Simultaneous Measurements of Estrogen Nuclear (E2Rn) and Progestin Cytosol (PRc) Receptors in the Hypothalamus and Pituitary Gland of Rats

Publisher Website
Google Scholar
Abstract
The present methods used to measure estrogen nuclear (E2Rn) and progestin cytosol (PRc) receptors in the hypothalamus and pituitary gland require that separate assays be performed to determine the concentrations of each receptor. In the present studies we describe a method which simultaneously measures both E2Rn and PRc in hypothalamic and pituitary tissue. Tissue samples were homogenized in tris-EDTA-glycerol-dithiothreitol buffer and centrifuged at 1500 × g for 5 min. The supernatant was purified for the PRc assay while the nuclear pellet was extracted for the E2Rn exchange assay. For the PRc assay, the supernatant was centrifuged at 106,500 × g for 30 min and aliquots from the resultant supernatant then were incubated with 3H-R5020. For the E2Rn exchange assay, the original pellet was purified further by successively resuspending and centrifuging it through several sucrose solutions. Estrogen-receptor complexes were extracted from the chromatin pellet with a 0.4M KC1 solution and aliquots of the final supernatant were incubated with 3H-estradiol. In both assays, the samples were placed onto Sephadex LH20 columns and the receptor bound 3H-steroid was eluted directly into scintillation vials. Scatchard analyses revealed that these assays measure a single class of binding sites for E2Rn and PRc with dissociation constants (KD) and maximal number of binding sites (Bmax) similar to those previously reported using a separate assay for each receptor.