| Hibernation is one of the important survival strategies that part of animals obtained to save energy, avoid the adverse environment, such as cold, hunger and disease in the long time of self-protection and evolution process. The traditional view is that the hibernation of variable temperature vertebrate, such as amphibians, is passive adaptation to the environment of low temperature and lack of food, belong to the simple phenomenon of cold paralysis. Amphibians such as variable temperature vertebrate hibernation is passive adaptation to the environment of low temperature and lack of food, belong to the simple phenomenon of cold paralysis. However, we consider that amphibians turning into hibernation is a process that one rhythm-specific homeostasis to another. And circadian rhythms is involved in the whole process of rhythm control in physiological and molecular level. This study proposes and tries to answer these scientific questions:if there is a difference in sensitivity between central and peripheral clocks of Bufo Bufo gargarigans to cold stimulation? If the clock can keep running or the characteristics of rhythmic oscillation is changed in hibernation of Toad by the condition of lack of zeitgebers?1. The circadian clock genes expression in brain tissuesIn the original natural environment, the Clock, Bmall, Per2 and Cry2 mRNA expression of brain tissue showed obvious diurnal variation (p<0.05). The Bmall mRNA expression showed significant circadian rhythm (p<0.05, SE/A(A)<0.3), and the peak was showed between 6:00 and 18:00, then expressing quantity began to gradually decline. Clock, Per2 and Cry2 mRNA expression patterns are similar, and their peaks appeared at 6:00. Perl and Cryl gene expression showed no obvious diurnal variation (p>0.05). At low temperature (4℃, darkness) within 24h, Clock, Bmall, Per2, Cryl and Cry2 expression obviously changed (p<0.05). Only Perl did not respond to low temperature (p>0.05). Bmall gene mRNA expression continued to rise, and Cry2 mRNA expression continued to decline. Per2 expression circadian rhythm patterns were obvious (p<0.05, SE/A(A)<0.3), and the peak appeared at 22:00 (p<0.05). Compared with the initial state, Cry2 average expression quantity is significantly reduced (p<0.05), but the average expression of other clock genes’ changes are not obvious (p>0.05). After cold acclimation 45 days, most of the clock genes did not change significantly, except Cryl (p<0.05). At the same time, Perl and Cryl average amount of the initial condition were significantly increased (p<0.05), while Per2 instead. In the initial state, the brain tissue ThrP gene expression quantity was no significant different statistically (p>0.05). And after being treated by cold exposure 24 h (4℃, darkness) or low temperature domestication 45 days, ThrP gene expression quantity was changed significantly (p<0.05), and average ThrP expression significantly was lower than the initial state (p<0.05).2. The circadian clock gene expression in liver tissuesInitially the natural environment, Clock, Bmall and Cry2 gene mRNA expression in liver tissue were significantly changed (p<0.05). Bmall mRNA expression showed a significant circadian rhythm patterns (p<0.05, SE(A)/A<0.3), and the peaks and valleys appear at 10:00 and 2:00 respectively. Clock mRNA expression peaked at 10:00 p<0.05). Both Bmall and Clock mRNA levels seems to be high during the day and low at night, exhibiting a certain synchronization. At a low temperature (4℃, darkness) cold exposure 24h, most liver clock genes have not responded (p>0.05), but only Per2 mRNA expression change significantly (p<0.05). In addition, compared with the initial state, all the average level of the clock genes mRNA did not change significantly (p>0.05). After cold acclimation 45 days, Clock, Bmal1, Per2 and Cryl mRNA expression restore a significant change (p<0.05), of which Bmall and Per2 peak appeared at 2:00, and their expression patterns almost were synchronized. Perl and Cry2 mRNA expression did not change significantly (p>0.05). Bmall, Perl, Cryl Cry2 mRNA and the average level in the initial state showed low expression, small amplitude. However, after cold acclimation, the four genes mRNA expression exhibit high levels and large amplitude on the average (p<0.05). In three experimental treatments in this study, Thrβ mRNA expression has no statistically significant difference in liver tissues (p>0.05), and the overall average levels of Thrβ mRNA expression are not different between the treatments (p>0.05).3. The changes of content of endocrine hormone levelIn the initial state and cold exposure under low temperature (4℃, darkness) for 24h, content of serum CORT had no obvious change (p>0.05), but the average content had been inhibited because of the cold exposure (p<0.05). After domestication under low temperature for 45 days, content of serum CORT had a significant change, and presented a peak at 10:00, then showed a trend of gradual decline. The average content of CORT was without significant difference compared with initial state (p>0.05). In the initial state and cold exposure under low temperature (4℃, darkness) for 24h, content of serum MT had significant change (p<0.05), and the peak appeared at 10:00. After domestication under low temperature for 45 days, the content of serum MT did not change significantly (p>0.05). In the initial state, the content of serum T3 had the significant change of circadian rhythm (p<0.05, SE/A(A)<0.3), presented a peak at 6:00, then showed a trend of gradual decline. In cold exposure under low temperature (4℃, darkness) for 24h, the content of serum T3 had no obvious changes (p>0.05), but the average content had significant change compared with initial state(p<0.05). After domestication under low temperature for 45 days, the content of serum T3 changed obviously (p<0.05), and the average content increased significantly compared to the initial state (p<0.05). In the initial state and after domestication under low temperature for 45 days, the content of serum T4 had obvious changes (p<0.05), but did not change significantly in cold exposure under low temperature (4℃, darkness) for 24h(p>0.05), and the average content was not obviously changed compared to in the initial state (p>0.05). After domestication under low temperature for 45 days, average content of serum T4 showed significantly lower compared to in initial state (p<0.05).4. The comparison of clock genes between tissuesThe initial state of the Clock, Bmal1, Cry2 mRNA expression remained oscillations in brain and liver, Per2 remained oscillation in the brain, there are no oscillation of Perl and Cryl in the two tissues. The most of the clock genes has made a quick response and obvious oscillation to cold stimulation in brain, but the overall average levels of Cry2 mRNA expression decreased obviously. On the contrary, in addition to the Per2, the vast majority of liver clock genes have not obvious oscillation. In addition, cold stress has no impact on Perl gene expression in the brain and liver. In cold acclimation clock genes oscillation of midbrain is not obvious, only Cryl is obvious, but Perl and Cryl appeared highly expressed genes. However, in the liver Bmal1, Per2, Cryl obvious oscillation and the amplitude increase obviously, expression peak occurred at 2:00, and most of the clock gene (Bmall, Perl fires our, Cryl, Cry2) appeared high expression. Cry1 in two organs showed obvious oscillation, and Bmal1 and Per2 is only obvious in the liver, but Clock lost oscillations. In addition, Perl and Cryl appeared high expression in two kinds of organs, only Bmall and Cry2 show highly expressed in the liver.In conclusion, although the sensitivity of the brain clock system to cold stimulation was significantly higher than the liver, clock system of brain was basically stopped or disordered in the cold acclimation while the liver clock system showed strong and orderly oscillation. |
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