ABSTRACT
Nuclei were isolated from porcine endometrium of castrated pigs either unexposed or exposed to estradiol in vivo by two techniques, one of which included a hypotonic step. Aliquots were analyzed for estradiol content. Receptor was extracted from buffered, Surfynol-stabilized suspensions by either (a) KCl alone, (b) in combination with dithiothreitol, or (c) by dithiothreitol with polypentosanesulfate and addition of KCl. The yields rose from a-->c. The same proportional gains with increasing extractant efficacies were obtained from nuclei of unstimulated and estradiol-treated cells. Receptor recovery with extractant "c" rose linearly over the range of 9-80 x 10(6) nuclei/mL and was independent of the technique used for isolation. Nuclear fractions isolated using steroid-free solutions contained more estrogen receptor than estradiol; the numerical excess in control nuclei persisted in the nuclei of stimulated cells featuring a stoichiometric rise of ligand and receptor contents. The increase of receptor contents in nuclei isolated from hormone-stimulated cells coincided with a decline in the cytoplasmic fractions. An excess of hormone over receptor was seen only when nuclei were isolated from untreated cells with media containing 10 nM estradiol. Our data strengthen earlier notions of an estradiol-promoted receptor translocation into the nucleus and are not compatible with the ligand-filling hypothesis of preexisting nuclear binding sites.
