Molecular Cloning And Gene Expression Of Insulin-like Growth Factors 1 And 2 And Regulation By Estradiol-17β In Spotted Scat (Scatophagus Argus)

Insulin-like growth factor(IGF)is a peptide hormone that secretes from liver,to regulate the proliferation and mitochysis of cell,that play an important role in animals.The synthesis and secretion of IGF influenced by variety of factors,Estrogen also stimulate the IGF product by estrogen receptors(ERs)in mammals.In our study,the partial c DNA sequences contain ORF of two IGF subtypes(IGF-1 and IGF-2)c DNA were cloned in S.argus.The expression pattern of IGF in various tissues was next characterized using RT-PCR(pituitary,stomach,hypothalamus,gonad,kidney,liver,intestines,spleen,gill,heart and muscle).The expression pattern of IGF during embryogenesis(four cell stage,blastula stage,archenteron stage,neurula stage,eye pouch formation,end tooth period,crystal formation,muscle effector phase,incubation period)and ontogenesis(Phase II with oocytes at perinuclear stage,phase III with oocytes forming the vitelline vesicle,phase IV with oocytes with lipidic;growth phase of spermatocytes,maturation phase of spermatocytes,initial appearance of spermatoblast)was next characterized using q RT-PCR.Effects of estradiol(E2)and estrogen receptor antagonists(Fulvestrent,MPP and PHTPP)(0.1,1 and 10μM)on IGFs,ERs,GHRs mRNA expressions in livers were examined using quantitative real-time PCR(q PCR).The main results were as follows:1.Molecular cloning and characterization of IGFs in S.argus.Two subtypes of IGFs were cloned in S.argus.The ORF of IGF-1 was 561 bp which encoded 186 amino acids.IGF-1 Contains 15 binding protein binding sites,four poly real nucleic acid binding sites,a signal peptide region active peptide and a family active peptide domain.The ORF of IGF-2 was 648 bp which encoded 215 amino acids.IGF-2Contains 14 binding protein binding sites,5 poly real nucleic acid binding site,a signal peptide regional active peptide and a family active peptide domain.Homology comparison revealed that S.argus IGF-1 and IGF-2 were the most conventional kind.The phylogenetic analysis also revealed two IGFs of S.argus to closely related to IGFs of closely related Japanese sea perch and Japanese sea perch share identify of 95% and 91% resrech the IGF-1 and IGF-2 of S.argus2.Temporal and spatial expression of IGFs in S.argus.In all the tissues of S.argus examined,the IGF-1 and IGF-2 were present of all tissues in both male and female.Significant greater IGF-1 mRNA expression was observed in the liver of S.argus;The IGF-2 mRNA was easily detected in most of the examined tissues,with abundant expression in the liver.Our results suggested that IGF-1 closely related to the growth of fish;IGF-2 may play a wider range functions.IGF-1 mRNAs were detected in nine embryonic stages by real-time PCR with obviously different expression patterns,IGF-1 mRNA level remained up-regulated from fertilized egg to newly hatched larva,our result suggested that IGF-1 is very important during early embryonic development in fish.In contrast to IGF-1,IGF-2 mRNA level was low from fertilized eggn to blastula,with a surge at archenteron stage and neurula stage,followed by a gradual decrease from eye pouch formation stage.IGF-2 might simultaneously function and interact in the regulation of embryo,nervus and eye development.During ontogenesis,IGF-1 was simultaneously up-regulated with maturation of female ovary and male testis in livers,while IGF-2 were contrary trend in female and male.Results suggested that IGF might involve in gonad development.3.Effects of E2 on the expression of GH/IGF in S.argusIn vivo,analysis E2 significantly promoted the expression of GH after 6h treatmeat in female pituitary,with no effect on male.E2 significantly promotes the expression of IGF-1after 6h treatmeat in female,and reduced.IGF-2.However the expression of IGF-1 and IGF-2 were both promoted in male.4.Effects of E2 on IGFs in S.argus in vitrothe expression of IGF-1 and ERα mRNA were promoted significantly(P<0.05)at 6hours incubated with E2(0.1、1and 10μM)in vitro in female,IGF-2 and ERβ1 were reduced,while ERβ2 and GHRs were not affected.the expression of IGFs and ERα mRNA were up-regulated significantly(P<0.05)in a dose-dependent manner at 6 hours incubation with E2(dose from 0.1 to 10μM),the expression of ERβ1 was inhibited,while ERβ2 and GHRs were not affected.5、Effects of estrogen receptor antagonists on the expression of IGFs、ERs and GHRs in S.argusEstrogen receptor broad-spectrum antagonist Fulvestrent(0.01,0.1 and 1μM)significantly inhibited the expression of IGFs and ERs with no effect on GHRs in both female and male.Estrogen receptor alpha antagonist MPP suppressed the expression of IGF-1and ERα and promoted the expression of IGF-2 and ERβ1 in female,while IGFs and ERα were reduced and promoted the expression of ERβ1 in male;However,estrogenreceptor beta antagonist PHTPP inhibited the expression of IGF-1 and ERβ,and promoted the expression of ERα in female.Up-regulation of IGF-2 and ERα,inhibited the expression of IGF-1 and ERβ in male.These results indicate that IGF-1 and IGF-2 play important role in the embryo and gonad development in S.argus.Estrogens regulate the expression of GH and IGFs mRNA.E2 regulate the expression of IGF-1 via both estrogen receptor α and β in S.argus.E2 regulates the expression of IGF-2 via ERβ1 and ERα in female and male,respectively.