| Seasonal production of Mammals is regulated by the complex feedback adjustment of hypothalamus-pituitary-gonad axis.The thyroid-stimulating hormone ?-subunit(TSHB)and thyroid-stimulating hormone receptor(TSHR)play an important role in its reverse pathway.The main function of TSHB and TSHR is to regulate the expression of DIO2(Type 2iodothyronine deiodinase,DIO2)and DIO3(Type 2 iodothyronine deiodinase,DIO3)in hypothalamus and then regulate the dynamic balance of thyroid hormone(T2,T3,T4,rT3),thereby regulating the key genes related to reproduction GnRH.Cricetulus barabensis is a typical seasonal breeding animal.Aiming to explore the molecular mechanisms of Cricetulus barabensis population dynamics interannual fluctuation,this study cloned the breeding candidate gene TSHB/TSHR through RT-PCR method,and analyzed the nucleotide and amino acid composition and physicochemical properties,then the TSHB/TSHR tertiary structures were predicted and do TSHB/TSHR amino acid sequence homology analysis by building system evolutionary trees.Fluorescence quantitative PCR method is used to explore TSHB/TSHR seasonal and photoperiod expression patterns and the influence of melatonin treatment on the two gene expression in the pituitary gland and the gonads.Using enzymelinked immunoassay to determine serum Follicle-Stimulating Hormone(Follicle-Stimulating Hormone,FSH),Luteinizing Hormone(Luteinizing Hormone,LH),Thyrotropic hormone(Thyrotropic hormone,TSH)and TSHR hormone concentration and do the correlation analysis of the striped hamsters serum hormone concentration and gene expression in four seasons.In this study we cloned TSHB and TSHR cDNA through RT-PCR.The sequence of TSHB is 470bp(Genbank registration number KX756570),including part of the 3 'noncoding region and all the CDS coding region 468 bp,155 amino acids are encoded totally.TSHR cDNA sequence is 2480bp(Genbank registration numbeKX756569),including part 5 'and 3'noncoding region and all the CDS coding region 2295 bp,encoding 764 amino acids.The bioinformatic analysis revealed that TSHB amino acid sequence contains the tag sequence of glycoprotein hormones family and TSHR belongs to the family of G protein coupled receptor family glycoprotein hormones subfamily.TSHR is a kind of seven transmembrane protein and the structures of TSHB and TSHR are relatively conservative in the process of evolution.It revealed that the seasonal expression patterns of TSHB and TSHR mRNA are completely consistent in the hypophysis of male individuals and increased significantly(P < 0.05)in spring,which was consist with the finding that DIO2 expression of hypothalamus and serum TSH,TSHR,FSH and LH concentrate were higher in spring.In winter,the expression of TSHB and TSHR reduced significantly comparing with other three seasons(P<0.05).It's speculated that TSHB and TSHR mRNA is closely related to RFRP-3 mRNA expression in hypothalamus in winter.TSHB and TSHR have a promote role in the expression of GnRH and RFRP-3 is an inhibitory factor of GnRH;For female individuals,the expression of TSHB and TSHR in hypophysis is higher in spring and autumn than that in summer and winter(P<0.05),which is in accordance with that TSH/TSHR serum concentration is higher in spring and autumn than in summer and winter,and that FSH/LH serum concentration is higher in autumn,and that the expression pattern of DIO2 in hypothalamus of female Cricetulus barabensis,indicating that there was a close relationship between TSHB/TSHR and deiodinase system.From an overall perspective,TSHB and TSHR expression in ovary and testes is higher in spring and autumn than that in summer and winter(P<0.05),which was consisting with the finding that TSHB and TSHR seasonal expression in hypophysis and the TSH/TSHR concentration in serum.This result consists with GnRH seasonal expression pattern,also consists with the fact that breeding peak of Cricetulus barabensis appears in spring and autumn.Therefore we speculated TSHB/TSHR expression in hypophysis and gonades of Cricetulus barabensis could enhance the activity of reproduction.TSHB and TSHR expression in ovary significantly reduced in summer,speculating that it is related with the increased RFRP-3 expression in summer.The results of the measured serum FSH,LH,TSH and TSHR hormone concentrations show that the four hormone concentrations are influenced by season,the relative expression of TSHB in hypophysis has significant positive correlation with the four hormone concentrations.The relative expression of TSHR in hypophysis has significant positive correlation with serum LH concentration.According with photoperiodic study,we found the expression of TSHB and TSHR in pituitary and testis of male individuals is significant higher under long day exposure than that under short day(P<0.05),which is consistent with the photoperiodic expression trend of DIO2 in hypothalamus and gonads.TSH,TSHR,FSH,LH concentration in serum is much higher under long day than that under short day,indicating the reproductivity could be promoted in the long day exposure but inhibited under short day.The expression of TSHB in Pituitary and ovary of female individuals is higher in middle day,which is inconsistent with the finding that DIO2 expression is highest under long day,but is consistent with the fact that the expressions of DIO3 in hypothalamus and serum TSH concentration of female individuals are the highest under middle day.It indicates that expression of TSHB in male individuals is probably more sensitive to middle day.For female individuals,serum TSHR concentration shows highest under middle day,which is inconsistent with TSHR expression in hypophysis but is consistent with TSHR expression in ovary.In order to study the effect of exogenous melatonin on the expression of TSHB and TSHR,Cricetulus barabensis with exogenous melatonin was conducted.It shows that the expression of TSHB/TSHR in pituitary and testis of male individual,TSHB in pituitary and ovary of female individuals and TSHR in pituitary of female individuals is significant higher in melatonin treatment group than that in vehicle control group(P<0.05),indicating the inhibitory effect of melatonin on the expression of TSHB and TSHR.Serum LH,FSH,TSH and TSHR concentration of male individuals and serum LH,FSH,TSH concentration of female individuals significantly decreased in melatonin treatment group(P<0.05).There were no significant differences in the serum TSHR of female individuals between melatonin treatment group vehicle control group(P>0.05),which is inconsistent with the effect of melatonin on TSHR expression in hypophysis of female,but is consistent with the fact that in ovary,indicating the melatonin inhibits Cricetulus barabensis reproductivity.This study elucidated TSHB/TSHR plays an important role on Cricetulus barabensis reproductivity regulation through treatment of natural season sampling,photoperiod and melatonin which indicates a photoperiod-Mel-TSHB-TSHR-DIO2/DIO3-TH-GnRH pathway in Cricetulus barabensis seasonal production. |
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