Circadian expression of DNA methylation and demethylation genes in zebrafish gonads

This research aimed at investigating the light synchronization and endogenous origin of daily expression rhythms of eight key genes involved in epigenetic mechanisms (DNA methylation and demethylation) in zebrafish gonads. To this end, 84 zebrafish were distributed into six tanks, each one containing 14 fish (7 males and 7 females). Animals were subjected to 12h light:12h dark cycles (LD, lights on at ZT0h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4h during a 24h cycle (ZT2, 6, 10, 14, 18, and 22h). The results revealed that most of the epigenetic genes investigated exhibited a significant daily rhythm. DNA methylation genes (dnmt4, dnmt5, dnmt7) exhibited a daily rhythm of expression with a nocturnal acrophase (ZT14:01 similar to ZT22:17h), except for dnmt7 in males (ZT2:25h). Similarly, all DNA demethylation genes (tet2, tdg, mb4, gadd45aa, and apobec2b) revealed the existence of statistically significant daily rhythms, except for gadd45aa in females. In females, tdg, mb4, and apobec2b presented a nocturnal peak (ZT14:20 similar to ZT22:04h), whereas the tet2 acrophase was diurnal (ZT4:02h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26 similar to ZT21:31h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02h, respectively). To determine the endogenous nature of gene expression rhythms, another experiment was performed: 12 groups of 14 fish (7 males and 7 females) were kept in complete darkness (DD) and sampled every 4h during a 48h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46h). Under DD, most of the genes (7 out of 8) presented circadian rhythmicity with different endogenous periodicities (tau), suggesting that the epigenetic mechanisms of DNA methylation and demethylation in the gonads follow an internal control, functioning as part of the translation network linking the environment into somatic signals in fish reproduction.