Duplicated membrane estrogen receptors in the European sea bass (Dicentrarchus labrax): Phylogeny, expression and regulation throughout the reproductive cycle

The numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb, were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction.info:eu-repo/semantics/publishedVersio

A fish scale in vitro bioassay to screen for endocrine disrupting compounds

A wide range of natural and anthropogenic compounds are accumulating in the aquatic environment, many of which can interact with and disrupt the endocrine system. Estrogenic endocrine disruptors (EDCs) are a particular problem with impact on humans, ecosystems and wildlife and are particularly relevant in aquatic organisms like fish that may experience life-long exposures. The effects of EDCs in fish have mainly been assessed using reproductive endpoints and in vivo animal experiments. We propose that using other potential endpoints, such as the effect of estrogens on mineralized tissue, would allow development of a simple non invasive assay using scales. Fish scales are mineralized tissues that express both membrane and nuclear estrogen receptors, and are targets for natural estrogens and EDCs. The in vitro bioassay optimized in this work includes sampling of fish scales, incubation in culture media containing the tested compounds and measurement of enzymatic activities related to calcium turnover (TRAP, tartrate-resistant acid phosphatase and ALP, alkaline phosphatase). Several variables were optimized including culture media, compounds concentrations and incubation conditions (e.g. temperature, time), using both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) scales. Significant effects of E2 and EDCs were detected, including both rapid (30 minutes) or slow (1day) changes in scale TRAP or ALP activities, but the responses were of low magnitude and varied with the individual, age, time of year, species and culture conditions. The in vitro fish scale assay is a promising non-invasive screening tool for E2 and EDCs effects, complying with the 3Rs of animal welfare. However, current technical limitations are its limited sensitivity for some parameters eg. TRAP/ALP activity and alternative, sensitive, robust and easy to measure endpoints are under investigation.info:eu-repo/semantics/publishedVersio

The effects of di-n-butyl phthalate and 4-tert-octylphenol in osteoclastic and osteoblastic activities in teleost fish scales

Di-n-butyl phtalate (DBP) and 4-tert-octylphenol (OP) are environmental pollutants with estrogenic activity that have been shown to have endocrine disruptive actions in reproduction of several fish species. However, their impact in bone and scale metabolism, which are estrogen-responsive tissues, remains unknown. In this study, we evaluated the impact of these compounds on mineral metabolism in fish scales that, like bone, are a dynamic tissue maintained by continuous cycles of formation and resorption mediated, respectively, by osteoblasts (OSB) and osteoclasts (OSC). Using an in vitro bioassay, Atlantic sea bass (a marine species) and Mozambique tilapia (a freshwater species) scales were incubated with a range of concentrations of OP and DBP in culture media for a short (30 minutes) or long (24 hours) incubation time. Effects on the activity of tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP), markers for OSC and OSB activities, respectively, were assessed using a colorimetric enzymatic assay. DBP (10-6 M) affected TRAP activity in both species. While in sea bass, TRAP activity increased with DBP after 30 min incubation but was unaffected after 24 h, in tilapia no alterations were observed at the short term but a significant decrease was observed after 24 h incubation with this compound. None of the tested concentrations (10-10 to 10-6 M) affected ALP activity in both species. On the contrary, OP effects were only observed on the activity of ALP, which was significantly decreased after a 24 h incubation with 10-8 M of OP in the scales of both species. These results suggest that the exposure to these compounds may have disruptive effects on the metabolism of mineralized tissues in both marine and freshwater species. Future studies will investigate the mechanisms involved in these responses and the consequences for fish health.Foundation for Science and Technology of Portugal (FCT), through projects PTDC/AAG-GLO/4003/2012 and PEst-C/MAR/LA0015/2011 and fellowship to PP (SFRH/BPD/84033/2012).info:eu-repo/semantics/publishedVersio

Tissue responsiveness to estradiol and genistein in the sea bass liver and scale

As in mammals, estrogens in fish are essential for reproduction but also important regulators of mineral homeostasis. Fish scales are a non-conventional target tissue responsive to estradiol and constitute a good model to study mineralized tissues effects and mechanisms of action of estrogenic compounds, including phytoestrogens. The responsiveness to estradiol and the phytoestrogen genistein, was compared between the scales and the liver, a classical estrogenic target, in sea bass (Dicentrarchus labrax). Injection with estradiol and genistein significantly increased circulating vitellogenin (for both compounds) and mineral levels (estradiol only) and genistein also significantly increased scale enzymatic activities suggesting it increased mineral turnover. The repertoire, abundance and estrogenic regulation of nuclear estrogen receptors (ESR1, 2a and 2b) and membrane G-protein receptors (GPER and GPER-like) were different between liver and scales, which presumably explains the tissue-specific changes detected in estrogen-responsive gene expression. In scales changes in gene expression mainly consisted of small rapid increases, while in liver strong, sustained increases/decreases in gene expression occurred. Similar but not overlapping gene expression changes were observed in response to both estradiol and genistein. This study demonstrates for the first time the expression of membrane estrogen receptors in scales and that estrogens and phytoestrogens, to which fish may be exposed in the wild or in aquaculture, both affect liver and mineralized tissues in a tissue-specific manner. (C) 2015 Elsevier Ltd. All rights reserved

Membrane and nuclear estrogen receptors in sea bass provide insight to explore genomic and non-genomic estrogenic actions: the mineralized scale example

The numerous estrogen functions across vertebrates have been classically explained by binding to nuclear estrogen receptors (ERs) regulating the transcription of responsive genes. It is now known that estrogenic compounds can also produce rapid non-genomic actions initiated by binding to plasma estrogen membrane receptors, such as the recently identified G protein-coupled estrogen receptor1 (GPER). Sea bass (Dicentrarchus labrax) express three ER subtype genes, one esr1 and two esr2 genes that appear to have been originated from the original esr2 gene in the teleost-specific whole genome duplication. We have recently identified two genes for GPER in the sea bass genome and phylogenetic analyses also suggest they are teleost-specific gene duplicates. Quantitative PCR revealed a wide tissue distribution for the five receptors in both male and female sea bass and expression throughout the reproductive cycle in brain and pituitary, although with subtype-specific and seasonal differences. When analyzing the sea bass scales, mineralized structures previously shown to be estrogen-responsive, a different receptor repertoire and regulation was detected compared to liver, a classical target gene. In juvenile sea bass scales, the main forms expressed were esr2a and gperb, which were also up regulated after injection with the natural estrogen estradiol (E2) and the phytoestrogen genistein (Gen). Both rapid (30 min) and slow (1 day or more) changes in the activities of enzymes related to mineral turnover were detected in fish scales in response to E2, Gen and xenoestrogens and the gene networks activated 1-5 days after injection of E2 and Gen are being characterized by transcriptomics, revealing both common and compound-specific effects at the transcriptional level. Functional characterization of the three sea bass ER subtypes and two GPERs is underway in mammalian cells, to allow to compare their signaling to different estrogenic compounds. These studies will help to understand the normal estrogen regulation of fish scale functions as well as its possible disruption by phytoestrogens and other xenoestrogens and the relative importance of genomic and non-genomic mechanisms of action of the five receptors.info:eu-repo/semantics/publishedVersio

Efeitos de fitoestrogénios no metabolismo mineral em escamas de robalo e de tilápia moçambicana

O rápido desenvolvimento da aquacultura nas últimas décadas fez aumentar a procura por fontes proteicas adequadas para incluir nas rações dos peixes. A soja tem sido muito utilizada com fonte proteica de origem vegetal mas é particularmente rica em fitoestrogénios, incluindo a genisteína (GEN) e a daidzeína (DAI), que são as principais isoflavonas presentes na soja. Os peixes podem estar expostos aos fitoestrogénios no ambiente ou através das dietas que os contêm, como é o caso da soja. Estes compostos podem ter atividades estrogénicas e efeitos disruptivos na reprodução mas o seu impacto nos tecidos mineralizados continua a ser desconhecido. As escamas de peixe são um tecido mineralizado que, tal como o osso de mamíferos, é mantido por ciclos de formação e reabsorção, mediado por osteoblastos (OSB) e osteoclastos (OSC), respetivamente. As escamas são um tecido responsivo aos estrogénios e expressam os recetores de estrogénio nucleares (ERs). As atividades das enzimas fosfatase alcalina (ALP) e fosfatase ácida resistente ao tartrato (TRAP) são usadas como marcadores das atividades dos OSB e OSC, respetivamente, e são modificadas pelo estradiol (E2) nas escamas de várias espécies de peixe. Usando um ensaio in vitro, investigámos o possível impacto da exposição a GEN e a DAI no metabolismo mineral em escamas. O efeito destes compostos foi avaliado através da determinação das atividades de TRAP e ALP em escamas de robalo (Dicentrarchus labrax), uma espécie marinha, e de tilápia moçambicana (Oreochromis mossambicus), mantida em água salgada (AS) e em água doce (AD).info:eu-repo/semantics/publishedVersio

Holography For a De Sitter-Esque Geometry

Warped dS$_3$ arises as a solution to topologically massive gravity (TMG) with positive cosmological constant $+1/\ell^2$ and Chern-Simons coefficient $1/\mu$ in the region $\mu^2 \ell^2 < 27$. It is given by a real line fibration over two-dimensional de Sitter space and is equivalent to the rotating Nariai geometry at fixed polar angle. We study the thermodynamic and asymptotic structure of a family of geometries with warped dS$_3$ asymptotics. Interestingly, these solutions have both a cosmological horizon and an internal one, and their entropy is unbounded from above unlike black holes in regular de Sitter space. The asymptotic symmetry group resides at future infinity and is given by a semi-direct product of a Virasoro algebra and a current algebra. The right moving central charge vanishes when $\mu^2 \ell^2 = 27/5$. We discuss the possible holographic interpretation of these de Sitter-esque spacetimes.Comment: 22 pages, 1 figure; v2: typos corrected, to match with published versio

Hierarchical information clustering by means of topologically embedded graphs

We introduce a graph-theoretic approach to extract clusters and hierarchies in complex data-sets in an unsupervised and deterministic manner, without the use of any prior information. This is achieved by building topologically embedded networks containing the subset of most significant links and analyzing the network structure. For a planar embedding, this method provides both the intra-cluster hierarchy, which describes the way clusters are composed, and the inter-cluster hierarchy which describes how clusters gather together. We discuss performance, robustness and reliability of this method by first investigating several artificial data-sets, finding that it can outperform significantly other established approaches. Then we show that our method can successfully differentiate meaningful clusters and hierarchies in a variety of real data-sets. In particular, we find that the application to gene expression patterns of lymphoma samples uncovers biologically significant groups of genes which play key-roles in diagnosis, prognosis and treatment of some of the most relevant human lymphoid malignancies.Comment: 33 Pages, 18 Figures, 5 Table

Serologically defined variations in malaria endemicity in Pará state, Brazil

BACKGROUND: Measurement of malaria endemicity is typically based on vector or parasite measures. A complementary approach is the detection of parasite specific IgG antibodies. We determined the antibody levels and seroconversion rates to both P. vivax and P. falciparum merozoite antigens in individuals living in areas of varying P. vivax endemicity in Pará state, Brazilian Amazon region. METHODOLOGY/PRINCIPAL FINDINGS: The prevalence of antibodies to recombinant antigens from P. vivax and P. falciparum was determined in 1,330 individuals. Cross sectional surveys were conducted in the north of Brazil in Anajás, Belém, Goianésia do Pará, Jacareacanga, Itaituba, Trairão, all in the Pará state, and Sucuriju, a free-malaria site in the neighboring state Amapá. Seroprevalence to any P. vivax antigens (MSP1 or AMA-1) was 52.5%, whereas 24.7% of the individuals were seropositive to any P. falciparum antigens (MSP1 or AMA-1). For P. vivax antigens, the seroconversion rates (SCR) ranged from 0.005 (Sucuriju) to 0.201 (Goianésia do Pará), and are strongly correlated to the corresponding Annual Parasite Index (API). We detected two sites with distinct characteristics: Goianésia do Pará where seroprevalence curve does not change with age, and Sucuriju where seroprevalence curve is better described by a model with two SCRs compatible with a decrease in force of infection occurred 14 years ago (from 0.069 to 0.005). For P. falciparum antigens, current SCR estimates varied from 0.002 (Belém) to 0.018 (Goianésia do Pará). We also detected a putative decrease in disease transmission occurred ∼29 years ago in Anajás, Goianésia do Pará, Itaituba, Jacareacanga, and Trairão. CONCLUSIONS: We observed heterogeneity of serological indices across study sites with different endemicity levels and temporal changes in the force of infection in some of the sites. Our study provides further evidence that serology can be used to measure and monitor transmission of both major species of malaria parasite