Simultaneous measurements of estrogen nuclear (e₂rn) and progestin cytosol (PRc) receptors in the hypothalamus and pituitary Gland of rats

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 (K_D) and maximal number of binding sites (Bmax) similar to those previously reported using a separate assay for each receptor.