Circadian Rhythm Of Clock Gene Expression And Peripheral Hormone Levels In Asiatic Toad Bufo Bufo Gargarigans

It remains ambiguous about the process of accurate molecular regulation maintaining circadian clock and there are major differences between species. Researches about molecular clock system mostly concentrate on model species such as rodents and fishes compared with amphibians and reptiles. Due to the transition status from aquatic to terrestrial in the process of evolution, amphibians occupies an important position in phylogenetics, thus, research on batrachians enables us to comprehend light-dark circadian rhythm about vertebrates more profoundly. However, fundamental knowledge on the circadian clock system of amphibians remains deficient.Accordingly, we selected Bufo bufo gargarigans as an experimental object, and cloned cDNA segments of core clock genes of molecular clock system, including positively regulatory element (Clock and Bmall) and positively regulatory element (Cryl/2and Perl/2), and then studied the relationship between circadian expression patterns of clock genes and rhythms of peripheral hormone levels in Asiatic toad Bufo bufo gargarigans under artificial zeitgeber conditions (12L:12D). These hormones include:(1) melation (MT) acting as the most crucial input signal of circadian clock (2) corticosterone (CORT) acting as a kind of glucocorticoid reflecting behavior and stress state of animals (3) thyroid hormones (T3and T4) reflecting metabolism levels of body. What’s more, we also cloned thyroid hormone receptor β (THRβ). The main results as follows:(1) Circadian expression patterns of clock genes mRNA levels in the neural retina of Bufo bufo gargarigans under artificial zeitgeber conditions (12L:12D):the expression pattern of Clock gene in the neural retina displayed significantly light-dark rhythm (SE(A)/A<0.3), but Bmall did not (SE(A)/A>03), whereas both showed similar trend with peak at ZT4; Perl transcript levels showed a significant daily variation (SE(A)/A<0.3), but Per2did not (SE(A)/A>0.3) and showed an reverse phase with Perl; The daily thythms of Cry land Cry2transcript levels were not examed (SE(A)/A>0.3), both mRNA levels were higher in the dark phase than in the photophase and displayed a synchronization.(2) Circadian expression patterns of clock genes mRNA levels in the brain of Bufo bufo gargarigans under artificial zeitgeber conditions (12L:12D):the expression patterns of Clock and Bmall in the brain were rhythmic (SE(A)/A<0.3) with peak (P<0.05) at ZT4and valley (P<0.05) at ZT16. Perl, Per2and Cryl, but not Cry2, transcript levels displayed significant daily rhythms (SE(A)/A<0.3), while expression patterns among all of them showed the homogeneous tendency.(3) Circadian expression patterns of hormone levels in the plasma of Bufo bufo gargarigans under artificial zeitgeber conditions (12L:12D):The plasma MT levels were rhythmic (SE(A)/A<0.3) with peak (P<0.05) prior to the dark phase at ZT12and with valley (P<0.05) close to the photophase at ZT24; The plasma CORT levels also displayed cyclic oscillation (SE(A)/A<0.3) when its peak level occurred at ZT4(P<0.05) and then it dropped to valley at the end of photophase (P<0.05), and gradually increased during the dark phase (P>0.05). In the neural retina and brain of Bufo bufo gargarigans respectively, THRβ transcript levels were no-rhythmic (SE(A)/A>0.3), while in the neural retina, THRβ peaked at ZT4(F<0.05) significantly and it bottom out at ZT21; Both T3and T4levels in the plasma of Bufo bufo gargarigans did not display a significant daily oscillation (SE(A)/A>0.3).From these findings, we are able to understand circadian rhythms of clock gene expression and peripheral hormone levels in Bufo bufo gargarigans, and also comprehend regulatory mechanism and function law of circadian clock on its behavior and metabolism, and thus acquire a deeper understanding of clock system of amphibians.