Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

Estrogen receptors in the wobbler mouse

Recent research has raised the interesting possibility that the neurological mutant mouse, wobbler (wr/wr), possesses an estrogen receptor deficit analogous to the androgen receptor deficiency found in androgen-resistant mice with testicular feminization. In the present report we examined estrogen-binding activity in cytosolic extracts of kidney, liver, and brain from wobbler mice, littermate control animals, and C57BL/6J mice, using DNA-cellulose chromatography. Estrogen binding components exhibiting properties of estrogen receptors were present in all tissues examined. Estrogen receptors adhered to DNA, displayed characteristic elution profiles from DNA-cellulose, and showed high affinity and limited capacity for estradiol, in contrast to non-receptor entities which bind estradiol. The qualitative elution patterns for estrogen receptors did not differ among groups within each tissue studied, and were similar to those reported previously in mouse kidney and brain. While estrogen receptors have been shown in mouse liver by other techniques, this is the first demonstration of putative estrogen receptors in mouse liver by DNA-cellulose chromatography. No consistent deficits in estrogen receptor concentration were found in wobblers compared to littermates. Thus, the data do not support the hypothesis that the wobbler mouse is an estrogen receptor-deficient mutant.     

Estrogen receptors in quail brain: a functional relationship to aromatase and aggressiveness

Estradiol (E2) mediates many of the activational effects of testosterone (T) on masculine reproductive and aggressive behaviors. Using Japanese quail (Coturnix coturnix japonica) as an animal model, together with a newly devised procedure for quantifying aggressiveness, we recently showed that aggression is E2-dependent and that individual differences in behavioral intensity are correlated with aromatase in the hypothalamus/preoptic area (HPOA). In this study we characterized estrogen receptors (ER) in quail brain and tested the hypothesis that aromatase in brain regulates T-induced behavioral responsiveness by regulating the quantity of E2 available for receptor binding. Based on standard binding assays and Sephadex LH-20 chromatography, quail brain ER was shown to be estrogen-specific, of high affinity (Kd = 0.88 nM), and of limited capacity with highest concentrations in limbic brain areas (Bmax 23-27 fmoles/gm HPOA). In addition, this ER adhered to DNA-cellulose under activating conditions. The quantitative relationship between aromatization, ER, and aggressiveness was tested in reproductively inactive (nonaggressive) males by treatment with T +/- the aromatase inhibitor 4-hydroxyandrostenedione (OHA). After 5 days, T markedly stimulated aggressiveness, and elevated aromatase and nuclear (occupied) ER in HPOA. Simultaneous treatment with OHA blocked effects on aggressiveness and aromatase, and lowered nuclear ER, but increased cytosolic (empty) ER. Total ER (nuclear plus cytosolic) was higher after T treatment whether or not OHA was administered, suggesting that androgen per se induces ER in quail HPOA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of short days on aromatization and accumulation of nuclear estrogen receptors in the hamster brain

Exposure of hamsters to short days increases sensitivity to the negative feedback effects of testosterone (T) but decreases responsiveness to the behavioral effects of the hormone. Since T is metabolized in the brain to 5 alpha-dihydrotestosterone (DHT) and estradiol, which differentially affect gonadotropin secretion and sex behavior, it is reasonable to postulate that daylength can modulate neural responses by quantitative or qualitative alterations in T metabolism and subsequent receptor binding of active hormone. Experiments reported here focused on aromatization and the nuclear accumulation of estrogen receptors. Adult male hamsters were maintained for 6-12 wk in long (14:10 LD) or short (8:16 LD) daily photoperiods. Both intact and castrated animals were used to assess direct effects of short days versus changes due to short-day-induced testicular regression. Discretely dissected regions of the brain (preoptic area, POA; hypothalamus, HTH; and corticomedial amygdala, CMA) or limbic blocks (LIM) comprised of all three regions were assayed for estrogen-synthesizing activity (aromatase) and estrogen-binding activity (receptors). Aromatase was estimated in vitro by conversion of [7-(3)H] androstenedione to [3H] estrogen and in vivo by measuring increases in nuclear estrogen receptor levels after injection of aromatizable androgen. Receptor-binding activity was assayed in crude cytosolic and nuclear extracts by incubating samples with [3H] estradiol +/- 100-fold excess inert estradiol, and separating free and bound steroids by Sephadex LH-20 gel filtration. When aromatase was assayed in homogenates prepared from discrete brain regions of individual hamsters, significantly lower activity was found in the HTH of short-day animals than in long-day controls. This effect was seen in both intact and castrated animals, which indicates that it was not mediated by the testis. Decreased enzyme activity in the POA and CMA of short-day hamsters was not significant, nor was there an effect of castration independent of short days. Low levels of nuclear estrogen receptors were present in LIM of intact males, but these were reduced after castration or concomitant with testicular regression after short-day exposure. This suggests that the hamster testis normally secretes estrogen or aromatizable androgen. A single injection of estradiol or aromatizable androgen (T or androstenedione) increased nuclear receptors in LIM of castrated animals. Cytosolic receptors were not different in short-day vs. long-day hamsters, nor were there differences in nuclear receptor levels after a single estradiol injection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endocrine aging in C57 BL mice--II. Dynamics of estrogen receptors in the hypothalamic-pituitary axis

Specific cytosolic and nuclear binding sites for estrogens were measured in the hypothalamic-pituitary axis (HPA) of young (4-8 months) and old (16-18 months) C57 BL mice in order to determine any age-related alteration in hormone-receptor interaction. Our results indicated no age differences in the affinity (KD = 0.89 +/- 0.03 (SEM) vs 1.09 +/- 0.2 X 10(-9) M), the specificity, the sedimentation profile (6 s) or in the number (98.9 +/- 4.9 vs 84.4 +/- 2.3 fmol/mg protein) of unoccupied estrogen binding sites in the cytosols. Estradiol administration to young mice induced a complete translocation of cytosolic estrogen receptors to the nucleus, and two types of nuclear binding sites were observed: Type I were specific for estrogens with high affinity (KD = 0.51 +/- 0.06 X 10(-9) M) and low binding capacity (115.1 +/- 22.7 fmol/mg DNA) and sedimented in the 4.0 s area, while Type II binding sites showed a much higher capacity and lower affinity for R2858. HPA nuclear suspensions of aged untreated mice showed undetectable (less than 50 fmol/mg DNA) levels of nuclear estrogen receptors and E2 pre-treatment resulted in a significant increase in both types of binding sites. While no significant changes in the physicochemical characteristics of these nuclear receptors were observed, when compared to young animals, aging was manifested by a translocation defect in the HPA of C57 BL mice. These results suggest aging changes in the endocrine regulating centers of the brain with defective activation of estrogen receptors.     

Estrogenic effect of phenol red in MCF-7 cells is achieved through activation of estrogen receptor by interacting with a site distinct from the steroid binding site

MCF-7 cells serially subcultured in media containing phenol red show poor stimulation of progesterone receptor (PR) synthesis in response to estradiol compared to cells grown in phenol red-free media. Phenol red, when added to cytosol, did not compete with [3H]estradiol for estrogen binding sites in concentrations ranging from 2 microM-1 mM. However 25 microM of the dye was sufficient to increase nuclear translocation of estrogen receptor (ER) in the intact cell. Phenol red activates cytoplasmic ER as indicated by DNA-cellulose binding studies. When cells grown in phenol red-free medium were exposed to phenol red for 48 h, PR levels increased in a dose dependent manner. From these data, it may be concluded that phenol red causes estrogenic effect in MCF-7 cells through activation of cytoplasmic receptor by interacting at a site distinct from the steroid binding site.     

Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.     

Progesterone treatment suppresses estrogen receptor in the sex skin of Macaca nemestrina

We measured tightly bound nuclear estrogen receptors (ER) in sex skin biopsies obtained from pig-tailed macaques (Macaca nemestrina) which were previously ovariectomized and treated with an estradiol-progesterone regimen. Incubation of fresh tissue slices with a saturating concentration of [3H]estradiol (E2) was done to determine the capacity of nuclear acceptor sites to bind activated ER with high affinity. The radiolabeled ER was extracted from nuclei with 0.5 M KCl, complexed with an anti-ER monoclonal antibody, and quantitated by analysis on sucrose gradients. Even though serum E2 levels were unchanged, 7 and 14 days of sequential progesterone (P) treatment decreased ER amounts below those found after 7, 14 and 21-23 days of E2 treatment. ER regulation in sex skin of this species is similar to that found in macaque reproductive tract; P suppresses ER levels even in the presence of continuous E2. The tissue responses of sex skin to the hormone treatments correlated well with the measured fluctuations of tightly bound nuclear ER, which suggests the functional significance of this ER component.     

Altered estrogen receptor system in estrogen-unresponsive human endometrial adenocarcinoma cells

Previous studies from our laboratories demonstrated that cells from a human endometrial adenocarcinoma cell line (Ishikawa) responded to estradiol whereas cells from another endometrial cancer line (HEC-50) did not. In an attempt to identify factors responsible for the observed estrogen insensitivity we compared the characteristics of the estradiol receptor (ER) systems in Ishikawa and HEC-50 cells. Saturation analyses of cytosolic estrogen binders were performed over a 0.1-70 nM range of [3H]estradiol concentrations. Equilibrium dissociation constants and number of binding sites were determined by graphic analysis of Scatchard plots or computed by applying Fourier-derived affinity spectrum analysis (FASA) of the binding data. No significant differences were noted in the dissociation constants (Kd approx. 0.6 nM) or number of binding sites (approx. 6-10 fmol/mg protein) for the single binder that could be evaluated by the graphic method in cytosol from the two cell lines. However, 2 binders in Ishikawa cells (Kd approx. 0.2 and 6 nM) could be detected by the FASA method; the higher affinity binder in HEC-50 cells could not be clearly demonstrated. Structural differences in the specific estrogen binders which might distinguish HEC-50 from Ishikawa cells or normal endometrial tissue were investigated by using the anti-ER monoclonal antibody JS 34/32. Interaction of the antibody with [3H]estradiol binders of estrogen-responsive cells and tissue was evident from the formation of labeled complexes that were shown to sediment faster in glycerol density gradients and could be immunoprecipitated with Protein A attached to Sepharose beads. In contrast, the antibody did not recognize labeled specific binders in the HEC-50 cells. Furthermore, [3H]estradiol receptors in Ishikawa cells could be transformed into a species that exhibited increased hydrophilicity, evident from its binding to DNA-cellulose, whereas binders from HEC-50 could not. These results indicate that the lack of responsiveness of HEC-50 cells to estrogens might be due to structural or functional alterations in the ER protein resulting in a loss of its capability to undergo estrogen-directed conformational changes required for biological activity.     

Enhanced inhibition of estrogen receptor nuclear binding in the uterus of aged mice

We have studied why uteri from aging mice show a decrease nuclear concentration of estrogen receptors (UER). While 50-60% of available cytosolic UER from ovariectomized (OVX) mice aged 4-8 months, upon physiochemical activation, are able to bind either to DNA-cellulose or nuclear suspensions from young animals, only 20-30% of comparable concentrations of cytosolic UER from mice aged 15-18 months did so under identical experimental conditions. Nuclear dilutions with uterine cytosolic fractions from estrogen treated OVX mice prior to determination of [3H] UER binding sites in nuclear suspensions decreased the number of nuclear ER sites in both age groups. However, we observed that cytosols from aged animals showed a greater ability to prevent [3H]E2 binding to nuclear sites when compared to young ones (inhibition index: 0.286 +/- 0.013 (SE) vs. 0.137 +/- 0.025, P less than 0.05). These changes occur independently of protein concentration and result from dilution of a specific endogenous inhibitor of [3H]E2 binding to nuclear sites. The significance of these observed differences is discussed.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

Androgen receptor in rat liver: characterization and separation from a male-specific estrogen-binding protein

Many liver processes are sexually dimorphic. In particular, the microsomal content of specific enzymes and the synthesis of specific proteins are under sex steroid hormone control. Because the liver of male rats is strikingly androgen responsive, we sought evidence for an androgen receptor in this tissue. We detected and characterized both cytosolic and nuclear androgen-binding proteins. Both forms bind [3H]R1881 (methyltrienolone, 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one) with the high affinity, low capacity, and specificity for androgens and antiandrogens characteristic of androgen receptors. No high-affinity binding of [3H]DHT could be detected in unfractionated cytosol because of the rapid metabolism of this ligand; however, binding of a DHT metabolite to the high-capacity male-specific estrogen binder (MEB) of cytosol was observed. Both gel filtration and heparin-Sepharose affinity chromatography separate the cytosolic androgen receptor from MEB. Incubation of cytosol in the absence of sodium molybdate resulted in androgen-binding activity which was retained by DNA-cellulose. Castration of male rats results in a time-dependent loss of both cytosolic and nuclear androgen binding, as well as a loss in MEB activity. Androgen-binding activity is low in livers from female rats, but can be induced by testosterone treatment. An intact pituitary is necessary for maintenance of androgen-binding activity, as hypophysectomy results in complete loss of activity.     

Effects of estradiol concentration on levels of nuclear estrogen receptors in MCF-7 breast tumor cells

We have measured the time-course of estrogen receptor levels in nuclei of the estrogen-responsive breast tumor cell line MCF-7 during 90-120 min exposure of the cells to estradiol at physiologic (10(-10)M), pharmacologic (10(-6)M), and an intermediate (10(-8)M) concentration. Cells were preincubated for one week in a serum-free defined medium resembling that of Barnes and Sato, and then incubated in estradiol-containing medium. Nuclei were isolated at various times during the incubation, and filled and unfilled nuclear estrogen receptor levels were assayed. Increasing the concentration of estradiol in the incubation medium from 10(-10)M to 10(-8)M yielded increasing levels of filled nuclear receptor at all times studied, while further increase of the estradiol concentration of 10(-6)M decreased filled receptor levels from 10(-8)M values. Unfilled receptor levels dropped rapidly to zero under 10(-6)M and 10(-8)M estradiol incubation, but remained unchanged under 10(-10)M estradiol incubation. Together these results suggest that high-concentration estradiol may lead to "down-regulation" of filled nuclear receptors, which may be a contributing factor in inhibition of tumor growth. On the other hand, the continued presence of unfilled receptors only under physiological concentrations of estradiol may suggest a role for these receptors in sustaining tumor growth.     

Estrogen receptors in colorectal adenocarcinomas and in other large bowel diseases.

Seventy-two patients with tumor and ten with non-neoplastic colon disease were studied for the presence of estrogen receptors (ER) by three different methods. Only seven specimens (six primary adenocarcinomas and one recurrent cancer) had an ER concentration above 3 fm/mg of cytosolic protein, with no sex, age and tumor stage correlation. Our results suggest that the large bowel does not contain a cytosolic receptor for estradiol.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.     

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol- 17beta. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.