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Attempts to identify estrogen target cells in bone by immunocytochemistry using antibodies to the receptor have proved to
be controversial. The aim of this study was therefore to determine whether immunogold labeling can be used as a technique
for the localization of estrogen receptors (ER) on a human osteoblast-like cell line. The aim was also to determine the distribution
of ER on the cell surface by using a scanning electron microscope (SEM) and intracellularly by using a transmission electron
microscope (TEM). Labeling of the cytoplasmic material was seen around areas that appeared to be a disrupted plasma membrane.
No nuclear or perinuclear labeling could be detected. The conclusion can be made that SEM immunogold labeling combined with
TEM can be regarded as a practical technique for localizing ER on human osteoblasts. This article clearly demonstrated that
osteoblast-like cells do express ER at low levels and that, although cytoplasmic immunoreactivity could be detected, no, nuclear
or perinuclear labeling was found. 相似文献
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Margeat E Bourdoncle A Margueron R Poujol N Cavaillès V Royer C 《Journal of molecular biology》2003,326(1):77-92
The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands. 相似文献
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Diethylstilbestrol was coupled to epoxy-activated agarose yielding an affinity resin which is highly efficient for the isolation of estrogen receptors. This resin, diethylstilbestrol-agarose (DES-agarose), bound two proteins (Mr = 50,000 and 65,000) from rabbit uterine cytosol that show a specific interaction with estradiol. A two step procedure--adsorption on DES-agarose followed by a selective elution with p-sec-amylphenol and NaSCN, yielded highly purified estrogen receptors which can be used in the studies of estradiol-receptor interactions with other cell constituents. 相似文献
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Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism. 相似文献
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The protein ER-alpha has been exhaustively characterized in estrogen-sensitive tissues and cell lines. However, little is known regarding the expression and cellular distribution of the newly identified ER-beta protein. We first quantified the specific estradiol binding site content in the estrogen-responsive cell lines MCF-7 (mammary) and SHM (myometrial). In the two cell types, these sites were associated to the expression of both ER-alpha and -beta isoforms. Native ER-beta was visualized to reside inside the nucleus by means of conventional indirect immunofluorescence. The cells expressed ER-beta as a tight approximately 50 kDa triplet when resolved by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and blotted using antibodies mapping different domains of the cloned ER-beta version. When the cells were subjected to homogenization and differential centrifugation, a substantial proportion of ER-beta immunolabeling was localized at membrane subfractions. ER-beta expression and partitioning was confirmed by Ligand blotting assays using estrogen derivatives coupled to different macromolecular tags. However, ER-alpha was expressed as the major estrogen binding protein in both cell lines. Similar localization experiments were performed on HeLa cells (cervix). Though usually considered ER-negative, this cell line displayed basal significant estrogen binding capacity and co-expression of both ER isoforms. Taken as a whole, the results indicate that ER-beta could be expressed as functional estrogen binding proteins among a dominant population of ER-alpha sites in the cell lines under study. 相似文献
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Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E2) causes a rapid stimulatory action. E2 (1 nM) stimulates firing activity and intracellular calcium ([Ca2+]i) oscillations of primate GnRH neurons within a few min. E2 also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E2-induced [Ca2+]i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E2 action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E2 by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E2-induced changes in [Ca2+]i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E2 in primate GnRH neurons. 相似文献
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Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line
The classical model of 17beta-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrial-microsomal localization of ER alpha in the C2C12 skeletal muscle cell line. We first established [(3)H]17beta estradiol binding characteristics in whole cells in culture. Specific and saturable [(3)H]17beta estradiol binding sites of high affinity were then detected in mitochondrial fractions (K(d) = 0.43 nM; B(max) = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17beta estradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17beta-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ER alpha proteins were confirmed in C2C12 cells transfected with ER alpha siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ER alpha in skeletal muscle cells suggests an alternative mode of ER localization/function. 相似文献
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The female sex steroid, estradiol 17, mediates its effect through its association with estrogen receptor present in the target cell. So far the major emphasis has been given to the genomic actions of the hormone mediated by the nuclear estrogen receptors. Recent years have seen a shift in the ideas revealing the existence of estradiol binding entities both in the plasma membrane and the endoplasmic reticulum. Though the true identity of this membrane associated receptors is far from being known, a functional role for the same have been implicated both at the genomic as well as the non-genomic level. The major focus of the review is to highlight the existence of membrane associated estrogen receptors and receptor-related proteins and the functional roles played by some of them. The signalling events exerted by this class of membrane associated estrogen receptor could partly explain the physiological significance of estrogen in cardiovascular disease, osteoporosis and breast cancer as well as the molecular mechanism associated with xenoestrogen action. 相似文献
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Laudański P Redźko S Przepieść J Koda M Wołczyński S Sulkowski S Urban J 《Reproductive biology》2004,4(3):305-311
We used immunohistochemistry to compare the expression of estrogen receptors (ERalpha and ERbeta) in term myometria of 32 pregnant women divided in two groups. Group I comprised of 16 women in labour and group II included 16 non-laboring gravidas. We observed cytoplasmatic localization of both ER isoforms and no differences in the ER expression between the two groups of patients. The abundance and specific localization of ERs in human term myometrium seems to be independent of its contractile activity which may point to the specific role of those receptors in late pregnancy myometrium. 相似文献
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Pepe GJ Billiar RB Leavitt MG Zachos NC Gustafsson JA Albrecht ED 《Biology of reproduction》2002,66(4):1054-1060
In adult mammals, estrogen regulates ovarian function, and estrogen receptor (ER) is expressed in granulosa cells of antral follicles of the adult baboon ovary. Because the foundation of adult ovarian function is established in utero, the present study determined whether ERalpha and/or ERbeta were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165-181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ERalpha protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERbeta protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165-180 of gestation, ERalpha expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERalpha expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERbeta expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERbeta, ERbeta protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ERalpha and ERbeta were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERalpha and ERbeta mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate. 相似文献
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Social and sexual incentive motivation, defined as the intensity of approach to a social and a sexual incentive, respectively, were studied in female Swiss Webster mice. In the first experiment, the social incentive was a castrated mouse of the same strain as the females, whereas the sexual incentive was an intact male mouse of the same strain. Ovariectomized females were first tested after oil treatment and then after administration of estradiol benzoate + progesterone in doses sufficient to induce full receptivity. The hormones increased sexual incentive motivation while leaving social incentive motivation unaffected. This suggests that sexual incentive motivation in the female mouse is dependent on ovarian hormones. In the next experiment, ovariectomized females were tested with an intact, male estrogen receptor α knockout and its wild type as incentives, first without hormones and then when fully receptive. There were no differences in incentive properties between the wild type and the knockout. In a similar experiment, we used an intact male estrogen receptor β knockout and its corresponding wild type as incentives. The wild type turned out to be a more attractive social incentive than the knockout, while they were equivalent as sexual incentives. Finally, an intact male oxytocin knockout and its wild type were used as incentives. The knockout turned out to be a superior incentive, particularly a superior sexual incentive. The fact that the estrogen receptor β and oxytocin knockouts have incentive properties different from their wild types may be important to consider in studies of these knockouts' sociosexual behaviors. 相似文献