Inhibition of rat uterine estradiol receptor by aurintricarboxylic acid

Estradiol-receptor complex from rat uterus has been shown to have an affinity for DNA-cellulose and ATP-Sepharose. This DNA and ATP binding of estradiol receptor was observed to be sensitive to low concentrations (0.01–0.2mM) of aurintricarboxylic acid. The inhibitor was more effective when added to preparations that contained activated estradiol-receptor complex. Steroid binding properties of the receptor remained intact under the above conditions as judged by charcoal adsorption assays and sucrose gradient analysis. In addition, a 40% inhibition in the nuclear translocation of cytosol estradiol receptor was observed when rat uteri were incubated with 10nM [³H] estradiol under an atmosphere of 95% O₂ and 5% CO₂ in the presence of aurintric-carboxylic acid. Our results suggest that aurintricarboxylic acid is an effective inhibitor of rat uterine estradiol receptor and that it may be acting by interfering with site(s) on the estradiol receptor which may be exposed upon activation and are subsequently involved in processes such as ATP binding, nuclear uptake and DNA binding.     

Dose-related action of estradiol on placental prostanoid prediction

Prostanoids play an important role throughout all of pregnancy and during the initiation and progress of labor. The human placenta at term produces large quantities of prostanoids, yet little is known of the factors that regulate their biosynthesis. Herein, we report the effect of estradiol or estradiol and progesterone on the basal release of placental prostanoids from fresh human term placental explants using a perifusion system.The basal release of prostaglandin E₂ (PGE₂, prostaglandin F_2α (PGF_2α), thromboxane (TxB₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) increased about 50% from the fifth to the ninth hour in culture, while the release of 13, 14-dihydro-15-keto-PGF (PGFM) remained constant and hCG release decreased. The dose-related effect of estradiol (20–2,000 ng/ml) in the perifusing medium starting at the fifth hour of perifusiOn (i.e., the zero treatment time) effected no change in the release of TxB₂, PGF_2α, PGFM or hCG. A biphasic action on the release of 6-keto-PGF,. was observed, i.e. it was significantly decreased when incubated with 20 ng/ml of estradiol, but effected an increase after exposure to 200 ng/ml. The concomitant addition of progesterone (2,000 ng/ml) with estradiol (200 ng/ml) significantly inhibited the stimulatory action of estradiol at this dose. The release of PGE₂ was inhibited in a dose-related fashion with increasing dose of estradiol. The addition of progesterone with estradiol (2,000 and 200 ng/ml, respectively) reversed the inhibition of PGE₂ by estradiol alone.These data demonstrate that physiologic levels of estradiol affect 6-keto-PGF_α and PGE₂ release from the human term placenta, but do not significantly alter production of TxB₂, PGFM or hCG under these conditions.     

Effects of estriol on PGF2α output by cultures of human endometrium and endometrial cells

We have shown that the rate of release of PGF_2α by monolayer cultures of epithelial cells from proliferative endometrium is markedly elevated by addition of estradiol to the medium. In cultures maintained in HAM F-10 medium containing charcoal stripped calf serum, estradiol (10⁻⁸M) increased the levels of PGF_2α several fold during the second and third days in culture. Similar responses were obtained with estradiol at 10⁻¹⁰M concentration.When this system was used to compare the effects of estradiol and estriol at equal concentrations (10⁻⁸M), similar elevation (10–16-fold) of PGF_2α levels were noted during 3 consecutive days in culture. When cultures of epithelial cells derived from secretory endometrium were used for these tests, estriol was as effective as than estradiol in elevating PGF_2α levels in the medium.When the effects of estradiol and estriol were compared using fragments of secretory endometrium in organ culture, the increases in PGF_2α levels noted in the medium were about equal (2- to 10-fold) for the two estrogens at the same concentration (10⁻⁹−10⁻⁸M).Exposure of the tissue to either estradiol or estriol for only 1 h resulted in increases in PGF_2α output for the following 3 days.These results clearly show that estriol is as effective as estradiol in stimulating PGF_2α output by human endometrial tissue.     

Hypothetical mechanism of sodium pump regulation by estradiol under primary hypertension

Causal relationship between sodium and hypertension has been proposed and various changes in Na⁺,K⁺-ATPase (sodium pump) activity have been described in established primary hypertension. A number of direct vascular effects of estradiol have been reported, including its impact on the regulation of sodium pump activity and vasomotor tone. The effects of estradiol involve the activation of multiple signaling cascades, including phosphatydil inositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44^MAPK). In addition, some of the effects of estradiol have been linked to activity of cytosolic phospholipase A₂ (cPLA₂). One possible cardioprotective mechanism of estradiol involves of the interaction between estradiol and the rennin-angiotensin system (RAS). Elevated circulating and tissue levels of angiotensin II (Ang II) have been implicated in the development of hypertension and heart failure. The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump, in vascular smooth muscle cells (VSMC). The aim of our investigation was to elucidate the signaling mechanisms employed by estradiol and Ang II in mediating sodium pump activity/expression in VSMC, with particular emphasis on PI3K/cPLA₂/p42/44^MAPK signaling pathways. Our primary hypothesis is that estradiol stimulates sodium pump activity/expression in VSMC via PI3K/cPLA₂/p42/44^MAPK dependent mechanism and, that impaired estradiol-stimulated sodium pump activity/expression in hypertensive rodent models (i.e. SHR), Ang II-mediated vascular impairment of estradiol is related to a decrease ability of estradiol to stimulate the PI3K/cPLA₂/p42/44^MAPK signaling pathways. An important corollary to this hypothesis is that in hypertensive state (i.e. SHR rats) the decreasing in ACE enzyme activity and/or AT1 receptor expression caused by administration of estradiol is accompanying with abrogated ability of Ang II to decrease IRS-1/PI3K association, and consequent PI3K/cPLA₂/p42/44^MAPK activity and associated sodium pump activity/expression.A clear characterization of how Ang II attenuates estradiol signaling may lead to a better understanding of the molecular mechanism(s) underlying pathophysiological conditions such as hypertension and to understanding how certain pathophysiological situations affect sodium pump activity/expression in VSMC.     

Metabolism of estradiol by uterine peroxidase: Nature of the water-soluble products

The nature of the water-soluble products formed by incubating labelled estradiol with uterine peroxidase in the presence of H₂O₂ and tyrosine was examined by two-dimensional thin-layer chromatography and high voltage electrophoresis. It was shown that the steroid and amino acid were associated in a 1:2 or 1:3 ratio and evidence was provided by ³H-exchange for the interaction of tyrosine with ring A of estradiol at C-2 and C-4. The possible role of estrogen-induced peroxidase in the uterus in vivo is discussed.     

Hormonal regulation of sialic acid-binding (SAS) protein synthesis of rat uterus

Sialic acid binding proteins (SAS) of rat uteri have been found in all three stages of the estrous cycle. To study the control of synthesis of these proteins two different animal models were used I-immature female rats (25 d) where the hormones estradiol (E₂) and progesterone (P₄) were given separately and together, and II-adult female rats where hormone treatment commenced 14 days after ovariectomy. The data indicated that E₂ initiated the synthesis of SAS proteins in the immature animals, while P₄ could inhibit SAS synthesis, either given alone or together with E₂. However, prior priming of the rat with E₂ and subsequent administration of P₄ stimulated SAS protein synthesis.     

Estradiol and tamoxifen stimulation of lapine articular chondrocyte prostaglandin synthesis

The effect of estradiol and tamoxifen on prostaglandin (PG) synthesis by rabbit articular chondrocytes in secondary monolayer cultures was investigated. Radioimmunoassay for PGE₂, PGF_2α, 6-oxo-PGF_1α and thromboxane B₂ was performed on media from cultures containing estradiol and tamoxifen (10⁻¹²M-10⁻⁷-M). Radiometric thin-layer chromatography was also carried out. The time course of estradiol/tamoxifen effect on chondrocyte PG synthesis was evaluated and its relationship to cell density in culture examined. Estradiol stimulated the synthesis of PGs by chondrocytes. Stimulation was noted at picomolar concentrations of estradiol without further stimulation at markedly higher concentrations. In time studies, after a lag, the effect of estradiol was present fully by 5 hrs, remained steady for 24 hrs and then declined by 48 hrs. Estradiol stimulation of PG synthesis was dependent upon chondrocyte culture plating density. Tamoxifen stimulated chondrocyte PG synthesis to relatively lower levels than estradiol. The characteristics of estradiol/tamoxifen stimulation of chondrocyte PG synthesis suggest a mechanism involving estradiol cytoplasmic receptors.     

Estradiol and progesterone receptor levels in human breast cancer in relation to cytosol and plasma estrogen level

Estradiol receptor (ER) and progesterone receptor (PR) content along with the cytosol and plasma estrone and estradiol levels in 15 premenopausal and 26 postmenopausal women with breast cancer in different clinical stages (T₁₂₃, N₀₁, M₀) were measured. ER-positive tumor frequency and the ER content tended to be higher in postmenopausal than in premenopausal patients. There was no evidence for a relationship between high cytosol estrogen levels and low receptor measurements. The estrogen concentration was higher in ER-positive tumor cytosols than in those of ER-negative tumors; the differences were significant in postmenopausal women, only, with P < 0.05 for estrone and P < 0.01 for estradiol values. Twelve pairs of tumor and normal tissue from the same breast, were also studied: seven of which contained ER-positive and five ER-negative tumors. The ER-positive tumors showed a clear trend to higher estradiol content as compared to the corresponding normal tissues. The circulating level of estradiol in postmenopausal women, was higher (P < 0.05) in ER-positive tumors than that in ER-negative tumors. Our results indicate that: (a) false negative ER assays are not likely to be due to the presence of endogenous estrogens, (b) higher amounts of estrone and estradiol are contained in ER-positive tumors than in negative ones.     

Effect of estrogen on egg production,shell quality and calcium metabolism in molted hens

Force-molted White Leghorn laying hens were implanted with 0.25, 0.5, 1 and 3 Compudose 200 pellets (24 mg 17β estradiol/pellet). Plasma estradiol increased with increasing E₂ dosages in a linear manner and decreased over time in a quadratic manner (P < 0.01). E₂ treatment had a nonlinear effect on total plasma calcium. Oviduct weight, shell thickness and egg weight were not significantly affected by exogenous estradiol whereas tibial bone ash percentage was increased at only one dose (P < 0.05:0.5 pellet group). Physiological supplementation with estradiol does not improve shell quality.     

Effect of prostaglandin inhibitor on the uterotrophic response of estradiol and gibberellic acid

An investigation was undertaken on the mode of estrogenic actions of gibberellic acid (GA₃), and estradiol (E₂) as determined by the mouse uterine bioassay. The prostaglandin inhibitor, indomethacin (I) was administered in order to learn whether E₂ and GA₃ activities were related to prostaglandin biosynthesis. E₂ and GA₃ stimulated a significant increase in the weight of the mouse uterus, whereas I inhibited the effects of E₂ and GA₃. These findings indicate an apparent involvement of the prostaglandins and their biosynthesis on the action of E₂ and GA₃.     

Oviduct response to prostaglandins: Influence of estradiol and progesterone

Oviductal motility was measured in the isthmus of ovariectomized New Zealand rabbits. The effects of estradiol and progesterone on spontaneous motility and on the response of the oviduct to exogenously administered prostaglandin E₁ (PGE₁) and PGF_2α were determined. Estradiol treatment significantly increased both the amplitude (P<0.05) and frequency (P<0.01) of spontaneous contractions. The amplitude of spontaneous activity was less following progesterone treatment than following estradiol treatment (P<0.05). Progesterone treatment increased the duration of oviduct response to PGE₁ (P<0.05). Estradiol treatment had no effect on the response to PGE₁. Increased oviductal activity caused by PGF_2α lasted significantly (P<0.01) longer in ovariectomized, untreated animals than in ovariectomized animals treated with estradiol or progesterone. Progesterone was more effective than estradiol in decreasing the duration of the response to PGF_2α. These effects of steroid hormones on the responsiveness of the oviduct to PGE₁ and PGF_2α could contribute to the physiological control of egg transport. The nadir of ovarian hormone influence, as in the recently ovariectomized animals and as occurs immediately after ovulation, is associated with a high responsiveness of the oviduct to PGF_2α. This could effectively increase isthmic occlusion and prevent the eggs from passing through the oviduct prematurely. The gradual increase in ovarian estradiol and progesterone secretion during the 3 days following coitus could result in decreased responsiveness to PGF_2α and increased responsiveness to PGE₁. These changes might cause relaxation of isthmic tone and allow movement of eggs through the isthmus into the uterus.     

The concentrations of urinary prostaglandins E and plasma prostaglandins F2α and E during induction of ovulation with human gonadotropins

Plasma prostaglandins F₂α and E (PGF₂α, PGE) and urinary PGE were measured in 10 women treated with human gonadotropins (HMG) and subsequently with human chorionic gonadotropins (HCG). Five women became pregnant (6 pregnancies). There was no correlation between concentrations of plasma PGF₂α or PGE and plasma estradiol or progesterone. Urinary PGE concentrations showed a positive correlation with estradiol before HCG and a negative correlation with progesterone after HCG, only in women who subsequently became pregnant.Higher urinary PGE concentrations before HCG suggest that either HMG or rising estradiol levels stimulate PGE renal production. The significant negative correlation.between urinary PGE and progesterone concentrations, after HCG, in those patients who became pregnant suggests that ovarian production of progesterone may decrease renal production of PGE.     

The comparative potency of various steroids to complete the priming process for lordosis in guinea pigs

Ovariectomized adult guinea pigs were treated with a regimen of estradiol benzoate (0.2 μg/animal estradiol benzoate at hr 0 and 19) that was shown to be minimally effective for the induction of lordosis. They were then treated with 10, 20, or 80 mg of enclomiphene, 5, 20, 40, or 100 μg of estradiol, or testosterone, cortisol, estrone, estriol, diethylstilbestrol, catechol estradiol, or catechol estrone (all at a dose equivalent to 5 μg of estradiol) at hr 28. At hr 39 all females were given 0.5 mg progesterone, and subsequently tested for lordosis behavior. Of the various agents injected at hr 28 only estradiol (at all doses given), estrone, estriol, and diethylstilbestrol were effective in supporting display of lordosis behavior. The results indicate that the antiestrogen enclomiphene, the catechol estrogens, and at least some C₁₉ and C₂₁ steroids are weaker than E₂ or ineffective in facilitating lordosis behavior when given late in the priming period. Because previous work had shown that enclomiphene has partial estrogenic effects on lordosis behavior when administered early in the priming period (i.e., at hr 0, 19), it is suggested the early and late phases of the priming process induced by E₂ entail qualitatively different neural processes.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF_2α the treatment with progesterone (4 mg.day⁻¹, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol- 17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE₂, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE₂ catabolism into 15-keto- PGE₂ as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Two sex steroid receptors in SC-115 mammary tumor cells

The growth of the SC-115 mammary carcinoma in mice is androgen dependent. Estrogens antagonize the androgen effect. The high affinity binding of androgens and estrogens has been studied in soluble extracts of the tumor, of primary culture cells and clone MI₁ cells.Results indicate that two distinct specific steroid hormone-binding sites (termed ‘receptors’) are found in all cytosol fractions. The androgen-receptor (A) binds testosterone, androstanolone, cyproterone (an anti-androgen), progesterone and estradiol, but only very weakly non-steroidal diethylstilbestrol. The estrogen-receptor (E) binds estrogenic substances such as estradiol and diethylstilbestrol, but no androgen. The apparent K_{D, eq} for A and E receptors of [³H]androstanolone and [³H]estradiol respectively, is identical (-0.5-1 nM at 4 °C). The affinity of estradiol for the A-receptor, when measured against [³H]androstanolone binding, indicates a K_i = 17.5 nM. The concentration of binding sites is of the order of 0.1 pmole/mg protein (somewhat higher for A than for E receptor) in MI₁ cell cytosol. Studies by ultracentrifugation through glycerol-Tris gradients (low salt medium) reveal the macromolecular nature of the cytosol A and E receptors (7–7.5 S). Evidence is presented of the transfer of the A and the E receptors to nuclei after incubation of tumor slices as well as of clone MI₁ cells with the corresponding hormones.Experiments suggest that the two different binding sites are present on two separated macromolecular moieties. After incubation at 37 °C of tumor slices with 10–20 nM [³H]testosterone, or with 10 nM [³H]estradiol, the corresponding radioactive hormone-receptor complexes are, as expected, found in the nuclear KCl extracts. In parallel experiments, where slices are incubated with non-radioactive hormones at the same concentration and the nuclear KCl extracts subsequently treated by radioactive steroids, no available androgen-binding sites are found in the nuclei after exposure to estradiol, nor estrogen-binding sites after exposure to testosterone.Therefore, in the same cell, two receptors are present which bind androgens and estrogens with high affinity, and one given hormone (estradiol) can be specifically bound (with different affinities) by two different receptors which, however, discriminate a synthetic analog (diethylstilbestrol). The data may give some molecular background for interpreting responses to the same hormone which may differ at various concentrations, for studying effects of analogs, and for analysing the control of tumor growth by antagonistic steroids.     

Determination of estradiol valerate in pharmaceutical preparations and human serum by flow injection chemiluminescence

A novel method for the detection of trace estradiol valerate (EV) in pharmaceutical preparations and human serum was developed by inhibition of luminol chemiluminescence (CL) by estradiol valerate on the zinc deuteroporphyrin (ZnDP)‐enhanced luminol‐K₃Fe(CN)₆ chemiluminescence system. Under optimized experimental conditions, CL intensity and concentration of estradiol valerate had a good linear relationship in the ranges of 8.0 × 10^‐8 to 1.0 × 10^‐5 g/mL. Detection limit (3σ) was estimated to be 3.5 × 10^‐8 g/mL. The proposed method was applied successfully for the determination of estradiol valerate in pharmaceutical preparations and human serum and recoveries were 97.0‐105.0% and 95.5‐106.0%, respectively. The possible mechanism of the CL system is discussed. Copyright © 2012 John Wiley & Sons, Ltd.     

Blood Rheology, Sex Hormones, and Cortisol in Athletes

The interrelations of blood rheology, plasma proteins and lipids, and steroid hormones (total testosterone, estradiol, and cortisol) were studied in athletes (N = 14). Blood (BV), plasma (PV), and erythrocyte suspension (ESV) viscosities; fibrinogen; total cholesterol (Ch); low density lipoprotein (LDL) Ch; and plasminogen activity were lower in the athletes than in control subjects (N = 10). The specific peripheral vascular resistance (SPVR) to BV ratio and PWC₁₇₀ were lower in the athletes. All these parameters correlated with decreased estradiol. Discriminant analysis indicated PWC₁₇₀, estradiol, and ESV to be the main parameters discriminating the groups. Decreased estradiol seemed to be associated with an increased physical activity in the athletes, as evident from its correlation with PWC₁₇₀ and differences in the body composition (a correlation with the body mass index). No significant difference was found in total Ch and cortisol. However, in the combined sample, decreased testosterone was associated with decreased BV, which was explained by direct correlations of the hormone with hematocrit (Ht) and PV through relationships with Ch and triglycerides (TG). Increased cortisol correlated with the higher plasma fluidity in the athletes as a result of inverse correlations of the hormone with TG and the increased suspension stability of the blood. Training-induced changes in hormonal and metabolic parameters were assumed to play an important role in the regulation of blood fluidity in athletes.     

Effect of estradiol-17ß and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17ß and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF_2α content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plama estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of ³H-estradiol into uterine tissues. Uterine PGF_2α content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF_2α content declined with repeated injections to values lower than that observed in controls. Prostaglandin F_2α content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Effects of fumonisin B1 alone and combined with deoxynivalenol or zearalenone on porcine granulosa cell proliferation and steroid production

Fumonisin B₁ (FB₁) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB₁, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B₁ had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB₁. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB₁. Regarding steroid production, FB₁ increased progesterone production, and FB₁ had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB₁ had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB₁ produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB₁. Fumonisin B₁ was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB₁ on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB₁ increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.