Transformation of the rat uterine estrogen receptor after partial purification

Warming crude rat uterine cytosol after the addition of [³H] estradiol accelerates the association of the 4-S estrogen-binding protein with a second macromolecule, resulting in the formation of the 5-S estrogen-binding protein. To determine whether the 5-S estrogen-binding protein consists of two similar or dissimilar subunits, uterine cytosol was subjected to a number of fractionation procedures that separate macromolecules by solubility, molecular gel sieving, sedimentation rate, ionic charge, and heat lability. Following each of these methods, the fraction containing the 4-S estrogen-binding protein was incubated at 28°C; each of these 4-S estrogen-binding protein-containing fractions retained its capacity to completely transform to the 5-S estrogen-binding protein. In samples subjected to partial purification procedures, it was necessary that the buffer contain 40 mM Tris, 60 mM KCI, 1–10 mM dithiothreitol, and 1 M urea at pH 7.4, in order to accomplish the 4-S to 5-S estrogen-binding protein transformation at 28°C. Formation of the 5-S estrogen-binding protein requires association of the 4-S estrogen-binding protein with a molecule identical to or very similar to itself.     

Estradiol down-regulation of the rat uterine estrogen receptor

We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.     

Continuous estrogen exposure in the rat does not induce loss of uterine estrogen receptor

Cytosol estrogen receptor (ERc) and nuclear estrogen receptor (ERN) levels were investigated in rat uteri under different conditions of hormonal exposure. The amount of directly assayable receptor was closely related to the serum concentration of 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2). A double injection technique was established to maintain serum levels of [3H]E2 which were sufficient to saturate receptor sites. Under these conditions, stable ERC and ERN levels are maintained throughout the study period. 30% of the total ER remains cytoplasmic in localization despite continuous hormonal exposure. Properties of ERC and ERN after 6 h of continuous hormonal exposure were investigated and found to be different from receptors found in these subcellular compartments 30 min after hormone injections. ERC from uteri 30 min after injection showed a faster sedimentation coefficient than ERC prepared 6 h after hormone treatment. ERC after 6 h of hormonal exposure showed a reduction of binding to calf thymus DNA adsorbed on cellulose in a cell-free system. ERC 30 min after [3H]E2 treatment had a biphasic dissociation pattern consistent with two different receptor populations, whereas uterine ERC obtained after 6 h of in vivo exposure to estradiol showed virtually no dissociation at 22 and 28 degrees C. In contrast to ERC, ERN 6 h after hormone injection sedimented faster than ERN obtained 30 min after treatment. KCl extractable ERN obtained either at 30 min or 6 h posthormone treatment showed biphasic dissociation kinetics at 22 and 28 degrees C, whereas KCl nonextractable ERN showed virtually no dissociation. Virtually all of the specifically bound ligand in cytosol and nuclear preparations was proven to be authentic E2. We conclude that total cellular receptor is quantitatively conserved during 6 h of continuous hormonal treatment. Nuclear receptor loss is not a requisite for receptor-mediated steroid function, although important time-dependent changes in receptor properties in both cytoplasmic and nuclear compartments do occur.     

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.     

Extraction and partial purification of mouse uterine alkaline phosphatase.

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0.2% (v/v) Triton X-100 and extracted wtih 20% (v/v) n-butanol. The procedure, which resulted in 182-fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex G200 gel filtration. Solubilization with Triton X-100 was an important step in the procedure since extraction with n-butanol alone only partially solubilized the enzyme and gave low extraction yields, much of the enzyme activity remaining in association with negatively charged residues. However, butanol extraction of Triton X-100-treated homogenates gave high yields of enzyme and eliminated p-nitrophenyl phosphatases which displayed activity in the pH range 3.0--7.5, together with a large proportion of inactive protein. The activity of the purified enzyme preparations was electrophoretically homogeneous on cellulose acetate membranes, suggesting that the alkaline phosphatase in the mouse uterus exists in a single isozymic form. Polyacrylamide-gel electrophoresis revealed that the purified preparations contained at least one protein as an impurity. Attempts to further purify the alkaline phosphatase by isoelectric focusing were unsuccessful since the enzyme was found to have an isoelectric point of about 5.0 and at this pH it was rapidly inactivated.     

Preliminary characterization and partial purification of rat uterine nuclear type II Binding sites

These studies represent the first biochemical characterization and purification of nuclear type II binding sites from the rat uterus. Uterine nuclei from estradiol-implanted rats were digested with DNA'se and RNA'se, washed with Na deoxycholate-Tween 40 and extracted with 0.4 M ammonium sulfate (AmSO4). Nuclear type II sites in the AmSO4 extract eluted as a single peak during DEAE ion exchange chromatography, HPLC (Waters DEAE 5PW column) and Sephadex G-100 chromatography with a molecular weight of approximately 37K. DEAE and quercetin-sepharose affinity chromatography resulted in significant purification (greater than 800-fold) of nuclear type II sites with a 49% yield. Type II sites were not recognized by rat ER antibodies (Abbot ER-EIA kit) which immunoadsorbed ER from these preparations. These biochemical and immunological studies suggest that the ER and type II sites are likely to be different proteins.     

Effect of estradiol on estrogen receptor expression in rat uterine cell types

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.     

Particulate nature of the unoccupied uterine estrogen receptor

Homogenization of rat uteri at 25 degrees C resulted in a particulate partitioning of the estrogen receptor. Homogenization at 0 degrees C, the use of frozen tissue, or the pre-exposure of the tissue to 0 degrees C prior to 25 degrees C homogenization induced soluble partitioning of the estrogen receptor. Binding of a radiolabeled monoclonal antibody indicated that, in absence of estradiol, the estrogen receptor is particulate and is associated with the nuclei-enriched fraction of the target cell. The presence of receptor in the soluble fraction thus appears to be an artifact of homogenization. The unoccupied receptor, loosely associated with the particulate fraction (cold-sensitive) represents the "native" form of receptor which, upon arrival of the hormone, becomes tightly associated (cold-insensitive). The transition from the cold-sensitive to the cold-insensitive status is accompanied by a modification of the electrical charge of the receptor.     

Immunoelectron microscopic localization of estrogen receptor on pre-mRNA containing constituents of rat uterine cell nuclei

The localization and quantitative changes of estradiol receptor (ER) were studied by means of immunogold-electron microscope methods using a polyclonal antibody directed against an amino acid sequence representing the DNA binding site of ER, a monoclonal antibody against hnRNP core protein, and anti-DNA antibody. The uteri of normal rats in estrus and those of ovariectomized females were used. Ovariectomized rats were studied 21 days after surgery at different times after the injection of normal saline or estradiol-17 beta. The density of labeling was measured in interchromatin space, compact chromatin, nucleolus, cytoplasm, and background of epithelial cells, muscle cells, and fibroblasts. In the three types of cells ER was found mainly on extranucleolar ribonucleoprotein (RNP) fibrils. In epithelial and muscle cells the nucleolus was labeled but compact chromatin was not labeled. In epithelial cells there was a low but significant labeling of the cytoplasm. Fibroblasts exhibited a low labeling of the compact chromatin. Ovariectomy did not change these distributions. The estradiol injection increased labeling in all compartments of epithelial and muscle cells but decreased the labeling of compact chromatin of fibroblasts. These results show: (a) that ER is mainly nuclear but it is also present in the cytoplasm, (b) that ER binds to the nuclear particles containing newly synthesized RNA, and (c) that the binding to RNPs does not block the DNA binding domain of the ER.     

Immunological analysis of the biochemical properties of the uterine estrogen receptor

Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to 1) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 17 beta-estradiol (E2), estriol (E3), and diethylstilbestrol (DES) exhibited almost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (postfixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCl, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCl or 0.5 M NaCl, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of immunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 min with PBS, 0.4 M KCl, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.     

Cellular turnover in the rat uterine cervix and its relationship to estrogen and progesterone receptor dynamics

Histoarchitectural changes of the uterine cervix allow its successful adaptation to different physiological conditions. In this study, we evaluated cell turnover in each cellular compartment of the uterine cervix in association with steroid hormone receptor expression in order to establish the range of physiological changes. Proliferation, apoptosis, and progesterone receptor (PR) and estrogen receptor alpha (ERalpha) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ERalpha and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ERalpha and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ERalpha were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ERalpha was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ERalpha and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.     

A nuclear transforming factor that converts the goat uterine nonactivated estrogen receptor to nuclear estrogen receptor II

A 62-kDa nuclear protein that transforms the goat uterine nonactivated estrogen receptor (naER) to nuclear estrogen receptor II (nER II) has been isolated and purified. This is being identified as the naER-transforming factor (naER-TF). The transformation is achieved through deglycosylation of the naER. It is observed that the naER-TF action on the naER introduces significant changes in the structural and functional features of the naER. The capacity of the naER to bind estradiol increases 8- to 10-fold, while its hormone binding affinity reduces to a considerable extent following its exposure to naER TF. There is a critical ratio in the concentration of the two proteins, the TF and the naER, that would ensure an optimum transformation process. The transformed naER is incapable of dimerization with the estrogen receptor activation factor (E-RAF).