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Estrogen Action in the Mouse Uterus: Characterization of the Cytosol and Nuclear Receptor Systems

Transplacental Toxicology Workgroup, Laboratory of Environmental Toxicology, National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709

Address requests for reprints to: Dr. Kenneth S. Korach, Laboratory of Environmental Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709.

Abstract

Cytosol receptor binding of 17β-estradiol in the mouse uterus was demonstrated in immature, ovariectomized, and intact animals. Quantification of specific binding by protamine sulfate precipitation was approximately 0.70 pmol estradiol bound/mg cytosol protein for all of the groups studied. Nuclear binding levels, assessed by the nuclear exchange assay, were 0.11–0.15 pmol/100 µg DNA. Stability of the nuclear receptorestradiol complex was evaluated at 25, 30, and 37 C and was found to be inversely related to the incubation temperature. Linearity of the binding assays was demonstrated for cytosol and nuclear receptors compared to milligrams of cytosol protein or micrograms of DNA, respectively. The mouse uterine cytosol receptor-estradiol interaction was shown to be second order by association rate analysis (k₁ = 2.0 x 10⁵ M^-1 sec^-1). Dissociation of estradiol from the receptor complex (k_-1) appeared to be first order, with a constant of 2.3 x 10^-5 sec_-1. Sucrose gradients performed in low salt buffer (10 mni Tris and 1.5 mM EDTA) resulted in the appearance of an 8S-sedimenting cytosol receptor. A cytosol receptor sedimenting at 6S was demonstrated on sucrose gradients in low salt buffer (10 mM Tris, 1 mM disodium EDTA, and 10% glycerol), while the same analysis in 0.4 M KC1 buffer gave a 4S component. Sedimentation profiles of KC1 extracts of nuclei showed a 5S moiety. A similar sedimenting component (5S) was demonstrable by a cell-free transformation of the cytosol preparation. Receptor specificity for estrogenic steroids was indicated from competitive equilibrium binding experiments with a variety of unlabeled steroids. Estrogenic compounds showed the following rank order: diethylstilbestrol 17β-estradiol estrone estriol 17-estradiol > R2858 [11β-methoxy- 19-nor-1,3,5- (10)pregnatrien-20-yne-3,17-diol] 3β,5-androstanediol. Other androgens, progestogens, and glucocorticoids had no appreciable effect on the binding. The distribution pattern of the immature mouse uterine cytosol and nuclear receptors after an injection of estradiol (10 µg/kg) showed initial translocation at 1 h, with the appearance of a second nuclear peak approximately 7–8 h postinjection. The properties of this estrogen receptor system and the possible role of this biphasic nuclear increase is discussed as it relates to estrogen action in the mouse uterus.

Received August 14, 1978.

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