Tissue and species specificity of unmasked nuclear acceptor sites for the estrogen receptor of Squalus testes

Abstract

The testicular estrogen receptor of the shark Squalus acanthias is restricted to nuclear subfractions when tissue is homogenized in low salt buffers and adheres tightly to nuclei and DNA-cellulose even when exposed to high salt conditions. Therefore, we examined the binding characteristics of this receptor to chromatin subfractions from homologous and heterologous tissues. Squalus chromatin linked to cellulose and partially deproteinized by 0-8 M guanidine hydrochloride (GuHCl) gave extraction patterns similar to those obtained with mammalian and avian chromatin. Chromatin as prepared in our laboratory contained no bound estrogen receptor. The binding pattern of the [3H]estradiol-labeled nuclear estrogen receptor to chromatin fractions from Squalus testicular zones I/II (containing spermatogonia, spermatocytes, and high receptor levels) revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 2-4 M GuHCl (350% increase over unextracted chromatin), with a 40% decrease from maximal binding at 5-8 M GuHCl. By contrast, binding to chromatin from zone III (containing spermatozoa and little or no detectable receptor) showed no major peak and 4 times less binding at 3 M GuHCl-extracted chromatin. We have previously shown that zones I and II contain the majority of testicular receptors and, presumably, are the primary sites of estrogen action, whereas receptor activity in zone III is minimal (less than 5%), indicating a secondary or nontarget tissue. Squalus testicular [3H]estradiol-receptor complexes bound minimally to rabbit uterine chromatin. Likewise, [3H]estradiol-receptor complexes from rabbit uterus, Squalus oviduct, or mouse testis bound minimally to Squalus testicular chromatin. Thus, maximal binding occurred only with Squalus zones I/II chromatin and Squalus testicular receptor. The binding of [3H]estradiol-receptor complexes to testicular chromatin (zones I/II) was of high affinity (Kd = 1.9 X 10(-10) M) and low capacity and was optimal in the presence of 150 mM KCl, but was unaffected by the addition of urea in an amount similar to that of Squalus body fluids (300 mM). [3H]Estradiol binding to chromatin required undenatured receptor and was competitively inhibited by radioinert estradiol-receptor complexes, confirming saturability of acceptor sites. The affinity of the Squalus estrogen receptor for homologous chromatin was in the same range as that reported for other systems, despite the unusual nuclear extractability characteristics of Squalus receptor. This study provides new evidence for tissue and species specificity of receptor binding to chromatin acceptor sites.