Nuclear Androgen Receptors in the Mouse Kidney: Validation of a New Assay*

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Abstract
A method for determining nuclear androgen receptors in mouse kidney was developed using [3H]methyltrienolone as the labeled ligand. Pyridoxal 5′-phosphate was used to extract receptors from nuclei isolated with the hexylene glycol technique. The pyridoxal 5′-phosphate extraction method yielded nuclear receptor concentrations about twice as high as those measured after 0.4 M KCl extraction or by a direct nuclear exchange reaction. The recovery of nuclear receptors was also increased 4-fold by the use of hexylene glycol as the initial homogenization medium rather than Tris-buffer containing glycerol, which is used in cytosol receptor assays. A complete exchange of endogenously bound testosterone with [3H]methyltrienolone was achieved during an 18-h incubation at 4 C. The nuclear androgen receptor had a KD of about 5 nM for [3H] methyltrienolone, was androgen specific (5α-dihydrotestosterone = methyltrienolone > testosterone > estradiol > rogesterone » dexamethasone = aldosterone) and sedimented as a 4S entity during sucrose gradient centrifugation. The nuclear receptor concentrations in intact male and female mice were 280 ± 100 and 90 ± 20 receptors/cell (mean ± SD), respectively, while the corresponding figures for the cytosol receptor concentrations were 1070 ± 80 and 1470 ± 390 receptors/cell (mean ± SD). After castration of male mice, the nuclear receptor concentration declined to the level found in females within 3 h. Administration of testosterone (1 mg, ip) increased nuclear receptor concentration 7-fold, with a maximum at 30–60 min. Nuclear receptors then subsided to the pretreatment level within 5 h. During the early hours after testosterone administration, nuclear accumulation accounted for only 50% of the receptors lost from the cytosol. Some synthetic progestins (6α-methylprogesterone, medroxyprogesterone acetate, and cyproterone acetate) known to mimic and modify androgen action in mouse kidney were capable of translocating androgen receptors to nuclei in vivo when administered in relatively high doses. The technique described here to improve the measurable androgen receptor in mouse kidney nuclei is also applicable to other tissues. This procedure, coupled with the previously described method for measuring cytosol androgen receptors, will permit a kinetic analysis of androgen receptors in the presence of a variety of exogenous and endogenous ligands.