Specific binding of oestradiol in human uterine tissue.

Oestradiol receptors in human uteri were measured by the uptake-competition technique with tissue slices and by agar gel electrophoresis of soluble cytoplasmic oestradiol receptor complexes. The concentration of spare receptor was found to be markedly higher during the proliferative phase as compared to the secretory phase of the cycle and was inversely correlated to the concentrations of free oestrogen in the blood. It is generally accepted that oestradiol requires binding to specific receptors in the target organ in order to exert its full biological activities. At present, a two-step mechanism of oestradiol fixation is proposed involving two different proteins (Gorski et al. 1968; Jensen et al. 1968, 1971; Jungblut et al. 1967, 1970). From recent experiments it is evident that the degree of oestrogen binding is dependent on the hormonal state of the animal (Eisenfeld 8c Axelrod 1966; McGuire 8c Lisk 1968; Whalen 8c Maurer 1969; Feherty et al. 1970; Lee 8c Jacobson 1971; de Hertogh et al. 1971). After the presence of specific oestrogen receptors had been demonstrated in human uterine tissue by Wyss et al. (1968), Meister et al. (1970), Wagner (1970, personal communication), Hähnel (1971), it was of interest to study whether the oestradiol binding capacity of the uterus varies at different stages of the menstrual cycle and during the postmenopausal period in relation to the plasma concentrations of Downloaded from Bioscientifica.com at 11/06/2018 10:19:48PM via free access endogenous oestrogen or of administered oestrogenic substances. It was hoped that the results of this study would provide guide lines for the characterization of target organ tumours by the assay of spare receptors. MATERIAL AND METHODS Chemicals [6,7-3H]17jS-oestradiol (spec. act. 48 Ci/mM) and [2,4,6,7-3H]17^-oestradiol (spec, act. 100 Ci/mM) were purchased from New England Nuclear Corp., Boston, Mass. The purity (> 94.0 %) was checked by thin layer chromatography. The anti-oestrogenic compound U. 11.100 (Nafoxidine) was provided by the Upjohn Company, Kalamazoo, Mich. All other chemicals were obtained from E. Merck AG, Darmstadt or Boehringer, Mannheim and were of analytical grade. Agar purum, human serum albumin and rabbit antiserum against human serum albumin were purchased from Behringwerke, Marburg (Lahn). Processing of tissue Human uterine tissue was placed on ice immediately after hysterectomy of Caesarian section. Tissue slices of 0.5 mm thickness were cut with a Stadie-Riggs-microtome within two hours after removal of the organ. Tissue specimens used for the prepara¬ tion of extracts were immediately dissected and stored at -20°C until processing. Receptor assay by the uptake-competition technique (Jensen et al. 1967) Uptake control: 14 tissue slices (5-10 mm in diameter) were incubated and stirred magnetically in 200 ml of a 10"10 M solution of [6,7-3H] 17/? -oestradiol in Krebs-Ringer NaHCO3~0.1% glucose buffer at 37°C. Uptake-competition: same conditions as in uptake control but with the addition of 10~5 M Nafoxidine. (Substance dissolved in 0.5 ml glycerol, the same amount of glycerol was added to the uptake control). Two tissue slices were removed from the incubation flasks at 15 min intervals (as indicated in Fig. 1), washed in cold buffer for 3 min, dried on filter paper, lyophilized and 2-3 mg aliquots were ashed in an oxygen atmosphere according to the procedure of Maurer (1968). After cooling the counting vials in a methanol-dry ice bath, the condensed tritiated water was dissolved in 10 ml of the scintillation fluid according to Hayes (1963) (7.0 g PPO, 0.3 g dimethyl-POPOP, 100 g naphthalene in 1000 ml dioxane). The actual radioactivity of the individual samples was measured in a Packard Tricarb scintillation counter, model 3380, and ranged from 800 to 3000 cpm. All counts were corrected to 100% efficiency by external standardization. Evaluation The radioactivity of the samples (DPM/mg dry weight) from the incubations with and without Nafoxidine were plotted against time of incubation. (Scale: abscissatime: 2 mm/min; ordinate radioactivity: 10 mm = 100 DPM/mg dry weight). The area within the two curves was then measured with a planimeter (Ott, Kempten, Germany) and was expressed as DPM/mg dry weight. A typical example of a "positive" and a "negative" tissue specimen is given in Fig. 1. Only those cases were taken as "positive" for oestrogen receptors in which the area was greater than 25 cm2. This borderline value was derived from the average plus one standard deviation of the DPM/mg dry weight values for "negative" tissue specimens. Downloaded from Bioscientifica.com at 11/06/2018 10:19:48PM via free access