| In the long process of evolution,animals form a fixed seasonal reproductive rhythm to ensure that their offspring are born in a relatively appropriate season.Circadian clock may play an important role in the regulation of reproduction.Asiatic toad(Bufo bufo gargarigans)are distributed widely in China,and the species migrates immediately to the spawning grounds after hibernation,and the breeding season is usually preceded by most of the other anuran amphibians in the same distribution areas.This specific breeding strategy may aims at avoiding interspecific competition.The aim of this study is to understand the changes of master and peripheral clocks of the toad at the different stages of the breeding season,and explore the role and function of the circadian clock in the breeding of this specie.The geographical population in Zhejiang generally begins to reproduce between February and March annually.This study sampled respectively in 2/27/2015(the breeding peak period)and 3/16/2015(the breeding late period)and set four sampling points per day(00:00,6:00,12:00,18:00).The expression of Clock,Bmall,Period 1/2,Cryptochrome 1/2 genes and the circadian rhythm changes of level of Corticosterone(CORT),Estradiol(E2),Testosterone(T)were examined and analyzed in male and female brain and liver tissues.The results are as follows:1.The circadian clock genes expression in brain tissuesThe Clock,Cry1/2 and Per1/2 mRNA expression of female toad brain tissue showed a significant diurnal variation(P<0.05)during the breeding peak period,and circadian oscillation patterns are synchronized highly,and appeared the peak at 12:00,but the Bmall gene expression showed no diurnal variation(P>0.05).However,these six clock genes expression patterns showed significant variation during the breeding late period compared with the peak period.The Cryl/2 and Perl/2 gene expression showed no obvious diurnal variation(P>0.05).Clock gene still showed obvious diurnal variation(P<0.05)while the peak time of clock gene(6:00)advanced 6 h compared with the breeding peak period.Bmall gene showed obvious diurnal variation(P<0.05),and the peak appeared at 18:00.The Clock,Bmall,Perl and Per2 mRNA expression of male toad brain tissue showed a significant diurnal variation during the breeding peak(P<0.05),among which the Clock and Perl/2 appeared the peak at 6:00 while Bmall appeared the peak at 12:00,and Cryl/2 gene expression showed no obvious diurnal variation(P>0.05).However,the Clock,Bmall,Cryl/2 and Per2 gene expression lost the diurnal variation at the breeding late period,except Perl(P<0.05),whose peak appeared at 00:00(24:00).In the breeding peak period,the Clock and Per1/2 gene expression of female and male brain showed obvious diurnal variation,but the expression patterns were different between male and female.Cryl/2 expressed rhythmically only in the female brain,while Bmall expressed rhythmically only in the male brain,which indicated that Clock and Per may have similar functions or roles at both sexes at the peak of the breeding,but there are gender differences in the function of the Bmall and Cry genes.However,most core clock genes of female and male brain showed no obvious diurnal variation in the late of the breeding,among which only Clock,Bmall showed significant diurnal variation in female,while only Perl among male.The above further illustrates the specific regulation of different core clock genes between sexes in the various stages of the breeding period,that's to say,the Cryl/2 and Perl/2 of female brain and Clock,Bmall and Per2 of male brain may act as a more special role in the breeding peak.In addition,these six clock genes average expression in brain of female and male were obvious higher during the breeding late period compared with the peak(P<0.05).The average expression of Cryl and Perl/2 of female brain were significant higher during the breeding peak period compared with the late(P<0.05),while only average expression of per2 was significant higher than male during the late(P<0.05).The average expression level of Clock,Bmall and Perl were significant lower than male during the late(P<0.05).2.The circadian clock genes expression in liver tissuesThe Clock,Bmall and Cry 1/2 mRNA expression of female toad liver tissue showed a significant diurnal variation(P<0.05)during the breeding peak period,and the peak appeared at 00:00(24:00),among which the Clock,Bmall and Cryl expression showed significant circadian rhythm(P<0.05,SE(A)/A<0.3).However,most of core clock genes expression showed no obvious diurnal variation during the breeding late period,except Clock(P<0.05),whose peak appeared at 00:00(24:00).These six clock genes expression of male toad liver tissue showed no obvious diurnal variation during the breeding peak period(P>0.05).In the breeding late period,only Per2 gene expression showed obvious diurnal variation(P<0.05),whose peak arrived at 18:00.In the breeding peak period,the Clock,Bmall and Cryl/2 mRNA expression of female toad showed a significant diurnal variation,while all of core clock genes of male showed no obvious diurnal variation.However,in the breeding peak period,only Clock still showed obvious diurnal variation in females,while only Per2 in males.It shows that the female liver circadian clock oscillation was stronger than the male during the breeding peak period,and that the liver acts as the largest metabolic organ in the body,indicating that Bmall and Cryl/2 may play a more special role in the metabolic regulation of female in the breeding peak period.That is to say,the rhythm of female metabolic may be stronger than male's during the breeding peak period.In addition,the Clock and Cryl average expression quantity of female and male were significantly reduced during the breeding late period(P<0.05),while Cry2 average expression quantity significantly increased(P<0.05),which present there is a significant difference in organ tissue compared with the increase of core clock gene expressions.Compared with the male,Clock and Cryl average expression quantity of female were significantly reduced at the peak and the late of breeding period(P<0.05),which present there is a significant difference with brain.It indicated that the expression of clock genes has a significant difference in gender and organ tissue ay the same breeding stage.3.The changes of serum hormone levelsThe content of serum CORT of female and male toad had no obvious change during the breeding peak period and late(P>0.05),but the average content was significantly increased during the breeding late period compared with the breeding peak period(P<0.05).The average content of female serum CORT had no significant different compared with male at the peak and the late of breeding period(P>0.05).The content of female serum E2 had a significant change during the breeding peak period(P<0.05,SE(A)/A<0.3),and presented a peak at 12:00.Although the diurnal variation of serum E2 during the breeding late period was similar to the breeding peak period,there was no obvious change in the statistic(P>0.05).The average content of E2 showed no obvious difference during the breeding peak period compared with late(P>0.05).The content of male serum T had no significant change during the breeding peak period and late(P>0.05),but the circadian patterns were similar.The average content of T was generally higher during the breeding peak period compared with late,while it was not statistically significant(P>0.05).In conclusion,the master and peripheral circadian clock systems of toad,which are during the breeding season,vary in degrees in the respective of clock gene expression patterns and its levels and endocrine regulated by the clock.Besides,a certain clock gene plays a role where it has obvious gender and organization differences.Therefore,circadian clock plays an essential part in seasonal reproduction and circadian rhythm of amphibians,and further research into related mechanisms is needed. |
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