Abstract
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.
Highlights
2.1 Intersex in wild fish exposed to ethynylestradiolThe issue of increased reproductive failure and intersexuality in wild fish due to the release of endocrine disrupting compounds (EDCs) in surface waters by sewage has attracted much attention in the scientific community over the last 20 years
Results obtained in the over-representation analysis methods (ORA) approach must be taken with caution regarding EE2 response and rainbow trout physiology
At several steps of the analysis, the results support an enrichment of true positives in the set of differentially expressed genes (DEGs) selected
Summary
2.1 Intersex in wild fish exposed to ethynylestradiolThe issue of increased reproductive failure and intersexuality in wild fish due to the release of endocrine disrupting compounds (EDCs) in surface waters by sewage has attracted much attention in the scientific community over the last 20 years. Of great concern is the ability of very low doses (in the range of ng to μg/L) of xenoestrogenic substances (molecules that mimic the natural estradiol) to alter gonad morphology, typically manifesting as the development of oocytes within a functional testis (ovotestis) [1,2,3] This feature has been correlated with reproductive dysfunctionalities and decreased population fitness in several fish species [4,5]. Their detection requires laborious histological analysis, with a limited area for investigation in the gonads, which renders the analysis unsuitable for quantitative evaluations In this context, the use of marker genes expressed in correlation with testis-ova development may provide early signature of gonad morphological disruptions and help in facilitating the environmental monitoring of xenoestrogenic exposure. Microarrays have been proven as efficient tools to determine the molecular mode of action (MOA) of environmental pollutants and to identify biomarkers as indicators of exposure for ecological risk assessments [10,11]
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