Reproductive Regulation Effect Of GPER1 In The Brain And Harderian Gland Of Cricetulus Barabensis

Striped hamster(Cricetulus barabensis)is a typical animal for the study of seasonal breeding.Their reproductive activity is mostly concentrated in the late spring and early autumn.In the winter,the reproductive activity stops.The reproductive activity of animals is mainly regulated by the hypothalamus-adenohypophysis-gonadal axis system.Estrogen plays an important regulatory role in reproductive activity.The biological function of estrogen is mainly achieved by combined with estrogen receptors.GPER1,also known as GPR30,is a recently discovered seven-transmembrane G protein-coupled receptor,consisting of 375 amino acid residues.It has no homology with classic estrogen receptor.It mainly mediates the rapid non-genetic effects of the protein kinase pathway after transactivation with the second messenger cAMP and EGFR.There are a few reports on the role of estrogen transmembrane receptor in the regulation of animal reproduction.In this study,the wild striped hamster was chosen as the animal model in order to explore the molecular mechanism of seasonal reproductive regulation of GPER1.Firstly,GPER1 cDNA sequence was cloned through RT-PCR method and the nucleotide sequence,amino acid composition and physicochemical properties were analyzed.Then the GPER1 tertiary structures were predicted.Based on the GPER1 amino acid sequence,evolutionary trees was build.Secondly,real-time fluorescence quantitative PCR and immunohistochemical methods were used to investigate the differential expression of GPER1 in the brain of different age's striped hamster,including gene level and protein level.Thirdly,through different photoperiod treatment,real-time fluorescence quantitative PCR and immunohistochemical methods were used again to explore the differential expression of GPER1 in the brain and the Harderian gland,including gene level and protein level.Using enzyme-linked immunoassay to determine serum FollicleStimulating Hormone(Follicle-Stimulating Hormone,FSH),Luteinizing Hormone(Luteinizing Hormone,LH),and estrogen(E)concentration,and measure body weight and organ coefficient for the striped hamster in different photoperiod modes.In this study we cloned GPER1 cDNA through RT-PCR.The sequence of GPER1 is 1806bp(Genbank registration number MF993526),including part of the 3 'noncoding region and all the CDS coding region 1107 bp,368 amino acids are encoded totally.The bioinformatic analysis revealed that GPER1 is a kind of seven transmembrane protein and the structures of GPER1 is relatively conservative in the process of evolution,suggesting that the function of GPER1 is significant.Study on the expression of GPER1 in brains of the striped hamsters of different ages have showed :(1)Among females,GPER1 mRNA levels of adult were significantly lower than that of subadult and old age(P<0.05),and there was no significant difference among young,subadult and old age.GPER1 protein levels of subadult were significantly lower than that of young,adult and old age(P<0.05).(2)Among males,GPER1 mRNA levels of subadult were significantly lower than that of young and old(P<0.05),and there was no significant difference among young and adult and old age,and there was no significant difference between subadult and adult.The GPER1 protein levels of old were significantly lower than that of subadult,and there was no significant difference among young and subadult and adult.(3)At the m RNA level,significant gender difference(P<0.05)was observed on the young and the old individuals while there was extremely significant gender difference(P<0.01)in the adult.(4)At the protein level,extremely significant gender difference(P<0.01)was observed in the young and the old individuals and significant gender difference(P<0.05)was observed on the adult individuals,while there was no significant difference between the adult(P>0.05).(5)The concentration of serum FSH,LH,and E have showed a tendency to decrease with age.These results indicate that GPER1 can be expressed in individuals of different ages and sexes,and there are gender differences and age differences in expression levels.These results indicate suggests that GPER1 is involved in the regulation of reproduction and development of the striped hamsters.Different expression principles of mRNA and protein suggest that expression of GPER1 may be regulated by post-translational levels.Study on the expression of GPER1 in the brains and the Harderian glands of the striped hamsters treated with different photoperiod have showed:(1)In the brain,the expression of GPER1 mRNA,both male and female,was up-regulated in LD and down-regulated in SD.And extremely significant gender difference(P<0.01)was observed on the LD groups while there was no significant gender difference(P>0.05)in the SD groups.At the protein level,GPER1 protein expression in the brains also showed significant difference in the LD-treated group compared to SD controls(P<0.05)on the striped hamsters.However,no significant gender difference(P>0.05)was observed in LD individuals and significant gender difference(P<0.05)was observed in SD individuals.(2)In the Harderian glands,GPER1 mRNA expression,both male and female,was higher in LD than in SD.However,no significant gender difference(P>0.05)was observed both LD and SD individuals.At the protein level,GPER1 protein expression in the brains showed significant difference in the LD-treated group compared to SD controls(P<0.05)on the female the striped hamsters.And no significant gender difference(P>0.05)was observed on the male individuals.Significant gender difference(P<0.01)was observed on the SD groups while there was no significant gender difference(P>0.05)in the LD groups.(3)LD increase increases body weight,uterine coefficient,ovarian coefficient,testis coefficient,epididymis coefficient,etc.SD reduces these indicators.(4)Serum concentrations of FSH,LH and E were significantly different between LD and SD.These results indicate that: Photoperiod is one of the important factors which affect the striped hamsters' seasonal breeding,photoperiod treatment can change the body weight and the viscera coefficient of reproductive organs in striped hamsters.LD can promote the growth of body weight and gonad,and SD can lead to body weight loss and gonad atrophy.According with photoperiodic study,we found GPER1 can be expressed in the brain and Harderian gland of the striped hamster.As a photoreceptor,Harderian gland participates in regulating seasonal reproduction.Photoperiod can affect GPER1 expressed in the brain to regulate reproduction.In summary,there are many internal and external factors involved in animal reproduction regulation.GPER1 is one of the new endogenous factors.This study revealed the role of GPER1 in the reproduction regulation of hamsters in the black line,but more detailed mechanisms still require further study.In addition,the unknown function of GPER1 remains to be discovered.