Characterization of an Estrogen Receptor in the Testis of the Urodele Amphibian Necturus maculosus 1

The exceptionally high rate of aromatization previously demonstrated in the testis of the urodele amphibian Necturus maculosus prompted us to investigate estrogen binding activity in this tissue. When crude cytosolic extracts were incubated with [3H] estradiol prior to DNA-cellulose affinity chromatography or sucrose gradient centrifugation, no estrogen binding was detected. Under these conditions, the presence of an endogenous estrogen-conjugating system in the crude cytosol resulted in extensive conversion of radiolabeled tracer to sulfoconjugates which presumably were poor receptor ligands; however, when this enzyme was first removed from the crude cytosol by fractionation through DNA-cellulose columns, DNA-adhering components could then be labeled with radioactive estradiol. This post-labeling method revealed an estrogen binding component with the physicochemical properties of a classical estrogen receptor: high affinity (Kd=10−10); low capacity (2–5 fmol/mg protein); and affinity for estrogenic steroids only. On sucrose gradients the estrogen receptor complex eluted from DNA-cellulose had a sedimentation coefficient of 4.7–5S. Estrogen receptors having properties similar to the cytosolic form were also demonstrated in nuclear extracts prepared from testes of untreated animals, suggesting that translocation of estrogen synthesized from endogenous precursors is a normal in vivo occurrence in this species. Preliminary measurements indicate that both cytoplasmic and nuclear estrogen receptors may show seasonal changes keyed to the annual reproductive cycle. We reported earlier that estrogen binding activity in the crude cytosol of Necturus testis is located mainly in regions comprised of differentiating or fully differentiated Leydig cells. Further studies of aromatization, the estrogen receptor system, and the estrogen-conjugating enzyme in Necturus testis may provide new insights into the role of intratesticular estrogen in male reproduction.