Meta-Analysis of Microarray Data of Rainbow Trout Fry Gonad Differentiation Modulated by Ethynylestradiol

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.

The Reversible Sex of Gonochoristic Fish: Insights and Consequences

“…In medaka some cyst germ cells were germline stem cells with bipotential activity that differentiate in to oogonia but in the absence of E2 are capable to differentiate into spermatogonia [Paul-Prasanth et al, 2013]. The mode of action of feminizing EE2 (ethinylestradiol) treatment was studied in rainbow trout fry/juveniles with a meta-analysis of microarray data showing a rapid and strong decrease of key testis genes, notably those involved in androgen synthesis [Depiereux et al, 2015]. It highlighted new pathways such as the progesterone-oocyte maturation pathway with several genes of the germline, cell division and oocyte meiosis represented and the peroxisome proliferator-activated receptor (PPAR) signaling pathway containing genes from the nuclear hormone (steroid/thyroid/retinoid) receptor family that perhaps regulate aromatase levels.…”

Integrated Genomic and Bioinformatics Approaches to Identify Molecular Links between Endocrine Disruptors and Adverse Outcomes

“…As an analog of E2, EE2 provides a clear example of how a XE can alter reproductive processes. An in vivo study in the fry of rainbow trout revealed that exposure up to 10 μg/L EE2 enriched pathways related to the cell cycle and DNA replication, and demonstrated the ability of EE2 to disrupt gonad differentiation [ 100 ]. While sex differentiation in fish is more labile than in mammals, the ability of EE2 to alter transcriptional patterns during female gonad differentiation in male fish demonstrates the estrogenic effects of EE2 [ 100 ].…”

Small RNA sequencing reveals sex-related miRNAs in Collichthys lucidus

“…To identify the potential sex-related miRNAs, we analyzed the DE-miRNAs targeting sex-associated genes, functions, or pathways, including progesterone-mediated oocyte maturation, the TGF-beta signaling pathway, and the Wnt signaling pathway, which are known to be involved in sex determination and differentiation in other fish species (Herpin et al, 2013;Depiereux et al, 2015;Jia et al, 2018). After establishing a DE-miRNA-mRNA-sex-related pathway network, we found that 22 DE-miRNAs potentially regulated the sex-related pathways by targeting 18 mRNAs (Figure 5A).…”