DNA methylation of gene promoters involved in sexual differentiation and stress of zebrafish (Danio rerio) subjected to high temperature during the early gonadal development

Abstract

Zebrafish (Danio rerio) is a gonochoristic species in which sex is determined by the combination of genetic and environmental influences. Thus, when zebrafish is exposed to critical abiotic stressors such as high temperature during the sex differentiation period (15-45 days post fertilization, dpf), there is an increase of the number of males in the population. This masculinization is likely mediated by epigenetic mechanisms, known to integrate environmental information into genomic functioning. In order to better understand the epigenetics of this process, we investigated whether high temperature affects the molecular mechanisms at the DNA methylation level in the promoter regions of 9 genes involved in sex differentiation, 7 genes in stress response, 2 genes linked to epigenetic regulation and 2 genes related to the immune response. We exposed eggs of two different zebrafish families (AB strain) either to normal (control; 28°C) or elevated (35°C) temperatures from 18 to 32 dpf. At 90 dpf, when fish were sexually mature, DNA from gonadal tissues was extracted, bisulfite treated and next-generation sequencing libraries performed. As expected, there were significant sex ratio shifts with a higher number of males in the high temperature groups. We found differences in the methylation level of the promoter of some genes between testis and ovaries. At low temperature, males showed significantly higher DNA methylation levels than females in some sex-related genes (cyp19a1a, amh), stress response genes (in3rc1, hsd17b3, hsd3b2, cyp11c1, hsd17b1, hsd11b2) and one immune-related gene (tnfα), while in dmrt1 and dmrt3a genes followed the opposite pattern. However, at high temperature, the DNA methylation levels increased in the above mentioned, especially in males. The results of this study contribute to our understanding of the role of epigenetic mechanisms and their involvement during the early gonadal development in zebrafish