蛙启动和维持冬眠的生物钟调节机制:外因诱导与节律调控

Hibernation is one of survival strategies for avoiding the risks from adverse conditions in winter. However, it is an interesting and profound question that how frogs adjust themselves to adapt to the environment that could induce them to hibernate. Here, we will investigate the initiating and sustaining hibernation mechanism in two temperate zone frog representative species, from the point of view of circadian clock genes control to circadian rhythms of sugar metabolism, immunity activity and hormone secretion. The main research focuses on the molecular system of master and peripheral clock and its feedback mechanisms of the frog hibernators in hibernating or non-hibernating state, mainly involving the relative clock genes including two key transcription factors including Clock and Bmal1, feedback regulators like Cryptochrome (Cry) and clock control factor like Arginine vasopressin. There are two key scientific questions we try to answer. One of the questions is how hibernation will be initiated, and the reason is due to that the organism could feel a mismatch between the external and its inner rhythms, or due to resetting of circadian clock according to environmental changes? The other question is that whether the frogs could inhibit metabolic level and block rhythms signal output by closing circadian rhythm clock or not,for sustaining hibernation and simultaneously saving energy ，and whether they could eliminate the bondage of circadian clock when they perceive environmental stimulation again? These results of our project will not only help improve theory knowledge about animal hibernation, but also provide a new theoretical basis for exploration on global amphibian endangered mechanisms.

冬眠是动物为规避冬季不利条件带来的风险而发展出的重要生存策略之一。那么，蛙类究竟是怎样在环境诱导和自我调节下进入冬眠的？这是一个有趣且深奥的问题。本项目以两种温带蛙类代表物种为研究对象，拟从生物钟基因对糖类代谢、免疫活性、激素分泌的昼夜节律控制的角度来研究蛙类冬眠的启动与维持机制。重点研究蛙在冬眠和非冬眠状态下母钟和子钟的分子模式系统（包括关键转录因子Clock与Bmal1、反馈调节子Cry和生物钟控制因子精氨酸加压素）和相关反馈环机制。拟解答的关键科学问题：（1）冬眠究竟是因为机体感知到环境变化与内在生物钟节律发生了错位还是因为生物钟被重调才启动的？（2）蛙是否会通过关闭生物钟来降低代谢水平和阻断节律信号输出以维持冬眠，同时达到保持维持体能的目的，以及当再次受到环境刺激后是否会解除对生物钟的束缚？对这些问题的研究有助于完善动物冬眠的理论知识，还可为两栖类濒危机制研究提供全新的理论依据。

为探索蛙类生物钟分子系统的运行特征以及在冬眠启动和维持中的角色和功能，研究了代表性物种在人工授时条件下和野外冬眠后繁殖期核心生物钟基因和激素分泌的节律变化，以及冬眠前后和人工冬眠初期和中期神经系统和外周系统生物钟、钟控基因以及激素分泌的节律变化，并尝试研究了特定光谱等对生物钟的调节机制。.1.中华大蟾蜍在人工授时下神经视网膜和脑内大多数核心生物钟基因存在明确的昼夜变化，但肝脏钟基因表达则无明显节律，而且血清褪黑素水平在黑暗前夕达到峰值。这表明蛙类应当存在与类似哺乳类的以正、负反馈环为核心的生物钟分子振荡机制，且具有夜行性特征。另外，在野外条件下发现蟾蜍雌雄个体在繁殖末期脑内生物钟基因表达水平和血清皮质酮水平均显著升高，但性激素水平则无明显变化，暗示繁殖期生物钟的变化可能与糖皮质激素系统密切相关。.2.蟾蜍作为地下冬眠代表物种，脑内分子钟系统对冷刺激的敏感性要明显高于肝脏，但冬眠中脑钟系统基本处于停止或处于无序状态，而肝脏钟系统和外周血皮质酮水平却表现出强劲而有序的振荡，说明当蟾蜍生物钟在接受到环境低温信号后未表现出温度补偿，且外周钟在冬眠状态下会脱离中枢生物钟的控制而自主运行。棘胸蛙作为水下冬眠代表物种，视网膜和脑内生物钟基因表达在冬眠中仍然具有明显节律，说明水环境中冬眠的蛙类神经系统生物钟仍受光的调控，而肝脏钟基因在冬眠中表达节律都不显著，这与蟾蜍明显不同。.3.蓝光和绿光对蟾蜍Per2的表达影响较大，而蓝光和红光对Cry1基因表达影响较大，说明光因子实现对两栖类生物钟的调控主要通过特殊波段的光谱发挥作用。. 综上所述，冬眠中生物钟的自主运行对温带蛙类的成功越冬发挥了至关重要的作用。冬眠可被看作是蛙类在生理上从一种节律内稳态进入另外一种节律内稳态的过程，由冬眠诱导的生物钟系统的适应性改变能够影响和协调包括抗氧化功能、糖代谢等多方面生理过程的节律性，从而提高冬眠者的越冬成活率。