Abstract
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.
Highlights
The four contrasts previously defined in section 2.4.3 for the 1st dataset represent 12,742 non-redundant differentially expressed genes (DEGs)
Both dataset analyses generated a high number of differentially expressed genes (DEG) following EE2 treatment, with nearly half of the array content displaying differential expression patterns in dataset 1 (12,742 non-redundant genes), and one third in dataset 2 (2,974 nonredundant genes)
We have previously shown that homology with Danio rerio retrieved several pathways associated with testis-ova development in juvenile rainbow trout exposed to low doses of EE2 [24]
Summary
Sex determination can be genetically controlled (for instance in XX/XY systems, the expression of Y-linked genes leads to testicular development), and the subsequent differentiation processes are supported by molecular and endocrine factors, such as sex steroid hormones [2]. While these processes are strictly genetically controlled in mammals, where the expression of a main male gene, SRY (Sex-determining Region of Y chromosome), irrefutably leads to testis development, gonadal sex differentiation in fish is more labile, which means that their phenotype may be different from their sex genotype. Considering that EDCs do not necessarily mimic the endogenous physiological processes [6,7,8], a clear understanding of these mechanisms is crucial
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