Estrogen exposure overrides the masculinizing effect of elevated temperature by a downregulation of the key genes implicated in sexual differentiation in a fish with mixed genetic and environmental sex determination

Abstract

Background: Understanding the consequences of thermal and chemical variations in aquatic habitats is of importance in a scenario of global change. In ecology, the sex ratio is a major population demographic parameter. So far, research that measured environmental perturbations on fish sex ratios has usually involved a few model species with a strong genetic basis of sex determination, and focused on the study of juvenile or adult gonads. However, the underlying mechanisms at the time of gender commitment are poorly understood. In an effort to elucidate the mechanisms driving sex differentiation, here we used the European sea bass, a fish species where genetics and environment (temperature) contribute equally to sex determination.

Results: Here, we analyzed the transcriptome of developing gonads experiencing either testis or ovarian differentiation as a result of thermal and/or exogenous estrogen influences. These external insults elicited different responses. Thus, while elevated temperature masculinized genetic females, estrogen exposure was able to override thermal effects and resulted in an all-female population. A total of 383 genes were differentially expressed, with an overall downregulation in the expression of genes involved in both in testicular and ovarian differentiation when fish were exposed to Estradiol-17ß through a shutdown of the first steps of steroidogenesis. However, once the female phenotype was imposed, gonads could continue their normal development, even taking into account that some of the resulting females were fish that otherwise would have developed as males.

Conclusions: The data on the underlying mechanisms operating at the molecular level presented here contribute to a better understanding of the sex ratio response of fish species subjected to a combination of two of the most common environmental perturbations and can have implications in future conservational policies.

Keywords: Climate change; Ecotoxicology; Estradiol; Fish; Phenotypic plasticity; Sex differentiation; Sex ratio; Temperature increase; Transcriptomics.

Fig. 1

Effects of thermal and estrogen exposure on one-year-old European sea bass gonads. a Percent females in each group, with a difference from a Fisherian 1:1 balanced sex ratio. b Female gonadosomatic index. Data as mean + SEM. Males were not included since no males were present in the E₂-treated group. * = P < 0.05; *** = P < 0.001

Fig. 2

Individual heatmap representation of the differentially expressed genes in European sea bass 170 dph gonads from HT-E₂ vs. HT group fish. Low to high expression is shown by a gradation color from red to green. On the right, the hierarchical clustering of genes by DAVID is shown (for a more tailed list of the altered pathways see Additional file 8: Table S3)

Fig. 3

Venn diagram representation of the common up- and down-regulated genes for the comparisons HT-E₂ vs. LT and HT-E₂ vs. LT-E₂)

Fig. 4

Quantitative real time PCR validation results for genes related to testis differentiation (a-c): anti-Müllerian hormone (amh), doublesex- and mab-3-related transcription factor 1 (dmrt1) and tescalcin (tesc); (d) cholesterol import: steroidogenic acute regulatory protein (star); ovarian differentiation (e-h): gonadal isoform of aromatase (cyp19a1a), SRY-related HMG-box transcription factor SOX17 (sox17), Wnt inducible signaling pathway protein 1 (wisp1), and vasa protein (vasa); (i) the neural isoform of aromatase (cyp19a1b), and (j) insulin-like growth factor 1 (igf1). Asterisks indicate significant statistical differences between groups (* = P < 0.05, ** = P < 0.01, *** = P < 0.001)

Fig. 5

A KEGG pathway-based figure depicting the ovarian steroidogenesis pathway. Microarray results are marked with arrows (green arrows indicate a fold change (FC) higher than 1.5, while red arrows indicate a FC lower than 1.5). Yellow stars note genes in the microarray HT-E₂ vs. HT comparison