The Role And Regulation Mechanism Of Somatostatin In The Puberty Development Of Zebrafish

SS was first isolated from the hypothalamus of sheep as a 14-amino acid peptide and found to inhibit the release of growth hormone（GH）from the pituitary gland.Somatostatin（SST）also known as growth hormone inhibiting hormone encoded by six genes（SST1-SST6）in zebrafish originating multiple bioactive isoforms of 14 and 28 amino acids through alternative cleavage of precursor preproproteins.These isoforms are produced in multiple tissues and bind to five receptors（SSTR1-SSTR5）resulting in a multitude of functions.Somatostatin（ss）plays important roles in growth,metabolism,development,reproduction and behavior.Puberty is a key developmental stage that enables individuals to produce viable germ cells.For puberty to occur individuals have to reach a certain size and body fat,and somatic signals acting on the brain mediate this process.Once the fish develops puberty,the individual’s growth slows down and the energy will supply the gonads,which is a bad signal for the fish farming industry,especially the precocious puberty will bring huge economic losses to the fish farming industry.For this,the project will use zebrafish as a model species to characterize the phenotyopes of the SST mutants produced using the CRISPR/Cas9 system,and determine the gene networks that are affected SST gene ablation in the mutant lines with a modified reproduction phenotype.It is expected that with the new knowledge strategies to minimize precocious puberty in aquaculture are devised.In order to avoid the economic loss caused by sexual precocity to the aquaculture industry,it can also provide scientific and effective management methods for aquaculture and promote the development of aquaculture.Firstly,the expression of somatostatin gene ss1-ss6 in zebrafish tissues was analyzed by qPCR.The ss1 and ss3 gene are mostly expressed in brain and liver.The ss2 gene is mostly expressed in brain and eye.The ss4 gene is mostly expressed in liver.The ss5 gene is mostly expressed in testis.The ss6 gene is mostly expressed in brain.To further determine whether the somatostatin gene has the specificity of spatiotemporal expression,we also used qPCR to analyze gene expression at various time points（24hpf,48hpf,72hpf,96hpf,120hpf）during zebrafish embryonic and pubertal development（25dpf,30dpf,35dpf,40dpf,45dpf,50dpf,55dpf,60dpf）.During the embryonic period,the expression patterns of ss1 and ss2 genes showed the same trend.The gene expression at 24hpf-72hpf increased,and 72hpf began to decline until 96hpf began to rise again.The ss3 gene is in a state of fluctuation during 24hpf-120hpf,and the ss4 gene has been on an upward trend,with a slight downward adjustment after 72hpf.The expression of ss5 gene decreased slightly at 24hpf-48hpf,at 72hpf reached the highest level and decreased after 72hpf,then began to rise after 96hpf.The expression level of ss6 was basically unchanged at 24hpf-48hpf.After48hpf,the expression level of ss6 increased,at 72hpf reached the highest level,at 72hpf-96hpf still decrease.After 96hpf,the expression level remained basically unchanged.During the puberty,the ss2 gene showed no obvious change in the time of 25dpf-60dpf.The expression levels of ss1,ss3 and ss4 genes were lower at 25dpf to 40dpf.When reaching45dpf,the gene expression was significantly up-regulated and at 50dpf reached the highest level.After 50dpf,the expression decreased and dropped to the lowest level at 55dpf,and then stabilized.The expression of ss5 start up-regulated at 40dpf-45dpf.At 60dpf the level reach the highest.The expression of ss6 up-regulated at 45-55dpf.At 60dpf the level reach the highest.We further verified the tissue expression and time expression,and found that the 45dpf-50dpf gene up-regulated mainly caused by the liver rather than the brain.The experiment results make us more convinced that somatostatin plays an indispensable role in the initiation of zebrafish puberty,and the main site of action may be liver tissue.In order to investigate the specific functions of SSTs,we have initiated the systematic production of knockout mutant zebrafish using the CRISPR/Cas9 system to edit somatostatin and its receptor genes.At present,we have obtained ss1^-/-mutant:-7bp,ss2^-/-mutant:-5bp,ss3^-/-mutant:-7bp,ss4^-/-mutant:+43bp,and ss5^-/-mutant:-8bp,sstr2l^-/-mutant:-11bp,sstr2a^-/-mutant:+2bp,+17bp,sstr2b^-/-mutant:+4bp.In order to further study the function and mechanism,in this study,two fish lines ss3^-/-and ss4^-/-with specific expression of brain tissue and liver tissue were selected based on the existing results.We mixed the offspring produced by F₁ heterozygous introversion to eliminate the effects of environmental conditions on zebrafish.The wild type was separated from the mutant by tail-cutting and sequencing at 45 dpf.The body length and body weight of the wild type and mutant zebrafish were measured at 45dpf,50dpf,55dpf.We found that the growth of the mutant was significantly faster than the wild type counterpart both in length and weight.To further validate our hypothesis that somatostatin regulates energy metabolism and affects the initiation of zebrafish puberty,we observed his liver and gonads histologically.Through frozen and paraffin sections,we found knockout ss3,ss4 promotes lipid metabolism of liver,while knockout ss3 led to slowing of ovarian development,and knockout ss4 led to testicular growth retardation.We analyzed the expression of upstream and downstream genes of the GH-IGF-1,an important pathway of the hypothalamic-pituitary-gonadal axis of growth and gonadal development,according to the literature.The expression analysis of downstream genes revealed that gh,igf1,igf2a,igf2b,igf3 were strongly up-regulated in the ss3^-/-mutant by qPCR,especially igf1 and igf3.The expression of gh gene was significantly up-regulated,and the expression of fshb and lhb genes showed significant down-regulation;the expression of igf1,igf2a,and igf2b genes in liver tissue showed significant up-regulation,which is consistent with previous reports;the expression of igf3,amh,insl3,hsd17b3,hsd11b2,cyp11b,cyp17 genes in testis showed significantly down-regulated;the expression of igf1,igf2a,cyp19a1a genes showed significantly down-regulated and igf2b,hsd20b showed significant up-regulation in ovary.These finding provide supporting data that somatostatin plays a important role in growth and development,as well as may has a direct and indirect role in the reproduction.