Cytoplasmic Estrogen Receptor Research Articles

Estrogen, Efferent Ductules, and the Epididymis

Estrogen's presence in the male reproductive system has been known for over 60 years, but its potential function in the epididymis remains an important area of investigation. Estrogen is synthesized by germ cells, producing a relatively high concentration in rete testis fluid. There are two estrogen receptors (ESR), the presence of which in the head of the epididymis is well documented and consistent between species; however, in other regions of the epididymis, their expression appears to be isotype, species, and cell specific. ESR1 is expressed constitutively in the epididymis; however, its presence is downregulated by high doses of estrogen, making the design of experiments complicated, as the phenotype of the Cyp19a1(-/-) mouse does not resemble that of the Esr1(-/-) mouse. Ligand-independent and DNA-binding Esr1 mutant models further demonstrate the complexity and importance of both signaling pathways in maintenance of efferent ductules and epididymis. Data now reveal the presence of not only classical nuclear receptors, but also cytoplasmic ESR and rapid responding membrane receptors; however, their importance in the epididymis remains undetermined. ESR1 regulates ion transport and water reabsorption in the efferent ducts and epididymis, and its regulation of other associated genes is continually being uncovered. In the male, some genes, such as Aqp9 and Slc9a3, contain both androgen and estrogen response elements and are dually regulated by these hormones. While estrogen pathways are a necessity for fertility in the male, future studies are needed to understand the interplay between androgens and estrogens in epididymal tissues, particularly in cell types that contain both receptors and their cofactors.

Abstract 5652: Cytoplasmic expression of estrogen receptors in breast cancers

Abstract Nuclear expression of estrogen receptor(ER)-A has been used as the prognostic and therapeutic marker for breast cancer. However, ER cytoplasmic expression (CyE) has not gained such support for its contribution to estrogenic response and clinical significance. Membrane ER or unliganded cytoplasmic hormone receptors may represent non-genomic actions of ER or subcellular dislocation during malignant transformation due to inappropriate /dysfunctional trafficking of transcription factors and cofactors. Here, we tested CyE of ERs in breast cancer tissues and breast cancer cell lines (BCC) to further define its role. Design; Tissue microarray sections of 271 cancers, 45 normals, 62 benign proliferative disease (BPD) of breast tissues and 11 BCC (MCF-7, T-47D, ZR-75, MB-361,MB 468, BT 474, MB-453, SK-BR-3,MB-231, Hs578T, BT-549) were tested by immunohistochemistry for the expression of ER-A (ID5, Dako), 5 different clones of ER-B (AR385-5R, Biogenex; PPG5/10, Dako;Ab288/14C8, ABCAM; EMR02, Novacastra; 57/3, AbD Serotec), Her-2 (HercepTest, Dako), Ki-67, AIB-1 (Clone 34, BD), TIF-2 (Clone 29, BD). Non-specific protein and peptide antigen were blocked by using serum block (Dako, CA) and peptides, respectively. Over 5% positive nuclear and cytoplasmic expression in each tissue core was considered positive. Results: ER-A-CyE was noted in 9.0% (27/271) of breast cancers; 6 were ER-A-/Her-2+, and 3 were ER-A+/Her-2+. One BCC, BT474 with ER-A+/Her-2+ showed ERA-CyE. ER-B-CyE was noted in 29.1 % (81/271) of cancers and 4 of 11 BCC. CyE of 5 ER-B clones were consistent expression in each breast tissue and BCC. The staining of ER-B-CyE in BCC showed diffuse, membranous or dotted appearance. In breast cancer, that of ER-B-CyE was diffuse and was prominent in high grade cancer with desmoplastic reaction and heavy lymphocytic infiltration. ER-B-CyE was noted in 47.0 % (47/100) of stage IV cancer including recurrent and tamoxifen resistant cancers, and 19.2 0% (33/171) stage I to III cancers. ER-B-CyE was associated with over expression of Her-2 in 22.2% (18/81), Ki-67 in 23.4% (19/81), TIF-2 in 46.5% (38/81) and AIB-1 in 21.0% (17/81). Only cytoplasmic ER-A and ER-B in breast cancer was noted in 3.3% (9/271) and in 8.4% (23/271), respectively. CyE levels in normals and BPD tissues were insignificant. Conclusion: Our study demonstrates the potential significance of cytoplasmic ER, particularly ER-B. Proteins translated from single and multiple exon deletion variant transcripts which lack nuclear localization signals may remain in the cytoplasm of breast cancer tissues and contribute to estrogen responses. Further study is in progress to test cytoplasmic expression of various ER isoforms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5652.

Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation

To determine the frequency of estrogen receptor alpha and beta and progesterone receptor protein immunohistochemical expression in a large set of non-small cell lung carcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports. Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors alpha and beta and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients' clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status. Estrogen receptors alpha and beta were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for alpha and 42-56% for beta; cytoplasm: <1-42% for alpha and 20-98% for beta) varied among the different antibodies tested. Progesterone receptor was expressed in the nuclei of malignant cells in 63% of the tumors. Estrogen receptor alpha nuclear expression significantly correlated with adenocarcinoma histology, female gender, and history of never smoking (P = 0.0048 to <0.0001). In NSCLC, higher cytoplasmic estrogen receptor alpha expression significantly correlated with worse recurrence-free survival (hazard ratio, 1.77; 95% confidence interval, 1.12, 2.82; P = 0.015) in multivariate analysis. In adenocarcinomas, estrogen receptor alpha expression correlated with EGFR mutation (P = 0.0029 to <0.0001). Estrogen receptor beta and progesterone receptor but not estrogen receptor alpha expressed in the normal epithelium adjacent to lung adenocarcinomas. Estrogen receptor alpha and beta expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor alpha expression correlates with EGFR mutations.

Effect of Estrogen-Receptor Interaction on the Levels of Fatty Acid-Binding Proteins and Spectra of Free Acids in Small Intestinal Mucosa of Rabbits

Data on the possibility of estrogens to regulate absorption of free fatty acids (FFA) during absorption of lipids in small intestine with associated estrogen-receptor interaction and changes in fatty acid-binding protein (FABP) levels are reported. In particular, characteristics of cytoplasmic estrogen receptors, estradiol metabolism, FABP levels and spectra of FFA in FABP fraction were studied in small intestinal mucosa of sexually mature and immature female rabbits, intact or after ovariectomy and estradiol dipropionate administration. The dissociation constants (Kd) and number of binding sites were determined in coordinates of Scatchard. FABP levels were demonstrated spectrophotometrically after ultracentrifugation (K-32M) and gel filtration (G-75). FFA composition of FABP fraction, chyme and enterocytes were analyzed by a chromatographic method (Tsvet-110). Levels of body estrogens were monitored by high-performance liquid chromatographic analysis (Tsvet-306) of estrogens in daily urine. The study has demonstrated a specific relation between FABP levels and their lipid component, FFA, on the one hand and endo- and exogenous estrogen levels on the other hand, in female rabbits maintained on a standard fat-free diet.

Prolactin regulation of estrogen and progesterone receptors in normal and neoplastic mouse mammary tissue.

The transplantable mouse mammary tumor, TPDMT-4, is pregnancy-dependent and requires prolactin (PRL), estradiol (E2) and progesterone (Pg) for growth. To examine the role of PRL in regulating tissue growth and the levels of estrogen receptor (ER) and progesterone receptor (PgR), tumor-bearing mice were ovariectomized, hysterectomized and then injected with ergocornine hydrogenmaleate (ERG), ERG + PRL, or ERG + PRL + E2 + Pg. Total (nuclear + cytoplasmic) ER and PgR in normal and neoplastic mammary tissues were measured. In addition, tumor size and tritium-labeled thymidine [( 3H]dThd) incorporation into nuclei of the tumor and mammary gland were determined. PRL alone caused a 2- to 3-fold increase in ER and PgR levels in normal mammary gland but not in the tumor. PRL alone caused a modest increase in the number of 3H-thymidine-labeled nuclei in both tissues. PRL combined with E2 and Pg increased the percent of labeled nuclei 5- to 10-fold in both tissues, and increased the PgR levels in normal but not in tumor tissue. Thus, PRL alone can increase ER in normal mammary tissue but this increase is not required for growth since ER levels are unchanged when PRL + E2 + Pg are injected and mammary cell growth is stimulated. The ability of PRL to up-regulate ER has been lost in the tumor. Since basal levels of ER and PgR are not altered in the tumor when PRL + E2 + Pg are given, an increase in ER and PgR levels is not required for the three hormones to stimulate tumor growth.

Evidence for the hormone dependency of hepatic hyperplastic nodules: inhibition of malignant transformation after exogenous 17 beta-estradiol and tamoxifen.

Hepatic hyperplastic nodules (HHNs) in rats were studied as an experimental prototype of oral contraceptive-related hepatic tumors. We have found cytoplasmic estrogen receptors in HHNs produced by acetylaminofluorene (AAF) (four cycles of 0.02% in diet). Rats with AAF-induced HHNs were randomized into four groups: (i) AAF-treated control; (ii) estrogen alone (estradiol-17 beta); (iii) tamoxifen alone, and (iv) estrogen + tamoxifen. After 8 months of treatment with estrogen (estradiol-17 beta) in combination with tamoxifen, there was regression of nodular involvement and no evidence of malignant transformation. Decreased nodular proliferation also occurred after 2 and 4 months treatment with estradiol-17 beta and after 8 months of tamoxifen administration. The incidence of hepatocellular carcinoma after 8 months of treatment was significantly less after treatment with estrogen (40%) or tamoxifen (42.9%) when compared to AAF-treated controls (87.5%). The number of gamma-glutamyltranspeptidase-positive foci were reduced in all treatment groups after 2, 4, and 8 months of treatment; these changes were most pronounced in the estrogen-treated group and did not directly correlate with the per cent inhibition of malignant transformation. Our results suggest that the malignant transformation of estrogen receptor-positive HHNs is hormone dependent.

O-34 Nuclear and cytoplasmic ER beta2 expression identifies distinct prognostic outcome in breast cancer patients

Cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma overexpressed gene (CCN) comprise a family of matricellular proteins that have multiple physiologic functions including development, tissue repair, cell adhesion, migration, and proliferation. The expression of CCN1, cyclin D1, β-catenin, and p53 was explored by immunohistochemistry in different grades of ductal carcinoma in situ (DCIS) cases. These cases did not contain any infiltrating carcinoma components. In addition, all cysteine-rich protein 61 gene exons (encoding the CCN1 protein) were sequenced in 30 samples. Allred and H-scores were calculated for expression in both DCIS and the surrounding benign breast tissue. All cases of DCIS showed degrees of cytoplasmic CCN1 staining with median H-scores of 170, 160, and 60 in grades 3, 2, and 1, respectively (P = .043). Twelve of 28 DCIS 3, 1 of 15 DCIS 2, and 0 of 18 DCIS 1 also showed nuclear staining for CCN1. The cytoplasmic staining difference was preserved when the cases were divided into estrogen receptor (ER)+/DCIS grade 1, ER+/DCIS 2 and 3, and ER−/DCIS 2 and 3 by the H-score (P = .037). Cyclin D1 expression was positively correlated with the CCN1 cytoplasmic H-score in all DCIS samples (P = .038). Membranous β-catenin expression correlated with the grade of intraepithelial carcinoma by both H-score (P = .047) and Allred score (P = .026). Our results suggest that CCN1 has a role in the development of intraepithelial carcinoma. CCN1 expression correlates with grade of DCIS independent of ER status. It can induce cell cycle progression through cyclin D1. It is warranted to study high expression of CCN1 in DCIS as an independent risk factor in a larger cohort.

Novel oestrogen receptor GPR30: Brain localisation and changes in expression during the rat oestrus cycle

Circulating levels of oestrogen are known to modulate autonomic function via the conventional nuclear oestrogen receptors (ERα and ERβ) localised in several areas of the brain. Recently several lines of evidence suggest that the orphan G protein-coupled receptor GPR30 may act as a cytoplasmic oestrogen receptor. This study was designed to examine the expression and localisation of GPR30 in the brains of male and female rats using immunohistochemistry on fixed brain sections with a rabbit antibody to GPR30 and reverse transcription polymerase chain reaction (RT-PCR) on tissue punches.

A study of cytoplasmic oestrogen receptors in patients with breast cancer in a district general hospital.

The tumors of 61 patients suffering from primary adenocarcinoma of the breast were assayed for oestrogen receptors (ER), using the dextran-coated charcoal method. A statistically significant relationship (P less than 0.05) was found to exist between ER status and the histological grade of the tumour. No relationship was found between ER status and either tumour size or the pathological state of the axillary lymph nodes. It is concluded that it is a practical proposition to establish an ER assay service in a district general hospital, with subsequent benefits for the individual patient.

Non-nuclear estrogen receptor ? and ? in the hippocampus of male and female rats

Estrogens play important roles in the brain, acting through two receptor types, ERalpha and ERbeta, both recognized as transcription factors. In this study, we investigated the ERbeta mRNA and protein expression in the male and female rat brain, focusing on the hippocampus, and comparing with well-known ERalpha expression patterns. Extranuclear ERbeta localization, as shown by light microscopic immunocytochemistry and tissue fractionation experiments, was noted in the hippocampus, whereas nuclear ERbeta was present in the amygdala. Despite these marked differences in subcellular localizations, similar expression levels of ERbeta proteins as well as the profile of ERbeta mRNA isoforms were observed in the two brain structures. ERalpha was localized to the nucleus more so than ERbeta, yet not without an extranuclear component. Our results suggest that cytoplasmic estrogen receptors may play an important role in hippocampal physiology.

Oestradiol rapidly inhibits Ca2+ signals in ciliary neurons through classical oestrogen receptors in cytoplasm.

Oestrogen plays a key role in a great variety of actions in the nervous system, either through classical or alternative pathways. The classical pathways are initiated after oestrogen binding to the oestrogen receptors ERalpha or ERbeta, which translocate from the cytoplasm to the nucleus and act there as transcription factors. Alternative pathways are initiated at the plasma membrane and cytoplasm, via binding to classical or non-classical ERs. Using isolated ciliary ganglion neurons from the chick embryo and Ca2+ imaging, we demonstrated that a 10-min exposure to 17beta-oestradiol reduces Ca2+ influx through the plasma membrane. This effect was not reproduced by oestradiol conjugated to bovine serum albumin, which does not cross the plasma membrane, indicating that 17beta-oestradiol was acting intracellularly. ERalpha was detected in the cytoplasm by immunostaining and its involvement in the regulation of Ca2+ influx by ICI182,780 inhibition. The phosphatidylinositol-3 kinase (Pi3-kinase) inhibitor wortmannin and the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) both blocked the oestradiol effect. The oestradiol effect was reproduced by 8Br-cGMP and abolished in the presence of the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Our study indicates that 17beta-oestradiol can regulate Ca2+ influx via PI3-kinase, NOS and PKG after activation of cytoplasmic ER.

Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance.

Introduced more than 100 years ago, endocrine therapy is still the most important systemic therapy for all stages of estrogen receptor (ER) -positive breast tumors. A major clinical problem limiting the usefulness of this therapy is tumor resistance, either de novo or acquired during the course of the treatment. Relatively new discoveries emphasize the complexity of ER signaling and its multiple regulatory interactions with growth factor and other kinase signaling pathways. Both genomic (nuclear) and nongenomic (membrane and cytoplasmic) ER activities contribute to this intimate cross-talk, which is probably a fundamental factor in endocrine resistance. New targeted therapies, especially against the epidermal growth factor receptor/HER-2 pathway, should be carefully evaluated in more (bio)logical strategies to enable them to be exploited appropriately. A strategy of combining endocrine therapy (particularly tamoxifen) with these inhibitors, to circumvent de novo and acquired resistance, will be discussed. We will also emphasize open questions and future challenges in the dynamic research field of molecular ER biology from the endocrine therapy perspective.

Minireview: A plethora of estrogen receptors in the brain: where will it end?

Until 1996, when estrogen receptor (ER)-beta was discovered, life seemed simple. The gonadal steroid hormone 17 beta-estradiol had one receptor, the ER, a ligand-inducible nuclear transcription factor. ER variants, the result of base pair insertions, transitions, and deletions, as well as alternative splicing, were considered abnormal and a prominent feature of breast cancer. Since then, like many other scientific beliefs, this concept has increased dramatically in complexity, and we are now faced with an ever-increasing array of estrogen-binding proteins, putative ERs, in the brain as well as in the extraneural targets of estrogen. The end is unlikely to be in sight. Some of these putative receptors have been localized to plasma or nuclear membranes, and others to the cytoplasm and/or nucleus. The molecular characteristics of membrane ERs are still in question, and, in most instances, the proteins have not been sequenced or cloned. However, based on transfection and immunohistochemistry, the generally held view, if not dogma, maintains that both nuclear and plasma membrane-associated ERs probably originate from the same gene and transcript that produce the classical intranuclear receptors ER-alpha and ER-beta. However, the physiological relatedness of this observation remains open to question. This review addresses evidence that, in addition to ER-alpha and ER-beta, there exist a variety of non-ER-alpha/non-ER-beta nuclear, cytoplasmic, and plasma membrane ERs in the brain, including G protein-coupled receptors; a novel, developmentally regulated, membrane-associated ER, ER-X; a functional, truncated ER-alpha variant, ER-46; and a putative ER that is immunochemically, structurally, and functionally completely distinct from ER-alpha and ER-beta.

Influence of the mycotoxins α- and β-zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells

The present study investigated the effects of the mycotoxins α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) at concentrations of 7.5, 15, and 30 μM, and deoxynivalenol (DON) at concentrations of 0.94, 1.88, and 3.76 μM on cell cycle distribution (propidium iodide, PI staining) in combination with the proliferating cell nuclear antigen (PCNA) by flow cytometry. The viability of porcine uterine cells was not impaired at 30 μM α-ZOL, whereas β-ZOL at this concentration and 3.76 μM DON significantly decreased cell number. Some cells showed ultrastructural features of cell death indicated by swollen mitochondria, disrupted cell membranes, and many vacuoles. After 24 and 48 h of exposure to α-ZOL (7.5, 15, or 30 μM), the cell cycle distribution was still comparable to the control groups. An anti-proliferative effect of β-ZOL and DON was detected by a significant reduction in the S-phase together with arrest of cells in the G 0/G 1-phase. The results show that β-ZOL (7.5, 15, or 30 μM) and DON (0.94, 1.88, or 3.76 μM) control the progression of cells through the cycle by decreasing S-phase and arresting cells in the G 0/G 1-phase of the cell cycle. A significant decrease in the expression of the proliferation marker PCNA amounts indicates that β-ZOL and DON disengaged cells from active cycling. We confirm that α-ZOL possesses a relative binding affinity to porcine uterine cytoplasmic estrogen receptor.

Estrogen receptor β is expressed in human endometrium and levels of expression vary during the menstrual cycle.

Objective: The recent discovery of a second distinct estrogen receptor (ER) β, in addition to the previously studied ER α, suggests the possibility of dual regulation of estrogen action in steroid-dependent tissues and the possibility for targeted therapeutics. Currently, there are conflicting data in the literature regarding the expression of ER β protein during the human menstrual cycle. The purpose of this study was to characterize the temporal and spatial localization of ER α and ER β in the endometrium of normally cycling women throughout the menstrual cycle. Design: Prospective cohort analysis with immunohistochemical analysis. Materials/Methods: Healthy ovulatory women (n = 15) gave informed consent for an endometrial biopsy. The paraffin-fixed tissue was stained with hematoxylin and eosin and dated using the Noyes’ criteria. Samples underwent immunohistochemical staining for ER if this histological date was within two days of a chronological date, assigned as the proliferative phase day from menses or the luteal phase day from the LH surge. Antibodies used were monoclonal antibody NCL-ER-6F11 (Vector Laboratories, Burlingame, California) directed against ER α (1:50) and a polyclonal antibody directed against ER β (1:250) previously described by Dr. J.-A. Gustafsson. Results: As noted in previous studies, ER α had a nuclear localization which was greatest in the epithelial cells during the proliferative phase, with a gradual decrease in staining during the luteal phase, to a minimum around days 26–28. ER β showed a similar nuclear and epithelial cell preference, with consistently less stromal cell staining. However, both epithelial and stromal cells showed cytoplasmic staining, except from days 19–22, when cytoplasmic staining was virtually absent. Additionally, stromal cells tended to show an increase in nuclear ER β at days 19–22, while epithelial cells tended to have consistent ER β staining at all stages in the cycle. Conclusions: ER β and ER α show different patterns of staining during the menstrual cycle, with nuclear down-regulation of ER α, but not ER β during the mid-luteal phase, and down-regulation of cytoplasmic ER β at that time. This differential expression may reflect differential roles for the two receptors.

Possible health impact of animal oestrogens in food

Oestrogens govern reproductive functions in vertebrates, and are present in all animal tissues. The theoretical maximum daily intake (TMDI) of oestradiol‐17β by consumption of cattle meat is calculated to be 4.3 ng. Following the use of oestradiol‐containing growth‐promoting agents, TMDI is increased by a factor of 4.6 to 20 ng oestradiol‐17β, assuming that single dosage and ‘good animal husbandry’ are observed. Pork and poultry probably contain similar amounts of oestrogens as untreated cattle. The mean concentration of oestradiol‐17β in whole milk is estimated at 6.4 pg/ml. Scarce data available on eggs report up to 200 pg/g oestradiol‐17β. The risk evaluation of oestrogenic growth‐promoting agents is limited by analytical uncertainties. Residues of oestradiol‐17α and the importance of oestrogen conjugates are widely unknown. The performance of mass spectrometry still needs to be improved for confirmation of oestrogen concentrations in most food. At present, the potential relevance of oestradiol acyl esters, the actual daily production rate of oestradiol in prepubertal children, and the role of oestradiol metabolites in cancer are obscure. The presence of different cytoplasmic oestrogen receptor subtypes and potential oestradiol effects in non‐reproductive functions require further examination.

Possible health impact of animal oestrogens in food.

Oestrogens govern reproductive functions in vertebrates, and are present in all animal tissues. The theoretical maximum daily intake (TMDI) of oestradiol-17beta by consumption of cattle meat is calculated to be 4.3 ng. Following the use of oestradiol-containing growth-promoting agents, TMDI is increased by a factor of 4.6 to 20 ng oestradiol-17beta, assuming that single dosage and 'good animal husbandry' are observed. Pork and poultry probably contain similar amounts of oestrogens as untreated cattle. The mean concentration of oestradiol-17beta in whole milk is estimated at 6.4 pg/ml. Scarce data available on eggs report up to 200 pg/g oestradiol-17beta. The risk evaluation of oestrogenic growth-promoting agents is limited by analytical uncertainties. Residues of oestradiol-17alpha and the importance of oestrogen conjugates are widely unknown. The performance of mass spectrometry still needs to be improved for confirmation of oestrogen concentrations in most food. At present, the potential relevance of oestradiol acyl esters, the actual daily production rate of oestradiol in prepubertal children, and the role of oestradiol metabolites in cancer are obscure. The presence of different cytoplasmic oestrogen receptor subtypes and potential oestradiol effects in non-reproductive functions require further examination.

Sex Steroid Receptors in Hodgkin's Disease

A case report of a dramatic therapeutic response of Hodgkin's disease (HD) to diethylstilbestrol (DES) in a man who was being treated for concurrent prostate cancer suggested that there also may be a role for sex steroids in the pathogenesis of HD (I). High levels of estrogen receptors (ER) comparable to those seen in breast carcinoma cells were detected in that patient's Hodgkin's biopsy specimen. In order to determine whether this patient was unique or whether sex steroid receptors commonly are present in HD specimens. we examined expression of ER and progesterone receptors (PR) in diagnostic tissue from pediatric (n=14) and adult (n=41) patients with HD using immunohistochemistry. None of the 55 samples expressed PR. 16/55 (29%) demonstrated weak nuclear 13 positivity, which was confined to germinal center and occasional mantle zone lymphocytes and was comparable to that seen in non-malignant control lymph nodes. 4/55 (7.3%) samples exhibited moderate positivity in Reed Sternberg cells, which in one case was nuclear. ER commonly are expressed weakly in some HD tumors unrelated to clinical stage or patient sex but arc generally limited to germinal center and mantle zone lymphocytes. A rare patient displays moderate cytoplasmic or nuclear ER in Reed-Sternberg cells.

Effect of estradiol and progesterone on daily rhythm in food intake and feeding patterns in Fischer rats

The product of meal number × meal size, over time, is food intake. Because estrogens modulate feeding activity via their action on the hypothalamus, and because there is a diurnal rhythm in the expression of cytoplasmic estrogen receptors and in estrogen binding activity, the present study examined the effects of ovariectomy and later hormone therapy on acute changes in body weight, and on the meal number-to-meal size relationship as reflected by food intake in the dark/light feeding patterns, in adult female rats in the intact state and after ovariectomy. Twelve female Fischer rats were randomized into ovariectomy and sham operation groups. A rat eater meter measured the feeding indexes for 15 days before and 25 days after ovariectomy, and later for 35 days with hormone therapy. We report: (a) mean body weight gain was linear before and up to ovariectomy, while exponential after ovariectomy; (b) increase in daily food consumption is mainly via an increase in food intake during the light phase; (c) light phase meal number remains unchanged, meal size significantly increases, with the resultant increase in overall food intake; (d) during the dark phase, meal size also significantly increases, but is accompanied by a proportional decrease in meal number, resulting in unchanged dark-phase food intake; and (e) estrogen restoration with either estradiol valerate or estradiol–progesterone combination, reversed the above changes. Data show that in the female Fischer 344 rat: (a) changes in daily rhythm in food intake are brought about by differential effects of the hormones on both meal size and meal number in both the total daily levels as well as in the dark-to-light distribution; (b) estadiol appears to have a tonic inhibitory effect on the light-phase meal size and a phasic effect on the dark phase meal size and number, but no significant effect on the light-phase meal number; and (c) in the Fischer rats, progesterone augments estradiol's effect on these indicies.

Immunogold labelling of estradiol receptor in MCF7 cells

The distribution of estradiol receptor in serial sections of estradiol-deprived and estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of estradiol-deprived cells, the receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antireceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen receptor in estradiol-deprived cells and support the premise of an estradiol-induced translocation of this ligand-dependent transcription regulator.