Overexpression Of IGF3in Gonad And Characterization Of Its Possible Binding Protein In Nile Tilapia

The Insulin-like Growth Factor system(IGF system) is an evolutionarily conserved signaling pathway, which includes the ligands, their corresponding receptors and binding proteins, and it is involved in the regulation of cell growth, survival, differentiation, migration and proliferation. IGF system consists of two ligands (IGF1, IGF2), two receptors (IGF1R, IGF2R) and six binding proteins (IGF binding proteins, IGFBP1-6) in mammals. The biological effects of the IGF system are mediated primarily by the interaction of IGF ligand with its receptor modulated through IGFBPs. The binding protein can extend the life cycle of the ligands through endocrine, paracrine or autocrine manner to regulate the biological functions of the ligand. Previous studies from our group have shown that a novel IGF3, which is distinct from the conventional IGF1and IGF2, is expressed exclusively in fish gonads, thus implying its possible roles in gonad development. Prilimilary studies on IGF3in tilapia (Oreochromis niloticus) and zebrafish(Danio rerio) supported the notion. Here in this study, we investigated its function by overexpression of IGF3in gonad and explored its possible binding protein by characterization of all IGFBPs in the Nile tilapia.The overexpression vector of pIRES-hrGFP-1a-IGF3was constructed and microinjected into XX fertilized eggs at one cell stage. Six positive transgenic fish were obtained from83injected fish by genomic PCR with the vector specific primers at120day after hatching (dah). The transgenic positive rate is7.2%. It was further identified by tracing the green fluorescent protein in gonad under the fluorescence stereo-microscope. Proliferation of granulosa-like cells and synchronized germ cell division similar to early spermatogenesis were observed in ovary with IGF3overexpression by histological analysis.All mammals have6IGFBPs. Theoretically, teleosts should have12IGFBPs because of the fish specific genome duplication. However, only11IGFBPs named IGFBPla,1b,2a,2b,3a,3b,4,5a,5b,6a and6b were isolated in Nile tilapia. Phylogenetic analysis further showed that only one IGFBP4existed in all teleosts examined. One paralog of IGFBP4might have lost before the divergence of major groups of modern teleosts. The gene structure analysis revealed that all IGFBPs consist of four exons. Sequences alignment revealed that all IGFBPs have3functional domains, the highly conserved N-and C-terminal domains and poorly conserved L domain. IGFBP2a, IGFBP3a, IGFBP3b, IGFBP4, IGFBP5a and IGFBP5b were found to be expressed in gonad by RT-PCR and transcriptome analysis of tilapia gonads. Ontogenic expression analysis of the above mentioned IGFBPs in gonad were performed by Real-Time PCR. IGFBP3b and IGFBP5a showed similar expression pattern with IGF3. The expression level of IGFBP2a at10,20dah and IGFBP3a at20dah, IGFBP4at70dah have a significant difference between XX and XYwith significantly higher expression in XY gonads than that of XX. In addition, the gonadal transcriptomtic analysis further confirmed the above results. ISH (in situ hybridization) results showed that both IGFBP3b and IGFBP5a were co-expressed with IGF3in the Leydig cell in the testis. IGFBP3a was shown to be expressed in the somatic cells in the testis. IGFBP4was expressed in oocytes and interstitial cells in ovary, as well as in the Leydig cells in testis. In addition, IGFBP5b was detected in Leydig cells, while IGFBP2a expression was detected in spermatids.In summary, IGF3overexpression led to the abnormality of ovary, suggesting that IGF3might play an important role in the growth and development of ovary. We have cloned11IGFBPs in the Nile tilapia, of which IGFBP2a, IGFBP3a, IGFBP3b, IGFBP4, IGFBP5a, IGFBP5b were expressed in the gonads. These results suggested that these genes might play an important role in gonadal growth and development. Taken together, transcriptomtic analysis, expression checked by gonadal ontogentic and in situ hybridization, and of IGF3and binding proteins suggested that IGFBP3b or IGFBP5a may be the possible binding protein of IGF3.