Estrogen Receptor Gene Expression Research Articles

High expression of the chemokine receptor CXCR4 is associated with estrogen receptor-negative breast cancer

642 Background: Chemokine receptor CXCR4 and its ligand CXCL12 (SDF-1alpha) are important in the metastatic spread of breast cancer. Previous studies have shown that CXCR4 is overexpressed in breast cancers as compared to normal breast tissue and that estradiol increases levels of CXCL12. The association between CXCR4 and estrogen receptor (ER) status is unclear. Methods: We performed genome-wide molecular analysis of 61 primary breast cancer tissue samples. Total RNA was extracted and hybridized to Affymetrix HG-U133 oligonucleotide microarrays, and differentially expressed genes between groups were identified using a Welch t-test. CXCR4 protein expression was evaluated by immunohistochemistry (IHC) using a monoclonal antibody (R&D Systems, Minneapolis, MN). We characterized CXCR4 nuclear staining as high vs low-medium. Several clinico-pathologic variables were correlated with CXCR4 expression. Results: A predominantly nuclear staining pattern was noted for CXCR4. Of 61 primary tumors, 15 had high nuclear expression and 46 had low-medium expression. High levels of nuclear CXCR4 expression were significantly correlated with ER-negativity (p =0.0005). CXCR4 protein expression had no association with Her-2 status (gene copy number or IHC 3+), tumor size, or lymph node status. Using p<0.001, protein expression of CXCR4 was associated with 105 significantly differentially expressed genes including relative under expression of ER and ER-related genes and over expression of VEGF. Conclusion: High nuclear protein expression levels of CXCR4 in primary breast cancers are significantly associated with ER-negativity and with low differential expression of ER and ER-related genes. Since estrogen may play a role in CXCR4 activation, further investigation of the association of high CXCR4 expression and ER-negativity is planned. No significant financial relationships to disclose.

Tissue preferential expression of estrogen receptor gene in the marine snail, Thais clavigera

Sex steroid hormones have been widely detected in molluscs, and experiments have shown the importance of sex steroids in sex determination, gonadal tissue maturation and gametogenesis. Nevertheless, the signaling pathways of sex steroids in invertebrates have not yet been elucidated. In order to gain insights into the mechanism of sex steroid signaling in molluscs, we have, therefore, tried to isolate molluscan estrogen receptors from the prosobranch mollusc Thais clavigera. Cerebral ganglia of T. clavigera (Mollusca, Gastropoda, Prosobranchia) were subjected to RNA extraction, and degenerate primers for amino acid sequences conserved in vertebrate estrogen receptors were designed. PCR amplification using cerebral RNA and degenerate primers followed by 5′- and 3′-RACE identified the cDNA encoding T. clavigera estrogen receptor 1 (tcER1). The deduced amino acid sequence showed 93% identity in the DNA-binding domain and 72% identity in the ligand binding domain when compared to Aplysia estrogen receptor. Reporter gene assay revealed that tcER1 is constitutively active and unresponsive to estrogen. Quantitative analysis of the tcER1 mRNA level demonstrated the preferential expression in the ovary. Furthermore, cerebral ganglia expressed tcER1 at a high level in the spring followed by subsequent enlargement of the ovary in later seasons. These results suggest importance of tcER1 in the seasonal development of reproductive organs in T. clavigera.

Molecules from Nature: Modulating the Expression of Estrogen Receptor Genes in Breast Cancer Cells

Estrogen receptors (ERα and ERβ) belong to the nuclear receptor superfamily. They mediate the effects of estrogens by binding (as homodimers or heterodimers) either directly to DNA at their estrogen-response elements (EREs) or by protein-protein interactions with other transcription factors (i.e., AP-1, NF-kβ) bound to their cognate DNA sequences, thus regulating the transcription of estrogen-responsive genes [1]. The ER mediates estrogenic activity in a variety of organs, including those in the reproductive, cardiovascular, immune and central nervous systems. Estrogen provides neuroprotection against neurodegenerative diseases, including Parkinsons disease. Its effects may stem from interactions with neurons, astrocytes and microglia. Human breast cancer cell lines expressing the (ERα), all-trans-retinoic acid (ATRA) receptor α (RARα) and cellular retinoic acid binding protein II (CRABPII) genes are sensitive to ATRA-mediated growth inhibition, as well. Several naturally found polyphenols like, flavonoids, phytoestrogens, etc., interact with the ERα and ERβ, exhibit estrogenic/antiestrogenic activities, and may play protective roles in cancer, inflammation, heart disease and osteoporosis, etc., and other disease conditions. These molecules are considered

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERα and ERβ) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERα, and ERβ from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERα, and ERβ genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ER α and Rm-ERβ cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERβ mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERα mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERα mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERβ mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 μg/l) and BisA (600 μg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERα, and Rm-ERβ mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERα, and Rm-ERβ genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.

Effects of 4-n-Nonylphenol and Tamoxifen on Salmon Gonadotropin-Releasing Hormone, Estrogen Receptor, and Vitellogenin Gene Expression in Juvenile Rainbow Trout

Alkylphenols such as nonylphenol (NP) are one of a wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of hepatic vitellogenin (Vg) gene expression is widely used as a biomarker for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the brain. To evaluate brain and liver effects of 4-n-nonylphenol (4-NP), juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne 4-NP or 17beta-estradiol (E(2)). Changes in mRNA levels of salmon gonadotropin-releasing hormone (sGnRH) and estrogen receptor alpha (ERalpha) isoforms in the brain and ERalpha isoforms and Vg in the liver were measured after 3 and 6 days of exposure, with the help of a relative RT-PCR-based quantification method. Fish were treated with increasing doses of 4-NP ranging from 0.01 to 10 microM (2.2 microg/l to 2.2 mg/l), and results were compared to the effect of E(2) or tamoxifen, a specific ER modulator. In liver, E(2) and the highest doses of 4-NP increased Vg and ERalpha long-isoform mRNA levels within 3 or 6 days of exposure, but 4-NP did not have any effect on ERalpha short-isoform transcription level. In the brain, 4-NP reduced sGnRH2 gene expression in a dose-dependent manner, but did not modify sGnRH1 or ERalpha mRNA levels.

Using Salmonid Microarrays to Understand the Dietary Modulation of Carcinogenesis in Rainbow Trout

The highlighted article in this issue by Tilton et al. (2005a) is an innovative approach to evaluate the modulation of estrogen receptor (ER) and aryl hydrocarbon (Ah)-receptor pathways as mechanisms underlying indole-3-carbinol (I3C) tumor promotion in rainbow trout (Onchorhynchus mykiss). I3C and its major in vivo component 3,3'-diindolylmethane (DIM) are potent tumor promoters that appear to target both of the aforementioned receptor pathways. However, the relative importance of I3C modulation of ER and AhR-dependent pathways in the promotion of rainbow trout hepatocarcinogenesis has not been established. Previously, researchers within this group reported that I3C promotes aflatoxin B1 (AFB1)-induced trout hepatocarcinogenesis post-initiation at low concentrations in the diet that induce the expression of vitellogenin, a downstream marker for the activation of ER-dependent pathways in fish. Furthermore, the promotional effects of I3C on AFB1 hepatocarcinogenesis in rainbow trout occur at concentrations that differentially induce vitellogenin, but not CYP1A expression. Interestingly, higher I3C concentrations induce the expression of both CYP1A and vitellogenin. Thus, the relative induction of vitellogenin and CYP1A expression, which are respective markers for activation of fish ER and AhR-mediated gene expression, suggest that these pathways may be important for tumor promotion by dietary I3C in trout. Understanding the complexities of I3C-mediated tumor promotion is essential from several perspectives. For example, there is the obvious need to increase our basic understanding of dietary modulation of carcinogenesis. In addition, I3C also exhibits significant antioxidant and cancer chemoprotective effects under certain experimental conditions and in certain models which have led to its recent marketing as a dietary supplement, as well as its development as a possible chemopreventive agent in humans.

Downregulation of Estrogen Receptor Gene Expression by Exogenous 17β-Estradiol in the Mammary Glands of Lactating Mice

The biological actions of estrogen are mostly conveyed through interaction with the nuclear estrogen receptor (ER). Previous evidence indicated that estrogen participates in self-regulation through the modulation of the expression of its own receptors. However, the self-regulation of estrogen against ER in the mammary gland during established lactation has not yet been investigated. The present study evaluated ER gene expression in the lactating gland activated by large doses of 17beta-estradiol (E(2)). Repeated E(2) treatments dose-dependently decreased the gene expression of ER, especially its subtype ER-alpha mRNA, which was decreased to 10% of the vehicle-injected control by 1 mug E(2) injection, whereas it was decreased by 73% for another subtype, ER-beta. A single injection of 5 mug of E(2) drastically downregulated both ER genes within 12 hrs of injection, and they did not recover to pretreatment level within 48 hrs. Western blot analysis verified that E(2) treatment inhibited the phosphorylation of Stat5, which is a potent transcriptional regulator for ER mRNA. The present findings demonstrate that E(2) treatment decreases the gene expression of its own receptor in the mammary gland during galactopoesis and induces an apparent transition of the ER profile in the mammary gland during lactation into postlactation.

Folate Intake and Risk of Breast Cancer Characterized by Hormone Receptor Status

Folate plays an important role in DNA methylation, and aberrant methylation of the estrogen receptor (ER) gene may be related to the loss of ER gene expression in breast tumors. Thus, deficient folate status has been hypothesized to be associated primarily with ER gene-negative breast tumors, but data relating folate intake to breast cancer risk according to ER status are sparse. We conducted a prospective cohort analysis of folate intake among 88,744 women in the Nurses' Health Study who completed a food frequency questionnaire in 1980 and every 2 to 4 years thereafter. During 20 years of follow-up, 2,812 ER+ and 985 ER- invasive breast cancer cases were documented. Higher total folate intake was significantly associated with lower risk of developing ER- but not ER+ breast cancer; the multivariable relative risks (RR) and 95% confidence intervals (95% CI) comparing the highest to the lowest quintile were 0.81 (0.66-0.99) for ER- tumors and 1.00 (0.89-1.14) for ER+ tumors. The inverse association between total folate intake and ER- breast cancer was mainly present among women consuming at least 15 g/d of alcohol (multivariable RR, 0.46; 95% CI,=0.25-0.86; top versus bottom quintile). These findings support the hypothesis that higher folate intake reduces the risk of developing ER- breast cancer. Ensuring adequate folate intake seems particularly important for women at higher risk of breast cancer because of alcohol consumption.

Use of DNA microarrays to determine estrogen and HER-2 receptor status in breast cancer

546 Purpose: To evaluate whether estrogen receptor (ER) and HER-2 receptor status in breast cancer is accurately determined using gene expression profiles. Patients and Methods: Gene expression profiles were obtained from 82 fine needle aspiration (FNA) samples and 40 frozen tissue samples in 122 patients with newly diagnosed breast cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were used to define ER and HER-2 status. The cohorts of FNA and tissue samples were evaluated in two laboratories using Affymetrix U133A GeneChip technology and all expression data were normalized using a single digital standard (dCHIP software). Thresholds of gene expression to determine ER and HER-2 status were defined in the cohort of 82 FNAs and then tested in the 40 tissue samples. Results: ER gene expression (probe ESR1 205225) ≥ 500 (arbitrary units) defined ER-positive status (IHC ≥ 10%) in 82 FNAs with 96% overall accuracy (95% CI, 90%–99%), and was validated with 90% overall accuracy (95% CI, 76%–97%) in 40 tissues. Considering ER IHC > 0% as positive, the overall accuracies were 98% (FNA) and 100% (tissue). ER reporter genes (55 probes) were identified by co-expression with ER (ESR1) (Spearman correlation, R ≥ 0.7) in the 82 FNAs, and their mean expression in the 40 tissue samples was correlated with ER (ESR1) expression (R = 0.84) and ER IHC status (p < 0.0001). HER-2 gene expression (probe ERBB2 216836) expression ≥ 1500 defined clinical HER-2-positive status (IHC = 3 and/or FISH ratio ≥ 2) in 82 FNAs with 96% overall accuracy (95% CI, 90%–99%), and was validated with 100% overall accuracy (95% CI, 91%–100%) in 40 tissues. Conclusion: Expression values of ESR1, ERBB2, and a set of ER reporter genes from Affymetrix GeneChips accurately determined ER and HER-2 status, and were comparable in FNA and tissue samples from two independent laboratories. DNA microarrays could be become a single assay to report ER and HER-2 status included with other clinically relevant predictions. No significant financial relationships to disclose.

Molecular cloning, gene expression and characterization of the third estrogen receptor of the Nile tilapia, Oreochromis niloticus

Estrogens are essential for many reproductive and non-reproductive functions. In teleosts, it is well-known that several subtypes of estrogen receptors are required for the precise action of estrogens. Present study describes the cloning of the third estrogen receptor, ER- beta2, from the Nile tilapia by EST sequencing coupled microarray. The cloned ER-beta2 showed 77.7% amino acid identity with the reported Atlantic croaker ER-beta. Three ERs, ER-alpha, ER-beta1 and ER-beta2, from the fugu genome were also isolated to analyze their gene structures. Comparison of the intron/exon boundaries and exon numbers of fugu, tilapia, rainbow trout and zebrafish, and phylogenetic analysis of 63 ER sequences revealed that ER-beta probably underwent two successive lineage-specific duplications in teleost. The former took place only in zebrafish lineage, and the latter took place in advanced teleosts without the zebrafish lineage, whereas no duplication of the ER-alpha gene has been detected. Tissue distribution analysis by RT-PCR revealed that tilapia ER-alpha and ER-beta1 were expressed ubiquitously, whereas ER-beta2 is expressed only in the pituitary, liver, intestine, kidney and gonads, with the highest expression in the testis and the lowest level in the ovary. Northern blot analysis detected a single transcript of about 3.4 kb in the testis but not in the ovary mRNAs. In transient transfection assays using human embryonic kidney 293 (HEK293) cells, tilapia ER-beta2 showed estrodiol-17beta dependent transactivation.

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.

Age-related changes of the hippocampal estrogen receptor gene expression in senescence-accelerated mouse

Estrogen receptor (ER) has two subtypes, ERα and ERβ. In this study, age-related changes of hippocampal gene expressions of ERα and ERβ were examined in senescence-accelerated mouse (SAM) with real-time reverse transcription-polymerase chain reaction (RT-PCR) technique, in an attempt to explore the relationship between estrogen receptor expression and age-related deficit of learning and memory in SAM. The results showed that the hippocampal ERα gene expressed constantly at the ages from 3 to 15 months in both SAMR1 and SAMP8. But the change of hippocampal ERβ gene expression was different from ERα gene during the aging process. In SAMR1, the expression of ERβ gene kept at a relatively high level at the ages from 3 to 12 months, and decreased significantly at the age of 15 months. In SAMP8, the expression of ERβ gene kept at a higher level at the ages of 3 and 6 months, and gradually decreased with advancing age, and reached the lowest level at the age of 15 months in both genders. The expression of ERβ gene decreased earlier in SAMP8 compared with SAMR1 during the aging process. These results suggested that the early decline of the expression of ERβ gene, but not ERα gene in the hippocampus of SAMP8 contribute to their age-related deterioration of learning and memory function.

Effects of estrogen on the vascular system.

The cardiovascular protective actions of estrogen are partially mediated by a direct effect on the vessel wall. Estrogen is active both on vascular smooth muscle and endothelial cells where functionally competent estrogen receptors have been identified. Estrogen administration promotes vasodilation in humans and in experimental animals, in part by stimulating prostacyclin and nitric oxide synthesis, as well as by decreasing the production of vasoconstrictor agents such as cyclooxygenase-derived products, reactive oxygen species, angiotensin II, and endothelin-1. In vitro, estrogen exerts a direct inhibitory effect on smooth muscle by activating potassium efflux and by inhibiting calcium influx. In addition, estrogen inhibits vascular smooth muscle cell proliferation. In vivo, 17beta-estradiol prevents neointimal thickening after balloon injury and also ameliorates the lesions occurring in atherosclerotic conditions. As is the case for other steroids, the effect of estrogen on the vessel wall has a rapid non-genomic component involving membrane phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, as well as the classical genomic effect involving estrogen receptor activation and gene expression.

Endometrial effects of selective estrogen receptor modulators (SERMs) on estradiol-responsive gene expression are gene and cell-specific

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10 −7 M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10 −7 M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.

C-fos and estrogen receptor gene expression pattern in the rat uterine epithelium during the estrous cycle.

Different studies in ovariectomized estrogen treated animals support the idea that c-fos plays a role in the proliferation of uterine epithelial cells. However, these studies invite us to reassess the role played by c-fos in epithelial cell types of the endometrium during the estrous cycle. The present study was undertaken to determine the c-fos and estrogen receptor (ER) gene expression pattern in the rat uterine epithelium during the estrous cycle in which natural and cyclic changes of steroid hormones occur, and correlate these changes with the proliferation status of this cellular types. Proliferation was assessed during the estrous cycle using bromodeoxyuridine incorporation to DNA. ERalpha and beta proteins were assessed by immunohistochemistry. The regulation of c-fos gene expression in the uterus of intact animals during the estrous cycle was evaluated using both in situ hybridization and immunohistochemistry. Estradiol (E(2)) and progesterone (P(4)) plasma levels were assessed by radioimmunoassay. The results indicated that luminal (LE) and glandular epithelia (GE) presented maximal proliferation during the metestrus (M) and the diestrus (D) days. However, during the proestrus (P) day only LE presented proliferation, and during the estrus (E) day only the stromal cells proliferated. A marked immunostaining for ERalpha was detected in both LE and GE cells during the early phases of the cycle but diminished on the P and the E day. In contrast, ERbeta was undetectable in both epithelia during all stages of the cycle. The highest c-fos mRNA level was detected in both epithelia on the M day, followed by a significant reduction during the other days of the cycle. The highest protein content was observed on the M and D days, and the minimal value was detected on the E day. The c-Fos protein level in LE was increased during M and D days, presenting a high correlation with the cellular proliferation pattern of this cell type. In conclusion, the overall results indicate that c-Fos protein presented a good correlation with uterine epithelial cell proliferation of LE. In the case of GE, the same tendency was observed, although no significant correlation was found. Both in LE and GE, c-fos mRNA did not strictly correlate with its protein levels. c-fos seems to have a postranscriptional regulation in uterine epithelial cells during the rat's estrous cycle.

Gene expression of estrogen receptors α and β during early sexual differentiation in the European sea bass (Dicentrarchus labrax)

A dimorphic expression pattern of ERα was found during sexual development in the European sea bass. It is therefore suggested that ERα plays an important role in sexual differentiation in this species.

Expression of Estrogen Receptor Gene in Degenerative Spinal Stenosis

Purpose : The purpose of this experiment was to investigate the relationship between sex hormones and degenerative spinal disorder. Materials and Methods : 22 ligamentum flavum were obtained from 22 patients who had undergone spinal surgery. Patients’specimens were divided into three groups. The experimental group contatining 16 postmenopausal female patients, diagnosed as having degenerative spinal stenosis. As a control group, two groups were formed. The first control group was composed of 4 male patients diagnosed as having same disease and the second group was two premenopausal females, who were diagnosed as having disc herniation and fracture. The relative amount of estrogen receptor expression at gene level was measured by reverse transcription-polymerase chain reaction (RT-PCR). The amounts of expressed gene in each group were compared: receptor subtypes (ER-, ER-), sex and age (pre, postmenopausal). The calculated amount of ER- and ER- gene expression was standardizised with respect to the mRNA of GAPDH. Results : The mean density of the estrogen receptor signals were 55.012.3, 143.032.1 in the experimental group, 39.68.0, 89.720.1 in the first control group and 34.12.0, 102.52.7 in the second control group. The mean density of the estrogen receptor signals were higher in the experimenal group than in the control group. A statistically significant difference was found between the experimental group and the control group (p gene in the human ligamentum flavum was confirmed. It was inferred from this study that a high expression of estrogen receptor might affect ligamentum flavum hypertropy in postmenopausal women and that this is likely to be a predisposing factor for degenerative spinal disorder.

Estradiol-induced gene expression in largemouth bass ( Micropterus salmoides)

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E 2). Single injections of E 2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ERα sequences. After acute E 2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36–42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E 2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E 2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen γ was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E 2 involves a complex network of responses over time.

Sequence and expression of androgen receptor and estrogen receptor gene in the sex types of protogynous wrasse, Halichoeres trimaculatus

Sex steroid hormones play important roles in sex change and behavior of wrasses. Their actions are considered to be mediated through the nuclear hormone receptors. In this study, to elucidate the roles of estrogen receptor (ER) and androgen receptor (AR) in the reproduction of the protogynous wrasse, Halichoeres trimaculatus, AR and ER genes were partially cloned using 5 ′- and 3 ′-RACE and their transcript levels in the gonads and the brain were measured by competitive RT-PCR. The amino acid sequence (563 a.a) deduced from 5 ′ truncated cDNA encoding wrasse AR shows about 81%, 69%, 66%, 64%, and 58% identity with those of red seabream ( Chrysophrys major) androgen receptor subtype, AR1, and rainbow trout ( Oncorhynchus mykiss) ARα, ARβ, Japanese eel ( Anguilla japonica) ARα, and ARβ, respectively. The amino acid sequence (458 a.a) deduced from 5 ′ truncated cDNA encoding wrasse ER shows about 81%, 79%, 73%, 66%, and 63% identity with those of red seabream estrogen receptor subtype, ERα, Atlantic croaker ( Micropogonias undulatus) ERα, tilapia ( Oreochromis niloticus) ERα, rainbow trout ERα, and catfish ( Ictalurus punctatus) ERα, respectively. Among the various tissues tested, AR and ER mRNAs were highly expressed in the gonads and brains. When the transcript levels of ER were measured in the gonads and the brains of females (F), initial phase male (IP), and terminal phase male (TP), no significant changes in the gene expression were observed. The transcript levels of AR in the gonads did not change among different sex types, while those in the brains of TP were higher than F and IP. These results suggest that higher expression of AR in the brains of TP is strongly correlated with behavioral change.