Rat uterine nuclei containing unlabelled progesterone–receptor complexes were incubated at 0 °C with the synthetic progestogen [3H]R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione). Complete exchange of steroid bound to the receptor was observed after 5–6 h. For longer times (up to 26 h) there was no further change in the concentration of steroid–receptor complexes, but the non-specific binding was increased. Saturation of nuclear receptors was obtained with a concentration of 5–10 nm-[3H]R5020. Competition with unlabelled steroids showed that only progestogens inhibited the binding of [3H]R5020 to nuclei. Optimum experimental conditions to reduce the non-specific binding to nuclei were established, and a method for the assay of nuclear progesterone–receptor complexes was devised, based on these characteristics. The concentration of nuclear receptors was low in oestradiol-primed ovariectomized rats; adrenalectomy gave rise to slightly lower values. Injection of the rats with progesterone resulted in a 5·5-fold increase in the number of nuclear receptors and a parallel decrease in the number of cytosol receptors. Similar injections of corticosterone and testosterone were without effect. Nuclear receptors were also shown to be stable in uteri kept in liquid nitrogen for up to 3 weeks. This assay may be used to study the correlation of a biological response to progesterone with the extent of receptor occupation in the nuclei.
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