Morpholino-mediated knockdown of ERα, ERßa, and ERßb mRNAs in zebrafish (Danio rerio) embryos reveals differential regulation of estrogen-inducible genes.

Genetically distinct estrogen receptor (ER) subtypes (ERα and ERß) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ERα) and 2 esr2 (ERßa and ERßb) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were microinjected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ERα mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ERßa with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ERα knockdown. Results indicate that ERα and ERßb, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ERßa, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.

Molecular cloning and expression analysis of estrogen receptor betas (ERß1 and ERß2) during gonad development in the Korean rockfish, Sebastes schlegeli.

Estrogen receptors (ER) play a crucial role in mediation of estrogen activities. Here we report the isolation and expression analysis of ERß1 and ERß2 from ovary Korean rockfish (Sebastes schlegeli). were isolated using reverse transcription-polymerase chain reaction (PCR) and rapid amplification of cDNA ends procedures. The cDNA of this study, ERß1 (588 amino acids) and ERß2 (659 amino acids) were identified using reverse-transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends procedures. Structural analysis showed both ERßs contain six typical nuclear receptor-characteristic domains. Phylogenetic analysis indicated that Korean rockfish ERßs were highly conserved among teleost. RT-PCR confirmed that the ERßs were widely distributed in both gonads and extra gonadal tissues. Further, we analyzed the expression patterns of male and female S. schlegeli during the reproductive cycle using quantitative real-time PCR (qRT-PCR). The results showed that the highest expression levels were observed in testis at immature sperm stage for both of KrERß1 and KrERß2. For female, the expressions of KrERß1 and KrERß2 were significantly higher in the ovary at the early-oocyte stage. Cloning these two ERß subtypes in the Korean rockfish, together with the information on expression levels in adult fish has given us the foundation to investigate their possible role in brain-pituitary-gonad neuroendocrine axis in future studies.

Involvement of estrogen receptor ß5 in the progression of glioma.

Emerging evidence suggests a decline of ERß expression in various peripheral cancers. ERß has been proposed as a cancer brake that inhibits tumor proliferation. In the current study, we have identified ERß5 as the predominant isoform of ERß in human glioma and its expression was significantly increased in human glioma as compared with non-neoplastic brain tissue. Hypoxia and activation of hypoxia inducible factor (HIF) increased ERß transcription in U87 cells, suggesting elevated ERß expression in glioma might be induced by the hypoxic stress in the tumor. Over-expression of either ERß1 or ERß5 increased PTEN expression and inhibited activation of the PI3K/AKT/mTOR pathway. In addition, ERß5 inhibited the MAPK/ERK pathway. In U87 cells, ERß1 and ERß5 inhibit cell proliferation and reduced cells in the S+G2/M phase. Our findings suggest hypoxia induced ERß5 expression in glioma as a self-protective mechanism against tumor proliferation and that ERß5 might serve as a therapeutic target for the treatment of glioma.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

Differential regulation of gonadotropin-releasing hormone neuron activity and membrane properties by acutely applied estradiol: dependence on dose and estrogen receptor subtype.

Gonadotropin-releasing hormone (GnRH) neurons are critical to controlling fertility. In vivo, estradiol can inhibit or stimulate GnRH release depending on concentration and physiological state. We examined rapid, nongenomic effects of estradiol. Whole-cell recordings were made of GnRH neurons in brain slices from ovariectomized mice with ionotropic GABA and glutamate receptors blocked. Estradiol was bath applied and measurements completed within 15 min. Estradiol from high physiological (preovulatory) concentrations (100 pm) to 100 nm enhanced action potential firing, reduced afterhyperpolarizing potential (AHP) and increased slow afterdepolarization amplitudes (ADP), and reduced I(AHP) and enhanced I(ADP). The reduction of I(AHP) was occluded by previous blockade of calcium-activated potassium channels. These effects were mimicked by an estrogen receptor (ER) beta-specific agonist and were blocked by the classical receptor antagonist ICI182780. ERalpha or GPR30 agonists had no effect. The acute stimulatory effect of high physiological estradiol on firing rate was dependent on signaling via protein kinase A. In contrast, low physiological levels of estradiol (10 pm) did not affect intrinsic properties. Without blockade of ionotropic GABA and glutamate receptors, however, 10 pm estradiol reduced firing of GnRH neurons; this was mimicked by an ERalpha agonist. ERalpha agonists reduced the frequency of GABA transmission to GnRH neurons; GABA can excite to these cells. In contrast, ERbeta agonists increased GABA transmission and postsynaptic response. These data suggest rapid intrinsic and network modulation of GnRH neurons by estradiol is dependent on both dose and receptor subtype. In cooperation with genomic actions, nongenomic effects may play a role in feedback regulation of GnRH secretion.

Characterization of estrogen receptor betab in sea bream (Sparus auratus): phylogeny, ligand-binding, and comparative analysis of expression.

Estrogens control many physiological processes in both female and male vertebrates, mostly mediated by specific nuclear estrogen receptors (ER). Two ER subtypes (ERalpha and ERbeta) are present in most vertebrates, including the sea bream (Sparus auratus) a hermaphrodite teleost fish. In the present study several variant cDNAs encoding a second sea bream ERbeta (sbERbetab) is reported. Phylogenetic and Southern blot analysis indicate that sbERbetab and the previously cloned sbERbetaa (formerly sbERbeta) are encoded by different genes, which may have arisen by duplication of an ancestral ERbeta gene. Competitive binding assays show that sbERbetab has high affinity for 17beta-estradiol (K(d) = 1 nM) and specifically binds estrogen agonists (diethylstilbestrol and ethynylestradiol) and antagonists (ICI 182,780). In Northern blot sbERalpha, sbERbetaa, sbERbetab produce several different transcripts in a variety of tissues. RT-PCR showed a partially overlapping but differential tissue distribution in both male and female sea bream.

Cloning, characterisation, and expression of three oestrogen receptors (ERalpha, ERbeta1 and ERbeta2) in the European sea bass, Dicentrarchus labrax.

Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERalpha, and two ERbeta subtypes, called ERbeta1 and ERbeta2 (or ERbeta and ERgamma in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERalpha cDNA) and ovary (partial sb-ERbeta1 and sb-ERbeta2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERalpha, ERbeta1 and ERbeta2. Deduced amino acid numbers for sb-ERalpha, sb-ERbeta1 and sb-ERbeta2 were 639, 517 and 608, respectively, representing in the case of sb-ERbeta1 and sb-ERbeta2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERalpha with eelpout ERalpha (88.7%), for sb-ERbeta1 with Atlantic croaker ERgamma (85.8%), and for sb-ERbeta2 with Atlantic croaker ERbeta (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERalpha was predominantly expressed in liver and pituitary, while sb-ERbeta1 and sb-ERbeta2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERalpha expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERbeta1 and sb-ERbeta2 highest expression levels were observed in gonads at 250 dph. For sb-ERbeta2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.