The Mechanisms Of Cholecystokinin-Regulated Feeding And Reproduction In Grass Carp

Grass carp（Ctenopharyngodon idella）is one of the four main domestic fishes cultivated in inland waters of our country,and its output is increasing year by year.However,the sexual maturity time of grass carp takes a too long,and it usually takes 4-5years for female fish to reach sexual maturity,which seriously restricts the process of artificial reproduction and breeding for elite species.Therefore,we shoud reveal the molecular mechanism of puberty onset in grass carp and find the key factors regulating gonadal development and reproductive regulation.Then,we will edit the key factors through genetic engineering technology to shorten the time of sexual maturity cycle in grass carp and realize the artificial control of the initiation of sexual maturity of fish,which is of great significance to grass carp breeding.This study found that cholecystokinin（CCK）as a satiety factor can not only inhibit the feeding of grass carp,but also significantly induce the synthesis and secretion of gonadotropin（Gt H）in the pituitary,indicating that CCK may be a coupling factor integrating the two systems of feeding and sexual maturity.Based on this,this study analyzed the relationship between CCK and key factors of feeding and reproduction regulation in grass carp pituitary,and elucidated the mechanism of CCK involved in fish feeding regulation and sexual maturation initiation.The main results are as follow:1.Using the genome database and pituitary transcriptome data of grass carp,CCK and its receptor sequences were analyzed and their functions in pituitary were scanned.Taking grass carp as the research object,this study first cloned the full-length c DNA sequences of CCKs（CCK1 and CCK2）and their receptors CCK-Rs（CCK1Ra,CCK1Rb and CCK2R）.The sequence comparison revealed that the CCK1 and CCK2sequences were highly conserved,and only the third amino acid of the mature peptide of CCK2 was mutated from"leucine"to"valine".In addition,the sequences of CCK1Ra and CCK1Rb are also highly conserved,both of which are subtypes of CCK1R.Then the results of receptor-ligand selectivity experiments showed that CCK1 had a higher activation rate for CCK1Ra and CCK1Rb,while the activation rate for CCK2R was the same as that of CCK2.Then,the tissue expression analysis revealed that CCK-Rs were highly expressed in grass carp pituitary,indicating that CCK might play a key role in grass carp pituitary.Finally,through transcriptome data analysis,it was found that CCK could significantly up-regulate the feeding-related genes expression of neuromedin-B（NMB）,cocaine-and amphetamine-regulated transcript（CART）,urotensin 1（UTS1）,adrenomedullin（ADM）,leptin receptor（LEPR）and cholecystokinin receptor 2（CCK2R）and reproduction related gene expression of follicle-stimulating hormone beta subunit（FSHβ）,inhibinβa（INHBβa）and secretogranin II（SN2）in grass carp pituitary cells.2.The mechanism of CCK regulating the feeding metabolism of grass carp.In this study,through feeding experiments,it was found that grass carp could significantly increase the expression of CCK1 and CCK2 m RNA in the hypothalamus after eating,and the blood glucose concentration of grass carp was also significantly increased.Then,intraperitoneal injection of glucose found that hypothalamus could significantly increase the expression of CCK m RNA.Then,CCK was injected intraperitoneally,and the results showed that CCK could significantly inhibit grass carp feeding and induce the expression of anorexia factor（NMB,CART,UTS1,ADM,LEPR and CCK2R）in the pituitary.Finally,pharmacological experiments found that the induction effect of CCK on the six anorexia factors were achieved in a time-and dose-dependent manner.The results of receptor selectivity and post-receptor signaling pathway experiments showed that CCK1R and CCK2R mediated the regulation of CCK on NMB,CART,UTS1,ADM,LEPR and CCK2R,and also activated adenylate cyclase/protein kinase A（c AMP/PKA）pathway,phospholipase C/inositol 1,3,5-triphosphate/protein kinase C（PLC/IP3/PKC）pathway and Ca²⁺/calmodulin/Ca M-dependent protein kinase II(Ca²⁺/Ca M/Ca MK-II)pathway.Based on the above experimental results,we can find that food intake increases the blood glucose level of fish,and high blood glucose levels significantly increase the expression of CCK m RNA in the hypothalamus,which in turn induces the high expression of anorectic peptides in the pituitary to participate in the regulation of bony fish feeding.3.The mechanism of CCK regulating the expression of reproduction-related genes in grass carp pituitary.First,intraperitoneal injection of CCK showed that the expression of four reproductive genes,FSHβ,LHβ,INHBβa and SN2,were significantly increased in the pituitary.Next,pharmacological experiments showed that CCK could induce the m RNA expression of FSHβ,INHBβa and SN2 in pituitary cells in a time-and dose-dependent manner,while inhibiting the m RNA expression of LHβ.Inerestingly,at low doses or within a short period of time,CCK treatment would stimulate the expression of LHβm RNA in pituitary cells.In addition,the results of WB and ELISA experiments showed that CCK could induce protein secretion of FSH and LH.Then,the results of receptor selectivity and post-receptor signaling pathway experiments showed that CCK could participate in the regulation of FSHβ,INHBβa,SN2 and LHβby activating CCK1R and CCK2R in the pituitary,and it also coupled c AMP/PKA,PLC/IP3/PKC and Ca²⁺/Ca M/Ca MK-II three pathways.4.The mechanism of CCK mediating feeding and reproduction regulation in fish.First of all,food intake could increase the high expression of CCK in the hypothalamus by raising blood glucose;secondly,on the one hand,CCK could descend to the pituitary and inhibit food intake by stimulating the expression of anorectic genes;on the other hand,CCK could also participate in reproductive regulation by significantly increasing the synthesis and secretion of FSH in pituitary.