Nuclear Estradiol Research Articles

A comparison of cytoplasmic and nuclear estradiol and progesterone receptors in human fallopian tube and endometrial tissue

Quantitative and qualitative aspects of the in vitro binding of 3H-estradiol and 3H-progesterone to receptor components from human endometrium and fallopian tube cytoplasmic and nuclear fractions were studied. The steroid binding macromolecules formed in vitro could be extracted from nuclei by 0.4 M KCl and detected by glycerol gradient centrifugation. Both estradiol- and progesterone-binding compounds formed only one peak (under high ionic strength conditions) with a sedimentation coefficient of about 4-5 S. The number of cytoplasmic and nuclear binding sites for both estradiol and R5020 varied dramatically throughout the menstrual cycle: the estradiol and progesterone receptor concentrations were highest during the proliferative phase and were very significantly lower in the second half of the menstrual cycle. Furthermore, measurement of both receptors in the cytosol revealed differences among the anatomic segments of the fallopian tube. The highest estradiol and progesterone binding could be detected in the ampullary region; significantly lower levels of estradiol and progesterone receptors were seen in the infundibulum and the isthmus.

Estrogens, estradiol receptors and peroxidase activity in human mammary carcinomas

Estrogens, cytoplasmic estradiol receptors, nuclear estradiol receptors and peroxidase activity were measured in human breast carcinomas. No correlation was found between either estrogens, cytoplasmic estradiol receptor or nuclear estradiol receptors and peroxidase activity. The lack of correlation between cytoplasmic estradiol receptors and peroxidase activity is not likely to be due to inhibition of estrogen-inducible peroxidase by endogenous progesterone or the presence of nuclear estradiol receptors in the absence of cytoplasmic receptors. It is concluded that peroxidase activity is not a specific marker for a functional cytoplasmic estradiol receptor in human mammary carcinomas.

The unoccupied nuclear oestradiol receptor in the rat uterus and hypothalamus during the oestrous cycle

The nuclear oestrogen receptor population in the rat uterus contained an unoccupied receptor component that bound oestradiol with the high affinity (Kd congruent to 0.5 nM) characteristic of oestrogen receptors. This unoccupied receptor was present at all phases of the oestrous cycle. Its content changed in parallel with that of the total nuclear receptor during the cycle. Oestradiol administration to the immature rat resulted in increases in the uterine content of long-term nuclear receptors (i.e., those still present 8 h after administration); these increases were due to occupied oestrogen receptors, since the content of unoccupied receptor was unchanged. Our previous experiments [White & Lim (1980) Biochem. J. 190, 833-837] have shown in contrast, that oestradiol administration results in an increase in the content of unoccupied nuclear receptor in the hypothalamus. However, as in the uterus, similar cyclic changes in the content of unoccupied nuclear receptor occurred in parallel with those of the total nuclear receptor population in the hypothalamus. Differences and similarities between the unoccupied nuclear receptor of the uterus and hypothalamus are briefly discussed.

The role of estradiol dehydrogenase in mediating progestin effects on endometrium from postmenopausal women receiving estrogens and progestins

Endometria from postmenopausal women taking estrogens exhibit little estradiol dehydrogenase activity and contain appreciable amounts of nuclear estradiol receptor. Ingestion of the synthetic progestin, norethisterone, markedly elevates the dehydrogenase activity and decreases the nuclear estradiol receptor content of the endometria. The quantitative relationship between enzyme activity and receptor binding has been studied in women receiving different daily dosages of norethisterone. Statistically, the best negative correlation is obtained when log receptor is plotted against log dehydrogenase. Thus, a small rise in dehydrogenase activity produces a disproportionate decrease in nuclear receptor. Estradiol dehydrogenase in postmenopausal endometria preferentially metabolizes estradiol into estrone. Separation of endometria into glandular and stromal elements indicates that although the dehydrogenase is present in both fractions it is concentrated in the epithelial glands. The functional significance of changes in receptor and dehydrogenase activity was studied by [ 3H]-thymidine autoradiography. Induction of the dehydrogenase was accompanied by an inhibition of DNA synthesis in both epithelial and stromal cells and the quantitative interrelationships are complex. The nuclear estradiol receptor content, estradiol dehydrogenase activity and DNA synthesis of the postmenopausal endometria have been compared with premenopausal samples from untreated women.

Steroid receptor levels in intact and ovariectomized estrogen-treated rats: an examination of quantitative, temporal and endocrine factors influencing the efficacy of an estradiol stimulus.

Cell nuclear estrogen receptors and cytosol progestin receptors were measured in the pituitary gland, preoptic area and hypothalamus throughout the estrous cycle of the rat. Cell nuclear estrogen receptor levels paralleled changes in serum estradiol concentrations with highest values on proestrus and lowest on diestrus. Proestrous values were 50-60% of capacity for each tissue. Cytosol progestin receptor number in these tissues was also highest on proestrus and lowest on diestrus. With these data as a guide, Silastic capsules filled with estradiol-cholesterol mixtures were used to generate physiologic levels of estrogen receptor occupation within the brain-pituitary complex of ovariectomized rats and to examine the kinetics of estradiol stimulation of lordosis behavior and cyclic gonadotropin release. Our results indicate that the effectiveness of an estradiol stimulus to elicit lordosis or luteinizing hormone release depends on at least three factors: the magnitude of the increment in serum estradiol and brain and pituitary cell nuclear estradiol receptor levels; the duration over which these increments area maintained; and the interval from previous exposure to estrogen.

Deficits in pituitary and brain cell nuclear retention of (3H)Estradiol in diabetic rats deprived of insulin: time course and metabolic correlates.

Withdrawal of exogenous insulin for 24 h in ovariectomized, streptozotocin-diabetic rats significantly impairs estradiol uptake in whole homogenate fractions of hypothalamus-preoptic area and pituitary gland. Significant reductions in cell nuclear fractions from the same tissues are seen after 36 h of insulin deprivation. Subsequent reinstatement of insulin treatment does not yield full recovery of estradiol uptake after 24 h of insulin replacement. Fat content of the diet has no effect on brain or pituitary estradiol uptake in diabetic animals deprived of insulin for 36 h. Circulating levels of triglycerides, ketones, glucose, glycerol and free fatty acids were found to predict uptake levels to a significant extent, but no single metabolite is reliably predictive across tissues. These data demonstrate that insulin-dependent changes in brain and pituitary uptake of estradiol in rats are slow to develop, and they support the hypothesis that at least some of the reproductive dysfunctions observed in diabetic rats may be the result of impaired cell nuclear estradiol binding in hypothalamus and pituitary.

Quality control in steroid hormone receptor analyses.

In this paper, standards for steroid hormone receptor determinations in cancer tissue have been outlined. Standardization of methods for receptor extraction and receptor assay are equally important; and it is a condition sine qua non, if comparable receptor values between different centers for multilocal studies are required. Immediate and proper cooling of the tissue samples after removal, homogenization at the temperatures of the liquid nitrogen (as well as the presence of a sulfhydryl-protecting agent in the extraction medium), and the use of a simple, reproducible assay procedure are most important. As a standard procedure for ER- and PR-assays, the charcoal adsorption method combined with a scatchard-plot has been suggested. Reference procedures, like protein with DNA-assays, must also be standardized. Quality control experiments can be performed with lyophylized calf uterus homogenates. Additional determination of the nuclear estradiol in the high-speed sediments by radioimmunoassay is highly recommended.

Induced protein and deciduoma formation in rat uterus.

Using the pregnant rat in which deciduomas were induced by injection of arachis oil into the uterus, it has been found that induced protein (IP) synthesis is greatly increased in the deciduomas. A similar increase was obtained in deciduomas induced in progesterone-maintained ovariectomized animals. The increased synthesis in the pregnant animals was not correlated either with levels of circulating estradiol or the concentration of nuclear estradiol receptor. Synthesis was generally correlated with the degree of cellular mitosis in the uterus. It is suggested that IP synthesis is suppressed in the uterus on Day 5 of pregnancy, but is released from suppression by the initiation of decidualization. It is also suggested that IP is involved in uterine cell division.

Studies on cytosol and nuclear binding of estradiol in human breast cancer

Defects in the translocation of the cytosol estradiol receptor (ERC)-estradiol complex to the nucleus could partly explain the failure of endocrine therapy in 40% of breast cancer patients with significant ERC. To examine this possibility, 98 cases of human breast cancer were investigated for cytoplasmic and nuclear (ERN) estradiol receptors. ERC was assayed by a charcoal method following incubation with [ 3H]-estradiol. Nuclei were isolated from the low speed sediment and incubated at 30°C with the ERC-[ 3H]-estradiol complex in the presence and absence of an estrogen competitor. ERN was extracted from the nuclei with 0.4 M KCl and expressed as specifically bound estradiol, fmol/mg DNA. The quantity of ERN ranged between 0–746 fmol/mg DNA. Of 50 cases with significant ERC activity, 11 had no ERN (24%). In the borderline ERC group of 17 tumors, 5 had no ERN (29%), while of the 31 cases with zero or negligible ERC, 27 eases had no ERN (87%). Characteristics of ERC are different from the classical model. Chromatography of ERC on Sephacryl-S-200 columns and sucrose gradient sedimentation values indicate that the original form has an approximate molecular weight of 35,600, which on warming is transformed into an even smaller form (mol. wt — 22,700). The larger form of ERC (mol. wt ∼- 250,000) is produced by aggregation in low ionic strength media. The major form of the ERN complex has a molecular weight of 35,600, the same as that of the cytosolic receptor complex. It appears that the absence of ERN in a significant proportion of ERC positive tumors could contribute to the failure of endocrine therapy in such patients. Results indicate that the determination of both ERC and ERN could improve the selection of patients for endocrine therapy.

Assay of nuclear estradiol receptor by exchange on glass fiber filters

A glass fiber filter exchange assay for nuclear estradiol receptor in human endometrium has been developed. It permits both exchange and measurement of bound radioactivity to be performed without any transfer of the nuclear preparations. Suspensions of nuclei containing estradiol-receptor complexes are adsorbed onto glass fiber filters. Receptor sites, both empty and occupied by endogenous hormone, are labelled by incubation with 20 nM [ 3H]estradiol without (total binding) or with 2 μM radioinert estradiol (nonspecific binding). Buffer containing unbound radioactive estradiol is then drained, the filters washed, transferred into vials and counted in toluene-based scintillator. Following removal from the scintillator, DNA content of the filters can be measured by the Burton procedure. This exchange technique is easy and specific, with accuracy and precision similar to those of the technique of Bayard et al. (Bayard, F., Damilano, S., Robel, P. and Baulieu, E.E. (1978) J. Clin. Endocrinol. Metad. 46, 635–648). It offers the advantage of increased sensitivity, allowing receptor determinations on nuclear samples containing more than 10 μg DNA. Approximately 20 measurements can be made from 50 mg tissue (wet weight).

Intracellular estradiol, estrone and estrogen receptor levels in endometria from postmenopausal women receiving estrogens and progestins

Estrone and estradiol have been measured in subcellular fractions of endometria from women taking estrogen either alone or in combination with norethisterone. Within the nucleus there was a four-fold excess of estradiol over estrone in endometria from women taking estrogen only. This ratio was diminished to near-unity when norethisterone was also taken. The diminished nuclear estradiol content in the norethisterone treated endometria correlated with lower nuclear estradiol receptor content and increased estradiol dehydrogenase activity. In all cases, there was an excess of estrone over estradiol in the plasma. It is suggested that in these postmenopausal women, estradiol is the most active intranuclear form of estrogen and that progestins such as norethisterone can diminish the nuclear estradiol content via a mechanism involving estradiol dehydrogenase. Some characteristics of the nuclear estradiol receptor from these estrogen-primed endometria have been studied. As judged from labelling with [ 3H]-estradiol at 4° and 30°C, the nuclear receptor behaves as if it were uncomplexed with estradiol. Highly purified nuclei behave in the same manner.

Unoccupied nuclear oestradiol-receptor sites in normal human endometrium.

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol--receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.

Improved biochemical characterization of breast cancer as a guide to hormonal treatment.

Theoretically, all estrogen-receptor positive (ER+) tumors of the breast should respond to hormonal therapy; however, only 50-60% do. Therefore, this study collected information on hormonal variations which affect the cytoplasmic receptor levels and offers other parameters besides estrogenicity for biochemically characterizing breast cancers. One reason ER+ does not insure hormonal responsiveness is that cytoplasmic receptor levels are influenced by endogenous hormones as well as by exogenous factors, specifically rhythmic fluctuations of unknown origin (circadian and seasonal). Because of these various influences, it is obvious that no normal baseline level of receptors in target tissue exists on which to place the decision for prognosis of hormonal therapy. In addition to quantitative assessment of ER by assay, determination of nuclear estradiol content and measurement of cytoplasmic progestin receptor must be employed. Experiments outlined have shown that the receptor can enter the nucleus of target tissue without the assistance of hormone. Hence, complete hormone withdrawal (as in ovariectomized and adrenalectomized subjects) is not a totally effective treatment for hormone-responsive cancers. Instead, this withdrawal must be supplemented by an antihormonal treatment that incapacitates the receptor. In this article, the virtue of tamoxifen for performing this blockade is pointed out. Since tamoxifen competes with estradiol at the receptor-binding site (according to pig uterus experiments outlined here), its biological effect might be explained by a blockade of the nuclear ER transfer, the parameter that hormonal withdrawal alone cannot affect.

Sex differences in serum testosterone and in exchangeable brain cell nuclear estradiol during the neonatal period in rats

Sex differences in serum testosterone and in exchangeable brain cell nuclear estradiol during the neonatal period in rats

Effect of progesterone pretreatment on the uptake of estradiol-17 beta by the uterine epithelium of the rat.

Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.

Estrogen binding proteins in rat skeletal and perineal muscles: In vitro and in vivo studies

Specific vitro binding of [ 3H]-estradiol was demonstrated in the cytosolic fraction of levator ani/bulbocavernosus (LA/BC) muscles and thigh muscles (TM) of 50-day-old rats. Using a charcoal assay, the apparent association constant ( K a ) for estradiol was found to be 1.6 × 10 9 M −1 in LA/BC and 0.9–2.0 × 10 9 M −1 in female and male TM. The K a was not affected by 24 h of castration but the number of binding sites increased significantly ( P < 0.05) from 7.8 ± 1.7 (SD) to 11.7 ± 2.8 (SD) fmol/mg protein in LA/BC and from 3.4 ± 1.6 (SD) to 5.8 ± 0.5 (SD) fmol/mg protein in male TM. In female TM, the level of binding sites of 24 h ovariectomized rats (4.3 ± 1.0 (SD) fmol/mg protein) was relatively similar to the one found in intact rats in diestrus phase (3.4 ± 0.8 (SD) fmol/mg protein) but higher than in proestrus phase (0.8 ±0.15 (SD) fmol/mg protein). In both types of muscles, this cytosol estradiol binding protein sedimented in the 9–11S region of sucrose density gradients. Competition studies with androgens, progestins and corticosteroids showed that the binding was highly specific for estradiol and was only partially decreased by 5α-androstan-3β, 17β-diol. In vivo binding of estradiol in muscles was also found after intravenous perfusion of [ 3H]-estradiol in castrated and functionally hepatectomized rats. Specific 8–10S binding peaks on sucrose gradients were present in LA/BC and in TM cytosols. Moreover in LA/BC muscles, in vivo nuclear estradiol binding proteins could be detected in a 4–5S binding peak on sucrose gradients obtained with 0.4 M KCl nuclear extract. These results show unequivocally the presence of cytosol estrogen binding proteins in skeletal and perineal muscles and suggest also the existence of nuclear estrogen-binding proteins complexes in LA/BC muscles.

Estradiol binding to nuclear receptors in human breast cancer tissue (MCF-7 cell line) and in dimethylbenz(a)anthracene-induced mammary carcinoma.

The presence or absence of estrogen receptors in the nuclei of human breast tumor may be a useful tool in determining whether the tumor will or will not respond to endocrine therapy. This paper describes an assay which measures both unoccupied and occupied nuclear receptors in human breast cancer tumors. The assay was predicated on the fact that at low salt concentration, the nuclear receptor is bound to chromatin particles and can be separated from the soluble components containing proteolytic acitivity. Nuclear estradiol receptors were measured in human breast cancer tissue (MCF-7 cell line) and in DMBA (dimethylbenz(a)anthracene-induced rat mammary carcinomas) tumors. Complete translocation of the cytoplasmic receptor in the MCF-7 cells was observed compared to only 35-50% of the cytoplasmic receptors seen in the nucleus of the DMBA tumor after estradiol injection. The study also showed 6 pmol/mg DNA for total unoccupied nuclei and cytoplasmic estrogen receptors, and 25% of it in the nucleus; this finding differed from Zava et al's finding of 2 pmol/mg DNA and 75% in the nucleus, probably because of differing methodology or use of a later passage of cell line. 29 out of the 34 tumors with cytoplasmic receptors were found to contain unoccupied nuclear receptors, indicating that free nuclear receptors are not exceptions. The assay used in this study is currently being used to determine the translocative ability of the cytoplasmic receptors in human breast carcinomas.

Regulation of Uterine Responsiveness to Injected Estradiol in Nutritionally Stressed Ovariectomized Rats by Adrenal-Derived Estradiol*

The effects of diet composition and starvation on the inducibility by estradiol of uterine glucose-6-dehydrogenase (G6PD) activity in mature ovariectomized rats has been evaluated. Starvation for 5 days and feeding a purified protein-free diet for 3 days caused a maximal increase in the inducibility of uterine G6PD activity relative to animals fed a basal diet. Inducibility of the enzyme was decreased as the amount of casein in the diet was increased from 0–45% and refeeding protein began to reverse the effect of protein deprivation on the inducibility of the enzyme within 24 h. The basal level of uterine G6PD activity was unchanged by diet or starvation, and the increased inducibility by estradiol in starved and/or proteindeprived rats was due to a 2-fold increase in the relative de novo rate of G6PD synthesis. The uterine binding and retention of [3H]estradiol given in vivo was increased by 40% in starved and/ or protein-deprived rats, and this increase appeared to be due to an increase in the estradiol receptor content of the uterus in the nutritionally deprived animals. Measurement of plasma estradiol levels revealed the presence of 12.7, 32.8, and 34.3 pg/ml in rats which were fed basal or protein-free diets or which were starved, respectively. The inducibility of uterine G6PD activity by estradiol was found to be statistically correlated with plasma estradiol levels before injection of exogenous steroid at r = 0.95 (P < 0.01), with cytosol estradiol receptor levels at r = 0.69 (P < 0.05), and with nuclear estradiol receptor levels at r = 0.86 (P < 0.01). The plasma estradiol levels appear to increase due to increased adrenal activity, since the dietary treatments caused increased adrenal weight and a decrease in thymus weight and adrenal suppression by administration of dexamethasone caused plasma estradiol levels in all groups to be suppressed and abolished the effect of starvation or a protein-free diet on the induciility of uterine G6PD. These results suggest that the uterine response to estradiol in starved and protein-deprived ovariectomized mature rats is increased due to elevated circulating levels of adrenalderived estradiol and that increased levels of plasma estradiol over the range of 13–35 μg/ml regulate uterine estradiol receptor levels in the uterus without initiating a complete uterine response. The magnitude of the uterine response after injection of estradiol appears to be directly related to the amount of estradiol receptor in the tissue.

The endometrial nuclear estradiol receptor of the pregnant cow has a molecular weight of 53000 in 6 M guanidine-HCl

Cell nuclei isolated from the endometrium of the 3 months pregnant cow were dialysed against 6 M guanidine-HCl + 0.1 M 2-mercaptoethanol. The resulting material (after eliminating the bulk of DNA by centrifugation) was filtered on a Sepharose 6/b column equilibrated in the above denaturing solvent. The molecular weight of the denatured “nuclear” estradiol receptor, estimated by a method described previously (T. Erdos and J. Friés (1974) Biochem. Biophys. Res. Commun. 58, 932–939), was about 53 000. This value is similar to that of the cytoplasmic receptor (55 000) but dissimilar to that of the cytoplasmic receptor transformed by the action of the Receptor Transforming Factor (35 000) estimated under the same experimental conditions, suggesting that the latter form of the receptor is not the biological precursor of the “nuclear” receptor. However, according to indirect estimates, probably not more than 10% of the “nuclear” receptor has been renatured in these experiments. Therefore the alternative that the results obtained are not representative for the “nuclear” receptor, cannot be excluded.

Hormonal control of nuclear estradiol receptor content and the luminal epithelium in the uterus of the golden hamster.

Uteri were removed and blood was drawn from hamsters during the estrous cycle, on the eighth day of pregnancy, and after different hormonal treatments. Serum estradiol (E2) and progesterone (P) levels were determined by RIA. Portions of the uteri were prepared for light and electron microscopy. Endogenous uterine nuclear E2 receptor was measured by exchange assay. Large increments of nuclear E2 receptor occurred when the level of serum E2 was rising and that of serum P was falling (diestrus day 2 through proestrus morning); decrements occurred when the serum E2 level was falling and the serum P level was rising (proestrus evening through diestrus day 1). Ovariectomy on proestrus morning led to a decline in the amount of nuclear E2 receptor, and this decline was prevented by administration of E2 at the time of ovariectomy. P depleted nuclear E2 receptor in the presence of high levels of serum E2. The uterine luminal epithelium passed through a phase of mitosis and hypertrophy (diestrus day 2 through proestrus morning), a brief phase suggestive of secretion (proestrus evening), a phase of degeneration (estrus), and a phase of quiescence (diestrus day I). Ovariectomy on proestrus morning led within 24 h to degenerative changes identical to those seen during estrus, and these changes were prevented by treatment with E2 at the time of ovariectomy. P induced these degenerative changes in the presence of high levels of serum E2. After 8 days of pregnancy, the epithelium resembled that seen during diestrus day 1. Elevated uterine nuclear E2 receptor levels were associated with growth and hypertrophy of the luminal epithelium, and severely depressed levels were associated with the degenerative and quiescent phases of the epithelium.