Study On The Reproductive Regulation Of Prolactin Releasing Peptide In Zebrafish

In mammals,PrRP has been found to induce luteinizing hormone secretion in the pituitary of mice.However,its specific functions in fish reproductive regulation are rarely studied.Therefore this paper took zebrafish as the research object and constructed a PrRP deletion model to explore its function and mechanism in fish reproductive regulation.The details are as follows:1.The potential functions of PrRP on the hypothalamic-pituitary-gonad axis of zebrafish were preliminarily verified by tissue expression analysis and immunofluorescence.In this study,two PrRPs subtypes（PrRP1 and PrRP2）and four receptor subtypes（PrRPR1,PrRPR2,PrRPR3 and PrRPR4）were cloned from zebrafish.Then,ligand-receptor selectivity experiments showed that PrRPR1 and PrRPR2 were universal receptors for PrRP1 and PrRP2,and PrRP1 and PrRP2 had stronger activation ability for PrRPR3.In addition,tissue expression analysis showed that prrp and its receptor were highly expressed in brain,pituitary and gonad of zebrafish,suggesting that PrRP may play a role in reproductive regulation.Finally,immunofluorescence technique was used to locate and analyze the position of PrRP in zebrafish gonads.The results showed that PrRP was expressed in zebrafish testis and ovaries to varying degrees,indicating that PrRP may play a direct role in fish gonads through parocrine/autocrine.2.Construct PrRP gene knockout model of zebrafish to verify its function in reproductive regulation of zebrafish.prrp1^-/-and prrp2^-/-knockout models of zebrafish were constructed using CRISPR/Cas9 technique.The results showed that the body length and body weight of males of prrp1^-/-and prrp2^-/-were larger than those of wild-type zebrafish,while the gonadal body index of knockout females was significantly lower than that of wild-type zebrafish.In addition,compared with wild-type zebrafish,knockout zebrafish had lower spawning rate,spawning number and fertilization rate.According to morphological and gonadal histological analysis,follicular development in the ovaries of prrp1^-/-and prrp2^-/-zebrafish was impaired to varying degrees,and the testis of prrp1^-/-zebrafish were also found to be dysplastic.In addition,prrp1^-/-and prrp2^-/-pituitary gland lhβ,fshβand cyp11a1a,cyp17a1a and cyp19a1a in gonads showed low m RNA levels.3.Wild zebrafish was used to further verify the mechanism of PrRP involved in fish reproductive regulation.In this study,wild zebrafish was intraperitoneally injected with PrRP,and it was found that PrRP could significantly induce m RNA expressions of cyp11a1a,cyp17a1a and cyp19a1a in gonads and lhβ,fshβin pituitary.In addition,Pr PR was used to treat gonadal tissues of zebrafish cultured in vitro,and it was found that PrRP could directly act on gonadal tissues and promote m RNA expression of cyp11a1a,cyp17a1a and cyp19a1a in gonadal tissues.