Estrogen action in the uterus: the requisite for sustained estrogen binding in the nucleus (original) (raw)
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Characterization of rat uterine estrogen receptors in vivo
The Journal of Steroid Biochemistry and Molecular Biology, 1993
In vivo binding of [3H]estradiol ([3H]E2) in the rat uterus was performed by an intraluminal perfusion of the ligand for different time periods. In this way the binding takes place in the intact organ before processing the tissue. In 10 min, with 10 nM [3H]E 2 apparent saturation or steady state incorporation of the [3I-I]E2 was achieved with a similar distribution of the label between cytosol and nuclear fractions. In vitro, the subcellular localization of the estrogen receptor (ER) is influenced by the extent of tissue damage. With the intact organ the ER subcellular distribution approaches that of the/n vivo perfusion. With increasing [3H]E2 in the perfusate it was possible to obtain a "saturation" curve and to derive the kinetic parameters. For cytosol: Kd 16 nM; Bm~ 235 fmol/mg prot. For nucleus: Kd 2.7 nM; Bm~ 103 fmol/mg prot. To follow the time course of the ER movement/n vivo, "pulse and wait" experiments were designed. Both uterine horns were peffused for 1 min. One of the horns was immediately processed (0 time) and the other was left in place after the peffusion for different periods. At 0 time 90% of the bound label appeared in the cytosol. At 5, 15 and 30 min, the label in the cytosol decreased and that of the nucleus increased approx, to 50%. Thus, translocation of the bound label from cytosol to nucleus was apparent. The role of the cytoplasm-nucleus ER traffic in the regulation of gene transcription by estrogens is discussed.
Teratogenesis, Carcinogenesis, and Mutagenesis, 1987
Estrogens are teratogens and developmental carcinogens in several species. We have used uterine growth to quantitate the potency of three estrogens [estradiol (E2), diethylstilbestrol (DES), ethynylestradiol (EE2)] during four postnatal periods (days 1–5, 10–14, 20–24, and 60–64) in the rat. Alphafetoprotein (AFP), present at high levels in neonatal serum, is thought to regulate estrogen bioavailability. Association constants for DES and EE2 were 2.7% and 4.9% of that for E2 binding to AFP, determined in a batch Sephadex equilibrium binding assay. On days 1–5, DES and EE2 were about 80‐fold more potent than E2 in increasing uterine weight. As AFP levels fell, potency differences between E2 and the synthetic estrogens decreased. In the adult, which essentially lacks AFP, the three estrogens were nearly equipotent. These data are consistent with AFP regulation of estrogen potency. On days 10–14, uterine growth was less sensitive than at other ages to all three estrogens, perhaps relat...
Localization of estrogen receptors in uterine cells* 1:: An appraisal of translocation
Experimental Cell …, 1979
The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The 'nuclei' which are obtained after lysis have a mediandiameter of 1 pm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a mode1 in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0°C provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressivelv accumulated in the nucleus; afterwards, estrogen receptor-was progres&ely lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction. which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.
Localization of estrogen receptors in uterine cells
Experimental Cell Research, 1979
The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The 'nuclei' which are obtained after lysis have a mediandiameter of 1 pm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a mode1 in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0°C provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressivelv accumulated in the nucleus; afterwards, estrogen receptor-was progres&ely lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction. which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.
Uterine Temporal Response to Acute Exposure to 17 -Ethinyl Estradiol in the Immature Rat
Toxicological Sciences, 2007
The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17a-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 mg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point ( p 0.0001), and at least 1.5-fold (up-or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively ( p 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogenresponsive genes involved in the uterotrophic response.
Estrophilic binding sites of the uterus. Relation to uptake and retention of estradiol in vitro
Biochemistry, 1973
The uptake and retention of [ 3H]estradiol by uterine and diaphragm tissue have been examined under initial velocity and equilibrium conditions, respectively. Evidence is presented to demonstrate that the estrogen receptor is not involved in the movement of estradiol into uterine tissue. Thus the rate of uptake of estradiol by the nontarget tissue, diaphragm, which possesses no estrogen receptor is the same as that for the uterus. In addition, inhibitors of the estrogen receptor fail to alter the rate of up-D ata on the uptake and retention of estradiol by the uterus have been reported by many investigators (for a review see . It is generally held that a twostep mechanism exists in which the estrogen receptor (R)' serves both an uptake function, i.e,, the receptor recognizes and binds estrogen which results in the increased uptake of estradiol in the uterus, and a retention function, i.e., the receptor-estrogen complex (RE) once formed acts to retain estrogen within uterine cells as a result of translocation of R E to the nucleus (Shyamala and . Unfortunately previous investigations have failed to differentiate clearly between uptake and retention so that it is difficult to evaluate these phenomena separately. Uptake is defined in this report as the initial rate of movement of estradiol into tissues whereas retention is defined by the amount of estradiol found in the tissue under equilibrium conditions.
Estradiol Regulation of Secretory Component in the Rat Uterus
Annals of the New York Academy of Sciences, 1983
Estradiol increases significantly the levels of IgA in rat uterine secretions.',' Recently, we found that this response involves strictly polymeric IgA, which accumulates against an apparent tissue to lumen concentration gradient.3 These results suggest that estrogen action may be mediated through effects on secretory component (SC), because at other mucosal sites, SC controls the movement of polymeric IgA into external secretions.' The purpose of the present study was to confirm quantitatively, and extend our previous qualitative finding5 that hormones regulate SC in the uterus. Uterine luminal secretions were obtained from either intact Sprague-Dawley rats (150-200 g) during the estrous cycle or hormonally treated, ovariectomized rats, as previously described: Ovariectomies were performed 7-10 days prior to experimentation. Uterine tissue cultures were established after three days of estradiol or saline injections to ovariectomized rats. Free SC and IgA levels in uterine secretions and culture media were measured by radioimmunoa~say.~,~~* As shown in FIGURE 1, SC levels in uterine secretions were highest at the proestrous and lowest at the diestrous stages of the estrous cycle. Secretory component content remained partially elevated during estrous. These variations in SC levels were paralleled by those of IgA and were most likely due to estrogen influence, because serum estrogen concentrations are maximal at proestrou~.~ This hypothesis is supported by our finding that estradiol, but not progesterone, treatment significantly increased SC levels in uterine lumina of ovariectomized rats (TABLE 11. In other studies, we have observed that the estradiol effect on uterine SC is both dose-and time-dependent. The time-course experiments, that demonstrated that luminal SC content was highest after three daily injections of estradiol to ovariectomized rats, also showed that increases in IgA levels occurred concomitantly with those of secretory component. Both SC and IgA responses were antagonized when rats received progesterone prior to estradiol. To determine whether estrogen stimulation of uterine SC content could be observed in vitro, individual uterine horns from rats administered estradiol in vivo were incubated in culture media. As demonstrated in FIGURE 2, incubation media containing uteri from estradiol-treated rats had significantly greater concentrations of SC than those of control uteri. In addition, the presence of cycloheximide in the culture significantly decreased the effect of estradiol on SC accumulation. These results indicate that estradiol regulates SC levels in the rat uterus. Moreover, given the close correlation between uterine SC and IgA accumulation *This work was supported by Research Grant A1 13541 from the National Institutes of ?To whom requests for reprints should be addressed.
Estren Behaves as a Weak Estrogen Rather than a Nongenomic Selective Activator in the Mouse Uterus
Endocrinology, 2006
A proposed membrane-mediated mechanism of rapid nongenomic response to estrogen has been the intense focus of recent research. Estren, a synthetic steroid, is reported to act selectively through a rapid membrane-mediated pathway, rather than through the classical nuclear estrogen receptor (ER)-mediated pathway, to maintain bone density in ovariectomized mice without uterotropic effects. To evaluate the mechanism and physiological effects of estren, we studied responses in adult ovariectomized mice. In a 3-d uterine bioassay, we found that 300 g estren significantly increased uterine weight; in comparison, a more maximal response was seen with 1 g estradiol (E2). The estren response was partly ER␣ independent, because ER␣ knockout (␣ERKO) uteri also exhibited a more moderate weight increase. Estren induced epithelial cell proliferation in wild-type, but not ␣ERKO, mice, indicating ER␣ dependence of the epithelial growth response. Examination of estren-regulated uterine genes by microarray indicated that early (2 h) changes in gene expression are similar to the early responses to E2. These gene responses are ER␣ dependent, because they are not seen in ␣ERKO mice. Later estren-induced changes in gene expression (24 h) are blunted compared with those seen 24 h after E2. In contrast to early genes, these later estren responses are independent of ER␣, because the ␣ERKO shows a similar response to estren at 24 h. We found that E2 or estren treatments lead to depletion of ER␣ in the uterine cytosol fraction and accumulation in the nuclear fraction within 30 -60 min, consistent with the ability of estren to regulate genes through a nuclear ER␣ rather than a nongenomic mechanism. Interestingly, estren, but not E2, induces accumulation of androgen receptor (AR) in the nuclear fraction of both wild-type and ␣ERKO samples, suggesting that AR might be involved in the later ER␣-independent genomic responses to estren. In conclusion, our studies suggest that estren is weakly estrogenic in the mouse uterus and might induce nuclear ER␣-and AR-mediated responses. Given its activity in our uterine model, the use of estren as a bone-selective clinical compound needs to be reconsidered.
In Vitro Cellular & Developmental Biology - Animal, 1994
Although estrogens have been shown to stimulate a variety of morphologic and biochemical changes in the uterus in vivo, no clear consistent demonstration of similar responses in vitro have been made; thus, a defined organ culture system using the immature mouse uterus was estabhshed to study the possibility of demonstrating estrogenic responses in vitro. Uterine tissue from immature outbred mice (17 to 24 days of age) were cut crosswise in 1-mm 3 coins and cultured in a defined medium in the absence of serum, phenol red, or growth factor supplements. Diethylstilbestrol (DES), a synthetic estrogen, was added to the media at doses ranging from 1 to 100 ng/ml. The effect of DES on uterine cell prohferation was assessed by morphologic changes in uterine epithelial and stromal cells, increase in number of epithelial cells per unit basement membrane, increase in height of luminal epithelial cells, and [3H]thymidine incorporation. Functional changes were determined by measuring the amounts of the estrogen-inducible uterine protein, lactoferrin, that was localized in the epithelial cells and secreted into the media, and the localization of the estrogen receptor in the cultured tissues. Results indicate that under the described conditions of culture, estrogens like DES can induce morphologic and biochemical responses in the uterus that are similar to those seen in vivo. This organ culture system will aid in the investigation of various mechanisms involved in the hormonal regulation of growth and differentiation of estrogen target tissues.
Journal of Veterinary Medical Science, 2003
The action of estrogen on target organs has been actively studied with the discovery of estrogen receptor (ER) β. This study was carried out to examine the expression of ERα and ERβ in the uterus and the vagina of immature Sprague-Dawley rats treated with 17-ethinyl estradiol (EE). Twenty days old rats were subcutaneously treated with EE at the doses of 0 (vehicle control), 0.03, 0.3, 1.0, 3.0, and 10.0 µg/kg/day for three consecutive days. The treatment of EE at the doses of 0.3, 1.0, 3.0 and 10.0 µg/kg/day significantly increased the weights of the uterus and vagina of rats (p<0.01) and retained fluid in the uterus of rats. At the high doses of 3.0 and 10.0 µg/kg/day, the treatment of EE caused an increase in the uterine height, hypertrophy, and a decrease in the expression of ER α and ERβ in the uterine luminal and glandular epithelium. The treatment of EE at the doses of 3.0 and 10.0 µg/kg/day also caused cornification and a decrease in the expression of ERα and ERβ in the vaginal epithelium. These results suggest that the EE treatment decrease the expression of ERα and ERβ in the uterus and vagina of immature rats and that may be associated with the morphological changes such as increase in the uterine height, hypertrophy of the uterine epithelium, and cornification of the vagina. KEY WORDS: estrogen receptor α and β, 17-ethinyl estradiol, uterus, vagina.