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- Tissue responsiveness to estradiol and genistein in the sea bass liver and scalePublication . Estêvão, M. Dulce; Andrade, André; Santos, Soraia; Power, Deborah; Pinto, Patricia ISAs 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.
- Identification of reliable reference genes for quantitative gene expression in the Mozambique tilapia, Oreochromis mossambicusPublication . Andrade, André; Canario, Adelino V. M.; Deloffre, Laurence A. M.A importância na análise de expressão genética tem vindo a crescer significativamente nas últimas década, sendo o real-time reverse transcription PCR (qRT-PCR) o método por excelência devido à sua grande eficiência e precisão. Um dos requisitos para medir a expressão genética através do qRT-PCR é utilização de um ou mais genes de referência, que funcionam como controlos internos para eliminar factores de variação inerentes a este método. Embora os genes de referência sejam a melhor ferramenta para a normalização, um dos grandes constrangimentos que se tem vindo a verificar é que cada vez mais estudos são reportados em que demonstram que muitos dos genes de referência até agora utilizados, são regulados em algumas circunstâncias tais como em diferentes estados de desenvolvimento ou em resposta a diferentes tratamentos. Indicando assim que não existe um gene de referência universal que tenha uma expressão constante em todas as células e diferentes situações. Neste estudo avaliámos a expressão genética de 10 possiveis genes de referência (18S RNA ribossomal, β-actina, HPRT-1, GADPH, Tubulina A, proteína de ligação TATA, factor de alongamento alpha 1, Beta-2 microglobulina, Catepsina D e Catepsina Z) através de qRT-PCR, em 13 tecidos de indivíduos adultos da espécie Oreochromis mossambicus e em cinco grupos de ovocitos em desenvolvimento de fêmeas desta espécie. A selecção de genes de referência é considerado um problema circular, já que mesmo estes genes precisam de ser normalizados. Diversas ferramentas informáticas baseadas em algoritmos têm sido desenvolvidas para contornar este problema. Duas destas ferramentas são os programas geNorm e Normfinder, os quais foram utilizados neste trabalho para analisar os dados fornecidos pelo qRT-PCR com a finalidade de validar a estabilidade destes genes e para permitir a determinação do(s) mais adequado(s). A análise efectuada por estes programas revelou que os genes mais estáveis ao longo dos tecidos foram os genes 18S RNA ribossomal, factor de alongamento alpha 1, Beta-2 microglobulina e o proteína de ligação TATA, e que nos cinco grupos de ovocitos em desenvolvimento foram os genes β-actina, factor de alongamento alpha 1 e Catepsina D. Outra conclusão obtida neste estudo foi o facto de o gene GADPH, muito usualmente usado como gene de v referência revelar-se pouco recomendado para a normalização no nosso caso, já que foi o gene que revelou uma maior variabilidade genética quer nos tecidos como nos ovocitos. Para testar e comparar o(s) gene(s) de referência apontados por estes dois programas, estes foram utilizados para a normalização da expressão genética de duas proteínas morfogenéticas ósseas antagonistas, BAMBI e Gremlin, nos cinco grupos de ovocitos em desenvolvimento. O teste revelou que o programa geNorm apresentou resultados mais satisfatórios que o programa Normfinder e que se deverá utilizar para a melhor normalização o conjunto de quatro genes CTSD, B-actin, EFa1 e CTSZ .
- Efeitos de fitoestrogénios no metabolismo mineral em escamas de robalo e de tilápia moçambicanaPublication . Estêvão, Dulce; Pinto, Patricia IS; Santos, Soraia; Andrade, André; Power, DeborahO 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).
- The effects of di-n-butyl phthalate and 4-tert-octylphenol in osteoclastic and osteoblastic activities in teleost fish scalesPublication . Pinto, Patricia IS; Estêvão, Dulce; Santos, Soraia; Andrade, André; Power, DeborahDi-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.
- Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrierPublication . Pinto, Patricia IS; Andrade, André; Moreira, Catarina; Zapater, Cinta; Thorne, Michael A.S.; Santos, Soraia; Estêvão, M. Dulce; Gomez, Ana; Canario, Adelino; Power, DeborahTeleost fish scales play important roles in animal protection and homeostasis. They can be targeted by endogenous estrogens and by environmental estrogenic endocrine disruptors. The phytoestrogen genistein is ubiquitous in the environment and in aquaculture feeds and is a disruptor of estrogenic processes in vertebrates. To test genistein disrupting actions in teleost fish we used a minimally invasive approach by analysing scales plucked from the skin of sea bass (Dicentrarchus labrax). Genistein transactivated all three fish nuclear estrogen receptors and was most potent with the Esr2, had the highest efficacy with Esr1, but reached, in all cases, transactivation levels lower than those of estradiol. RNA-seq revealed 254 responsive genes in the sea bass scales transcriptome with an FDR < 0.05 and more than 2-fold change in expression, 1 or 5 days after acute exposure to estradiol or to genistein. 65 genes were specifically responsive to estradiol and 106 by genistein while 83 genes were responsive to both compounds. Estradiol specifically regulated genes of protein/matrix turnover and genistein affected sterol biosynthesis and regeneration, while innate immune responses were affected by both compounds. This comprehensive study revealed the impact on the fish scale transcriptome of estradiol and genistein, providing a solid background to further develop fish scales as a practical screening tool for endocrine disrupting chemicals in teleosts.
- Estradiol and genistein effects on the sea bass (Dicentrarchus labrax) scales: transcriptome datasetPublication . Pinto, Patricia IS; Andrade, André; Thorne, Michael A.S.; Estêvão, M. Dulce; Canario, Adelino; Power, DeborahFish scales are mineralized structures that play important roles in protection and mineral homeostasis. This tissue expresses multiple estrogen receptor subtypes and can be targeted by estrogens or estrogenic endocrine-disrupting compounds, but their effects are poorly explored. The transcriptome data here presented support the findings reported in the research article "Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrier" [1]. Juvenile sea bass were exposed to estradiol and the phytoestrogen genistein for 1 and 5 days, by intraperitoneal injections, and the effects on scale transcript expression were analysed by RNA-seq using an Illumina Hi-seq 1500. The raw reads of the 30 libraries produced have been deposited in the NCBI-SRA database with the project accession number SRP102504. Mapping of RNA-seq reads against the sea bass reference genome using the Cufflinks/TopHat package identified 371 genes that had significant (FDR<0.05) differential expression with the estradiol or genistein treatments in relation to the control scales at each exposure time, 254 of which presented more than a 2-fold change in expression. The identity of the differentially expressed genes was obtained using both automatic and manual annotations against multiple public sequence databases and they were grouped according to their patterns of expression using hierarchical clustering and heat-maps. The biological processes and KEGG pathways most significantly affected by the estradiol and/or genistein treatments were identified using Cytoscape/ClueGO enrichment analyses.
- Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax)Publication . Pinto, P.; Velez, Zélia; Sousa, Carmen; Santos, Soraia; Andrade, André; Alvarado, M. V.; Felip, A.; Zanuy, S.; Canario, A.V.M.The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29 Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29 Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates. (C) 2017 Elsevier Inc. All rights reserved.
- Duplicated membrane estrogen receptors in the European sea bass (Dicentrarchus labrax): Phylogeny, expression and regulation throughout the reproductive cyclePublication . Pinto, Patricia IS; Andrade, André; Estêvão, M. Dulce; Alvarado, M. Victoria; Felip, Alicia; Power, DeborahThe 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.
- A fish scale in vitro bioassay to screen for endocrine disrupting compoundsPublication . Pinto, Patricia IS; Estêvão, Dulce; Santos, Soraia; Andrade, André; Power, DeborahA 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.
- Identification of asymptomatic plants infected with Citrus tristeza virus from a time series of leaf spectral characteristicsPublication . Afonso, Andreia; Guerra, Rui Manuel Farinha das Neves; Cavaco, A. M.; Pinto, Patricia IS; Andrade, André; Duarte, Amílcar; Power, Deborah; Marques, N T.Citrus tristeza virus (CTV) affects citrus crops with differing severity, depending on the viral strain, the citrus cultivar and the scion/rootstock combinations. In this study we address the problem of identifying asymptomatic infected plants using reflectance spectra of the leaves in the visible/near infrared region. Sixteen young citrus plants (8 Citrus x clementina hort. ex Tanaka ‘Fina’ and 8 Citrus sinensis (L.) Osbeck ‘Valencia Late’) were split into control and T318A isolate infected groups. Measurements of reflectance in the 400-1100 nm range, in two leaves per plant, were performed monthly over 6 months and the presence of the virus was confirmed by IC/RT-PCR and real-time PCR. The spectra acquired in a single day of measurements was inconsistent for inoculated and control plants. However, by monitoring the same leaves over 6 months it was possible to identify infected plants on the basis of the spectra time evolution. In order to achieve this a simple unfolding implementation of 3-way PCA was applied such that group separation in the scores plot was spontaneous and not forced by any a priori assumption. The model was tested through leave-one-out cross validation with a good rate of correct classification for the left out sample. A real situation was simulated by applying the NPCA algorithm to healthy plants only and checking if the infected ones would be projected on the model scores plot as outliers. Again, a good rate of classification was obtained. Finally, we discuss the spectral features that may be associated with the clustering obtained through NPCA and their physiological significance. Reflectance measurements between infected and healthy samples of two citrus cultivars and their correlation with real-time PCR results for the presence of CTV suggest reflectance spectra of the leaves in the visible/near infrared region is a promising tool for plant stress monitoring linked to the presence of CTV infection prior to symptom expression.