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A number of synthetic and naturally occurring steroids were studied for competitive binding to two progesterone receptors, one in chick oviduct and another in human uterus. The results show a good correlation of relative (to progesterone) binding affinity with progestational activity as measured by subcutaneous Clauberg assay. 17α-Ethynyltestosterone and closely related compounds compete very well in binding to both receptors; 19-nor derivatives show dramatic enhancement of binding affinity to the human receptor. Of the substituted progesterones tested, 21-fluoro- and 6αfluoroprogesterone competed best with progesterone for binding to both receptors. 17α-Acetoxyprogesterone and the substituted 17α-acetoxyprogesterones tested were fairly good competitors for the human receptor but did not compete at all for the chick. The binding data are assessed in terms of structural requirements for formation of a steroid-receptor complex. The data suggest that the receptors are able to modify the mode of binding at ring D to accommodate several different types of C-17 substitution. Of the steroids with reactive halo, thio, diazo or chloromercuri substituents which were tested as possible substrates for affinity labeling of the receptor active site, only 6α- and 6β-bromoprogesterone and 17β-thiol- and 17β-methylthio-4-androsten-3-one competed sufficiently well with progesterone as to be considered good candidates.

A novel androgen receptor was observed in estrogen-stimulated chick oviducts but not in unstimulated oviducts. This binding component showed a preference for androgens and could therefore be distinguished from oviduct receptors for estadiol and progesterone. Testosterone was tightly bound having a dissociation constant (Kd) of 2.7 × 10 −10 M. Sucrose gradient centrifugation, under low ionic strength conditions, showed testosterone to be bound as an 85 complex. These binding properties, plus the estrogen dependency of this component, suggest its role as a biological receptor for androgens.

A receptor with specificity and high affinity for glucocorticoid hormones has been identified and partially characterized in the cytoplasmic fraction of AtT-20 mouse pituitary tumor cells grown in tissue culture. The binding of triamcinolone acetonide, a potent synthetic glucocorticoid, was analyzed using the high speed supernatant or cytosol fraction from homogenates of these cells. The interaction with [3H]triamcinolone acetonide had an apparent dissociation constant (Kd) of about 1.0 x 10-9 m and a second order association rate constant of 2.2 x 105 m-1s-1 at 0°. The triamcinolone-receptor complex was stable at 0°, but the binding activity of the receptor was labile in the absence of bound steroid. The receptor activity was destroyed by heating to 60° and by treatment with Pronase and N-ethylmaleimide, but not by deoxyribonuclease or ribonuclease. The size of the cytoplasmic binding component was estimated by sucrose gradient centrifugation studies. The complex had a sedimentation coefficient of about 5.4 S in 0.01 m KCl and 4 S in 0.4 m KCl. The binding of [3H]triamcinolone acetonide to the cytosol receptor was largely inhibited by unlabeled corticosterone and dexamethasone and partially reduced by cortisol, progesterone, deoxycorticosterone, and aldosterone, but unaffected by estradiol, testosterone, or dihydrotestosterone at the concentrations tested. A functional role for these receptors in the regulation of adrenocorticotropic hormone (ACTH) production is supported by the correlation between effectiveness of the glucocorticoids in competing with triamcinolone binding and their ability to reduce ACTH levels in cell incubation studies.

We have demonstrated the existence of a highly specific binding component for 17²– hydroxy–5±–androstane–3–one (5±DHT) in cytosol obtained from the caput epididymidis of sexually mature intact rabbits. Using sucrose gradient analysis and charcoal assay methods we were not able to obtain evidence that free receptor activity exists in the corpus or cauda epididymidis. All three epididymal segments contain a component that binds cortisol. The cortisol binding molecule is distinct from the 5±DHT receptor as shown by competition studies and sucrose gradient analysis. Unlabeled testosterone is able to compete with [3H] 5±DHT for sites on the receptor molecule, but is approximately 1/5 as effective as unlabeled 5±DHT. I7²estradiol, 53DHT, cortisol, and progesterone are weak or ineffective competitors. The 5±DHT receptor sediments as a single symmetrical peak with a sedimentation coefficient of approximately 4S on linear 5–20% sucrose gradients. At the concentration of labeled hormone used, only a single high aff...

The effects of estrogen treatment on the specific progesterone binding protein (receptor) in the chick oviduct were investigated. The progesterone receptor is present in non-estrogen treated oviducts but the tissue concentration of this protein increases several fold during a 2- week-period of estrogen treatment. In addition, qualitative changes in the receptor protein are indicated by its sedimentation properties on sucrose gradients. Following estrogen treatment, the 4-5S progesterone-receptor complex is apparently altered to a form which sediments in the 6-8S region on sucrose gradients. These results suggest that the enhanced responsiveness of the oviduct to progesterone following estrogen treatment may arise through quantitative and qualitative changes in progesterone receptor molecules. (Endocrinology 90: 1041, 1972)

Components A and B of the chick oviduct progesterone-binding protein system have been prepared by chromatography on DEAE-cellulose. Both components are shown to bind [3H]progesterone in vivo and to accumulate in oviduct nuclei in vitro. Neither component preparation contains detectable enzymatic activity for metabolizing progesterone in vitro. Each component can enter nuclei separately where both are strongly retained bound to nuclear chromatin. In vitro assays for the interaction of the components with DNA and chromatin acidic proteins were used to study binding specificity of the components. Component A binds to DNA from several sources but Component B does not bind to DNA. Conversely, Component B binds to purified oviduct chromatin and retains target organ specificity, but Component A does not bind to purified chromatin. The two types of binding activity characteristic of crude cytosol progesterone receptor, namely, the association with DNA and with target cell chromatin proteins have thus been resolved and shown to reside in receptor Components A and B, respectively.

Recent investigations have indicated that administration of corticosteroids affects the maturation of lung tissue in utero. To determine the receptivity of lung to glucocorticoids at the molecular level, the direct interaction of this hormone with lung tissue components was analyzed. Hormone binding was measured in the highspeed supernatant fraction of lung homogenates using the potent synthetic glucocorticoid, triamcinolone acetonide (TA). [ 3 H]TA binds to a macromolecular component of lung cytosol which has a sedimentation constant of 7S on sucrose gradients. This binding is of a high affinity with a dissociation constant of about 10 −9 m and can be clearly distinguished from corticoid binding in rat serum. Both natural and synthetic glucocorticoids (triamcinolone, dexamethasone, cortisol, and corticosterone) compete with the binding of [ 3 H]TA, but other steroids (estradiol, progesterone, testosterone, and dihydrotestosterone) are inactive in this respect. Binding activity was observed in lungs from adult, fetal, and newborn rats, and also in the lungs of adult rabbits and fetal lambs. These results demonstrate a binding protein in lung with properties one would expect of a target tissue receptor for glucocorticoids.

Progesterone—binding components in the uterine cytosol from the mouse and rat have been detected using sucrose—glycerol gradient analysis. The binding components in mouse uterine cytosol had sedimentation values of 4S and 6.5S and for the rat, values of 4S and 7S were determined. The binding components in both species were found to be specific for the uterus and to be protein in nature. The amount of binding was dependent upon the endocrine status of the animal. Castration destroyed the binding components, but they could be restored to intact levels following estrogen stimulation. Competition studies showed a high specificity for progesterone and that little of the progesterone binding was due to contamination by corticosteroid binding globulin (CBG). Equilibrium measurements have indicated a high affinity of the receptor—hormone interaction (Kd 10-8 – 10-9M) while kinetic measurements have revealed a very rapid dissociation of the receptor—hormone complex which could be stabilized somewhat by glycerol. T...

The high-speed supernatant fraction of rat liver, lung, kidney, testis, and intestinal mucosa contains a component capable of binding [ 3 H]retinol in vitro when binding is analyzed by sucrose density gradient centrifugation or gel filtration. This binding component can be distinguished from one identified in rat serum. Whereas the tissue component sediments in the 2S region of sucrose gradients, the serum component sediments in the 4.6S region. Molecular weight estimations by gel filtration indicate molecular weights of 16,000 and 67,000 for the tissue and serum binding components, respectively. Unlabeled retinol, but not retinoic acid, competes for the binding of [ 3 H]retinol in tissue cytosols. Competition for the binding of [ 3 H]retinol by unlabeled retinal has also been observed in tissue cytosols, but may result from the in vitro reduction of retinal to retinol. Unlabeled retinol, retinal, and retinoic acid fail to compete for the binding of [ 3 H]retinol in serum under the conditions used. The tissue binding component (testis) is sensitive to digestion with Pronase, but not with RNase or DNase, indicating a protein nature for this component.

This report demonstrates that the chick oviduct, a specific target organ for progesterone, contains both cytoplasmic and nuclear macromolecules which bind progestins. These binding molecules can be clearly distinguished from transcortin by centrifugation through sucrose gradients of low ionic strength and by agarose gel filtration. The cytoplasmic progesterone-binding molecules also bind 5-α-pregnane-3,20-dione, but have significantly lower affinity for cortisol, estrone, or aldosterone. They are absent from blood and nontarget organs such as lung and spleen. The tissue-specific binding components appear to be heat-labile proteins with an average dissociation constant for progesterone of about 8 × 10 -10 M at 2°C. These results are consistent with the identification of the progesterone-binding molecules as the functional hormone receptors. In further support of this concept is the finding that treatment of the chicks with estrogen coordinately induces a 20-fold increase in the number of progesterone-binding molecules and enhances the capacity of progesterone to induce avidin synthesis. A progesterone-„receptor” complex can be detected in both the cytoplasm and nuclei of oviduct tissue after an injection of [ 3 H]progesterone to estrogen-treated chicks. By contrast, incubation of oviduct tissue with [ 3 H]progesterone in vitro at 2°C for 5 min leads to labeling of the cytoplasmic „receptor” only. Transfer of the „receptor”-steroid complex into the nucleus then appears to occur upon subsequent incubation in vitro at 37°C. This observation suggests that the transfer of bound progesterone across the nuclear membrane may be an energy-requiring enzymatic process.

Researchers at the Vanderbilt University School of Medicine have used the chick oviduct in many of their efforts to understand the mechanism of steroid hormone action. In this paper they describe their efforts to establish the existence and characteristics of a specific receptor for progesterone in target cells and to relate the initial hormone-receptor interactions to subsequent alterations in RNA synthesis induced in the oviduct cell nucleus. Areas treated include progesterone binding to macromolecules following in vivo administration characterization of macromolecular-progesterone complex formed by oviduct cytosol in vitro partial purification of cytoplasmic complex evidence that binding components mediate progesterone action transfer of steroid-receptor complex from cytoplasm to nucleus and receptor interaction with the genome. OMalley leads a discussion further considering the nature of the progesterone receptor.