An ovarian independent population of uterine estrogen receptors

Estrogen receptor levels in uteri from ovariectomized mice varied markedly with time after surgery; these changes were not due to nuclear translocation. Receptor cyclicity was abolished in ovariectomized-adrenalectomized mice, and receptor levels remained low. This indicates that the adrenals significantly influenced uterine estrogen receptor levels. However, these adrenal-mediated changes did not alter uterine responsiveness to estrogen stimulation.     

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.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Interaction of calf uterine estrogen receptors with chicken target cell nuclei

The calf uterine estrogen receptor (ER) was used to study the capacity and the characteristics of the acceptor sites in chicken target cell nuclei. The temperature-activated ER is bound at 0 degrees C with a high affinity to all chicken cell nuclei tested (Kd = 0.4-1.0 nM). The nuclear binding displayed tissue specificity: oviduct greater than liver, heart greater than spleen greater than erythrocytes and was salt-dependent. ER binding to liver nuclei measured in 0.15 M KCl varied between 3000 and 6000 acceptor sites per nucleus. Liver nuclei isolated from estrogen-treated cockerels showed a 2-fold lower binding capacity than nuclei from non-treated chickens. When nuclei were incubated with [3H]ER from embryo liver and increasing concentrations of uterine non-radioactive-ER a progressive inhibition of the binding of the liver ER was found. These experiments suggest that liver and uterine ER compete for a common acceptor site. Liver nuclei charged in vitro with calf uterine ER were digested at 0 degree C with DNAase I and micrococcal nuclease. Both enzymes excised the ER in the form of a chromatin-ER complex. A considerable portion was associated with nucleosomal subunits and a minor fraction was associated with a nuclease-sensitive, protein-poor fraction of the chromatin.     

Diurnal variation in uterine estrogen receptors in immature female rats--inhibition by arginine vasotocin

A diurnal variation in uterine estrogen receptors from immature female rats was observed with the peak occurring during the mid-light phase (noon) of the cycle. Although no dose response was noted, all concentrations of arginine vasotocin ranging from 5 x 10(-7) to 5 x 10(-9) significantly inhibited binding of 3H-estradiol to estrogen receptors in the cytosol fraction of uteri obtained from immature female rats either during the light or dark phase of the photoperiod.     

Characterization of uterine estrogen receptors by size-exclusion and ion-exchange high-performance liquid chromatography

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.     

Immunocytochemical detection of estrogen receptors in mammary Paget cells

Immunocytochemistry was used to analyze the estrogen receptor (ER) content in mammary Paget cells obtained by scraping the nipples of six patients. The Paget cells in the smears were ER positive in four cases and ER negative in two cases. Five of the patients underwent a modified radical mastectomy; histologic study of the excision specimens showed three invasive ductal carcinomas and two intraductal carcinomas. Analysis of the ER status of the three invasive tumors, analyzed both by immunohistochemistry and by the radioligand technique, showed that the ER content in the Paget cells reflected that in the tumor in the breast parenchyma. This finding lends support to the hypothesis that Paget cells originate from an epidermotropic cancer in the parenchyma of the breast.     

Localization of estrogen and progesterone receptors in the endometrium of common marmosets Callithrix jacchus

In the present study, changes in the immunohistochemical localization of endometrial estrogen receptor (ER) and progesterone receptor (PR) during various stages of the ovarian cyclicity in common marmoset, have been reported. Ovarian cyclicity was monitored by estimating plasma estradiol and progesterone. During the early follicular phase, weak ER immunolocalization was observed in the endometrial stroma. During the late follicular phase under the influence of rising estradiol levels, stromal ER localization was intense. During the luteal phase, ER localization was absent in the stroma indicating that high concentrations of progesterone suppressed ER. PR localization was not observed in the stroma during the early follicular phase, while weak staining was seen in the stroma during the late follicular phase. PR localization was maximum during the mid luteal phase. However in marmoset, endometrial ER and PR localization was restricted only to the stroma. This unique feature may be due to the characteristic reproductive profile of this nonmenstruating species and needs to be studied further. Thus it can be hypothesized that in the marmoset endometrium, steroid hormone mediated effects possibly occur directly in the stroma and are then transmitted to the epithelium by autocrine/paracrine action of growth factors and cytokines.     

The relationship between ovarian steroids and uterine estrogen receptors during late pregnancy

Although a direct interdependence exists between the ovarian steroids, estrogen and progesterone, the exact role of these two hormones during pregnancy, especially late pregnancy, is not completely understood. Investigations have been conducted to determine whether the circulating levels of progesterone and estrogen or changes in the ratio of progesterone/estrogen in relation to the concentration of uterine estrogen receptors are associated with triggering parturition. Ninety-day old female rats were sacrificed at gestation days 14, 16, 18, 20 and two days post-partum. The plasma levels of estradiol and progesterone were measured by solid-phase radioimmunoassay. Uterine cytosol was subjected to a charcoal binding assay to determine the concentration of estrogen receptors. Our findings demonstrate that there is a significant (p less than 0.05) drop in both plasma progesterone and estradiol during late pregnancy. Also indicated is a significant (p less than 0.05) increase in uterine estrogen receptors throughout late pregnancy. Finally, during this period there is a direct correlation between the shift in the progesterone/estrogen ratio and the increase in the concentration of uterine estrogen receptors in late pregnancy.     

Localization of androgen receptors in ram epididymal principal cells

Androgen receptor was immunolocalized in the epididymal epithelium of rams and in isolated cells using an antibody against a synthetic polypeptide representing a portion of the androgen receptor. Immunostaining was predominant in the epithelium in tissue sections. Concentrations of androgen receptor were determined in cells from the central caput, distal caput, and central corpus epididymidis enzymically dissociated and elutriated to provide two fractions. On the average (n = 18), Fraction I contained 8% principal cells while Fraction II contained 71% principal cells; the stromal cells in each fraction were primarily smooth muscle and fibroblasts. For each sample, the number of DHT receptors (fmol) per 10(6) total cells was greater in Fraction II than in Fraction I. Few cells in Fraction I were immunostained for androgen receptor, whereas most cells in Fraction II were intensely stained. The numbers of DHT receptors per cell, or per principal cell, were similar for the central caput and distal caput, but lower in the central corpus epididymidis. The results support our hypothesis that most epididymal DHT receptors are localized in principal cells and confirm that the region between the central caput and proximal corpus of the ram epididymis is most dependent on androgen stimulation.     

An acute exercise-induced translocation of beta-adrenergic receptors in rat myocardium

The effect of acute exercise (treadmill running) on rat myocardium beta-adrenergic receptors (beta-AR) was studied. beta-AR was identified in purified sarcolemmal membrane fractions and light vesicle fractions. In control hearts, the number of beta-AR was 21.25 +/- 2.25 and 20.89 +/- 2.89 fmol/mg protein (mean +/- SE) in sarcolemmal membranes and light vesicles, respectively. Immediately after a single bout of dynamic exercise, about 35% of beta-AR was transferred from light vesicles to sarcolemmal membranes (p less than 0.05); concomitantly, isoproterenol-stimulated adenylate cyclase activity also significantly increased in sarcolemmal membranes (p less than 0.05). These results suggest that acute exercise provokes the translocation of beta-AR from a presumably intracellular site (light vesicles) to functional membrane fractions (sarcolemmal membranes) in rat myocardium.