Identification of an androgen receptor in the zonal testis of the salamander ( Necturus maculosus)

Abstract

The testis of the salamander, Necturus maculosus, is advantageous for studying biochemical changes during spermatogenesis because germ cells and associated Sertoli and Leydig cells are topographically separated by stage of development. Using extracts of staged tissue samples and [ 3H]testosterone (T) in a standard binding assay, followed by Sephadex LH-20 or DNA-cellulose chromatography to separate free and bound steroid, we have identified a T-binding protein having physicochemical characteristics of a classical androgen receptor (AR): high affinity ( K d = 10 −9 M), limited capacity ( B max) = 10 −10 M or 350 fmol/g tissue) and androgen specificity (T = 5α - dihydrotestosterone > progesterone = corticosterone > estradiol). AR was present in nuclear extracts, where >80% of binding sites were occupied by endogenous ligand, but was not detectable in cytosol. On linear sucrose gradients, nuclear AR sedimented at 3–4 S in both low and high ionic-strength buffers and, by this and other criteria, was distinguishable from the nonreceptor androgen binding protein (ABP) of the same species. The diffuse distribution of AR in germinal and nongerminal (glandular) tissues at all developmental stages is consistent with a dual localization in Sertoli cells and Leydig cells, as previously reported in mammals, and further suggests a regulatory role of androgen throughout spermatogenesis.