Formation of Kiss1R/GPER Heterocomplexes Negatively Regulates Kiss1R-mediated Signalling through Limiting Receptor Cell Surface Expression.

Kisspeptin receptor (Kiss1R) is an important receptor that plays central regulatory roles in reproduction by regulating hormone release in the hypothalamus. We hypothesize that the formation of heterocomplexes between Kiss1R and other hypothalamus G protein-coupled receptors (GPCRs) affects their cellular signaling. Through screening of potential interactions between Kiss1R and hypothalamus GPCRs, we identified G protein-coupled estrogen receptor (GPER) as one interaction partner of Kiss1R. Based on the recognised function of kisspeptin and estrogen in regulating the reproductive system, we investigated the Kiss1R/GPER heterocomplex in more detail and revealed that complex formation significantly reduced Kiss1R-mediated signaling. GPER did not directly antagonize Kiss1R conformational changes upon ligand binding, but it rather reduced the cell surface expression of Kiss1R. These results therefore demonstrate a regulatory mechanism of hypothalamic hormone receptors via receptor cooperation in the reproductive system and modulation of receptor sensitivity.

Determination and analysis of agonist and antagonist potential of naturally occurring flavonoids for estrogen receptor (ERα) by various parameters and molecular modelling approach.

Most estrogen receptor α (ERα) ligands target the ligand binding domain (LBD). Agonist 17ß-estradiol (E2) and tamoxifen (TM, known SERM), bind to the same site within the LBD. However, structures of ligand-bound complexes show that E2 and TM induce different conformations of helix 12 (H12). During the molecular modelling studies of some naturally occurring flavonoids such as quercetin, luteolin, myricetin, kaempferol, naringin, hesperidin, galangin, baicalein and epicatechin with human ERα (3ERT and 1GWR), we observed that most of the ligands bound to the active site pocket of both 3ERT and 1GWR. The docking scores, interaction analyses, and conformation of H12 provided the data to support for the estrogenic or antiestrogenic potential of these flavonoids to a limited degree. Explicit molecular dynamics for 50 ns was performed to identify the stability and compatibility pattern of protein-ligand complex and RMSD were obtained. Baicalein, epicatechin, and kaempferol with 1GWR complex showed similar RMSD trend with minor deviations in the protein backbone RMSD against 1GWR-E2 complex that provided clear indications that ligands were stable throughout the explicit molecular simulations in the protein and outcome of naringin-3ERT complex had an upward trend but stable throughout the simulations and all molecular dynamics showed stability with less than overall 1 Å deviation throughout the simulations. To examine their estrogenic or antiestrogenic potential, we studied the effect of the flavonoids on viability, progesterone receptor expression and 3xERE/3XERRE-driven reporter gene expression in ERα positive and estrogen responsive MCF-7 breast cancer cells. Epicatechin, myricetin, and kaempferol showed estrogenic potential at 5 µM concentration.

Estrogen receptors in immunohistochemically stained slides: comparing subjective and computerized evaluation methods / Receptores de estrógeno en cortes teñidos inmunohistoquímicamente: comparación de métodos de evaluación subjetivos y computarizados

Immunohistochemistry is a specific and sensitive staining method for detection of several proteins. One important function of this method is to help us on the diagnosis of the presence of specific receptors as the estrogen receptors. The aim of this study was to compare two different methods to evaluate immunohistochemical staining intensity to detect the presence of ER in the nasal mucosa tissue: one using a digital system and the other through conventional direct microscopy observation. Sixty two stained samples were observed and analyzed under optic microscopy by three specialized professionals, who have graded intensity from: absent, mild, moderate and intense staining. Afterwards, an objective measurement was obtained by a relative optical density (ROD) reading, through the MCID 7.0 system of densitometric digital analysis (Inter Focus Imaging LTDA, Linton, England). Subjective and objective classifications were compared under statistical analysis (Kolmogorov-Smirnov and Spearman Correlation Test, p<0.05). We found a positive correlation between the subjective findings of observers and digital analysis in all the categories of beta receptor. For the alpha receptor, there was a correlation only in extreme categories. The subjective evaluations by observers and digital method by measuring the relative optical density show statistically significant correlations in quantifying the estrogen receptors by immunohistochemistry staining. This indicates that both methods show accuracy for the proposed study.

La inmunohistoquímica es un método de tinción específico para la detección de varias proteínas. Este método es importante en el diagnóstico de la presencia de receptores específicos, tales como los receptores de estrógeno. El objetivo de este estudio fue comparar dos métodos diferentes para evaluar la intensidad de la tinción inmunohistoquímica para detectar la presencia de RE en el tejido de la mucosa nasal: el primero utilizando un sistema digital y el segundo a través de la observación directa de microscopía convencional. Tres profesionales especializados observaron sesenta y dos muestras teñidas las que fueron analizadas con microscopio óptico con la siguiente intensidad de tinción: ausente, leve, moderada e intensa. Posteriormente, se obtuvo una medición objetiva a través de lectura de densidad óptica relativa (DOR) del sistema MCID 7.0 de análisis de densitometría digital (Inter Enfoque de imágenes LTDA, Linton, Inglaterra). Clasificaciones subjetivas y objetivas fueron comparadas bajo análisis estadístico (Kolmogorov-Smirnov y prueba de correlación de Spearman, p<0,05). Encontramos una correlación positiva entre los resultados subjetivos de los observadores y el análisis digital en todas las categorías de los receptores beta. Para el receptor alfa, hubo una correlación solamente en categorías extremas. Las evaluaciones subjetivas de los observadores y método digital midiendo la densidad óptica relativa muestran correlaciones estadísticamente significativas en la cuantificación de los receptores de estrógeno por tinción inmunohistoquímica. Por tanto ambos métodos demostraron ser correctos para el estudio propuesto.

Structural, ultrastructural and immunohistochemical analysis of the vesicular gland in the male greater cane rat (Thryonomys swinderianus)

This study reports the structure, ultrastructure, morphometry and distribution patterns of the two estrogen receptors in the vesicular glands of the male greater cane rat. Samples of vesicular glands from 15 sexually mature male greater cane rats raised in captivity were routinely processed for histological, ultrastructural and morphometric analysis, while immunohistochemistry was also carried out using rabbit polyclonal antibodies against estrogen receptors.The vesicular gland in the greater cane rat is a paired transparent elongated branched tube that presents a characteristic Y-shaped outline. The tube is made up of three histological layers: mucosa, muscularis and adventitia with the mucosa thrown into branching and anastomosing folds that form cavities and recesses within it. Though the epithelium is lined by principal and scarce basal cells, the principal cells are, however, of two types - light and dense based on their electron density and cytoplasmic characteristics. A prominent ultrastructural feature of the light principal cells is the presence of abundant mitochondria surrounded by well-developed cisternae of rough endoplasmic reticulum that have dilated edges and small vesicular extensions. The epithelial cells exhibited different patterns of expressions of estrogen receptor alpha (ERalfa) and estrogen receptor beta (ERbeta). The findings highlight the peculiarities in the structure, ultrastructure and distribution of the estrogen receptors of the vesicular gland of greater cane rat

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Hippocampal dynorphin immunoreactivity increases in response to gonadal steroids and is positioned for direct modulation by ovarian steroid receptors.

The hippocampal formation (HF) is involved in modulating learning related to drug abuse. While HF-dependent learning is regulated by both endogenous opioids and estrogen, the interaction between these two systems is not well understood. The mossy fiber (MF) pathway formed by dentate gyrus (DG) granule cell axons is involved in some aspects of learning and contains abundant amounts of the endogenous opioid peptide dynorphin (DYN). To examine the influence of ovarian steroids on DYN expression, we used quantitative light microscopic immunocytochemistry to measure DYN levels in normal cycling rats as well as in two established models of hormone-treated ovariectomized (OVX) rats. Rats in estrus had increased levels of DYN-immunoreactivity (ir) in the DG and certain CA3 lamina compared with rats in proestrus or diestrus. OVX rats exposed to estradiol for 24 h showed increased DYN-ir in the DG and CA3, while those with 72 h estradiol exposure showed increases only in the DG. Six hours of estradiol exposure produced no change in DYN-ir. OVX rats chronically implanted with medroxyprogesterone also showed increased DYN-ir in the DG and CA3. Next, dual-labeling electron microscopy (EM) was used to evaluate the subcellular relationships of estrogen receptor (ER) alpha-, ERbeta and progestin receptor (PR) with DYN-labeled MFs. ERbeta-ir was in some DYN-labeled MF terminals and smaller terminals, and had a subcellular association with the plasmalemma and small synaptic vesicles. In contrast, ERalpha-ir was not in DYN-labeled terminals, although some DYN-labeled small terminals synapsed on ERalpha-labeled dendritic spines. PR labeling was mostly in CA3 axons, some of which were continuous with DYN-labeled terminals. These studies indicate that ovarian hormones can modulate DYN in the MF pathway in a time-dependent manner, and suggest that hormonal effects on the DYN-containing MF pathway may be directly mediated by ERbeta and/or PR activation.

Subcellular relationships between cholinergic terminals and estrogen receptor-alpha in the dorsal hippocampus.

Cholinergic septohippocampal neurons are affected by circulating estrogens. Previously, we found that extranuclear estrogen receptor-alpha (ERalpha) immunoreactivity in presynaptic profiles had an overlapping distribution with cholinergic afferents in the rat hippocampal formation. To determine the subcellular relationships between cholinergic presynaptic profiles and ERalpha, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and ERalpha and examined by electron microscopy. Within the hippocampal formation, immunoreactivities for VAChT and ERalpha both were presynaptic, although their subcellular targeting was distinct. VAChT immunoreactivity was found exclusively within presynaptic profiles and was associated with small synaptic vesicles, which usually filled axon terminals. VAChT-labeled presynaptic profiles were most concentrated in stratum oriens of the hippocampal CA1 region and dentate inner molecular layer and hilus. In contrast, ERalpha immunoreactivity was found in clusters affiliated either with select vesicles or with the plasmalemma within preterminal axons and axon terminals. ERalpha-immunoreactive (IR) presynaptic profiles were more evenly distributed between hippocampal lamina than VAChT-IR profiles. Quantitative ultrastructural analysis revealed that VAChT-IR presynaptic profiles contained ERalpha immunoreactivity (ranging from 3% to 17%, depending on the lamina). Additionally, VAChT-IR presynaptic profiles apposed ERalpha-IR dendritic spines, presynaptic profiles, and glial profiles; many of the latter two types of profiles abutted unlabeled dendritic spines that received asymmetric (excitatory-type) synapses from unlabeled terminals. The presence of ERalpha immunoreactivity in cholinergic terminals suggests that estrogen could rapidly and directly affect the local release and/or uptake of acetylcholine. The affiliation of cholinergic terminals with excitatory terminals near ERalpha-labeled dendritic spines or glial profiles suggests that alterations in acetylcholine release could indirectly affect estrogen-modulated structural plasticity.

Estradiol and testosterone have opposite effects on microtubule polymerization.

We have reported earlier the purification of tubulin from a plasmalemmal-microsomal fraction derived from rat hippocampus using an estradiol (E(2)) affinity column and the specific binding of tubulin to both E(2) and testosterone (T). To further investigate the effect of E(2) and T on the function of this protein, changes in microtubule polymerization as a result of exposure to the steroids were examined in this study, using both pure tubulin and rat hippocampal primary cell cultures. First, pure tubulin was incubated with or without steroids for 30 min on ice followed by polymerization at 37 degrees C. The numbers of microtubules formed were counted from electron microscopic pictures. The results showed that at 30 min of polymerization, 10 nM, 30 nM and 30 microM of E(2) inhibited microtubule assembly by -70%, -94%, and -92%, respectively (p < 0.01), while T at the same three concentrations stimulated it by +83%, +66%, and +121%, respectively (p < 0.05). The inhibitory effect of E(2) and the stimulatory effect of T were observed at 15, 30 and 60 min of the polymerization process. Next, primary cell cultures from 17-day rat fetus hippocampal tissues were treated with the steroids and polymerized microtubules (Triton X-100 resistant) were examined by immunocytochemistry. The results demonstrated that 60 min of E(2) treatment (10 nM) decreased the intensity of the immunolabeling of polymerized microtubules. The effect of T at nM concentration was not significant though it increased the immunolabeling at microM concentration. Of great significance was a remarkable inhibition by T of the well-established depolymerization effect of colchicine in both the pure tubulin assay and the cell culture model, while E(2) was not effective. In an effort to pursue the possible mechanism(s) of the effect of E(2) and T on microtubule formation, we found that T only inhibited the microtubule depolymerization process without affecting the rate of polymerization. In contrast, E(2) modifies only the polymerization process without altering the depolymerization. Overall, these data indicate that E(2) and T may be considered as novel regulators of microtubule dynamics and thereby controlling cytoskeleton function in cells.

Ependimoma de ovario: comunicación de un caso con receptores de estrógenos y progestágenos / Ovarian ependymoma: A case with estrogen and progestin receptors

Los tres primeros casos de ependimomas puros de ovario se describieron en 1984. En la actualidad al menos se encuentran 12 casos descritos en la literatura inglesa. Presentamos un nuevo caso de ependimoma puro ovárico diagnosticado como hallazgo incidental en una paciente de 44 años de edad a quien se realizó una histerectomía total y anexectomía bilateral por leiomiomas uterinos múltiples. Material y métodos: Sobre la superficie del ovario izquierdo se encontró un nódulo de 2x1,5x1,5 cm, de consistencia sólida y coloración amarillenta. Se realizaron estudios de inmunohistoquímica y microscopia electrónica en el tejido fijado en formol e incluido en parafina. Resultados: El diagnóstico de ependimoma se basó en los hallazgos microscópicos similares a los de los ependimomas papilares dei sistema nervioso central, inmunohistoquimicos como la positividad para GFPA y ultraestructurales como la presencia de microfilamentos intracitoplasmáticos, microvellosidades, cilios y complejos de unión en la superficie. Además, como en los casos descritos previamente, el tumor mostró positividad para receptores de estrógenos y progesterona. Conclusiones: El diagnóstico de ependimoma de ovario puede ser establecido por el patólogo mediante los hallazgos morfológicos e inmunohistoquimicos. La presencia de receptores hormonales ha permitido sugerir la administración de un tratamiento hormonal en los raros casos que se acompañen de recidiva (AU)

Localization of androgen and estrogen receptors in rat and primate tissues.

There is now evidence that estrogens and androgens are exerting their effects in different tissues throughout the body. In order to determine the sites of action of these steroids, studies have been performed to identify at the cellular level the localization of androgen receptor (AR) and the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, specially in the rat, monkey and human. In the prostate, AR was observed in the secretory and stromal cells. In the testis, Sertoli, Leydig and myoid cells were labelled. In the epididymis and seminal vesicles, both epithelial and stromal cells contained AR. In the ovary, AR was detected in granulosa and interstitial cells. In the uterus, epithelial, stromal and muscle cells were all immunopositive for AR. In the central nervous system, AR-containing neurons were found to be widely distributed throughout the brain. In the mammary gland, epithelial cells in acini and ducts and stromal cells were demonstrated to express AR. In the skin, AR was detected in keratinocytes, sebaceous and sweat glands, and hair follicles. In addition, AR was also found in anterior pituitary, thyroid, adrenal cortex, liver, kidney tubules, urinary bladder, cardiac and striated muscle, and bone. The ER subtypes are in general differentially expressed. While ERalpha has been predominantly found in anterior pituitary, uterus, vagina, testis, liver and kidney, ERbeta is predominant in thyroid, ovary, prostate, skin, bladder, lungs, gastro-intestinal tract, cartilage and bone. In tissues which contain both receptor subtypes, such as ovary, testis and various regions of the brain, a cell-specific localization for each ER subtype has been generally observed. Altogether, the recent results on the cellular localization of sex steroid receptors will certainly contribute to a better understanding of the specific role of these steroids in different target organs.

El receptor de estrógenos y la glándula mamaria / Estrogen receptors and the mammary gland

Desde hace varias décadas se sabe que las hormonas esteroides, como la progesterona y el estradiol, son importantes en la regulación de algunos genes involucrados en los procesos de crecimiento, proliferación y diferenciación de las células de la glándula mamaria en diferentes especies animales y en el humano. La presencia o ausencia del receptor de estrógenos es utilizado en la clínica como un marcador de malignidad, evaluación pronóstica, o como parámetro de decisión para el tratamiento con antiestrógenos de algunos tipos de cáncer dependientes de hormonas en la glándula mamaria. La presente revisión tiene por objeto el mostrar algunos de los avances en el conocimiento de la estructura del receptor y su interacción con protooncogenes y factores de crecimiento. Por último, se hace referencia a la relación que tiene la presencia de este receptor en la fisiopatología del cáncer de mama

Localization of estrogen receptor protein and estrogen receptor messenger RNA in peripheral autonomic and sensory neurons.

The presence of estrogen receptor protein and estrogen receptor messenger RNA was revealed in peripheral ganglionic neurons of the rat. The pelvic parasympathetic autonomic ganglion and lumbosacral dorsal root sensory ganglia were examined for estrogen receptor-containing neurons because they have known projections to the uterus and uterine cervix. The vagal nodose ganglia were studied for estrogen receptor-containing neurons because they are suspected sources of influence on the uterus. Immunohistochemistry. in situ hybridization histochemistry and retrograde tracing were utilized. Immunoreactivity for estrogen receptors was evident in the nuclei of a subpopulation of neurons in the pelvic ganglia, sixth lumbar and first sacral dorsal root ganglia and nodose ganglia. Some estrogen receptor-positive neurons also contained the retrograde tracer FluoroGold that previously had been injected into the uterus and uterine cervix. Estrogen receptor messenger RNA was also evident in a subpopulation of ganglionic neurons. These data suggest that a certain population of neurons in autonomic and sensory ganglia are capable of synthesizing estrogen receptors and these receptors can serve as binding sites for estrogen. Thus, certain aspects of the structure, function and neurochemistry of some autonomic and sensory neurons may be influenced by the sex steroid estrogen.

Oestrogen receptor analysis of paraffin sections and cytosol samples of primary breast cancer in relation to outcome after adjuvant tamoxifen treatment. The South Sweden Breast Cancer Group.

The aim of the present study was to compare oestrogen receptor (ER) analysis results obtained in cytosols of frozen breast cancer tissue (using biochemical assay) with those obtained in paraffin-embedded tissue (using immunoperoxidase staining with monoclonal antibodies (DAKO-ER, 1D5), and an ER positivity cut-off level of >10% stained nuclei). In 86% (84/98) of the samples the same ER status (28 negative and 56 positive) was obtained with both procedures. In eight cases, the paraffin section was ER positive but the corresponding cytosol sample ER negative, whereas six cases showed the opposite pattern. The ER positive subgroup manifested better outcome after adjuvant treatment than the ER negative subgroup (p = 0.003 (cytosol), and p = 0.004 (paraffin)). As compared with the percentage of stained nuclei, staining intensity yielded no additional information. Although the results of ER analysis of paraffin-embedded material seem promising, it is too early to prefer it to frozen tissue, though this would be useful when no frozen tissue is available.

Quantitative imaging of estrogen and progesterone receptors, estrogen-regulated protein, and growth fraction: immunocytochemical assays in 52 meningiomas. Correlation with clinical and morphological data.

Quantitative imaging of estrogen receptors (ER's), progesterone receptors (PR's), estrogen-regulated protein (pS2), and growth fraction (Ki67) immunocytochemical assays were performed in 52 meningiomas. The results were correlated with clinical (age, sex, hormonal status, and tumor volume and location) and morphological (histological types and grades) data. The authors observed a lack of ER's in all meningiomas but the presence of PR's in 53% of these meningiomas. The immunoreactivity was restricted to tumor cell nuclei. The PR immunocytochemical assay was correlated with tumor location, histological type, histological grade, and pS2 immunocytochemical assay, but not with Ki67 immunocytochemical assay; high PR content was observed in cisternae, transitional, meningothelial, and low-grade meningiomas. Only 11 meningiomas showed more than 1% Ki67 immunoreactive nuclei. These meningiomas were usually located in the convexity and were of high histological grade. Estrogen-regulated protein immunoreactivity was observed in 34 meningiomas but the number of immunoreactive nuclei was low. The pS2 immunocytochemical assay was not related to clinicopathological features but was preferentially observed in PR-negative meningiomas. The results of this study are compared with those previously reported, and the function and regulation of PR's in meningiomas is discussed. The results indicate that 1) regulation of PR's and pS2 proteins in meningiomas differs from regulation in estrogen-dependent tissues such as breast or endometrium; 2) interruption of hormonal therapy in women presenting with a meningioma is not absolutely necessary; 3) meningiomas have different biological properties according to their clinicopathological features; and 4) future studies of hormonal clinical trials should be performed on well-defined meningioma subgroups.

Solution structure of the DNA-binding domain of the oestrogen receptor.

Steroid hormone receptors control gene expression through binding, as dimers, to short palindromic response elements located upstream of the genes they regulate. An independent domain of approximately 70 amino acids directs this sequence-specific DNA binding and is highly conserved between different receptor proteins and related transcription factors. This domain contains two zinc-binding Cys2-Cys2 sequence motifs, which loosely resemble the 'zinc-finger' motifs of TFIIIA. Here we describe the structure of the DNA-binding domain from the oestrogen receptor, as determined by two-dimensional 1H NMR techniques. The two 'zinc-finger'-like motifs fold to form a single structural domain and are thus distinct from the independently folded units of the TFIIIA-type zinc fingers. The structure consists of two helices perpendicular to each other. A zinc ion, coordinated by four conserved cysteines, holds the base of a loop at the N terminus of each helix. This novel structural domain seems to be a general structure for protein-DNA recognition.

Sexual dimorphism characterizes baboon myocardial androgen receptors but not myocardial estrogen and progesterone receptors.

Using biochemical methods we established that estrogen receptor content and distribution and progesterone receptor content in female and male baboon myocardium did not differ between sexes. In contrast, myocardial androgen receptor distribution between cytosolic and nuclear compartments was sexually dimorphic. Female baboon myocardial androgen receptors were restricted to the cytosolic compartment, whereas male myocardial androgen receptors were distributed between the cytosolic and nuclear compartments. Using human estrogen receptor cDNA we showed that baboon aorta, myocardium and uterus contain a 6.3 kb estrogen receptor transcript. Analyses performed with human progesterone receptor cDNA established that baboon aorta and uterus contain an 8 kb progesterone receptor transcript; however, progesterone receptor transcripts were not demonstrable in baboon myocardial RNA preparations. Because relative hybridization signal intensity reflected known uterine and aortic progesterone receptor content, failure to detect progesterone receptor transcripts in myocardial preparations may reflect sensitivity limitations and the fact that aortic progesterone receptor content is 5-fold greater than that of myocardium. Immunocytochemical analyses demonstrated that baboon myocardial progesterone receptors were present in greater than 25% of myocytes and generally absent from other myocardial cells. Our studies establish that: (1) gonadal steroid hormone receptor gene transcription occurs in cells of the baboon cardiovasculature, (2) these steroid hormone receptors may be physiologically functional, and (3) gonadal steroid hormone receptors may be restricted to specialized cells of the cardiovasculature.

Studies on the non-activated and activated forms of the estrogen receptor.

The activation of the steroid receptor is a necessary process for the biological role of the receptor. Many factors are involved in this mechanism; in addition to time, temperature, and salt concentration, RNA and RNAase can also affect the transformation of the non-activated to the activated form of the receptor. Using as a model the estrogen receptor of fetal uterus of guinea-pig, the studies of the interaction with three different monoclonal antibodies (D547, H222 and H226) reveal structural transformation during the process of the receptor activation. These conformational transformations suggest that a change in the exposure of the functional domains of the estrogen receptor occurs during activation.

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain.

Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement. These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.

Hormonal influences on the middle-affinity estrogen-binding sites in the liver of the newt Pleurodeles waltl.

The effects of hormonal changes on the male-specific, middle-affinity, estrogen-binding component (MEBC) were investigated in the Pleurodele. Induction of MEBC was shown to be under androgen control, similar to that observed for the cytoplasmic middle-affinity estrogen-binding sites in rat liver and human hepatoma cells. But, in contrast to the male-specific middle-affinity estrogen-binding sites identified in the rat, the administration of estrogen to male Pleurodeles did not lead to the disappearance of MEBC but raised levels significantly. The MEBC displays the properties of type II middle-affinity estrogen-binding sites, which are characterized by an oestrogen-dependent rise, a sensitivity to reducing agents, a specificity for diethylstilbestrol, and a binding capacity enhanced by increasing dilutions of cytosol. In female Pleurodeles, MEBC can be induced by treatment with androgens. This induction appears to be modulated by the estrogen/androgen ratio. The induction of MEBC and the estrogen-dependent increase in the male were not found to be correlated with hepatocyte proliferation.

Effect of phospholipids on estrogen receptor of rat uterine cytosol.

Phospholipids such as phosphatidyl inositol and phosphatidyl serine inhibit the binding of R5020 and progestin receptors. The effect of phospholipids on the binding of estrogen and estrogen receptors of rat uterine cytosol was studied. Phosphatidyl choline, sphingomyelin, phosphatidyl inositol, phosphatidyl ethanolamine, cardiolipin, and phosphatidic acid inhibited the binding of estradiol and estrogen receptors. This inhibitory effect of phosphatidyl inositol and cardiolipin was dose dependent.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.