Social and sexual incentive properties of estrogen receptor alpha, estrogen receptor beta, or oxytocin knockout mice

Social and sexual incentive motivation, defined as the intensity of approach to a social and a sexual incentive, respectively, were studied in female Swiss Webster mice. In the first experiment, the social incentive was a castrated mouse of the same strain as the females, whereas the sexual incentive was an intact male mouse of the same strain. Ovariectomized females were first tested after oil treatment and then after administration of estradiol benzoate + progesterone in doses sufficient to induce full receptivity. The hormones increased sexual incentive motivation while leaving social incentive motivation unaffected. This suggests that sexual incentive motivation in the female mouse is dependent on ovarian hormones. In the next experiment, ovariectomized females were tested with an intact, male estrogen receptor α knockout and its wild type as incentives, first without hormones and then when fully receptive. There were no differences in incentive properties between the wild type and the knockout. In a similar experiment, we used an intact male estrogen receptor β knockout and its corresponding wild type as incentives. The wild type turned out to be a more attractive social incentive than the knockout, while they were equivalent as sexual incentives. Finally, an intact male oxytocin knockout and its wild type were used as incentives. The knockout turned out to be a superior incentive, particularly a superior sexual incentive. The fact that the estrogen receptor β and oxytocin knockouts have incentive properties different from their wild types may be important to consider in studies of these knockouts' sociosexual behaviors.     

Estrogen-mediated neuroprotection against beta-amyloid toxicity requires expression of estrogen receptor alpha or beta and activation of the MAPK pathway

It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non-classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) alpha and/or beta to mediate such responses is lacking, the hippocampal-derived cell line HT22 was stably transfected with either ERalpha (HTERalpha) or ERbeta (HTERbeta). In HTERalpha and HTERbeta cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17beta-estradiol treatment. The ER antagonist ICI 182, 780 (1 microm) and the MEK inhibitor, PD98059 (50 microm) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERalpha and HTERbeta cells with the beta-amyloid peptide (25-35) (10 micro m) resulted in a significant decrease in cell viability. Pre-treatment for 15 min with 10 nm 17beta-estradiol resulted in a 50% increase in the number of living cells in HTERalpha and HTERbeta cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17beta-estradiol-mediated protection. This study demonstrates that both ERalpha and ERbeta can couple to rapid signaling events that mediate estrogen-elicited neuroprotection.     

Distinct effects of 4-nonylphenol and estrogen-17 beta on expression of estrogen receptor alpha gene in smolting sockeye salmon

Xenoestrogens such as 4-nonylphenol (4-NP) have been shown to affect the parr-smolt transformation, but their mechanisms of action are not known. We therefore examined effects of 4-NP and estradiol-17beta (E2) on expression of estrogen receptor (ER) alpha gene in the liver, gill, pituitary and brain of sockeye salmon to elucidate molecular mechanisms of 4-NP and E2 and developmental differences in response during smolting. Fish were treated twice within a week with 4-NP (15 and 150 mg/kg BW), E2 (2 mg/kg BW) or only vehicle at three stages of smolting, pre-smolting in March, early smolting in April and late smolting in May. The absolute amounts of ERalpha mRNA were determined by real-time PCR. The basal amounts of ERalpha mRNA peaked in April in the liver, gill and pituitary. In March, E2 extensively increased the amounts in the liver, while 4-NP had no effects at this stage. In contrast, 4-NP (but not E2) decreased liver ERalpha mRNA in April. 4-NP also decreased the amount of ERalpha mRNA in the gill in April. In the pituitary, 4-NP increased ERalpha mRNA in March but decreased it in May. There were no significant effects in the brain. Changes in basal ERalpha mRNA observed in this study indicate that estrogen responsiveness of tissues may change during salmon smolting. Furthermore, 4-NP and E2 have different effects on expression of ERalpha gene in the liver and gill during smolting, and the response is dependent on smolt stage.     

The expression of aromatase, estrogen receptor alpha and estrogen receptor beta in mouse Leydig cells in vitro that derived from cryptorchid males

A broad expression of aromatase and estrogen receptors (ERs) in the testis suggests an important role for estrogens in regulating testicular cell function and reproductive events. The aim of the present study was to show whether Leydig cells in vitro isolated from cryptorchid testes of two inbred strains of mice, KE and CBA, are a site of estrogen synthesis. Using immunocytochemistry, aromatase, estrogen receptor alpha(ERalpha), and estrogen receptor beta(ERbeta) were localized in cultured Leydig cells. Immunoreactive aromatase was found in the cytoplasm of control Leydig cells and those isolated from cryptorchid males, however the intensity of immunostaining was different, being stronger in Leydig cells deriving from cryptorchid mice. The strongest aromatase immunostaining was found in cryptorchid-KE Leydig cells. Strong immunoexpression of ERalpha was detected in the nuclei of both KE-and CBA-Leydig cells. The intensity of ERalpha immunostaining was stronger in cultured cells deriving from cryptorchid testes. ERbeta immunoexpression was detected predominantly in KE-Leydig cells. Control CBA-Leydig cells were negative for ERbeta or the result was inconclusive, whereas in cryptorchid CBA-Leydig cells a weak immunostaining was present in their nuclei. Western blot analysis confirmed the results obtained by immunocytochemistry. In KE- and CBA-Leydig cells aromatase as a band of 55 kDa protein was present, whereas ERalpha molecular weight was 67 kDa on Western blots. No band was detected for ERbeta. Radioimmunological analysis revealed that androgen and estrogen levels secreted by Leydig cells in vitro were strain-dependent. Additionally, in KE-Leydig cells that derived from cryptorchid mice estrogen level was distinctly higher in comparison with that of the respective control.     

Impaired estrogen sensitivity in bone by inhibiting both estrogen receptor alpha and beta pathways

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways.     

Polymorphisms of estrogen receptor alpha gene in endometrial cancer

It is hypothesized that polymorphisms of estrogen receptor-alpha (ERalpha) gene are involved in endometrial cancer. To test this hypothesis, the genotype distributions of six different loci (codon 10 T-->C, codon 87 G-->C, codon 243 C-->T, codon 325 C-->G, codon 594 G-->A, and intron 1 C-->G) of the ERalpha gene were investigated and their association with endometrial cancer was determined. The DNA from 113 cases of human endometrial cancer was analyzed by sequence-specific polymerase chain reaction. The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The frequency of variant genotype on codon 10 was significantly lower in endometrial cancer patients as compared to controls. Nine of 113 endometrial cancer patients (8.0%) showed genotype 10C/C compared to 27 of 200 healthy controls (13.5%). The relative risk of genotype 10C/C was calculated as 0.44, compared to wild-type. Forty-five of 113 endometrial cancer patients (39.8%) showed genotype T/C on codon 10 compared to 111 of 200 healthy controls (55.5%). The relative risk of genotype 10T/C was calculated as 0.67, compared to wild-type. The polymorphism on codon 87 was not detected both in endometrial cancer patients and in healthy control. Other loci, intron 1, and codons 243, 325, and 594, did not show a correlation with endometrial cancer. The frequency of alleles on codon 10 was also significantly lower in endometrial cancer patients as compared to controls. Sixty-three of 226 alleles (27.9%) of endometrial cancer patients showed allele C compared to 165 of 400 (41.2%) of healthy controls. The relative risk of allele 10C was calculated as 0.67, compared to wild-type. Other loci, intron 1, and codons 243, 325, and 594, did not show a difference between cancer patients and controls. All genotype and allelic distributions were in accordance with the Hardy-Weinberg equilibrium. The present study demonstrates for the first time a protective effect of 10C allele against endometrial cancer. Thus, inherited alterations in ERalpha may be associated with changes in estrogen metabolism and thereby may possibly explain inter-individual differences in disease incidences of endometrial cancer.     

Rapid action of estradiol in primate GnRH neurons: the role of estrogen receptor alpha and estrogen receptor beta

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E₂) causes a rapid stimulatory action. E₂ (1 nM) stimulates firing activity and intracellular calcium ([Ca²⁺]_i) oscillations of primate GnRH neurons within a few min. E₂ also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E₂-induced [Ca²⁺]_i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E₂ action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E₂ by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E₂-induced changes in [Ca²⁺]_i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E₂ in primate GnRH neurons.     

Alpha beta lineage-specific expression of the alpha T cell receptor gene by nearby silencers

T cells expressing either the alpha beta or gamma delta antigen receptor (TCR) are distinct cell lineages. The single locus encoding the TCR alpha and delta genes requires special regulation to avoid alpha gene expression in gamma delta T cells. We show here that the minimal alpha enhancer is active in the gamma delta T cell lineage but gains alpha beta lineage specificity through negative cis-acting elements 3' of the C alpha gene that silence the enhancer in gamma delta T cells. The negative elements at the C alpha locus consist of several silencers that work in an orientation- and distance-independent fashion. These silencers also act on a retroviral enhancer that is normally ubiquitously expressed, restricting its activity to alpha beta cells. The alpha silencers are active in non-T cell lines, suggesting that the decision of a cell to differentiate into the alpha beta T cell lineage may involve specific relief from these silencers. Silencers are likely to be as important as enhancers in establishing lineage-specific gene expression in many systems.     

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

In various species, androgens and estrogens regulate the function of testicular Leydig, Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), and their localization and function in testicular cells varies depending on the species, developmental stage of the cell and type of receptor. The localization of AR in an immature and mature stallion has been reported but estrogen receptors have only been reported for the mature stallion. In the present study, the localizations of AR and ERα/ERβ were investigated in pre-pubertal, peri-pubertal and post-pubertal stallions. Testes were collected by routine castration from 21 horses, of light horse breeds (3 months-27 years). Animals were divided into the following age groups: pre-pubertal (3-11 months; n=7), peri-pubertal (12-23 months; n=7) and post-pubertal (2-27 years; n=7). Testicular tissue samples were fixed and embedded, and the presence of AR, ERα and ERβ was investigated by immunohistochemistry (IHC) using procedures previously validated for the horse. Primary antibodies used were rabbit anti-human AR, mouse anti-human ERβ and rabbit anti-mouse ERα. Sections of each region were incubated with normal rabbit serum (NRS; AR and ERα) or mouse IgG (ERβ) instead of primary antibody to generate negative controls. Androgen receptors were localized in Leydig, Sertoli and peritubular myoid cells of all ages. Estrogen receptor alpha was localized in Leydig and germ cells of all ages but only in pre- and peri-pubertal Sertoli cells and post-pubertal peritubular myoid cells. Estrogen receptor beta was localized in Leydig and Sertoli cells of all ages but in only pre-pubertal germ cells and absent in peritubular myoid cells of all ages. Taken together, the data suggest that estrogen regulates steroidogenesis by acting through ERα and ERβ in the Leydig cells and promotes gametogenesis by acting through ERβ in the Sertoli cells and ERα in the germ cells. In contrast androgen receptors are not found in germ cells throughout development and thus are likely to support spermatogenesis by way of a paracrine/autocrine pathway via its receptors in Leydig, Sertoli and peritubular myoid cells.     

Hepatic stellate cells contain the functional estrogen receptor beta but not the estrogen receptor alpha in male and female rats

In an earlier study, we showed that estradiol (E2) inhibits proliferation and transformation in cultured rat hepatic stellate cells (HSCs) and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on an investigation of the cellular localization of ER subtypes ERalpha and ERbeta using immunohistochemistry in experimental fibrotic liver rats and of each ER subtype expression in cultured rat HSCs by evaluating the produced mRNA and protein. The results indicate that high levels of ERbeta expression and low or no levels of ERalpha expression were observed in normal and fibrotic livers and in quiescent and activated HSCs from both males and females. The specificity of E2-mediated antiapoptotic induction through the ERbeta was shown by dose-dependent inhibition by the pure ER antagonist ICI 182,780 in HSCs which were undergoing early apoptosis. These findings demonstrate for the first time that rat HSCs possess functional Erbeta, but not Eralpha, to respond directly to E2 exposure.     

Gene expression profile of estrogen receptor alpha and beta in the ovaries of Zi geese (Anser cygnoides)

The profile of ERalpha and ERbeta gene expression in the ovaries of Zi geese at 1 day and 1,2, 3, 4, 5 and 8 months of age (n=8, respectively) was examined by quantitative real-time PCR (qRT-PCR). The results showed that the expression of ERalpha and ERbeta mRNA was greater at 1 to 5 and 8 months compared with that observed at 1 day. In particular, the level of expression of ERalpha and ERbeta at 8 months was greater, 2.47 +/- 0.23 fold and 29.07 +/- 1.25 fold, respectively, compared with that at 1 day (P<0.05). The expression of ERalpha mRNA was not significantly different at 1, 2, 3 and 4 months (P>0.05). The level of expression of ERalpha mRNA at 5 months was 1.86 +/- 0.17 fold higher than at 1 day (P<0.05). The level of expression of ERbeta mRNA at 2, 3, 4, 5 and 8 months (1.96 +/- 0.13, 2.58 +/- 0.08, 2.08 +/- 0.05, 3.25 +/- 0.11 and 29.07 +/- 1.25 fold, respectively, P<0.05) was significantly higher than at 1 day. In summary, the expression of ERalpha and ERbeta mRNA in the ovaries of geese was increased between newborn and the laying stage. These results suggest that ERalpha and ERbeta mediate the process of ovarian development and egg laying in geese. In addition, ERbeta may play a more important role in regulating the response of the ovary to estrogen during the developmental and egg-laying stages.     

The ligand binding profiles of estrogen receptors alpha and beta are species dependent

Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-alpha and ER-beta. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-alpha and ER-beta. Certain compounds show species dependent binding preferences for ER-alpha or ER-beta, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.