| The gonadal development of fish is a complex process,which is regulated by the brain/hypothalamus-pituitary-gonad(HPG)axis,including gonadal histogenesis and germ cells maturation.Insulin-like growth factors(IGFs)are key factors that regulate the growth and reproduction axis,they are a kind of single-chain polypeptide with insulin-like metabolism and mitotic function.IGFs consist of three ligands(igfl,igf2,and most recently,igf3),two receptors(igf1r,igf2r),and six IGF-binding proteins(igfbp1-igfbp6).IGFs have many physiological functions such as regulating cell metabolism,promoting cell growth,differentiation and division,inhibiting cell death,regulating osmotic pressure,etc.They are important regulators of animal growth and development,and mediate growth hormone as a major growth-promoting factor.Since the insulin-like growth factor 3(igf3)gene was recently discovered in fish ovary,its function in the gonads has received much attention,and the perspective of the fish GH-IGFs axis has shifted from growth to reproduction.Currently,igf3 was found only in a few fish and amphibians,and highly expressed in gonadal tissues.Studies have shown that,igf3 plays an important role in the proliferation and differentiation of spermatogonia,spermatogenesis,oocyte maturation and ovulation in fish.Nevertheless,although there are some relevant studies on igf3,the function behind its regulation in fish gonadal development remains unclear.Thus the function of igf3 on gonad will become an important research field.The common carp(Cyprinus carpio)is the dominant fish in freshwater aquaculture in China,and it is also one of the model organisms used to study the endocrine regulation.The FFRC strain common carp,Cyprinus carpio,is a new aquaculture variety derived from the original parents Jian carp(C.carpio var.jian)and Huanghe carp(C.carpio haematopterus).It is a new species of freshwater aquaculture fish promoted by the Ministry of Agriculture in the 12th Five-Year Plan.It has been popularized in most areas of China and plays an important role in aquaculture.It has become one of the important ways to increase agricultural efficiency and farmers'income.After further breeding,a new variety of FFRC No.2 strain common carp was obtained in 2017 with growth rate improved again.However,the improvement of growth traits is only an appearance,and the molecular mechanism involved is not very clear.In this study,FFRC No.2 strain common carp was used for the study of the role of igf3 gene in C.carpio gonadal development.In this study,the full-length cDNA of igf3 was obtained by RACE,its tissue expression and subcellular localization in gonad was analyzed.A living model of igf3 knockdown C.carpio was established via double-stranded RNA(dsRNA)injection,and then a high-throughput RNA sequencing(RNA-seq)was employed to profile the primordial gonad transcriptome of igf3 knockdown and control C.carpio.The aims of the experiment were to discover and characterize lncRNAs in carp gonad tissue and identify key genes,lncRNAs,and pathways that are associated with igf3.Aprokaryotic expression system of igf3 was constructed and its recombinant protein was expressed and purified.The GST Pull-down and mass spectrometry were used to study the interaction between Igf3 protein and upstream or downstream proteins,then the Igf3 recombinant protein was verified in vivo.The main results are as follows:1.In this study,two igf3 subtypes were isolated from C.carpio,the sequence analysis,tissue expression and location analysis were performed.Igf3 mRNA was mainly expressed in the gonads.In the gonad development stage,igf3a mRNA expression was highest in the maturity and recession stage of the ovary,the recession stage of the testis,igf3b was highest in the recession and fully mature stages of ovary and testis both.Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes,no signal was detected in the ovary.The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids.17?-Ethinylestradiol treatment increased igf3 mRNA expression in both ovary and testis.2.The dsRNA of igf3 in C.carpio was successfully synthesized,and the optimal injection position,injection concentration and duration of effects were explored.Finally,an igf3 knockdown common carp(Cyprinus carpio)model was established by RNA interference.After 60 days of continuous interference experiment,significant effect was not found on the growth,but was found on the development of ovary and testis of juvenile C.carpio.The interference affected the secretion of gonadotropin and sex steroid hormones,significantly reduced the expression of IGFs in HPG axis.3.In this study,an igf3 knockdown common carp(Cyprinus carpio)model was established by RNA interference.Then RNA sequencing of transcriptome in C.carpio after igf3 knockdown was performed.A total of 327,169,410 and 306,305,018 clean reads were identified from control and igf3-dsRNA interference group,respectively.After a stringent filtering,RNA-seq yielded 14199 lncRNA and 106932 mRNA transcripts with 124 and 353 differentially expressed lncRNAs and mRNAs.Gene Ontology(GO)analysis revealed these genes were associated with intracellular estrogen/androgen receptor signaling pathway and male/female gonad development.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis indicated that these genes were related to three specific signaling pathways,i.e.Wnt,PI3K-Akt and TGF-? signaling pathway.These three signaling pathways including the same differentially expressed genes,which may play a role of connector,suggest that igf3 may play its role through IGF/Wnt/PI3K-Akt/TGF-? signal pathway.LncRNA-mRNA co-expression network showed that,igf3 knockdown up-regulated expression of lncRNA MSTRG.42108,subsequently,induced expression of cyclin-dependent kinase,Tbx18,Fgfr2,serine/threonine protein kinase gene family etc.,and then affected gonadal cells signal transduction pathway,cell cycle,differentiation,sertoli cell and germ cell differentiation,and ultimately affected gonadal development.The result suggested that lncRNA MSTRG.42108 might be a negative modulator of igf3 and then plays an important role in the gonadal development after igf3 knockdown.4.One prokaryotic expression system of igf3 was successfully constructed.The protein was low expressed in the supernatant but high expressed in the inclusion body after induced by IPTG.The purified recombinant protein purity was about 80%and verified by Western Blot.The GST Pull-down and mass spectrometry were used to study the interaction between Igf3 protein and upstream or downstream proteins.A total of 84 proteins were identified,GO and KEGG analysis indicated that,Igf3 protein may play its role in cells through PI3K-Akt-mTOR-AMPK signaling pathway,and involves multiple organelles,which can regulate cell growth,development and cell metabolism.5.Intraperitoneal injection with Igf3 recombinant protein into C.carpio could decrease the expression of igf3 in the gonad,and continuous reduction to 48 or 72h to the lowest value and then began to rise.Twice-a-week for 60 consecutive days of Igf3 recombinant protein injection could significantly inhibited the development of ovary and the expression of IGFs in HPG axis,and the growth rate of C.carpio was significantly increased in the first two weeks. |
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