Nuclear Estrogen Binding Sites Research Articles

Nuclear and Extranuclear Estrogen Binding Sites in the Rat Forebrain and Autonomic Medullary Areas

Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating (125)I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting (125)I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of (125)I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting (125)I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received (125)I-estradiol combined with cold estradiol. The association of (125)I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.

Neuroprotection by estrogen in animal models of global and focal ischemia.

Estrogen has been demonstrated to protect against brain injury, neurodegeneration, and cognitive decline. Furthermore, estrogen seems to specifically protect cortical and hippocampal neurons from ischemic injury. Here our data evaluating the neuroprotective effects of estrogens, the selective estrogen receptor modulators (SERMs), and estrogen receptor alpha- and beta-selective ligands in animal models of ischemic injury are discussed. In rats and mice, the middle cerebral artery occlusion (MCAO) model was used as models representing cerebrovascular stroke, while in gerbils the two-vessel occlusion model, resenting acute heart attack, was used. Using focal ischemia in ovariectomized ERalphaKO, ERbetaKO, and wild-type mice, we clearly established that the ERalpha subtype is the critical ER-mediating neuroprotection in mouse focal ischemia. Because of the characteristic blood supply of the gerbil, the gerbil global ischemia model was used to evaluate the neuroprotective effects of estrogen, SERMs, and ERalpha- and ERbeta-selective compounds in the hippocampus. Analysis of neurogranin mRNA, a marker of viability of hippocampal neurons, with in situ hybridization, revealed that estrogen treatment resulted in a complete protection in the CA1 regions not only when administered before, but also when given 1 hour after occlusion. Our in vivo binding studies with (125)I-estrogen in gerbils revealed the presence of nuclear estrogen binding sites primarily in CA1 neurons, but not in the CA3 region, as we saw in rats and mice. Together, these observations demonstrate that estrogen protects from ischemic injury in both the focal and global ischemia models by acting primarily via classical nuclear receptors.

Estrogen prevents the loss of CA1 hippocampal neurons in gerbils after ischemic injury

Estrogen replacement therapy is thought to attenuate the incidence of Alzheimer’s disease in women and enhance cognitive functions. In rodents, estrogen protects cerebral cortical neurons from ischemic injury and cultured neurons from a variety of perturbations. Because few nuclear estrogen receptors have been detected in the dorsal hippocampus, the present studies used a global ischemia model to evaluate the neuroprotective actions of estrogen in this region. Ovariectomized gerbils were treated with placebo, 0.5 mg or 1 mg pellets of estradiol for 13 days. On day 7, the common carotid arteries were occluded for 5 min and on day 13 the animals were killed. Analysis of neurogranin mRNA, a marker of hippocampal neurons, with in situ hybridization revealed a dramatic and selective loss of CA1 neurons in the placebo-treated ovariectomized gerbils, whereas both 0.5 mg and 1 mg pellets of 17β-estradiol prevented cell loss. Subsequent studies showed that a variety of estrogens, including diethylstilbestrol, estrone and 17α-estradiol as well as vitamin E, also protected CA1 neurons from ischemic injury in ovariectomized gerbils, whereas treatment with the estrogen antagonist tamoxifen was ineffective. The results of in vivo binding studies with 17α-iodovinyl-11β-methoxyestradiol revealed a concentration of nuclear estrogen binding sites in the CA1 region of the ovariectomized gerbil brain, whereas binding in other hippocampal regions was limited. Moreover, the binding studies revealed that the regional pattern of binding was not altered after ischemic injury, with the exception of the hippocampus, where the binding sites were attenuated in ovariectomized animals with time after ischemic injury. Together, these data demonstrate that a variety of steroidal and non-steroidal estrogens are potent neuroprotective agents in an animal model of global ischemia, agents that protect neurons critical for learning and memory and susceptible to neurodegenerative diseases.

Chapter 2 Estrogen modulates oxytocin gene expression in regions of the rat supraoptic and paraventricular nuclei that contain estrogen receptor-β

Oxytocin is an important modulator of female reproductive functions including parturition, lactation and maternal behavior, while vasopressin regulates water balance and acts as a neurotransmitter. For decades, it has been suggested that estrogen regulates the production and/or release of oxytocin and vasopressin in the rodent brain. Although several studies demonstrated that estrogen can modulate vasopressin mRNA levels in regions known to contain estrogen receptor (ER), such as the bed nucleus of the stria terminalis and medial amygdala, data from the paraventricular and supraoptic nuclei were inconclusive. Since early immunohistochemical and in situ hybridization studies revealed few, if any, ER containing cells in these hypothalamic nuclei, it was thought that oxytocin and vasopressin were not directly regulated by estrogen. The discovery of a second ER (ER-beta) in the late 1990s suggested that estrogen could act in many brain regions heretofore not considered targets for estrogen action. Initial in situ hybridization studies revealed a wide distribution of ER-beta mRNA in the rat brain including neurons of the supraoptic nucleus and the parvocellular and magnocellular divisions of the paraventricular nucleus. Subsequent double-label in situ hybridization/immunocytochemistry studies showed that ER-beta mRNA was present in oxytocin and vasopressin neurons, with the degree of colocalization being both neuropeptide and region specific. In an attempt to demonstrate that ER-beta mRNA was translated into a biologically active protein, a series of in vivo binding studies were conducted in rats with 125I-estrogen. These data revealed the presence of nuclear estrogen binding sites in neurons of the magnocellular system indicating that ER-beta mRNA was translated into protein. Concurrent studies in mice found that the distribution of ER-beta mRNA and 125I-estrogen binding was similar to rats, although there were some notable differences. For example, ER-beta mRNA and binding were not detected in the mouse supraoptic nucleus and although ER-beta was the principle ER in the paraventricular nucleus, ER-alpha was also present. The prevalence of ERs in the mouse paraventricular nucleus was further investigated using ER-alpha and ER-beta knockout mice for in vivo binding studies with 125I-estrogen. The results of these studies showed that ER-beta was the predominant ER in the paraventricular nucleus and confirmed the presence of ER-beta in other brain regions. Moreover, our group recently generated and characterized several polyclonal antisera raised against the C-terminus of ER-beta. Through the use of these antisera, we have confirmed the presence of ER-beta in the rat paraventricular and supraoptic nuclei and shown that ER-beta is colocalized, in part, with oxytocin and vasopressin. To assess the ability of estrogen to modulate the expression of oxytocin mRNA, ovariectomized rats were treated with vehicle or estradiol and the brains processed for in situ hybridization. The results of these studies revealed that estradiol down-regulated oxytocin mRNA in the rat paraventricular nucleus within 6 h of treatment. Together these data and the observation that some of the oxytocin and vasopressin neurons contain ER-beta suggest that estrogen, acting through ER-beta, may directly regulate oxytocin gene expression. However, since the paraventricular nucleus has many subdivisions with different projections and the degree of colocalization of ER-beta with oxytocin/vasopressin varies among subdivisions, the effects of estrogen treatment on gene expression requires further study to ascertain the role of estrogen action in this neuronal systems.

Evidence that Estrogen Binding Sites are Present in Bone Cells and Mediate Medullary Bone Formation in Japanese Quail

Studies were carried out in Japanese quail to characterize the binding of estrogen to bone cells and to determine whether induction of medullary bone by estrogen is mediated by estrogen receptors. First, attempts were made to identify specific, high affinity nuclear binding sites for estrogen with a nuclear exchange assay in oviduct and femora from laying female quail and in liver, kidney, and femora from adult male quail treated with estradiol valerate (2 mg per bird). High-affinity nuclear estrogen binding sites were detected in each of the tissues except femora. Second, estrogen binding sites in femora from male quail were localized by radioautography after treatment with 100 μCi of [3 H]-17β-estradiol ([3 H]-E). Estrogen binding sites were present at 1 and 3 h after administration of [3 H]-E, and binding of the radiolabeled hormone was prevented by the simultaneous administration of an excess of radioinert estrogen. Third, estradiol valerate (.4 mg) was given as a bolus to adult male Japanese quail. Five days later, the quail had hypercalcemia due to accumulation of phosvitin in serum, had an extensive network of medullary bone at the femur midshaft, and had reduced cortical bone area. The nonsteroidal, anti-estrogen tamoxifen (2 mg/day) prevented estrogen-induced hypercalcemia, medullary bone formation, and reduction of cortical bone area. Fourth, the estrogen target cells in femora from adult male quail that were induced to differentiate to osteoblasts by estrogen treatment were located by [3 H]-proline radioautography as early as 12 h following administration of the hormone. These results are interpreted as evidence that induction of medullary bone is a process mediated by estrogen receptor.

Nuclear Type II Estradiol Binding Sites and Type I Estrogen Receptors in Human Endometrial Cancer: A 5-Year Follow-Up Study

Type II nuclear-estrogen binding sites (type II EBS) have been postulated to play a crucial regulatory role in estrogen-stimulated uterine growth. The pathogenesis of endometrial cancer is known to be connected to estrogens. However, there are no data on type II EBS in human endometrial cancer. Therefore, we investigated the presence of nuclear type II EBS and nuclear type I estrogen receptors (ER) by radioligand-binding assays in endometrial tumorous tissues ( n = 135). Determination of cytoplasmic ER and progesterone receptor (PR) concentrations were also included in the study. Estradiol (E 2) and progesterone (P) hormone levels in sera of patients were determined by RIAs. In addition, all data were analyzed on 5-year survival rates. Saturation analyses of 3H-E 2 binding in crude nuclear pellets ( n = 5) showed two types of estradiol binding: type I ER having high affinity and low capacity and type II EBS binding estradiol with lower affinity and high capacity in a positive cooperative fashion. The nuclear type II EBS and type I ER concentrations were significantly higher in cancers of increasing grade. In contrast, a significant decrease of cytoplasmic 3 H-E 2 binding was detected. Cytoplasmic PR binding capacities were high in G1 and G2, but low in G3 tumors. The 5-year survival data showed nuclear type I ER to have the best correlation for prognostic value (cut-off, 0.5 pmol/mg DNA), while type II EBS had no significant impact on it. Serum E 2 concentrations decreased with tumors of higher grade, but were generally still higher than the control values. The serum P levels did not alter. None of the parameters investigated here differed from control values in adenoacanthoma ( n = 6), suggesting a separate pathomechanism. As type II EBS are known to be stimulated by E 2 under conditions that cause uterine hyperplasia, we concluded that the higher "runaway" nuclear type II EBS levels, in contrast with the lower serum estradiol concentrations, might be connected with the strong proliferative and invasive character of higher grade endometrial adenocarcinomas. This is the first demonstration of the presence of nuclear type II EBS and their possible pathological role in human endometrial cancer, and of the high prognostic significance of nuclear type I ER to identify a subgroup having a fatal form of the disease in a 5-year survival study

Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins.

The stimulation of chicks or embryos with estrogen results in transient, hepatic expression of the vitellogenin gene, as well as long-term, propagatable alterations in the rapidity with which the gene can be reactivated. We examined the possibility that nuclear, type II estrogen-binding sites are involved in this long-term change in response characteristics. We demonstrate that the primary induction kinetics of type II sites in embryos and chicks correlated with the expression of the vitellogenin gene and that once their induction was triggered by estrogen, they accumulated, were propagated, and persisted for months after withdrawal of the hormone. We also show that their accumulation in the embryo was accompanied by prolonged expression of both the vitellogenin and very low-density apolipoprotein II genes, in the absence of elevated levels of type I receptor, and that the type II sites, like the classical receptor, appear to be preferentially associated with active or potentially active chromatin. Finally, we describe a regulatory mechanism, tested by computer modelling, that simulated the behavioral characteristics of these nuclear estrogen-binding sites and which may explain their role in mediating the long-term effects of estrogen.

Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins.

The stimulation of chicks or embryos with estrogen results in transient, hepatic expression of the vitellogenin gene, as well as long-term, propagatable alterations in the rapidity with which the gene can be reactivated. We examined the possibility that nuclear, type II estrogen-binding sites are involved in this long-term change in response characteristics. We demonstrate that the primary induction kinetics of type II sites in embryos and chicks correlated with the expression of the vitellogenin gene and that once their induction was triggered by estrogen, they accumulated, were propagated, and persisted for months after withdrawal of the hormone. We also show that their accumulation in the embryo was accompanied by prolonged expression of both the vitellogenin and very low-density apolipoprotein II genes, in the absence of elevated levels of type I receptor, and that the type II sites, like the classical receptor, appear to be preferentially associated with active or potentially active chromatin. Finally, we describe a regulatory mechanism, tested by computer modelling, that simulated the behavioral characteristics of these nuclear estrogen-binding sites and which may explain their role in mediating the long-term effects of estrogen.

The role of estrone in porcine blastocyst development

In target tissues for estrogen, including breast cancer cells, the synthesis of progesterone receptors (PRs) is controlled by estradiol acting through estrogen receptors (ERs). We describe studies with T47D human breast cancer cells, whose PRs are not regulated by estradiol, though present in extraordinary amounts (300,000 sites per cell). These cells have no ERs sedimenting at 8S on sucrose density gradients, and no unfilled cytoplasmic or nuclear ERs; some apparently hormone-filled nuclear sites, with KD ⋍ 0.7 nM, can be demonstrated by exchange. The nuclear ER sites are not processed after estradiol treatment. Nafoxidine, however, doubles nuclear estrogen binding in 6 hr, in a cycloheximide-insensitive step that may represent a reversal of processing. T47D cells are profoundly resistant to estrogens and antiestrogens; estradiol does not stimulate PRs, and nafoxidine concentrations that are cytotoxic to ER-positive cells have no effect on cell growth or on PR levels. Yet the PRs are normal by several criteria, and they can be stoichiometrically translocated to, and extracted from, nuclei in the first 3 min after progesterone addition. If progesterone treatment exceeds 10 min, rapid nuclear turnover prevents quantitative PR recovery. Cytoplasmic PRs are replenished in 10 to 24 hr, and this cycloheximide-sensitive step is also estrogen- and nafoxidine-resistant. However, despite their insensitivity to estradiol or antiestrogen, PRs are not constitutively synthesized; 5-bromodeoxyuridine and sodium butyrate can selectively inhibit PR production. Thus, since PRs retain some characteristics of inducible proteins, the persistent nuclear estrogen-binding sites may be stimulating PRs continuously, even in the absence of exogenous estradiol.

Immunohistologic detection of estrogen receptors in paraffin-embedded breast cancers: Correlation with cytosol measurements

Paraffin-embedded sections of 76 human breast tissue specimens were analyzed for estrogen receptors (ER) and endogenous bound estrogen (ER-E). Preincubation of sections with polyestradiol phosphate was followed by stabilization of the complex with glutaraldehyde. The bound hormone was then visualized by the peroxidase-antiperoxidase (PAP) technique with antiestradiol as the primary antiserum. Normal breast tissue and benign proliferations were consistently positive for ER and ER-E. All specimens were examined for free and bound receptors in cytoplasm and nuclei. Among the carcinomas examined, a high correlation was found between the presence of ER by the PAP method and by the biochemical analysis of cytosol preparations. The PAP method, requiring no special preparation of surgical specimens, overcomes many of the disadvantages of the cytosol method and adds the advantage of independent evaluation of nuclear and cytoplasmic estrogen binding sites.

Kinetics of estrogen-dependent modulation of apolipoprotein A-I synthesis in human hepatoma cells.

Apolipoprotein (apo) A-I is the principal protein component of high density lipoproteins and the major in vivo activator of lecithin-cholesterol acyltransferase. We have used the human hepatoma cell line, HepG2, as a model to examine the ability of estrogen to modulate hepatic synthesis of apo-A-I. Primer extension studies have demonstrated that the major transcriptional initiation site of the apo-A-I gene utilized in the hepatoma cells is the same as that used in human liver. The kinetics of induction of high- and low-affinity estrogen-binding sites, rates of secretion of apolipoproteins, and apo-A-I mRNA levels were examined following treatment of the cells with estrogen. Initial concentrations of 20 nM 17 beta-estradiol resulted in a 14-15-fold increase in the levels of high-affinity nuclear estrogen-binding sites within 8 h, while the level of low-affinity sites increased by only 10%. During the same period, the levels of apo-A-I mRNA and the rate of accumulation of the secreted protein increased by 55 and 50%, respectively. New steady state levels of apo-A-I mRNA and rates of accumulation of protein, approximately twice those in control cultures, were established within 24-48 h of exposure to hormone. Experiments with a 50-fold higher concentration of estrogen resulted in only an additional 10% increase in mRNA levels. The increase in mRNA levels following estrogen treatment was adequate to account for 85-90% of the elevation observed in the rate of accumulation of secreted apo-A-I. Comparison of the apo-A-I mRNA levels in HepG2 cells with those present in human liver revealed that the concentration of the mRNA was approximately 3-fold lower than that found in vivo.

An endogenous ligand for type II binding sites in normal and neoplastic tissues.

The rat uterus contains two classes of specific nuclear estrogen binding sites which may be involved in estrogen action. Type I sites represent the classic estrogen receptor (Kd 1 nM) and type II sites (Kd 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]-estradiol exchange results in an increase (3-4 fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogenous inhibitor of [3H]-estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20-beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggest the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both alpha and beta inhibitor components whereas estrogen-induced rat mammary tumors contain very low to non-measurable quantities of the beta-peak inhibitor activity.

Multiple estrogen binding sites in malignant mouse leydig cells and their role in cell proliferation

Incubation of dispersed cells derived from an estrogen-responsive mouse Leydig cell tumor with [ 3 H]estradiol in the presence or absence of unlabeled estradiol showed the existence of two types of binding components with high ( K d ∼ 10 −9 M) and low ( K d ∼1.5 × 10 −8 M) affinity respectively. The use of unlabeled diethylstilbestrol as a competitor, however, abolished the low-affinity binder, resulting in the demonstration of the high binding site. This diethylstilbestrol-suppressible binding site was exclusively located in the nuclear fraction, even without hormonal stimuli. A long (5-hr) exposure of these cells to estrogen caused the decrease in the number of nuclear estrogen binding sites, which was similar to so-called ‘processing’ of putative nuclear estrogen receptor. The direct stimulative effect of estrogen on the proliferation of these cells was demonstrated by measuring cell numbers as well as thymidine incorporation into DNA in the cultured condition. These results would indicate that estrogen directly exerts its effects on this malignant Leydig cell through this unique nuclear binding site.

The binding of estrogen to liver nuclei from Japanese quail

Specific binding sites for estrogen were characterized in liver nuclei from adult Japanese quail. A single class of high-affinity binding sites ( K D = 2 × 10 −10 M) was revealed by saturation analysis using an exchange assay. Estradiol (E 2), estrone, and estriol competed effectively with [ 3H]E 2 for the nuclear binding sites but progesterone and testosterone were ineffective. The concentration of sites was 0.15 ± 0.05 pmol/mg DNA (mean ± 1 SD) in sexually immature females and 0.47 ± 0.11 pmol/mg DNA in layers. Nuclear estrogen binding sites were not detected in liver nuclei from male Japanese quail. However, after treatment of male birds with E 2 there was a dose-dependent accumulation of binding sites. The maximum number of occupied sites was obtained with 50 ng E 2/g body wt and equaled that found in laying females. The estrogen binding sites were retained in nuclei of estrogentreated male quail with a T 1 2 of 2.5 hr. Thus, an estrogen binding component was characterized in quail liver nuclei that had features consistent with an estrogen receptor, including high affinity, limited capacity, structural specificity, tissue specificity, nuclear translocation, and nuclear retention.

Nuclear Estrogen Binding Sites in Human Endometriosis

With a technique of in vitro steroid autoradiography, the localization of nuclear estrogen binding sites has been studied in ovarian endometriotic foci from untreated patients in both the follicular and luteal phases of the cycle. Unlike the uterine endometrium, which displays cyclic changes in estrogen binding sites, the endometriotic foci show no such changes in the localization of estrogen binding sites. Throughout the cycle, a marked degree of estrogen binding is present in the stromal cells of the endometriotic foci, while in the uterine endometrium stromal binding sites are seen only during the proliferative phase and not during the secretory phase of the cycle. The glandular epithelium of the endometriotic foci displays a patchy localization of nuclear estrogen binding sites at all stages, while the glandular epithelium of the uterus is strongly positive during the proliferative phase but displays no estrogen binding sites during the secretory phase of the cycle. Thus, the endometriotic foci appear to respond differently to ovarian hormones, both in terms of the modulation of estrogen binding and in terms of glandular histology.

The autoradiographic demonstration of estrogen binding in normal human cervix and vagina during the menstrual cycle, pregnancy, and the menopause.

Using the technique of in vitro steroid autoradiography, the localization and modulation of nuclear estrogen binding sites has been studied in normal human cervix and vagina during the menstrual cycle, pregnancy, and the menopause. Marked differences occur in nuclear estrogen binding between these two organs. Nuclear estrogen binding varies throughout the menstrual cycle in the vaginal epithelium, whereas vaginal stromal cells consistently exhibit nuclear estrogen binding throughout the cycle. In contrast, the cervical squamous and columnar epithelia show much less cyclic variability in nuclear estrogen binding sites. As in the vagina, the cervical stroma consistently binds estrogen. High levels of nuclear estrogen binding sites are found in the vagina of the postmenopausal patient, and lower levels of binding occur postpartum. The implications of these localizations, with special reference to the role of the cervical and vaginal stroma, are discussed.

Autoradiographic demonstration of high affinity nuclear binding and finite binding capacity of [3H]estradiol in mouse vaginal cells.

Vaginae from adult C57Bl/6J mice were analyzed for nuclear estrogen-binding sites by biochemical as well as in vitro steroid autoradiographic methods. Finite binding capacity (saturability) and high affinity binding were demonstrated in stromal cell nuclei (Kd = 1.0 nM) by autoradiographic methods and in nuclear extracts of vaginal homogenates (Kd = 1.9 nM) by biochemical techniques. The results of this study demonstrate that all criteria considered definitive for estrogen receptors can be met by autoradiographic analysis, which makes feasible the assessment of estrogen receptor activity in the individual cell types that comprise fetal and neonatal estrogen target organs.

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei.

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.

Nuclear estrogen binding sites in human endometriosis

With a technique of in vitro steroid autoradiography, the localization of nuclear estrogen binding sites has been studied in ovarian endometriotic foci from untreated patients in both the follicular and luteal phases of the cycle. Unlike the uterine endometrium, which displays cyclic changes in estrogen binding sites, the endometriotic foci show no such changes in the localization of estrogen binding sites. Throughout the cycle, a marked degree of estrogen binding is present in the stromal cells of the endometriotic foci, while in the uterine endometrium stromal binding sites are seen only during the proliferative phase and not during the secretory phase of the cycle. The glandular epithelium of the endometriotic foci displays a patchy localization of nuclear estrogen binding sites at all stages, while the glandular epithelium of the uterus is strongly positive during the proliferative phase but displays no estrogen binding sites during the secretory phase of the cycle. Thus, the endometriotic foci appear to respond differently to ovarian hormones, both in terms of the modulation of estrogen binding and in terms of glandular histology.

Autoradiographic analysis of nuclear estrogen binding sites during postnatal development of the genital tract of female mice

Autoradiographic analysis of [ 3H]-estrogen nuclear binding sites was performed on developing genital tracts (uterus, cervix and vagina) of mice 1 to 90 days postpartum. During days 1 to 15 postpartum, nuclear estrogen binding sites were observed exclusively within mesenchymal cells; epithelial cells did not exhibit nuclear labelling. At 18 days postpartum vaginal and cervical epithelial cells exhibited nuclear estrogen binding sites for the first time, whereas the initial appearance of estrogen receptor activity in the epithelium of the uterus was detected at 20 days postpartum. Thereafter, nuclear estrogen binding sites were maintained in both epithelial and stromal cells into adulthood. The acquisition of nuclear binding sites within epithelium of female genital organs at 18 days is discussed in terms of epithelial-mesenchymal interactions and the acquisition of growth responsiveness.