which to study the role of the androgen receptor in the maintenance of spermatogenesis.