The Cloning And Expression Patterns Analysis Of Igf3 In Medaka

Insulin-like growth factors(IGFs)can regulate the growth,development,maturation and metabolism of vertebrates,as well as participate in the regulation of cell survival and differentiation.Migration and the process of cell proliferation.The IGFs system consists of three ligands(IGF1 and IGF2),as well as recently discovered IGF3,two receptors(IGF1R and IGF2R)and six highly binding proteins IGFBP1-6.The IGF ligand needs to bind to the binding protein through the receptor,regulating a series of biological processes in the organism by paracrine,autocrine and endocrine mechanisms.IGF1 and IGF2 have been well studied in a variety of teleost fish,and our laboratory have been reported them.The expression patterns of medaka IGF1 in the gonads has well studied,and the function of IGF2 in the development of the embryos is also reported.However,reports of the novel ligand IGF3 is limited to a few species of fish.These related studies have shown that gonadal specifical IGF3 plays an important role in growth and reproduction of teleost fish.In current study,IGF3 is involved in growth,development and oocyte maturation in zebrafish.Similarly,IGF3 can regulate the proliferation and differentiation of spermatogonia,and spermatogenesis and maturity.Other fish-related IGF3 studies are lacked.Oryzias latipes is a small freshwater fish which living in rice fields.It has a series of characteristics,such as embryonic transparency,small individual size,easy reproduction and complete genome sequence.In addition,there are many mutants of barley,including diploid embryonic stem cell lines and haploid stem cells have been established.Moreover,medaka is the only individual with male and female sex determining genes-Dmy and Folx3.In previous studies,many germ cell marker genes have been reported,such as dazl,dnd,piwi,and vasa.And medaka has established transgenic lines,such as nanos and vasa.In addition,many leading technologies such as Morpholino,Talen,and Cas9 have been successfully established in the medaka,which providing a good technical platform for gene functional research of.Now,igf1 and igf2 have been studied,including expression patterns and embryo function verification in the medaka.Gonad-specific growth factor igf3 is only reported in tilapia,zebrafish,organe-spotted grouper and common carp.Because of the unique gonad expression of igf3,it plays a more important roles in gonadal development than other igfs ligands.The expression patterns and function of igf3 become more important.In medaka,the expression patterns and functions of igf1 and igf2 have been studied,respectively.In this paper,the sequence of igf3 was cloned and sequence alignment,protein structure and phylogenetic tree analysis were performed.The expression levels in tissues and embryos were detected by RT-PCR,then the temporal and spatial expression patterns of gonads and embryos was analyzed by in situ hybridization,the RNA levels of igf3 in different stages of oocytes was detected by Q-PCR.Finally,expression patterns of igf3,igf1 and germ cell marker gene vasa were compared during gonadal development by fluorescence in situ hybridization.In this study,the open reading frame of igf3 is 501 bp and encoding 166 amino acids.From protein sequence analysis and phylogenetic tree analysis,the structure of igf3 was similar to igf1 and igf2,but the analysis on the phylogenetic tree was not in the same evolutionary branch,which further testifying that igf3 is a new member of the igf family.By RT-PCR and Q-PCR analyzed shown that igf3 was restricted to the gonads of both sexes and the expression of testis was much higher than that of the ovary.In the embryonic development stage.In the embryonic development stage,igf3 was persistently occurred in all embryogenesis ranging from 2 cell stage to fry form stage.In situ hybridization of the whole embryo showed that the signal of igf3 existed in the classification sphere of 128 cells in the early stage of embryonic development,and the signal of igf3 was found in the gonads which developed for seven days.In the ovary,chemical in situ hybridization revealed that the igf3 signal is expressed in the oocytes from stages I-V,as well as high expression in the somatic cells of the oocytes from IV-V stage,such as granulosa cells and theca cells.In order to compare the expression level of igf3 in oocytes,the level of igf3 was detected in oocytes from I-V stages by Q-PCR.the RNA expression level of igf3 showed an increasing trend,and the high expression of igf3 in the oocytes stage V.In the testis,the signal of igf3 was detected in Leydig cells and Sertoli cells.To precisely identify the the subcellular localization of igf3,the expression patterns of igf3 and the germ cell marker gene vasa by fluorescence in situ hybridization were compared.We shown that igf3 was found in oogonia compared with vasa,and other expression patterns in ovary and testis were consistent with chemical in situ hybridization.To further compare the different expression patterns of igf3 and igf1 during gonadal development,two-color fluorescence in situ hybridization was performed,igf3 was present in the oogonia but igf1 was absent.The signal of igf1 was weak in the oocytes V stages,and the signal of igf3 was stronger than of igf1 in the ovary.Igf3 and igf1 was expressed mutually in granulosa cells and theca cells in the oocytes from stage IV-V.Interestingly,igf3 and igf1 could identify distinct types of theca cells in the ovary.In the testis,the expression patterns of igf3 and igf1 in Leydig cells and Sertoli cells were similar.