Lower E2 Concentrations Research Articles

Ultrasonographic evaluation of endometrial thickness near timed AI as a predictor of fertility in high-producing dairy cows

The objectives were to evaluate changes in endometrial thickness (ET) near the time of a synchronized ovulation and to assess the relationship of ET and fertility in lactating Holstein cows, with or without estrogen supplementation near timed ovulation. In Experiment 1, eight cows were examined with transrectal ultrasonography, once daily for 5 d, starting concurrent with PGF 2α (PGF) treatment during an Ovsynch protocol (GnRH - 7d - PGF - 72h - GnRH). The ET increased rapidly after PGF (from ∼7 to ∼9.5 mm), remained > 9 mm for the next 2 d, then decreased to ∼8 and 7.4 mm, 1 and 2 d, respectively, after the second GnRH. In Experiment 2,642 cows (total of 758 breedings) were subjected to an Ovsynch protocol (GnRH - 7d - PGF - 56h - GnRH - 16h - timed AI); cows received either no further treatment (Ovsynch) or 1 mg of estradiol-17β im 8 h before the second GnRH (Ovsynch + E2). For both uterine horns, ET was measured (∼2 cm from the internal uterine body bifurcation) before E2 treatment (48 h after PGF). In cows with ET ≤ 8 mm vs > 8 mm, rates of ovulation were 86.0% (n = 136) vs 98.1% (n = 472; P < 0.01), respectively, and percentage pregnant per AI (P/AI) were 26.7% (n = 146) vs 42.7% (n = 524; P < 0.01). Treatment with E2 increased P/AI in cows with lower ET (Ovsynch + E2 = 37.0% vs Ovsynch = 23.3%; P = 0.07), but did not significantly improve P/AI in cows with ET > 8 mm (Ovsynch + E2 = 43.4% vs Ovsynch = 42.1%). In conclusion, a single ultrasonographic evaluation of ET in Holstein cows 48 h after PGF treatment in an Ovsynch program was a good predictor of ovulation failure and pregnancy success. Perhaps poor fertility in cows with reduced ET was low peripheral E2 concentrations near AI, poor P4 priming, or luteolysis failure during timed AI procedures.

Estrogenic Activities of Sesame Lignans and Their Metabolites on Human Breast Cancer Cells

Sesame lignans (sesamin, sesamolin) and their metabolites (enterodiol, ED; enterolactone, EL; and sesamol) have been evaluated for their estrogenic activities. ED and EL have been indicated to have estrogenic/antiestrogenic properties on human breast cancer cells; however the estrogenic activities of sesamin, sesamolin and sesamol have not been reported. In the present study, estrogenic potencies of sesame lignans and their metabolites were determined by estrogen responsive element (ERE) luciferase reporter assay in T47D cells stably transfected with ERE-luc (T47D-KBluc cells) and quantifying pS2 and progesterone receptor gene expression in T47D cells. All tested compounds except ED possessed ability of ERE activation with a very low potency compared to estradiol (E2). These effects were abolished by coincubating tested compounds with 1 μM ICI 182 780, suggesting that estrogen receptors were directly involved in their ERE activations. Among tested compounds, sesamol showed the highest ability in ERE induction. The coincubation of increasing concentration of E2 (10(-12)-10(-6) M) with 10 μM of tested compounds resulted in a downward shift of E2-ERE dose-response curves. In contrast, at the low concentration of E2 (10(-12) M), sesamin and sesamol significantly exhibited additive effects on the E2 responses. The inhibitory effect in a dose-dependent manner was also observed when 1-100 μM sesamol was coincubated with 1 nM E2. Sesamin, sesamol and EL significantly induced pS2 gene expression whereas only sesamol could significantly induce progesterone receptor gene. The data obtained in this study suggested that sesame lignans and their metabolites possess weak estrogenic/antiestrogenic activity.

Molecular cloning, tissue distribution of two estrogen receptor β cDNAs of blue tilapia (&lt;I&gt;Oreochromis aureus&lt;/I&gt;) and effects of estrogen on their expression

Two sub-type estrogen receptor β (ERβ1/β2) cDNAs were obtained from blue tilapia,Oreochromis aureus by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RACE. The full length cDNA sequence of ERβ1 was 4 262 bp in length,containing a 5' Untranslated Regions (UTR) of 239 bp,3' UTR of 2 349 bp and an open reading fame (ORF) of 1 674 bp which encoded a putative protein of 557 amino acids with an estimated molecular weight of 62 ku. The ERβ2 cDNA was 2 506 bp in length with 393 bp of 5' UTR,109 bp of 3' UTR and an ORF of 2 004 bp which encoded a putative protein of 668 amino acids with an estimated molecular weight of 74 ku. The deduced amino acid sequence of ERβ1 of blue tilpia possessed 99.1% similarity with the counterpart of nile tilapia,O. niloticus,and similarities with other Perciformes fishes were from 82.6% to 94.2%. The ERβ2 amino acid sequences of blue tilpia shared 98.7%,81.8%,76.3%,64.7% and 55.0% identities with nile tilapia,Micropterus salmoides,Oncorhynchus mykiss,Fundulus heteroclitus and Danio rerio respectively. Blue tilpia ERβ1/β2 were clustered with nile tilapia ERβ1/β2 respectively in the phylogenetic tree. ERβ1/β2 mRNA expressions between male and female were compared by Real-Time PCR (RT-PCR). ERβs were expressed ubiquitously and differently in ten tested tissues. ERβ1 was significantly expressed in ovary,testis,liver,kidney and intestinal tissues compared with other tissues (P0.05). ERβ2 was highly expressed in testis,liver and kidney (P0.05). In ovary,testis,intestine and hypothalamus,expression of ERβ1 was significantly higher than that of ERβ2,and it was 164 times of ERβ2 in ovaries. After 17 beta-estradiol (E2) was injected for 24 h,ERα/β1 mRNA in hypothalamus of male blue tilpia was increased,of which ERα increased significantly. Expression of ERα in hypothalamus increased significantly in 7.5 mg/kg E2 treated group,while no significant change was observed for ERβ2. In pituitary,expression of ERα/β1/β2 in the low and middle E2 concentrations (2.5,5.0 mg/kg) was down-regulated,but up-regulated in high E2 concentration group (7.5 mg/kg),and significant difference was found for ERα (P0.05). ERβ2 expression was increased in testis significantly (P0.05) in middle and high E2 concentration groups (5.0,7.5 mg/kg). No significant change was observed for ERα/β1 expression. The different distribution of the ERβ1/β2 mRNAs and different regulation of ERα/β1/β2 mRNA in hypothalamus,pituitary and testis by E2 indicated that various biological functions existed among these three ER subtypes in blue tilpia.

Clomiphene citrate elicits estrogen agonistic/antagonistic effects differentially via estrogen receptors .ALPHA.and .BETA.

Clomiphene citrate (CC) is known to possess dual actions as an estrogen agonist and an estrogen antagonist. To see how the dual actions of CC are exerted through estrogen receptor alpha (ER alpha) and/or ER beta we developed a cell-based transcription assay system in which 293T cells were transfected with the luciferase reporter plasmid with estrogen responsive element and either human ER alpha or ER beta expression plasmid. CC at lower doses (10(-10) M and 10(-12) M), but not higher doses (10(-6) M and 10(-8) M) elicited estrogenic activity via ER alpha. However, CC at concentrations between 10(-6) M and 10(-12) M did not elicit any estrogenic activity via ER beta. In the presence of 17beta-estradiol (E2), CC behaved either as an agonist or as an antagonist via ER alpha depending on the concentrations of E2, i.e., antagonistic when combined with the higher E2 concentrations, agonistic with the lower E2 concentrations. On the other hand, via ER beta, CC acted as an estrogen antagonist regardless of the concentration of E2 added together. In conclusion, CC acts as an estrogen agonist/antagonist via ER alpha in a coexisting estrogen concentration-dependent way whereas it acts as an estrogen antagonist via ER beta whether or not estrogen is present.

17β-Estradiol at Low Concentrations Acts through Distinct Pathways in Normal Versus Benign Prostatic Hyperplasia-Derived Prostate Stromal Cells

The aim of this study was to identify differential responses to low concentrations of 17beta-estradiol (E2) in primary stromal cell cultures derived from either normal organ donors or benign prostatic hyperplasia or hypertrophy (BPH) specimens. Furthermore, we sought to identify the potential mechanism of E2 action in these cell types, through either a genomic or nongenomic mechanism. We initially treated stromal cells derived from five normal prostates or five BPH specimens with low concentrations of E2 (0.001-1.0 nM) and analyzed their growth response. To determine whether genomic or nongenomic pathways were involved, we performed studies using specific estrogen receptor antagonists to confirm transcriptional activity or MAPK inhibitors to confirm the involvement of rapid signaling. Results of these studies revealed a fundamental difference in the mechanism of the response to E2. In normal cells, we found that a nongenomic, rapid E2 signaling pathway is predominantly involved, mediated by G protein-coupled receptor-30 and the subsequent activation of ERK1/2. In BPH-derived prostate stromal cells, a genomic pathway is predominantly involved because the addition of ICI 182780 was sufficient to abrogate any estrogenic effects. In conclusion, prostate stromal cells respond to far lower concentrations of E2 than previously recognized or examined, and this response is mediated through two distinct mechanisms, depending on its origin. This may provide the basis for new insights into the causes of, and possible treatments for, BPH.

Gonadal Restructuring During Sex Transition in California Sheephead: a Reclassification Three Decades After Initial Studies.

California Sheephead, Semicossyphus pulcher, is a monandric protogynous hermaphrodite and a commercially and recreationally valuable labrid. Gonadal functionality of Sheephead through sex change was reclassified into nine classes using current criteria for categorization. Female ovaries were classified as immature, early maturing, mature, and regressing/recovering classes. Transition from female to male and subsequent male development was divided into early, mid and late transitional, developing/active male and regressing/recovering male. Reproductive states in Sheephead were correlated with estradiol (E2) and 11-keto testosterone (11-KT) concentrations in the blood plasma. All sexes had low E2 concentrations in the fall /winter seasons; in transitional and male individuals, levels remained low throughout the year. In contrast, female E2 concentrations were elevated in spring and peaked in the summer. Concentrations of 11-KT were variable throughout the year; however, females had significantly lower levels in the summer. This study allows a better understanding of the current state of California Sheephead in a heavily fished area. Knowledge of a species' reproductive characteristics is important in evaluating the sustainability of a population as it can set a baseline for reproductive potential. This research takes a critical step in gathering and organizing reproductive data such that it may be used in future studies for comparing reproductive potential across the range of the California sheephead.

Abstract 3770: Concentration-dependent Divergent Hypertrophic Effects of Estrogen in Adult Ventricular Myocytes

High concentrations of estrogen have been shown to attenuate myocardial hypertrophy and left ventricular remodeling. However, the effects of low concentration of estrogen observed in postmenopausal women on cardiac hypertrophy have not been studied. In the present study we examined the effects of high (0.1 and 1 nM) and low (1 and 10 pM) concentration of the synthetic analog of estradiol, 17β-estradiol (E2) on adult cardiomyocytes (CMs). CMs were isolated from adult male and female Sprague-Dawley rats. The cells were used immediately after isolation to measure pH i or cultured to assess hypertrophic phenotype (cell surface area), gene markers (atrial natriuretic peptide, ANP), and protein activation. Low concentration of E2 (1 and 10 pM) increased cell surface area (females: 20%, P &lt;0.05; males: 28%, P &lt;0.05) and ANP expression (females: 394%, P &lt;0.05; males: 497%, P &lt;0.05) after 24 h. However, high concentrations (0.1 and 1 nM) of E2 did not induce cell hypertrophy but instead blocked the hypertrophic effect of the α 1 -agonist phenylephrinerophy. The pro-hypertrophic effect of low concentration of E2 was prevented by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 μM) suggesting involvement of NHE-1 in mediating the E2-induced hypertrophy. Fluorometric measurements with the pH i -sensitive dye BCECF demonstrated that a 1 pM E2 increased the pH i (females: +0.05 pH units; males +0.12 pH units, P &lt;0.05) by a rapid non-genomic mechanism that was blocked by AVE. On the other hand, 1 nM E2 decreased the pH i (females: −0.24 pH units, P &lt;0.05; males: −0.07 pH units, P &lt;0.05) and this effect was also prevented by AVE. The NHE-1-mediated pro-hypertrophic effect of 1 pM E2 was dependent on phosphorylation of ERK1/2 MAPK since the effect was blocked with the ERK1/2 inhibitor PD98059 (10 μM) and there was no gender difference on ERK1/2 activation. E2 has a dual concentration-dependent role in adult CMs as manifested by a pro-hypertrophic effect at low concentrations (1 and 10 pM), and conversely, an anti-hypertrophic effect at high concentrations (0.1 and 1 nM). The pro-hypertrophic effect of E2 is mediated, at least in part, through ERK1/2/NHE-1 activation.

Estradiol and the Estradiol Metabolite, 2‐Hydroxyestradiol, Activate AMP‐activated Protein Kinase in C2C12 Myotubes

Systemic loss of estradiol (E2) during menopause is associated with increased adiposity which can be prevented with E2 replacement. Rodent studies suggest that E2, or lack of, is a key mediator in menopause-related metabolic changes. We have previously demonstrated that E2 treatment produces a rapid, dose-dependent activation of AMP-activated protein kinase (AMPK) in murine skeletal muscle. Activation of AMPK is implicated in the therapeutic benefits of many insulin sensitizing agents including metformin and thiazolidinediones. Here, we expand our observations and provide novel data which demonstrate that in addition to E2, its metabolite 2-hydroxyestradiol (2-HE2), activate AMPK in C2C12 myotubes. C2C12 myotubes were used to examine the effects on E2 and the by-products of its metabolism on AMPK activation. Low concentrations of E2 (10 and 100 nmol/l) were found to increase AMPK phosphorylation by approximately 1.6-fold, while a higher concentration (10 micromol/l) resulted in a approximately 3.0-fold increase. In comparison to E2 treatment alone, incubation of myotubes with E2 and 1-aminobenzotriazole (ABT) (a CYP450 inhibitor that blocks metabolism of E2) caused AMPK activation to be enhanced at low E2 concentrations, but attenuated at higher concentrations. The effects of ABT suggested that one or more E2 metabolites contribute to the maximal activation of AMPK at high E2 concentrations. Indeed, the estrogen metabolite 2-HE2, but not 2-methoxyestradiol (2-ME2), directly activated AMPK in C2C12 myotubes. We propose a model where E2, acting through its metabolite 2-HE2 and the estrogen receptors (ERs), activates AMPK in myotubes. Finally, activation is abolished when all E2 is metabolized to 2-ME2.

Hepatic and extrahepatic expression of estrogen-responsive genes in male adult zebrafish (Danio rerio) as biomarkers of short-term exposure to 17β-estradiol

Growing concern over reproductive hormones in the environment demands sensitive and efficient methods by use of molecular biomarkers to detect these contaminants in oviparous vertebrates. In this study, a real-time quantitative RT-PCR was adopted to investigate the expressions of three estrogen-responsive genes, Vtg I, Vtg II and ERalpha, in hepatic and extrahepatic tissues of male adult zebrafish exposed to varying concentrations of 17beta-estradiol (E2) for selected periods. Without exposure to E2, all the genes were expressed in the tissues of male controls with their levels being much lower than those in the respective tissues of female controls. The expressions of hepatic Vtg I and II mRNAs were induced significantly after 1-day exposure to E2 at as low as 0.25 microg L(-1). Significant induction in the expression of hepatic ERalpha mRNA required a higher E2 concentration (> or = 0.5 microg L(-1)) and a longer exposure (> or = 2 days), suggesting that Vtg I and Vtg II are more sensitive to E2 exposure. The induction of Vtg mRNA in the skin of zebrafish was also significant following a short exposure (1 day) to low E2 concentration (0.25 microg L(-1)), with the levels of Vtg I and Vtg II mRNA being increased by 25 and 5 times, respectively. These results suggest that Vtg I mRNA is a highly sensitive biomarker for determining the estrogenic effects of E2 and that the skin of zebrafish may be an appropriate substitute for liver for such a determination.

Low concentrations of prostaglandin E2 reduces type II collagen cleavage in OA

Low concentrations of prostaglandin E2 reduces type II collagen cleavage in OA

Highly Sensitive Second-Antibody Enzyme Immunoassay for Determination of Estradiol-17β Concentration in Blood Plasma of the Mithun (Bos frontalis)

The objective of the present study was to develop and validate a simple, sensitive, quick and economic enzyme immunoassay (EIA) for estradiol-17beta (E2) in mithun (Bos frontalis) plasma on microtiter plates using a second-antibody coating technique and hormone-horseradish peroxidase as a label. For the assay, the wells of microtiter plates were coated with affinity-purified goat anti-rabbit IgG that binds the hormone-specific antibody. One milliliter of mithun plasma was extracted using benzene and 50 microl of 300 microl volume reconstituted with assay buffer was run in the assay along with standards ranging from 0.10-100 pg/well prepared in assay buffer. The sensitivity of the assay was 0.72 pg/ml. The intra- and inter-assay coefficients of variation were below 10%, and the extraction efficiency was >93%. Linearity of recovery of the added hormone concentrations was recorded. The assay developed was further validated biologically by estimating the hormone concentrations in six female and five male mithun calves, 12 cyclic mithuns for the entire reproductive cycle, and four pregnant mithun cows. The EIA developed can estimate low concentrations of E2 (2.2-5.2 pg/ml) in growing calves as well as very high concentrations of the hormone during pregnancy (E2=85.6-143.5 pg/ml). Apart from being non-radioactive, the assay developed has several advantages over conventional radioimmunoassays: it is more sensitive, less labor intensive, simpler to perform, and less time consuming. In conclusion, the EIA procedure described herein is sufficiently reliable, economic, safe, quick and sensitive to estimate the hormone at all physiological levels in bovine plasma.

Differential Effect of Follicle-Stimulating Hormone and Estradiol on Expressions of Vascular Endothelial Growth Factor (VEGF) 120, VEGF164 and Their Receptors in Bovine Granulosa Cells

Vascular endothelial growth factor (VEGF) isoforms (VEGF 120 and VEGF 164) secreted by granulosa cells are involved in thecal angiogenesis during follicular development in the bovine ovary. However, whether the transcript of the VEGF120 and VEGF164 isoforms differs during follicular development in the ovary is still unknown. We first examined the gene expression of VEGF120, VEGF164, fms-like tyrosine kinase (Flt-1), and fetal liver kinase (Flk-1) in the granulosa cells (GCs) and theca cells (TCs) of pre-selection and post-selection follicles (PRF and POF respectively) from the bovine ovary. Then we examined the effects of FSH and estradiol (E2) on these factors in cultured bovine GCs. Messenger RNA (mRNA) expression was quantified using real-time PCR methods. The concentrations of E2 and P4 in the follicular fluid (FF) of the PRF and POF were estimated using an enzyme immunoassay (EIA). The concentrations of E2 and P4 in the FF were significantly higher in the POF than in the PRF. The ratio of E2/P4 in PRF and POF was 0.37 and 3.8, respectively. The expression levels of the VEGF120, VEGF164, and Flk-1 mRNAs in the GCs of POF with high E2 concentration were higher than those of PRF. The levels of the Flt-1 and Flk-1 mRNAs in the TCs were not different between PRF and POF. Since E2 in the FF of the POF used in the present study was high compared with the PRF, we examined the effects of E2 and FSH on the expression of the above genes using cultured GCs. Expression of VEGF120 mRNA was induced by a low concentration (1 ng/ml) of E2, whereas the levels of VEGF164 and Flk-1 mRNAs were not affected by E2. FSH stimulated the expression of the VEGF isoforms and Flk-1 genes. Moreover, the expression of those genes was enhanced when low E2 (1 ng/ml) was added to FSH. In conclusion, our data indicates that the VEGF isoforms have a follicle stage-dependent expression pattern. Thus, our results suggest that the expression of VEGF isoforms may be associated with characterization of the preovulatory phenotype during follicle development in the bovine ovary.

Estrogen Up-Regulates Mismatch Repair Activity in Normal and Malignant Endometrial Glandular Cells

Impaired mismatch repair (MMR) is reportedly crucial in the early stages of endometrial carcinogenesis. Although estrogen exposure is considered an important risk factor for endometrial carcinoma, the relationship between estrogen and MMR activity remains undetermined. The present study was undertaken to elucidate the effect of estrogen on MMR activity in normal and malignant endometrial cells. The expression of MMR proteins, hMLH1 and hMSH2, and its correlation with estrogen was examined using immunohistochemical and immunofluorescent techniques. The effect of estradiol (E2) on the expression of hMLH1/hMSH2 protein/mRNA and in vitro MMR activity using two types of heteroduplex (G/T mismatches, 2-base insertion-deletion loops) was examined in cultured normal endometrial glandular cells and estrogen receptor-positive endometrial carcinoma Ishikawa cells. Immunohistochemical expression of hMLH1 and hMSH2 in normal endometrial glands was positively correlated with the serum E2 levels. The expression of hMLH1/hMSH2 protein and mRNA was increased in normal endometrial glandular and Ishikawa cells by E2 treatment. In vitro MMR activity was up-regulated by E2 in both types of cell and heteroduplex. Immunofluorescent analysis demonstrated that E2 enhanced proliferation and hMLH1/hMSH2 expression in both cells; however, proliferating cells without hMLH1/hMSH2 expressions implying high-risk cells were more frequently observed under low E2 concentrations. Collectively, the E2-induced up-regulation of MMR activity in endometrial cells suggests that high estrogen levels act as an intrinsic defense against endometrial carcinogenesis, whereas the imbalance between cell growth and MMR under low E2 environment as seen at postmenopause is vulnerable to carcinogenesis.

High tea consumption diminishes salivary 17β-estradiol concentration in Polish women

We hypothesized that among reproductive-age women consuming large quantities of tea, the production of estradiol would be suppressed. It has been shown that catechins and theaflavines, the major constituents of tea, inhibit aromatase, an enzyme which catalyses the conversion of androgens to oestrogens. Our study included Polish women living in urban (n 61) and rural (n 48) areas. Women collected daily saliva samples for one complete menstrual cycle and filled out dietary questionnaires. Saliva samples were analysed by RIA for concentration of 17beta-estradiol (E2). Women with high (above the median) average daily consumption of black tea had reduced levels of salivary E2 in comparison with women who drank less black tea (below the median). This effect was observed within the whole study group, as well as separately within urban (P=0.0006) and rural (P=0.013) groups. High intake of the sum of subclasses of tea catechins and epigallocatechin gallate, assessed using the United States Department of Agriculture database (http://www.nal.usda.gov), was also associated with lower concentrations of E2 within all women (P=0.01 and P=0.0001, respectively) and within the urban group (P=0.0001 and P=0.004, respectively). Similar relationships were observed between the sum of subclasses of theaflavines and thearubigines and E2 levels for the whole group (P=0.002) and for urban women (P=0.02). Women with high consumption of tea had lower levels of E2 concentration throughout the entire menstrual cycle. These results may have implications for reducing hormone-related cancer risk by a relatively easy dietary intervention.

Development of Estrogen-Responsive Transgenic Medaka for Environmental Monitoring of Endocrine Disrupters

To develop a transgenic fish system to monitor environmental pollution, we generated a mvtg1:gfp transgenic medaka line, in which the gfp reporter gene was under the control of medaka vitellogenin1 (mvtg1) gene promoter. In this transgenic line, GFP was exclusively expressed in the liver of the mature adult female. Male and juvenile transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17-beta-estradiol (E2). Concurrent accumulation of mvtg1 and gfp mRNAs was observed during both development and estrogen treatment, indicating that the gfp transgene was faithfully expressed under the mvtg1 promoter. Dose- and time-dependent induction of GFP expression by E2 was investigated in male transgenic fish. The lowest-observed-effect concentration (LOEC) of E2 to induce GFP expression was 0.5 microg/L by observation of live fish and 0.05-0.1 microg/L by observation of dissection-exposed liver in a 30 day exposure experiment. GFP expression was observed within 36 h after treatment in high concentrations of E2 (5 microg/L), and it took longer to detect GFP expression under lower concentrations of E2. By removal and readdition of E2, we demonstrated that GFP expression was repeatedly induced. Finally, we also demonstrated that GFP expression could be induced by other estrogenic compounds, including 17-alpha-ethynylestradiol (EE2, 0.05 microg/L), diethylstibestrol (DES, 5 microg/L), estriol (10 microg/ L), and bisphenol A (BPA, 1 mg/L), but not by weak estrogenic chemicals such as nonylphenol (NP, up to 1 mg/ L) and methoxychlor (MXC, up to 20 microg/L). Our experiments indicated the broad application of the transgenic line to monitor a wide range of estrogenic chemicals.

Estradiol levels in prepubertal boys and girls--analytical challenges.

Increasing evidence points at an important function of low concentrations of estradiol (E2) in prepubertal boys and girls. E2 serum levels in prepubertal children are, however, often immeasurable in conventional E2 assays. This strongly hampers further investigation of the physiological relevance of E2 in children. In addition, there is an increasing concern of the potential effect of exposure to endocrine disrupters with estrogenic or antiandrogenic activity on pubertal development. A requirement of assessing the instance for this concern, adds further to the demands for applicable methodologies for the evaluation of the sensitivity of the organism to low E2 concentrations. Traditionally, E2 is measured by use of the radioimmunoassay (RIA). As an ultrasensitive alternative to the RIA, a recombinant cell bioassay has been developed. In this review, methodological aspects for these methods of analysis are examined and their applicability for evaluation of low E2 serum concentrations in children is estimated. Furthermore, available data on E2 levels in prepubertal boys and girls are evaluated and discussed, taking into consideration the limitations of the methods of analysis. In conclusion, there is a pronounced demand for new and improved methods of analysis for accurate and sensitive evaluation of low concentrations of E2.

Estrogen receptor expression in a human primitive neuroectodermal tumor cell line from the cerebral cortex: estrogen stimulates rapid ERK1/2 activation and receptor-dependent cell migration

Primitive neuroectodermal tumors (PNETs) are the most common form of pediatric brain tumor. Most often these malignant childhood brain tumors arise from neuroepithelial precursor cells in the cerebellum, and less frequently in the cerebral cortex. Because the normal PNET precursor cells from the cerebrum and cerebellum transiently express high levels of estrogen receptors (ERs), we hypothesized that the PNET cells of the cerebrocortical-derived cell line PFSK1 may also express ERs and would be responsive to estrogen. Results of immunoblot studies using ER-specific antiserum indicate that both ERα and ERβ are expressed in PFSK1 cells. The ability of estrogen to rapidly activate MAPK signaling was tested; low physiological concentrations of E2 stimulated ERK1/2 phosphorylation and nuclear translocation within 15min of exposure. Exogenously added 17β-estradiol (E2) could not stimulate PFSK1 growth, however E2 significantly increased PFSK1 cell migration, suggesting that rapid actions of E2 and ER-mediated processes might contribute to the metastatic phenotype of some PNETs.

Impacts of 17β-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish ( Danio rerio)

Zebrafish ( Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17β-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Long-term exposure to tamoxifen induces hypersensitivity to estradiol.

In women with hormone-dependent breast cancer, tamoxifen (TAM) frequently induces tumor regression, but regrowth occurs with continuation of antiestrogen therapy. Studies of breast xenografts in nude mice suggest that this secondary resistance to TAM may reflect the development of enhanced sensitivity to the estrogenic properties of TAM. In the current study, we examined the hypothesis that TAM could also induce a state of hypersensitivity to estradiol (E(2)) itself. Oophorectomized nude mice with MCF-7 cell xenografts received 25-mg implants of TAM [long-term TAM treated (LTTT) mice] or cholesterol (C-MCF-7) over a 5-month period (phase 1). The LTTT group regressed to a lesser extent than did C-MCF-7 tumors. After 4 months of TAM exposure, the LTTT tumors begin to regrow, as did the C-MCF-7, as assessed by slope analysis. At 5 months, TAM or vehicle implants were removed, and the LTTT and C-MCF-7 subgroups were given vehicle or two doses of E(2) to test estrogen sensitivity (phase 2). We used our "E(2) clamp" technique to maintain levels of plasma E(2) at either 1.25 or 20 pg/ml. Neither group responded to the very low concentrations of E(2) (1.25 pg/ml) or vehicle. The LTTT tumors but not C-MCF-7 tumors exhibited a growth response on exposure to 20 pg/ml E(2) during 7 weeks, as demonstrated with mixed models analysis. These studies provide evidence that long-term TAM exposure enhances sensitivity to the estrogenic effects of TAM and also to E(2) itself.

Lipoproteins during the estrous cycle in swine

The purpose of this study was to examine lipoprotein values at high versus low 17β-estradiol (E2) concentrations in Yucatan miniature swine. Estrous cycles were measured by heat checking the female on a daily basis using a boar. All swine were fed a 1,050-g low-fat, standard chow diet (8% kcal from fat) once per day. Fasted (24 hours) blood samples were collected during low (early luteal, day 5) and high (late follicular, day 18) E2 concentrations to determine differences in concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein cholesterol (HDL-C), and subfractions. Concentrations of E2 differed significantly from day 5 (3.5 ± 0.7 pg/mL) to day 18 (14.2 ± 1.8 pg/mL) of the estrous cycle. Except for HDL 3-C, all lipoprotein parameters examined were significantly elevated during high E2 versus low E2. TC/HDL-C and LDL-C/HDL-C ratios were significantly lower during the high E2 phase. These results suggest that lipoprotein concentrations fluctuate during the estrous cycle of swine, with high E2 concentrations associated with elevated lipoprotein concentrations.