Treatment with a DNA methyltransferase inhibitor feminizes zebrafish and induces long-term expression changes in the gonads

Abstract

BackgroundThe role of epigenetic modifications such as DNA methylation during vertebrate sexual development is far from being clear. Using the zebrafish model, we tested the effects of one of the most common DNA methyltransferase (dnmt) inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), which is approved for the treatment of acute myeloid leukaemia and is under active investigation for the treatment of solid tumours. Several dose–response experiments were carried out during two periods, including not only the very first days of development (0–6 days post-fertilization, dpf), as done in previous studies, but also, and as a novelty, the period of gonadal development (10–30 dpf).ResultsEarly treatment with 5-aza-dC altered embryonic development, delayed hatching and increased teratology and mortality, as expected. The most striking result, however, was an increase in the number of females, suggesting that alterations induced by 5-aza-dC treatment can affect sexual development as well. Results were confirmed when treatment coincided with gonadal development. In addition, we also found that the adult gonadal transcriptome of 5-aza-dC-exposed females included significant changes in the expression of key reproduction-related genes (e.g. cyp11a1, esr2b and figla), and that several pro-female-related pathways such as the Fanconi anaemia or the Wnt signalling pathways were downregulated. Furthermore, an overall inhibition of genes implicated in epigenetic regulatory mechanisms (e.g. dnmt1, dicer, cbx4) was also observed.ConclusionsTaken together, our results indicate that treatment with a DNA methylation inhibitor can also alter the sexual development in zebrafish, with permanent alterations of the adult gonadal transcriptome, at least in females. Our results show the importance of DNA methylation for proper control of sexual development, open new avenues for the potential control of sex ratios in fish (aquaculture, population control) and call attention to possibly hidden long-term effects of dnmt therapy when used, for example, in the treatment of prepuberal children affected by some types of cancer.Electronic supplementary materialThe online version of this article (10.1186/s13072-017-0168-7) contains supplementary material, which is available to authorized users.

DNA Methylation Reprogramming during Sex Determination and Transition in Zebrafish

“…Then we checked the male/female ratio in each group (see Methods). The male sex ratio in treated group from 35 dpf to 90 dpf is lower than that in the control (Figure 2E and Table S5), indicating that fish treated with DNA methylation inhibitor are prefer to develop into females, which is consistent with recent study [42]. Therefore, these results suggested that DNA methylation plays a role during sex determination.…”

Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations

“…Thus, regardless of the exposure, some females resisted masculinization even though their amh levels were upregulated in the mature ovary. The fact that the upregulation of some promale genes and pathways was also observed in ovaries of zebrafish treated with a demethylation agent (decitabine) during sex differentiation, which skewed the sex ratios toward females (Ribas, Vanezis, Imués, & Piferrer, ), may indicate that activation of some promale genes is the consequence of the presence of external disturbances during sex differentiation rather than the masculinization per se brought by these perturbations. The results presented here open the possibility that these well‐known reproduction‐related genes, cyp19a1a , amh , and sycp3, but also other genes with other functions such as faima , ccm2l , ankrd6b , lgals9l1 , or ubxn2a , might be good markers of environmental perturbation independent of conspicuous alterations in sex ratios or gonadal morphology.…”

Gonad Transcriptome Analysis of High-Temperature-Treated Females and High-Temperature-Induced Sex-Reversed Neomales in Nile Tilapia

“…Our previous study also showed that high-temperature treatment during early sex differentiation stages increased cyp19a1a promoter DNA methylation levels in four-month-old Nile tilapia [ 36 ]. Collectively, DNA methylation-mediated mRNA expression changes of many important sex differentiation-related genes during temperature treatment possibly play an important role in fish TSD or GSD + TE [ 16 , 37 ].…”