A study of the protamine precipitable cytoplasmic female sex-steroid receptors in human endometrial tissue
In Part 1 of the study methods for estimating the concentrations of the total and free specific steroid receptors for oestradiol and progesterone in the cytoplasm from endometrial tissue are assessed and novel features integrated. The cytosol is treated with Dextran coated charcoal (DCC), and after removing the DCC the steroid receptor proteins are precipitated from the cytosol with a protamine sulphate solution prior to incubation of the precipitate with 3H-labelled steroids in a phosphate buffer. Conditions for the optimum binding of the 3H-labelled steroids are investigated. These include the constituents of the DCC and protamine sulphate solution, and the pH and molarity of the incubation buffer and the concentrations of the 3H-labelled and non-labelled steroids for maximum specific binding. Temperature and duration of incubation are established for the maximum binding of the 3H-labelled steroids to their respective receptors for the estimation of the total and free receptor concentrations. The differences between the two is a measure of the endogenously bound or filled receptor. A novel feature of the methods is that the precipitation of the cytosol, incubation of the precipitate with 3H-labelled steroid and the subsequent counting of the bound radio-activity are performed in the same vial. In Part 2 methods are used to measure the concentrations of the total and free cytoplasmic receptors for oestradiol and progesterone in normal and pathological human endometrial tissue. Also the relationship between the receptor fractions for the two steroids is examined. The binding of 3H-oestrone to the receptor is studied and it is established that this binding is separate from the oestradiol binding. Even though statistically significant differences are found between the receptor concentrations for different types of endometrial tissue, the level of the bound oestradiol receptor, particularly in cases of high oestrogenic stimulation do not agree with those expected from the accepted idea of translocation of oestradiol and receptor from the cytoplasm to the nucleus. To explain the values for the two oestrogen receptors it is suggested that oestradiol and its receptor enter the nucleus separately before combining and binding to the nuclear acceptor. The action of the binding alters the oestradiol receptor to a receptor which does not recognise oestradiol but binds oestrone. The free oestrone receptor is reconverted into an oestradiol receptor in the cytoplasm. Therefore the total available receptor for binding oestradiol is the sum of the concentrations of the free receptor of oestradiol and oestrone. Therefore, if no oestrone receptor is found then the attachment of oestradiol to the nuclear acceptor is not functioning properly.