Localization and measurement of occupied androgen receptors in thaw-mounted rat and human prostate tissue sections by in vitro autoradiography

Abstract

In this paper we present an in vitro exchange binding assay procedure for measurement of androgen receptors in slide-mounted tissue sections. This method allows quantitative autoradiographic determinations with an anatomical resolution approaching the cellular level. Tissue sections are incubated with the synthetic androgen [ 3H]R1881 in the presence of triamcinolone acetonide to suppress possible binding of the radioligand to the progestin receptor. Adjacent tissue sections are incubated with [ 3H]R1881 in the presence of excess unlabeled 5α- dihydrotestosterone or R1881 to assess nonspecific binding. Following incubation, the tissue sections are washed to remove unbound radioligand and either scraped for immediate determination of androgen receptor binding or placed against emulsion-coated film for the production of an autoradiographic image. In validation experiments with rat prostate sections from castrated, gonad-intact, and androgen-supplemented animals, maximum levels of androgen binding were observed with incubation at 4°C for 72 h. Markedly less binding was detected with shorter incubations or with incubations at even slightly elevated temperatures. Very little androgen receptor binding was detected in castrated animals whereas receptor levels in intact and androgen-supplemented animals were 79.3 fmol/mg and 143.6 fmol/mg protein, respectively, suggesting that the method is selective for occupied receptors. Saturation binding analysis revealed binding to a single class binding site with high affinity (k d = 1.475 ± 0.12 nM). Autoradiographic images of androgen binding in the prostate reflected the findings with the scraped sections: essentially no specific binding was present in sections from castrated animals whereas much heavier labeling was present in sections from intact animals. This method enables measurement of androgen receptor in regions of androgen-responsive tissues under various physiological conditions, with a high degree of anatomical resolution. Moreover, the method appears to be selective for endogenously occupied receptor.