Intersex occurrence in rainbow trout (Oncorhynchus mykiss) male fry chronically exposed to ethynylestradiol.

This study aimed to investigate the male-to-female morphological and physiological transdifferentiation process in rainbow trout (Oncorhynchus mykiss) exposed to exogenous estrogens. The first objective was to elucidate whether trout develop intersex gonads under exposure to low levels of estrogen. To this end, the gonads of an all-male population of fry exposed chronically (from 60 to 136 days post fertilization--dpf) to several doses (from environmentally relevant 0.01 µg/L to supra-environmental levels: 0.1, 1 and 10 µg/L) of the potent synthetic estrogen ethynylestradiol (EE2) were examined histologically. The morphological evaluations were underpinned by the analysis of gonad steroid (testosterone, estradiol and 11-ketotestosterone) levels and of brain and gonad gene expression, including estrogen-responsive genes and genes involved in sex differentiation in (gonads: cyp19a1a, ER isoforms, vtg, dmrt1, sox9a2; sdY; cyp11b; brain: cyp19a1b, ER isoforms). Intersex gonads were observed from the first concentration used (0.01 µg EE2/L) and sexual inversion could be detected from 0.1 µg EE2/L. This was accompanied by a linear decrease in 11-KT levels, whereas no effect on E2 and T levels was observed. Q-PCR results from the gonads showed downregulation of testicular markers (dmrt1, sox9a2; sdY; cyp11b) with increasing EE2 exposure concentrations, and upregulation of the female vtg gene. No evidence was found for a direct involvement of aromatase in the sex conversion process. The results from this study provide evidence that gonads of male trout respond to estrogen exposure by intersex formation and, with increasing concentration, by morphological and physiological conversion to phenotypic ovaries. However, supra-environmental estrogen concentrations are needed to induce these changes

Adaptation of a bioinformatics microarray analysis workflow for a toxicogenomic study in rainbow trout

Sex steroids play a key role in triggering sex differentiation in fish, the use of exogenous hormone treatment leading to partial or complete sex reversal. This phenomenon has attracted attention since the discovery that even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies on statistical analysis, species genomic resources, and bioinformatics tools. The goals of this study are to improve the knowledge of feminisation in fish, by the analysis of molecular responses in the gonads of rainbow trout fry after chronic exposure to several doses (0.01, 0.1, 1 and 10 μg/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. We successfully adapted a bioinformatics microarray analysis workflow elaborated on human data to a toxicogenomic study using rainbow trout, a fish species lacking accurate functional annotation and genomic resources. The workflow allowed to obtain lists of genes supposed to be enriched in true positive differentially expressed genes (DEGs), which were subjected to over-representation analysis methods (ORA). Several pathways and ontologies, mostly related to cell division and metabolism, sexual reproduction and steroid production, were found significantly enriched in our analyses. Moreover, two sets of potential ovotestis biomarkers were selected using several criteria. The first group displayed specific potential biomarkers belonging to pathways/ontologies highlighted in the experiment. Among them, the early ovarian differentiation gene foxl2a was overexpressed. The second group, which was highly sensitive but not specific, included the DEGs presenting the highest fold change and lowest p-value of the statistical workflow output. The methodology can be generalized to other (non-model) species and various types of microarray platforms

Meta-analysis of microarray data of rainbow trout fry gonad differentiation modulated by ethynylestradiol

Sex differentiation in fish is a highly labile process easily reversed by the use of exogenous hormonal treatment and has led to environmental concerns since low doses of estrogenic molecules can adversely impact fish reproduction. The goal of this study was to identify pathways altered by treatment with ethynylestradiol (EE2) in developing fish and to find new target genes to be tested further for their possible role in male-to-female sex transdifferentiation. To this end, we have successfully adapted a previously developed bioinformatics workflow to a meta-analysis of two datasets studying sex reversal following exposure to EE2 in juvenile rainbow trout. The meta-analysis consisted of retrieving the intersection of the top gene lists generated for both datasets, performed at different levels of stringency. The intersecting gene lists, enriched in true positive differentially expressed genes (DEGs), were subjected to over-representation analysis (ORA) which allowed identifying several statistically significant enriched pathways altered by EE2 treatment and several new candidate pathways, such as progesterone-mediated oocyte maturation and PPAR signalling. Moreover, several relevant key genes potentially implicated in the early transdifferentiation process were selected. Altogether, the results show that EE2 has a great effect on gene expression in juvenile rainbow trout. The feminization process seems to result from the altered transcription of genes implicated in normal female gonad differentiation, resulting in expression similar to that observed in normal females (i.e. the repression of key testicular markers cyp17a1, cyp11b, tbx1), as well as from other genes (including transcription factors) that respond specifically to the EE2 treatment. The results also showed that the bioinformatics workflow can be applied to different types of microarray platforms and could be generalized to (eco)toxicogenomics studies for environmental risk assessment purposes

gViz, a novel tool for the visualization of co-expression networks

<p>Abstract</p> <p>Background</p> <p>The quantity of microarray data available on the Internet has grown dramatically over the past years and now represents millions of Euros worth of underused information. One way to use this data is through co-expression analysis. To avoid a certain amount of bias, such data must often be analyzed at the genome scale, for example by network representation. The identification of co-expression networks is an important means to unravel gene to gene interactions and the underlying functional relationship between them. However, it is very difficult to explore and analyze a network of such dimensions. Several programs (Cytoscape, yEd) have already been developed for network analysis; however, to our knowledge, there are no available GraphML compatible programs.</p> <p>Findings</p> <p>We designed and developed gViz, a GraphML network visualization and exploration tool. gViz is built on clustering coefficient-based algorithms and is a novel tool to visualize and manipulate networks of co-expression interactions among a selection of probesets (each representing a single gene or transcript), based on a set of microarray co-expression data stored as an adjacency matrix.</p> <p>Conclusions</p> <p>We present here gViz, a software tool designed to visualize and explore large GraphML networks, combining network theory, biological annotation data, microarray data analysis and advanced graphical features.</p

PathEx: a novel multi factors based datasets selector web tool

<p>Abstract</p> <p>Background</p> <p>Microarray experiments have become very popular in life science research. However, if such experiments are only considered independently, the possibilities for analysis and interpretation of many life science phenomena are reduced. The accumulation of publicly available data provides biomedical researchers with a valuable opportunity to either discover new phenomena or improve the interpretation and validation of other phenomena that partially understood or well known. This can only be achieved by intelligently exploiting this rich mine of information.</p> <p>Description</p> <p>Considering that technologies like microarrays remain prohibitively expensive for researchers with limited means to order their own experimental chips, it would be beneficial to re-use previously published microarray data. For certain researchers interested in finding gene groups (requiring many replicates), there is a great need for tools to help them to select appropriate datasets for analysis. These tools may be effective, if and only if, they are able to re-use previously deposited experiments or to create new experiments not initially envisioned by the depositors. However, the generation of new experiments requires that all published microarray data be completely annotated, which is not currently the case. Thus, we propose the PathEx approach.</p> <p>Conclusion</p> <p>This paper presents PathEx, a human-focused web solution built around a two-component system: one database component, enriched with relevant biological information (expression array, omics data, literature) from different sources, and another component comprising sophisticated web interfaces that allow users to perform complex dataset building queries on the contents integrated into the PathEx database.</p

Functional Analysis: Evaluation of Response Intensities - Tailoring ANOVA for Lists of Expression Subsets

Background: Microarray data is frequently used to characterize the expression profile of a whole genome and to compare the characteristics of that genome under several conditions. Geneset analysis methods have been described previously to analyze the expression values of several genes related by known biological criteria (metabolic pathway, pathology signature, co-regulation by a common factor, etc.) at the same time and the cost of these methods allows for the use of more values to help discover the underlying biological mechanisms. Results: As several methods assume different null hypotheses, we propose to reformulate the main question that biologists seek to answer. To determine which genesets are associated with expression values that differ between two experiments, we focused on three ad hoc criteria: expression levels, the direction of individual gene expression changes (up or down regulation), and correlations between genes. We introduce the FAERI methodology, tailored from a two-way ANOVA to examine these criteria. The significance of the results was evaluated according to the self-contained null hypothesis, using label sampling or by inferring the null distribution from normally distributed random data. Evaluations performed on simulated data revealed that FAERI outperforms currently available methods for each type of set tested. We then applied the FAERI method to analyze three real-world datasets on hypoxia response. FAERI was able to detect more genesets than other methodologies, and the genesets selected were coherent with current knowledge of cellular response to hypoxia. Moreover, the genesets selected by FAERI were confirmed when the analysis was repeated on two additional related datasets. Conclusions: The expression values of genesets are associated with several biological effects. The underlying mathematical structure of the genesets allows for analysis of data from several genes at the same time. Focusing on expression levels, the direction of the expression changes, and correlations, we showed that two-step data reduction allowed us to significantly improve the performance of geneset analysis using a modified two-way ANOVA procedure, and to detect genesets that current methods fail to detect

New insights on the male-to-female transdifferentiation processes in rainbow trout (Oncorhynchus mykiss) fry gonads following exposure to ethynylestradiol at the morphological and transcriptional level by in vivo and in silico approaches

Gonads are unique among all organs given their bipotential nature: they can develop either into an ovary or a testis from a single germ cells primordium. In mammals, the way by which cells choose between either fate is genetically controlled by the expression of a main male gene SRY (situated on the Y chromosome) that leads irrefutably to testis development. Sex differentiation is the physical achievement of events leading to the development of functional gonads. These steps allow the expression of the genetic sex into the appropriate phenotypic sex. Sex steroids are involved in all aspects of the regulation of reproductive processes in vertebrates, estrogens acting as feminizing factors and inversely androgens acting as masculinizing factors. The release of endocrine disrupting compounds (EDCs) in the aquatic environment disrupts gonads development in numerous fish species. For example, xenoestrogens (molecules that mimics the natural female hormone estradiol), even at very low doses (ng to μg/L range), induced the production of a female protein (vitellogenin) by male fish and gonads morphological disruptions such as intersex induction (e.g. ovotestis: oocyte development into the testis), which can adversely impact species reproductive success. The mechanisms underlying this issue are still largely unknown. The aim of this study was to gain insights into the molecular mechanisms involved in the male-to-female transdifferentiation processes induced by exogenous estrogenic treatment in fish. To this end, we have chronically exposed an all-male rainbow trout fry population to several concentrations (0, 0.01, 0.1, 1 and 10 μg/L) of ethynylestradiol (EE2, the most potent xenoestrogen in vivo) and investigated the alterations induced by this treatment at the morphological level by an extensive histological analysis of the gonads and at the transcriptomic level through the use of microarray. This modern genomic-based technology has enhanced opportunities to find out mechanisms of actions (MOA) and identify biomarkers related to the toxic action of a compound. However, high throughput data interpretation relies sorely on statistical analysis, genomic resource of the model species used, and bioinformatics tools. To obtain differentially expressed genes (DEGs), we have successfully adapted a bioinformatics workflow (including a re-analysis and a meta-analysis with another dataset) previously implemented for human microarray platforms, to our model fish species. The histological investigations revealed the occurrence of intersex features in rainbow trout juvenile gonads exposed to EE2, which appear more complex than those usually observed in other fish species. Three types of ovotestis were observed from the lowest concentration used, and complete sex reversal occurred at higher doses. Gene expression investigations allowed highlighting several pathways/GO Terms associated with the EE2 exposure. It represents mostly pathways involved in Cell division e.g. Cell cycle, DNA replication, and the Lipid metabolism, along with newly associated pathways such as Oocyte meiosis. A similar pattern is highlighted by the GO terms enrichment, with however more significance for terms related to Sexual reproduction (e.g. ‘Female gonad development’, ‘Gametogenesis’), ‘Cell proliferation’ and ‘Sex steroids biosynthesis’. The filtering steps of our workflow, anchored to the morphological investigations, allowed selecting several specific (e.g. Foxl2, spon2, cdkn1b) and/or sensitive (Theg, thrap3, dnaaf2) potential biomarkers of the intersex phenotype observed. Among them, Foxl2 (forkhead box protein L2), is known as an early ovarian differentiation marker. The meta-analysis of our study with a dataset investigating gene expression patterns following EE2 exposure (one high dose, 20 mg/kg of food) in a time-series encompassing earlier developmental stages gives insights into the pathways/GO Terms potentially involved in the response of EE2, by performing the intersection between the lists of differentially expressed genes (DEGs) at early stages and low concentrations. The results encompassed most of the pathways highlighted in the first experiment (e.g. Cell cycle, DNA replication), confirming their implication in the response of EE2. Two interesting pathways emerged, the PPAR signalling and the Progesterone-mediated pathways, and further studies are needed to assess their implication in the appearance of intersexuality. The application of the workflow allowed us to select 20 potential key genes implicated in the process of rainbow trout transdifferentiation. Among them, the expression of 10 genes was found to respond similarly to genes naturally expressed in developing females (e.g. genes encoding enzymes implicated in the production of androgens: cyp17a1, cyp11b and hsd17b6), and the other half show patterns of expression specific to the treatment (e.g. VTG, the gene encoding the vitellogenin, a well-known marker of xenoestrogenic exposure). We show that our workflow can be generalised to other species and different types of microarray platforms, even if the species is not yet completely sequenced. Overall, the results obtained are very promising in the field of environmental risk assessment related to EE2 exposure and the approach allowed generating new insights on the mechanistic basis of the testis feminization induced by EE2. Further studies are needed to confirm the numerous hypotheses generated by our work.Les gonades sont des organes bipotentiels qui se différencient en ovaire ou en testicule à partir d’un même primordium de cellules germinales. La destinée de l’organe est déterminée génétiquement, notamment chez les mammifères par l’expression du gène SRY (situé sur le chromosome Y) qui mène au développement du testicule. La différenciation du sexe comprend toutes les étapes menant au développement fonctionnel des gonades. Les hormones sexuelles jouent un rôle prédominant dans la régulation de ces processus, les estrogènes agissant comme facteurs féminisant, et les androgènes comme facteurs masculinisant. Il existe chez les poissons une diversité des mécanismes de détermination du sexe, ainsi qu’une labilité des processus de différenciation, qui peuvent être sensibles à des facteurs environnementaux externes. La présence accrue de perturbateurs endocriniens dans l’environnement aquatique conduit à des perturbations du développement des gonades chez de nombreuses espèces de poissons. Par exemple, les xénoestrogènes (molécules mimant l’effet de l’hormone femelle estradiol), même à de très faibles doses (de l’ordre du ng au μg/L), conduisent à la production d’une protéine femelle, la vitellogénine, par les poissons mâles, ou encore à des perturbations de la morphologie des gonades en induisant de l’intersexe (ou ovotestis : développement d’ovocytes au sein du testicule), pouvant être délétère à la survie de l’espèce. Les mécanismes par lesquels ces molécules agissent restent largement inconnus. Le principal objectif de notre travail est de mettre en évidence les voies métaboliques impliquées dans la transdifférenciation mâleKfemelle des gonades de poissons soumis à de faibles concentrations en ethynylestradiol (EE2 – un xénoestrogène puissant). Dans ce but, nous avons soumis de façon chronique des truitesKarcKenKciel mâles juvéniles à une large gamme de concentrations en EE2 (de 0.01 à 10 μg/L) afin de caractériser l’apparition d’ovotestis dans les gonades, ainsi que de mesurer leur transcriptome par la technique des microarrays. Cette technique repose sur l’utilisation d’outils statistiques et bioinformatiques puissants. Les résultats de cette approche dépendent également des connaissances génomiques disponibles pour l’espèce modèle utilisée. Nous avons adapté avec succès une méthodologie précédemment mise au point sur le génome humain à notre modèle poisson. Notre étude a mis en évidence une altération de la morphologie des gonades dès la plus faible dose utilisée, avec l’apparition de 3 types d’ovotestis, ainsi qu’une réversion complète des gonades en femelle aux plus fortes doses. Notre approche microarray nous a permis de ressortir plusieurs pathways et ontologies (GO terms) potentiellement impliqués dans la réponse des gonades à l’EE2. Le profil général des pathways implique la Division cellulaire, avec notamment le Cycle cellulaire et la Réplication de l’ADN qui apparaissent significativement enrichis et ont déjà été corrélés à des réponses d’organismes à une exposition à des composés oestrogéniques. De façon originale, la Méiose ovocytaire apparaît dans nos résultats. Un pattern similaire est observé dans l’enrichissement en GO terms, avec cependant l’émergence de termes reliés à la Reproduction sexuelle (par exemple le Développement des gonades femelle et la Gamétogenèse), la Prolifération cellulaire et la Synthèse des stéroides sexuels. Plusieurs étapes de filtration des résultats, ainsi que l’intégration des observations morphologiques nous ont permis de sélectionner plusieurs biomarqueurs potentiels, spécifiques (par exemple Foxl2,* spon2,* cdkn1b) et/ou sensibles (Theg,* thrap3,*dnaaf2) du phénotype d’intersexe. Parmi ceuxKci, Foxl2 est un marqueur de la différenciation ovarienne précoce. Une métaKanalyse de notre étude a été réalisée avec un autre jeu de donnée investiguant l’expression génique de truites arcKenKciel juvéniles exposées à de l’EE2 (une seule dose élevée : 20mg/kg de nourriture) à différent temps d’exposition, au départ de stades de développement très précoces (Jour 0 = 55 jours postKfertilisation). L’intersection des gènes différentiellement exprimés (DEGs) aux stades précoces et aux faibles doses nous a permis de mettre en évidence plusieurs pathways et GO terms potentiellement impliqués dans la réponse précoce à EE2. De façon intéressante, les résultats obtenus sont très similaires aux précédents, la majorité des pathways significatifs étant impliqués dans la Division cellulaire (dont le Cycle cellulaire et la Réplication de l’ADN). Cela renforce leur rôle dans la réponse à EE2. De plus, deux pathways originaux émergent de cette analyse, le ‘PPAR signalling’ ainsi que le ‘Progesterone mediated’ pathways. Des études complémentaires doivent être réalisées pour évaluer leur implication dans la transdifférenciation des gonades. De plus, l’application de notre méthodologie nous a permis de nous focaliser sur 20 gènes potentiellement impliqués dans les mécanismes précoces de transdifférenciation maleKfemelle. Nous avons pu mettre en évidence que dix d’entre eux présentent des profils d’expression similaires à ceux observés chez des femelles contrôles (par exemple plusieurs gènes codant pour des enzymes impliquées dans la production d’androgènes : cyp17a1,* cyp11b* et* hsd17b6). La seconde moitié des gènes présentent des profils d’expression spécifique au traitement (par exemple VTG, le gène codant pour la vitellogénine). Les résultats obtenus sont très prometteurs dans l’évaluation des risques environnementaux liés à l’exposition aux xénoestrogènes, ainsi que pour la mise en évidence de nouvelles hypothèses sur les bases mécanistiques de la féminisation des testicules soumis à l’EE2. Notre méthodologie s’avère applicable à différents types de plateformes microarray ainsi qu’à différentes espèces, même non entièrement séquencées. Des études complémentaires sont nécessaires pour confirmer les nombreuses hypothèses générées dans le cadre de ce travail.(DOCSC03) -- FUNDP, 201