FundingLaia Navarro-Martín and Noelia Díaz were supported by predoctoral scholarships and Jordi Viñas and Laia Ribas by postdoctoral grants from the Spanish Ministry of Science and Innovation (MCINN).

Research was funded by the MCINN projects “Sexgene”, “Aquagenomics”, and “Epigen-Aqua” to Francesc Piferrer and by a MICINN project and AGAUR grant to Luciano Di Croce.

CitationDNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass. Laia Navarro-Martín, Jordi Viñas, Laia Ribas, Noelia Díaz, Arantxa Gutiérrez, Luciano Di Croce, Francesc Piferrer. PLos Genetics 2011; 7(12): e1002447. doi:10.1371/journal.pgen.1002447

Abstract

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.

Author Summary

Temperature changes during early embryonic and/or larval stages are able to modify sex ratios in fish and reptiles. However, the underlying mechanism by which temperature is able to modify the molecular pathways that developing gonads follow to become ovaries or testes is still unknown. One of the most interesting questions raised from previous studies with our model species, the European sea bass, was how temperature could affect the developmental fate of the gonads at a time when they were not even formed in the most rudimentary manner. This was the telltale sign of an epigenetic mechanism. In this study, DNA methylation levels of the aromatase promoter were analyzed in European sea bass exposed to different temperatures during early developmental stages. Aromatase is the enzyme that converts androgens (male hormones) into estrogens (female hormones), which are essential for ovarian development in all non-mammalian vertebrates. We show that increased temperature during a critical period in early development is able to increase DNA methylation of the aromatase promoter, preventing aromatase gene expression. We conclude that gonadal aromatase promoter methylation is most likely part of the long-sought-after mechanism connecting temperature and environmental sex determination in vertebrates.