Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.
While the field of epigenetics is increasingly recognized to contribute to the emergence of phenotypes in mammalian research models across different developmental and generational timescales, the comparative biology of epigenetics in the large and physiologically diverse vertebrate infraclass of teleost fish remains comparatively understudied. The cypriniform zebrafish and the salmoniform rainbow trout and Atlantic salmon represent two especially important teleost orders, because they offer the unique possibility to comparatively investigate the role of epigenetic regulation in 3R and 4R duplicated genomes. In addition to their sequenced genomes, these teleost species are well-characterized model species for development and physiology, and therefore allow for an investigation of the role of epigenetic modifications in the emergence of physiological phenotypes during an organism's lifespan and in subsequent generations. This review aims firstly to describe the evolution of the repertoire of genes involved in key molecular epigenetic pathways including histone modifications, DNA methylation and microRNAs in zebrafish, rainbow trout, and Atlantic salmon, and secondly, to discuss recent advances in research highlighting a role for molecular epigenetics in shaping physiological phenotypes in these and other teleost models. Finally, by discussing themes and current limitations of the emerging field of teleost epigenetics from both theoretical and technical points of view, we will highlight future research needs and discuss how epigenetics will not only help address basic research questions in comparative teleost physiology, but also inform translational research including aquaculture, aquatic toxicology, and human disease.
Bisphenol A (BPA), perfluorooctane sulfonate (PFOS), and tributyltin (TBT) are emerging endocrine disruptors (EDCs) with still poorly defined mechanisms of toxicity and metabolic effects in aquatic organisms. We used an untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomic approach to study the effects of sub-lethal doses of these three EDCs on the metabolic profiles of zebrafish embryos exposed from 48 to 120hpf (hours post fertilization). Advanced chemometric data analysis methods were used to reveal effects on the subjacent regulatory pathways. EDC treatments induced changes in concentrations of about 50 metabolites for TBT and BPA, and of 25 metabolites for PFOS. The analysis of the corresponding metabolic changes suggested the presence of similar underlying zebrafish responses to BPA, TBT and PFOS affecting the metabolism of glycerophospholipids, amino acids, purines and 2-oxocarboxylic acids. We related the changes in glycerophospholipid metabolism to alterations in absorption of the yolk sack, the main source of nutrients (including lipids) for the developing embryo, linking the molecular markers with adverse phenotypic effects. We propose a general mode of action for all three chemical compounds, probably related to their already described interaction with the PPAR/RXR complex, combined with specific effects on different signaling pathways resulting in particular alterations in the zebrafish embryos metabolism.
novel PFOS alternatives, 6:2 chlorinated polyfluorinated ether
sulfonate (F-53B) and sodium p-perfluorous nonenoxybenzenesulfonate
(OBS), are emerging in the Chinese market, but little is known about
their ecological risks. In this study, zebrafish embryos were exposed
to PFOS, F-53B, and OBS to evaluate their bioconcentration and acute
metabolic consequences. Per- and polyfluoroalkyl substances (PFASs)
accumulated in larvae in the order of F-53B > PFOS > OBS, with
bioconcentration factors ranging from 20 to 357. Exposure to F-53B
and PFOS, but not OBS, increased energy expenditure, and reduced feed
intake in a concentration-dependent manner and the expression of genes
involved in metabolic pathways at the transcriptional and translational
levels. Molecular docking revealed that the binding affinities of
PFASs to glucokinase were decreased in the following order: F-53B
> PFOS > OBS. Finally, the results of Point of Departure (PoD)
that metabolic end points at the molecular and organismal level are
most sensitive to F-53B followed by PFOS and OBS. Collectively, F-53B
has the highest bioconcentration potential and the strongest metabolism-disrupting
effects, followed by PFOS and OBS. Our findings have important implications
for the assessment of early developmental metabolic effects of PFOS
alternatives F-53B and OBS in wildlife and humans.
The European sea bass is a teleost fish that lacks sex chromosomes and for which temperature influences sex ratios. However, correlation between temperature, developmental stage at a given age and sex-specific gene expression is hampered by the lack of sex markers. To study this correlation, fish were exposed to feminizing (15 degrees C) or masculinizing temperature (21 degrees C) from 0-120 days post fertilization, throughout the thermosensitive period (TSP). Aromatase (cyp19a1a), 11beta-hydroxylase (cyp11b), androgen receptor (arb) and estrogen receptors (era, erb1 and erb2) were assessed by qPCR prior and during sex differentiation. Canonical discriminant analysis (CDA), with length--as proxy for developmental stage--and cyp19a1a expression as predictors, was validated and used to reliably assign gonadal sex to fish sampled within and outside the TSP. Differences in cyp19a1a and cyp11b expression could be detected 1-month before the first signs of histological sex differentiation. Cyp19a1a and cyp11b were significantly higher in future females and males, respectively, and revealed as robust molecular markers to predict future ovarian and testicular differentiation. In contrast, no association between phenotypic sex and arb, era, erb1 and erb2 expression was found, suggesting that these genes do not contribute to the differentiation of a particular sex. The CDA-based approach implemented here could be used to sex undifferentiated animals in species where genetic sex cannot be known owing to the lack of simple sex determining systems, as it is the case of many fish and reptiles with or without temperature-dependent sex determination, and provide a useful tool to relate gene expression and phenotypic sex.
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