ERα mRNA Levels Research Articles

Cloning of three estrogen receptors (ER) from killifish ( Fundulus heteroclitus): Differences in populations from polluted and reference environments

Epidemiological, wildlife, and laboratory studies support the hypothesis that chemicals released into the environment through anthropogenic activities are responsible for abnormalities of reproduction and development. Although the New Bedford Harbor (NBH) killifish population has survived and reproduced successfully for >50 yr (∼20 generations), fish have high body burdens of the major NBH contaminants (polychlorinated biphenyls); elevated levels of P450 aromatase B and vitellogenin mRNA (markers of estrogen effect); and evidence of endocrine disruption. To investigate possible adaptive changes in the estrogen response system of NBH killifish, we cloned the estrogen receptors (ER) from killifish populations resident in NBH and a relatively unpolluted reference site (Scorton Creek MA, SC). ERα, -βa, and -βb cDNAs encoding full-length polypeptides of 620, 543, and 672 amino acids, respectively, were identified. Each ER subtype had multiple splice variants, single nucleotide polymorphisms (SNPs), and a characteristic tissue distribution and developmental profile. As measured by real-time quantitative RT-PCR analysis, the overall tissue distribution of each ER was similar in NBH and SC fish, implying that the regulatory pathways which maintain tissue-specific expression are largely unchanged by long term pollutant exposure. Nonetheless, a striking difference was seen in the quantity of mRNA of the estrogen-inducible gene ERα, which was significantly lower in brain, liver and ovaries of reproductively active NBH as compared to SC females. Paradoxically, despite the “estrogenic” NBH environment, ERα mRNA levels did not differ in reproductively inactive NBH and SC females, or in males at the two sites at any time of year. We interpret results in NBH fish as due in part to a deficit of circulating estrogen, and in part to pollutant-mediated hyporesponsiveness of the ERα gene. In marked contrast to adult fish, ERα was elevated ∼5-fold in NBH as compared to SC embryos/larvae, perhaps indicative of estrogenic chemicals in yolk. We conclude that contaminants in the NBH environment impact molecular components of the estrogen signaling pathways in resident killifish populations. Whether these changes are transient or heritable requires further study.

Persistent sex-reversal and oviducal agenesis in adult Xenopus ( Silurana) tropicalis frogs following larval exposure to the environmental pollutant ethynylestradiol

It is known that estrogen-like environmental pollutants can feminise gonadal differentiation in frogs resulting in female-biased sex-ratios at metamorphosis. The long-term effects on reproductive function in frogs following larval exposure to pollutants are less known. Amphibian test systems which allow life-cycle studies are therefore needed. The aim of the present study was to characterise long-term estrogenic effects on the reproductive system of the emerging model species Xenopus ( Silurana) tropicalis following larval exposure to ethynylestradiol (EE 2). EE 2 is a synthetic estrogen that has been detected in sewage effluents and in surface waters. Newly hatched tadpoles (Niewkoop Faber (NF) stage 48) were exposed to the nominal EE 2 concentrations 0 (control), 1, 10, and 100 nM (with analytical chemistry support) until complete metamorphosis (NF stage 66). Effects on the reproductive organs were determined in juveniles (1 month after metamorphosis) and in 9-month-old frogs. Larval exposure to EE 2 caused female-biased phenotypic sex-ratios in both juvenile and adult frogs, which is in agreement with previous work on other frog species. Nearly all (97%) of the 63 EE 2-exposed 9-month-old frogs had ovaries. Histological evaluation of the gonads of the 9-month-old frogs showed that they were sexually mature. Among the adult frogs with ovaries there was a dose-dependent increase in the frequency of individuals lacking oviducts. Adult frogs exposed to 100 nM EE 2 that had ovaries but no oviducts had lower levels of estrogen receptor alpha (ERα) mRNA in the brain than control animals and those exposed to 100 nM EE 2 that had ovaries as well as oviducts. EE 2 exposure did not cause any significant changes in ERα mRNA levels in the ovaries of the adult frogs. The reduced level of ERα mRNA in the brain of individuals with ovaries lacking oviducts suggests an organizing effect of EE 2 on the central nervous system. The results show that transient early life-stage exposure to an environmental pollutant can induce effects on the reproductive organs and the central nervous system that persist into adulthood. Overall, our data suggest that X. tropicalis, which has a shorter generation time than the well-established model species Xenopus laevis, is a suitable model organism for research on developmental reproductive toxicity in anuran species.

Expression of estrogen receptors‐α and ‐β in bladder cancer cell lines and human bladder tumor tissue

Estrogen receptors (ERs) are known to mediate important physiologic responses as well as the growth of some tumors in response to estradiol stimulation. In a previous study the selective ER modulator raloxifene was shown to induce apoptosis in an ERbeta-positive bladder cancer cell line. However, the expression of ERbeta in human bladder cancer has not been thoroughly investigated. ERalpha and ERbeta expression in 224 bladder tumor samples was evaluated using tissue microarray and immunohistochemistry. Levels of ERalpha and ERbeta protein and mRNA expression were determined in several bladder cancer cell lines using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of estradiol and antiestrogen treatments on RT4 bladder cancer cell growth was determined by cell proliferation assays. Analyses revealed that only 2 human bladder cancers weakly expressed ERalpha. In contrast, the expression of ERbeta was detected in 141 tumors (63%). ERbeta was expressed in 58% of WHO Grade 1 and 2 tumors, whereas 70% of Grade 3 tumors demonstrated expression (P = .085). Importantly, although only 53% and 55% of Ta and T1 tumors demonstrated ERbeta expression, 80% of T2, 81% of T3, and 75% of T4 tumors showed ERbeta expression. The differences in ERbeta expression between Ta/T1 and T2/T3/T4 tumors were found to be highly significant (P < .001). Metastatic transitional cell carcinomas had ERbeta expression (80%) comparable to that of muscle invasive bladder cancers. Western blot analysis detected ERbeta protein expression in each of the 5 bladder cancer cell lines tested, whereas no or very low levels of ERalpha were found. Quantitative RT-PCR revealed that higher levels of ERbeta than ERalpha mRNA were present in 5637, T-24, TSU-Pr1, and TCC-Sup bladder cancer cells, whereas ER-alpha mRNA levels were greater than ERbeta in RT4 cells. Treatment with 17beta-estradiol modestly increased RT4 cell growth, whereas the antiestrogens, 4-hydroxtamoxifen, raloxifene, or ICI 182,780 inhibited the growth of RT4 cells. ERbeta is the dominant receptor expressed in bladder cancer cell lines and in the majority of human bladder tumors. Moreover, the degree of ERbeta expression increases with increasing stage and grade of differentiation. Antiestrogens have an inhibitory effect on the growth of bladder cancer cells in vitro.

Differential expression of the estrogen receptors alpha and beta during postnatal development of the rat cerebellum

Estrogen receptor (ER) β is a dominant ER subtype in the adult cerebellum. However, it is not known if this is also the case for the developing cerebellum. In the present study, quantitative real-time RT-PCR demonstrated that levels of cerebellar ERα mRNA in neonatal pups were significantly higher than in adults. In contrast, expression levels of cerebellar ERβ mRNA remained significantly unchanged during postnatal development. In situ hybridization and immunohistochemistry demonstrated that ERα mRNA and protein were predominantly expressed by Purkinje cells at all ages examined. ERα-expressing Purkinje cells were confined to the anterior lobes at postnatal day 7 (P7) but distributed in most lobes at P14 and P21. In the adult cerebellum, however, only a few ERα-immunoreactive Purkinje cells were observed. Thus, ERα expression was transiently increased during the time when Purkinje cell dendritic growth and synapse formation proceed, suggesting that a role for ERα in Purkinje cell differentiation. ERβ expression occurred in Golgi type neurons in the granular layer at P7, Purkinje cells at P14, and basket cells in the molecular layer at P21 and was detected in all the cell types in the adult cerebellum, suggesting a role for ERβ associated with neuronal differentiation and maintenance. Furthermore, double-labeled immunofluorescence for ERα and ERβ demonstrated their colocalization in Purkinje cells at P14, suggesting a possibility of their interaction. The discrete expression profiles for ERα and ERβ in the developing cerebellum suggest the two ERs play distinct roles in cerebellar development.

Efficient in vivoknock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA

BackgroundAdenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor α (ERα).ResultsTwo different shRNA constructs derived from the murine ERα coding sequence were designed (shERα). In vitro, transfection of three mouse cell lines with pSUPER-shERα constructs resulted in up to 80% reduction of endogenous ERα activity. A single mismatch in the target sequence eliminated the reduction of ERα activity, demonstrating the specificity of shERα. The subsequently generated Ad.shERα vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERα resulted in 70% reduced hepatic mouse ERα mRNA levels. Co-injection of Ad.shERα with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection.ConclusionAd mediated transfer of shERα expression constructs results in efficient and specific knockdown of endogenous ERα transcription both in vitro and in vivo.

Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage

Although 17beta-estradiol (E2) administration after trauma-hemorrhage (T-H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T-H are tissue and receptor subtype-specific. Male Sprague-Dawley rats underwent sham operation or T-H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 microg/kg), ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN; 5 microg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and intercellular adhesion molecule (ICAM)-1 levels in the liver, intestine, and lung were measured (n = 6 rats/group). ER-alpha and ER-beta mRNA levels in sham-operated rats were also determined. T-H increased MPO activity, CINC-1, CINC-3, and ICAM-1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T-H. Administration of the ER-alpha agonist PPT but not the ER-beta agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER-alpha mRNA expression was highest in the liver, whereas ER-beta mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T-H are receptor subtype and tissue-specific.

Transcriptional Regulation of the Estrogen Receptor α Gene by Testosterone in Cultures of Primary Rat Sertoli Cells

Background: We wanted to identify the presence of the estrogen receptor (ER) α in Sertoli cells and gain insight on the regulation of the ERα gene expression by testosterone in Sertoli cells. The transcriptional regulation of the ERα gene was investigated in primary Sertoli cell cultures by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). Methods: Primary Sertoli cell culture was performed. The expression levels of ERα and ERβ mRNA in Sertoli cells were detected by Northern blot, RT-PCR, immunocytochemistry and in situ hybridization. Results: The ovary, testis and epididymis showed a moderate to high expression of ERα while the prostate, ovary and LNCap cells showed the ERβ expression. ERα mRNA and protein were detected in the germ cells and Sertoli cells by in situ hybridization and immunocytochemistry. The level of ERα mRNA was gradually decreased in a time-dependent manner after testosterone treatment, and the changes of ERα mRNA were dependent on the concentration of testosterone. Androgen binding protein and testosterone-repressive prostate message-2 (TRPM-2) mRNA were reduced at 24 hour by estradiol, while the transferrin mRNA was not affected. ERα mRNA was strongly detectable in the testes of 7 days-old-rats, but it was gradually decreased from 14 to 21 days of age. The primary Sertoli cells also showed the same pattern. The ERα gene expression was also regulated by testosterone in the Sertoli cells prepared from the 14- and 21-day old rats. Conclusions: These results suggest that ER α is transcriptionally regulated by testosterone and it may play some role in the Sertoli cells. (J Kor Soc Endocrinol 21:106~115, 2006)

Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Estrogen receptor α as a target for indole-3-carbinol

A wealth of preclinical evidence supports the antitumorigenic properties of indole-3-carbinol (I3C), which is a major bioactive food component in cruciferous vegetables. However, the underlying molecular mechanism(s) accounting for these effects remain unresolved. In the present study, estrogen receptor alpha (ER-α) was identified as a potential molecular target for I3C. Treating MCF-7 cells with 100 μM I3C reduced ER-α mRNA expression by approximately 60% compared to controls. This reduction in ER-α transcript levels was confirmed using real-time polymerase chain reaction. The I3C dimer, 3,3′-diindolylmethane (DIM), was considerably more effective in depressing ER-α mRNA in MCF-7 cells than the monomeric unit. The suppressive effects of 5 μM DIM on ER-α mRNA was comparable to that caused by 100 μM I3C. DIM is known to accumulate in the nucleus and is a preferred ligand for aryl hydrocarbon receptor (AhR) to I3C. The addition of other AhR ligands, α-naphthoflavone (α-NF, 10 μM) and luteolin (10 μM), to the culture media resulted in a similar suppression in ER-α mRNA levels to that caused by 5 μM DIM. Thus, it is likely that the binding of ligands to AhR inhibits nuclear ER-α transcript. The results from these experiments suggest that the antitumorigenic effects of I3C in MCF-7 human breast cancer cells may arise from its ability to reduce ER-α expression through the binding of its metabolite, DIM, to the nuclear AhR.

Distinct mRNA, protein expression patterns and distribution of oestrogen receptors α and β in human primary breast cancer: Correlation with proliferation marker Ki-67 and clinicopathological factors

To elucidate the molecular profile of oestrogen receptors α and β (ERα, ERβ) we studied ERα and ERβ expression at the mRNA and protein levels using real-time polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemical (IHC) methods in 41 primary breast cancers and surrounding tissues. ERα mRNA and ERβ mRNA were detected in all of the breast cancer and normal matched tissues analysed. ERα mRNA levels showed greater diversity than ERβ mRNA levels and the range of amount of ERβ transcripts was far smaller than that of ERα. At the protein level, the percentage of ERα- or ERβ-positive cases changed. Seventy percent of the tumours studied produced full-length 65 kDa ERα protein in Western blot analysis and 67% of assessed cases were positive in IHC. Full-length 57 kDa ERβ protein was detected by Western blotting in 97% of analysed breast cancers, while 67% were ERβ-positive using IHC. ERα was localised in the nucleus, while cytoplasmic and perinuclear localisation of ERβ was observed in normal as well as in breast cancer cells. The amount of ERα (but not ERβ) increased with age. The expression of ERα correlated positively with progesterone receptor and negatively with proliferation marker Ki-67. These results confirm the previous observations that the lack of ERα protein expression is not due to lack of ERα gene expression or methylation of ERα promoter, but due to post-transcriptional or post-translational mechanisms. Our investigation also suggests that ERα is more dysregulated in breast cancer, and thereby ERβ is more tightly regulated in the tumour.

Neural steroid hormone receptor gene expression in pregnant rats

Estrogen and progesterone play important roles during pregnancy in stimulating the onset of maternal behavior at parturition. The status of receptor expression of these hormones during pregnancy in neural regions that regulate maternal behavior is unclear. The objective of the present study is to characterize changes in neural gene expression of the estrogen receptors α and β (ERα and ERβ) and the progesterone receptor (PR) during the latter part of pregnancy. Brains from primigravid Sprague–Dawley rats were collected on days 15 and 21 of pregnancy. Micropunches of the olfactory bulb (OB), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BnST), hypothalamus (HYP), medial amygdala (MeA), and the temporal cortex (TCx) were analyzed by real-time RT-PCR (Taqman™) for levels of gene expression. No changes in either ERα or ERβ mRNA levels were detected in any brain region between days 15 and 21 of pregnancy: however, the MPOA had higher levels of both ERα and ERβ than other brain regions. Progesterone receptor mRNA levels, in contrast, declined significantly in the MPOA, HYP, and TCx, between days 15 and 21 of pregnancy ( P < 0.05). In addition, the levels of PR mRNA were significantly higher in the HYP and TCx compared to both the OB and MeA. These data indicate that there is a downregulation of PR prepartum and suggest that this decrease may play a role in the disinhibition of maternal behavior at parturition.

The estrogen receptor α Σ3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus

The gene for estrogen receptor alpha (ER alpha) has been shown to be under complex hormonal control and its activity can be regulated by mRNA alternative splicing. Here we examined the regulation of ER alpha transcription and translation in the rat uterus by ovarian steroid hormones. We examined whether expression of ER alpha mRNA splice isoforms is hormonally regulated in ovariectomized (OVX) and cycling rats. Adult OVX female rats were treated daily with 17-beta estradiol (E2) (0.05 microg/rat or 5 microg/rat), progesterone (P4) (1 mg/rat) or a combination of both hormones for 4 days. Animals were killed 24 h after the last injection and uterine horns were removed. In order to determine whether ER alpha mRNA isoforms are differentially expressed under various physiological conditions, animals were evaluated at proestrus, estrus and diestrus. The ER alpha protein and mRNA were detected by immunohistochemistry and comparative RT-PCR analysis respectively. The presence of ER alpha mRNA isoforms was evaluated using a nested RT-PCR assay. In OVX control rats, ER alpha mRNA and protein levels were high, demonstrating a constitutive expression of the ER alpha gene in the uterus. When animals received P4 or the high dose of E2, a significant decrease in both ER alpha mRNA and protein was observed in the uterus. However, when rats were protein was treated with the low dose of E2, only the ER alpha down-regulated; no changes were observed in ER alpha mRNA expression. In addition to the full-length ER alpha mRNA, OVX control rat uteri expressed three shorter transcripts: sigma3, sigma4 and sigma3,4 (lacking exon 3, exon 4, or both 3 and 4 respectively). Surprisingly, when OVX animals were treated with P4, the low dose of E2 or a combination of both steroids, expression of the sigma3 isoform was completely abolished. During the estrous cycle, all ER alpha mRNA splicing variants were detected at proestrus and estrus. However, in diestrus, significant low levels of the sigma3 isoform were observed. In summary, our results suggest a dose-dependent relationship between E2 concentrations and the level of control in the ER alpha transcription-translation cascade. Moreover, the alternative splicing of the ER alpha primary transcript is influenced by the hormonal milieu, suggesting that these events could affect the estrogen responsiveness of the rat uterus during the estrous cycle.

Oestrogen receptor-related receptor alpha (ERRα) and oestrogen receptors (ERα and ERβ) exhibit different gene expression in human colorectal tumour progression

We have investigated the presence of oestrogen receptor-related (ERR) mRNA in human colorectal tumour tissues and adjacent normal mucosa by reverse transcriptase and nested-polymerase chain reaction. ERRα was found in 100% of the patients and ERRγ in approximately 30% while ERRβ was not detected at all. The multiplex PCR analysis showed elevated levels of ERRα mRNA in tumour tissue compartment as compared to normal mucosa, whereas ERRγ mRNA was found in lower levels but in both tissue compartments. In contrast, oestrogen receptor (ERα and ERβ) mRNA levels were shown to be decreased in tumour tissues. A positive correlation was observed between ERα and ERβ and between ERα and ERRα, respectively, in normal mucosa but not in tumour tissue. ERRα expression in tumour tissues significantly increased from TNM stages II to IV, whereas both ERs progressively declined. These findings suggest that ERRα, as well as the two ERs, might play a critical role in the progression of the colorectal cancer.

Gene expression changes in rat prostate after activation or blocking of the androgen and estrogen receptor

Several endpoints of different molecular complexity were studied in the Hershberger assay in order to evaluate the specificity and suitability of this test as a broad screening model. Androgen and estrogen receptors were activated or blocked, and expression of typical estrogen- or androgen responsive genes (complement C3, ERα, ERβ, AR, TRPM-2, PBP C3, ODC, and IGF-1 mRNA) was analyzed in rat ventral prostate by real time RT-PCR. Administration of estradiol benzoate (EB) to castrated testosterone-treated rats had no effect on reproductive organ weights or gene expression levels and the anti-estrogen, ICI 182780, only affected ODC expression. Therefore, estrogenic or anti-estrogenic compounds would not be expected to seriously affect the outcome of a Hershberger test. However, EB given alone to castrated rats resulted in various effects. EB increased seminal vesicle weight, an effect reversed by ICI 182780, and affected TRPM-2, PBP C3, ODC, IGF-1, AR, and ERα mRNA levels. AR expression in the prostate seemed to be under regulation of both estrogens and androgens, as ICI 182780 inhibited the testosterone-induced AR expression, and flutamide inhibited the EB-induced AR expression. These data indicate that estrogens have various effects in castrated male rats and that expression of several genes is under multi-hormonal control in the ventral prostate. However, interactions between estrogens and androgens do not play a major role in the Hershberger assay, as simultaneous TP administration abolished the effects of EB. First choice of gene expression profiles in the Hershberger assay to study androgenic or anti-androgenic effects would be the traditional, TRPM-2 and PBP C3, supplemented with the new complement C3.

Effects of the putative xenoestrogens Benzophenone-2 (BP2) and Octyl-methoxycinnamate (OMC) on gene expression in the rat pituitary

Based on the rat uterotrophic assay, Benzophenone-2 (BP2) and Octyl-methoxycinnamate (OMC), two frequently used UV-filters in cosmetics, have been recently accused as estrogenic endocrine disrupters. An estrogenic action requires induction of the estrogen receptor (ER) signalling pathway comprising ERα and ß and their splice variants like the truncated estrogen receptor protein 1 (TERP1). Other known modulators of the ER signalling pathway are the aryl hydrocarbon receptor (AhR) and the estrogen receptor-related receptor 1 (ERR1). To further characterise the suggested estrogenic activity of OMC and BP2, the expression pattern of these nuclear receptors were measured in the pituitary of ovariectomized (ovx) rats. As reference compounds Estradiol-valerate (E2, ER ligand), Diethylstilbestrol (DES, ERR1 ligand) and 3-Methylcholanthrene (3MC, AhR ligand) were used. Adult ovx rats were treated orally once per day for 5 days with either E2, DES (0.6mg/kg each), 3MC (40mg/kg), or BP2 or OMC (10, 33, 100, 333mg/kg dissolved in olive oil). The control group received olive oil only. Gene expression was determined with real-time RT-PCR. A strong uterotrophic effect, cornification of the vaginal epithelium and a significant reduction of LHß-expression was observed in E2, DES and BP2 but not in OMC and 3-MC treated rats. Pituitary mRNA levels of ERα and ERR1 remained unaffected in all treatment groups, while levels of ERß and AhR were reduced to about 50% of the control group by E2 and DES (p<0.01). A profound increase in TERP1 expression occurred in the E2, DES and BP2 groups (about 200 fold, p<0.01).

Differential estradiol effects on estrogen and progesterone receptors expression in the oviduct and cervix of immature ewes

In order to study the effect of estradiol-17β (E2) on estrogen (E) and progesterone (P) receptors expression in oviduct and cervix of lambs, their respective transcripts (ERα mRNA and PR mRNA) were determined by solution hybridization and the receptor proteins (ER and PR) by binding assays after E2 treatments. Lambs ( n = 4 in each group) were not treated or treated with one, two or three i.m. injections of E2 (1 μg/kg) at 24 h of interval. Tissues were obtained 12 or 24 h after the last E2 injection. Estradiol treatments increased ERα mRNA and PR mRNA concentrations in an organ-dependent manner: transitory in the oviduct while maintained in the cervix. The E2 effect on the oviductal and cervical ER and PR concentrations were biphasic, with an initial reduction of receptors content that was followed by restoration. The ER restoration in oviduct was earlier than in the cervix. In summary, this study shows that E2 treatments may exert an inductive effect in ERα mRNA and PR mRNA levels and a biphasic effect in ER and PR concentrations in oviduct and cervix of immature ewe. These E2 effects varied in timing and strength depending on the organ of the reproductive tract.

Prospective evaluation of estrogen receptor-β in predicting response to neoadjuvant antiestrogen therapy in elderly breast cancer patients

It has been proposed that knowledge of estrogen receptor beta (ER-beta) expression may refine estrogen receptor alpha (ER-alpha) predictivity of response to endocrine therapy. We challenged this hypothesis in ER-alpha-positive breast cancers subjected to preoperative antiestrogen treatment. Forty-seven elderly (> or =65 years old) women with nonmetastatic, ER-alpha-positive (by immunohistochemistry) primary breast cancers (> 2 cm in diameter) entered a neoadjuvant hormone therapy protocol (60 mg/day toremifene for 3 months). ER-alpha and ER-beta (ERs) mRNA was determined by semiquantitative RT-PCR, before (on core needle biopsy) and after (on surgical specimens) neoadjuvant treatment. Study end points included: (1) relation between treatment response and ER mRNA expression; and (2) changes in ER expression after treatment. The response was clinically assessed as tumor size change at the end of the preoperative treatment. ER mRNA expression was assessable before and after treatment in 38 and 20 cases respectively. ER-beta was co-expressed with ER-alpha at variable levels and significantly correlated only with progesterone receptor (P = 0.0285). Objective clinical response, including patients with minor change (> or =25-<50% tumor shrinkage after treatment), was documented in 68.4% of cases and was independent of ER-beta levels or changes. ER-alpha levels were higher in tumors from patients in complete remission than in those from women achieving partial response or minor change compared with non-responsive patients (median expression values: 801 versus 516 versus 320 arbitrary units) and were consistently down-regulated by preoperative treatment. We conclude that in this elderly patient population with ER-alpha-positive tumors, ER-beta mRNA was neither predictive of response to preoperative toremifene nor provided additional information to the knowledge of ER-alpha mRNA levels, which, conversely, were directly correlated with likelihood of response.

Expression of Estrogen Receptors-α and -β in the Pregnant Ovine Uterine Artery Endothelial Cells In Vivo and In Vitro1

Estrogen is recognized to be one of the driving forces in increases in uterine blood flow through both rapid and delayed actions via binding to its receptors, ER alpha and ER beta at the uterine artery (UA) wall, and especially in UA endothelium (UAE). However, information regarding estrogen receptor (ER) expression in UAE is limited. This study was designed to test whether ERs are expressed in UAE in vivo, and if they are, whether these receptors are maintained in cultured UA endothelial cells (UAECs) in vitro. By using immunohistochemical and Western blot analyses, we clearly demonstrated ER alpha and ER beta protein expression in pregnant (Days 120-130) sheep UA and UAE in vivo and as well as cultured UAECs in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) amplified both ER alpha and ER beta mRNAs in UA, UAE, and UAECs. Of interest, a truncated ER beta (ER beta2) variant due to a splicing deletion of exon 5 of the ER beta gene was detected in these cells. Quantitative RT-PCR analysis revealed that ER alpha mRNA levels are approximately 8-fold (P < 0.01) higher than that of ER beta in UAECs, indicating that ER alpha may play a more important role than ER beta in the UAEC responses to estrogen. Fluorescence immunolabeling analysis showed that ER alpha is present in both nuclei and plasma membranes in UAECs, and the latter is also colocalized with caveolin-1. The membrane and nuclear ER alpha presumably participate in rapid and delayed responses, respectively, to estrogen on UAE. Taken together, our data demonstrated that UAE is a direct target of estrogen actions and that the UAEC culture model we established is suitable for dissecting estrogen actions on UAE.

Differential regulation of nuclear receptors, neuropeptides and peptide hormones in the hypothalamus and pituitary of food restricted rats

Food restriction is associated with a number of endocrine disturbances. We validated the experimental conditions for several house-keeping genes and determined the effects of 12 day 50% food restriction on hypothalamic and pituitary transcription of genes involved in different neuroendocrine systems, using real-time quantitative polymerase chain reaction (PCR). A total of 7 nuclear receptors and 12 neuropeptides and peptide hormones were investigated in the dorsal and ventral hypothalamus and the pituitary gland in rats. In the hypothalamus, food restriction reduced mRNA levels of estrogen receptor α (ERα), progesterone receptor, glucocorticoid receptor, thyroid hormone receptor α and β, pro-opiomelanocortin (POMC), growth hormone-releasing factor (GHRF), corticotropin-releasing factor (CRF), thyrotropin-releasing factor (TRF), somatostatin, and increased that of neuropeptide Y (NPY). In the pituitary, the treatment reduced growth hormone (GH), luteinizing hormone β (LHβ) and thyrotropin β, but increased ERα mRNA levels. The study provides a map of how food restriction affects the regulation of a number of transcripts involved in neuroendocrine control.

Differential estrogen receptor gene expression in human peripheral blood mononuclear cell populations

Estrogens have been shown to modulate immune responses. Several studies have demonstrated the capacity of T cells, B cells, and monocytes to respond to estrogens and estrogen receptor (ER) expression in these cell types has been reported. However, little is known regarding the relative expression in these cells of ERα and the more recently identified ERβ. In the present study, results of quantitative TaqMan™ RT-PCR analyses indicate that ERs are differentially expressed in PBMC subsets. CD4 + T cells express relatively high levels of ERα mRNA compared with ERβ, whereas B cells express high levels of ERβ mRNA but low levels of ERα. Peripheral blood CD8 + T cells and monocytes express low but comparable levels of both ERs. This quantitative analysis of ER expression in distinct PBMC subsets may provide a basis for dissecting the mechanisms of immune modulation by estrogens and identifying therapeutic targets for the treatment of inflammatory and immunologic disorders.