Paredes Juan Fernando, Lopez-Olmeda Jose Fernando, Muñoz-Cueto Jose A, Sánchez-Vázquez F
a Physiology , Murcia University , Murcia , Spain.
b Department of Physiology, Faculty of Biology , University of Murcia , Murcia , Spain.
Chronobiol Int. 2018 Jul;35(7):920-932. doi: 10.1080/07420528.2018.1440403. Epub 2018 Mar 6.
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 12 h light:12 h dark cycles (LD, lights on at ZT0 h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20 days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4 h during a 24 h cycle (ZT2, 6, 10, 14, 18, and 22 h). 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 (ZT14:01ZT22:17 h), except for dnmt7 in males (ZT2:25 h). 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 ~ ZT22:04 h), whereas the tet2 acrophase was diurnal (ZT4:02 h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26ZT21:31 h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02 h, 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 4 h during a 48 h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 h). 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.
本研究旨在调查斑马鱼性腺中参与表观遗传机制（DNA甲基化和去甲基化）的八个关键基因的日常表达节律的光同步性和内源性起源。为此，将84条斑马鱼分布到六个水箱中，每个水箱中有14条鱼（7条雄性和7条雌性）。动物接受12小时光照：12小时黑暗周期（LD，在ZT0小时开灯），并在光照阶段每天随机喂食三次。记录每个水箱中运动活动节律20天，以测试它们与光的同步性。然后，斑马鱼禁食一天，并在24小时周期（ZT2、6、10、14、18和22小时）内每4小时收集一次性腺样本。结果显示，所研究的大多数表观遗传基因表现出显著的日常节律。DNA甲基化基因（dnmt4、dnmt5、dnmt7）表现出每日表达节律，夜间峰值相位（ZT14:01ZT22:17小时），雄性中的dnmt7除外（ZT2:25小时）。同样，所有DNA去甲基化基因（tet2、tdg、mb4、gadd45aa和apobec2b）均显示存在统计学上显著的日常节律，雌性中的gadd45aa除外。在雌性中，tdg、mb4和apobec2b呈现夜间峰值（ZT14:20ZT22:04小时），而tet2的峰值相位是白天（ZT4:02小时）。在雄性中，tet2、tdg和gadd45aa有夜间峰值相位（ZT18:26~ZT21:31小时），而mb4和apobec2b显示白天峰值相位（分别为ZT5:28和ZT4:02小时）。为了确定基因表达节律的内源性本质，进行了另一项实验：将12组14条鱼（7条雄性和7条雌性）置于完全黑暗（DD）中，并在48小时周期（CT2、6、10、14、18、22、26、30、34、38、42和46小时）内每4小时取样一次。在DD条件下，大多数基因（8个中的7个）呈现出具有不同内源性周期（tau）的昼夜节律，这表明性腺中DNA甲基化和去甲基化的表观遗传机制遵循内部控制，作为将环境转化为鱼类繁殖中体细胞信号的翻译网络的一部分发挥作用。
