Expression, Transcriptional Regulation And Function Of Insulin-like Growth Factor Acid-Labile Subunit (Igfals) In Zebrafish

IGFs play important roles in regulating development, growth and reproduction. Insulin-like growth factor acid-labile subunit (IGFALS), one of the important members of IGF system, forms ternary complex with IGFs and IGFBP3/5 and significantly increases the half-lives of both IGFs and IGFBP3/5 in serum and maintains a circulation pool of these molecules. Until now, almost all reports about IGFALS has been limited to mammals. Little is learned about IGFALS in non-mammal vetebrates. In order to elucidate the role of IGFALS in lower vertebrates, we employed molecular developmental biological techniques, such as PCR, RT-PCR, Q-PCR, RACE, western blot, in situ hybridization, microinjection and overexpression to explore developmental and physiological actions of IGFALS in zebrafish.Zebrafish igfals gene was located in chromosome 3 with a whole sequence of 3694bp, including two exons (58bp and 3466bp) and one intron (170bp). The open reading frame, which coded 614 amino acids (20 kinds) was limited in the second exon with a sequence of 1845bp. The leucine was the richest amino acid in the sequence with a ratio of 20.5%. First 19 amino acids in the N-terminal were signal peptides. There were 19 leucine rich repeats (LRR) in mature IGFASL peptide and every LRR contained 24 amino acids. The LRR was showed as xLxxLxLxxNxLxxLxxxxFxxLx (L, N, F represent leucine, asparagine and phenylalanine respectively in most time). IGFALS contains 11 cysteine residues and 5 N-linked glycosylation sites. The secondary structure was formed by extension chain,β-turn, random coil and a-helix, and the latter (50.98%) was the richest type. The tertiary structure of IGFALS was just like a horse hoof. The electrostatic potential was negative in C-terminal and N-terminal and positive in the surface of LRR. The difference in amino acids of IGFALS between zebrafish and other animals was 43.9-85.8%. In phylogenetic tree analysis with amino acids of IGFALS, zebrafish was close to Xenopus laveis.A 2910bp 5'flanking sequence of igfals was cloned from zebrafish. It contained double TATA-boxes, CAAT-boxes and GC-boxes. Two hundred and fifteen potential sites identified in the 5'flanking sequence can be binded by 54 transcription factors. GAS2 instead of GAS1 was the responding element to GH in zebrafish igfals. Transcription activities of zebrafish igfals promoter were significantly improved when sequence between-2041 to-2910 were added to vector in zebrafish embryoes and HepG2 cells. EGFP was first observed in the surface cells in 19hpf zebrafish embryos, which were injected with pEGFP enhancer vector including igfals promoter.In adult zebrafish, igfals mRNA expressed in many tissues and mainly expressed in liver tissue, modestly expressed in intestine, spleen, kidney, muscle and gill, and weakly expressed in eyes, brain and heart. Interestingly, the level of igfals was significant different between male and female gonad tissue. The expression patterns of igfals and igfbp5 between male and female zebrafish were similar in a reproduction cycle. However the expression patterns of igfs and igfbp3 was quite different between male and female. Expressions in ovary were significantly affected by photoperiod in female in spawning, but photoperiod had no effects on these genes expressions of female out of spawning cycle. Weak igfals mRNA could be detected in 6hpf embryoes, and then igfals mRNA gradually increased, but not significantly different between 24 hpf and 4dpf embryos. The level of igfals mRNA was obviously increased in 5dpf embryoes.Overexpression of igfals caused defects in midline, notochord and somite development of zebrafish embryos. Expressions of igf2a,igf2b,igfbp3,gh and ghr mRNA were significantly increased by overexpression of igfals, but expressions of igf1ra,igf1rb,igfbp5 and gh mRNA were unaffected. Western blot analysis indicated a marked increase in the level of phosphorylated JAK2 in igfals overexpression zebrafish embryoes, but the level of phosphorylated Akt was not significantly changed.In a spawning cycle, the igfals mRNA levels from 3-5d female zebrafish after ovulation were higher than other days in ovary.igfals mRNA was exclusively detected in the follicle cells. Overexpression of igfals could significantly increased the number of GVBD follicles and igfals potently promotes maturation of follicles. The expression patterns of igfs and igfbps were quite different from igfals in ovary. Level of igfals mRNA of female was not influenced by sex pheromone from male zebrafish, but igf2b and igfbp3 mRNA levels were negatively regulated by sex pheromone from male zebrafish. In addition, photoperiod could also affect expression of igfals in zebrafish ovaries. DES, DHP, hCG significantly contributed to increase ovary igfals mRNA expression with a dose-dependent manner, but no obviously impact was observed in ovary treated with E2 on igfals mRNA expression. Injection igfals vector and then treatment with DES, hCG and DHP can obviously enhanced maturation of follicle cells.Fasting strikingly increased expression of igfals and igfbp3 in zebrafish liver, but have no significant effects on igf1. After refeeding 3 weeks, levels of igfals and igfbp3 mRNA were reduced to control level. Intraperitoneal injection of GH to adult zebrafish influenced expression of igfals, igfl and igfbp3 in a similar manner. Their expressions were decreased at first few days, but increased to the level of control gradually. After intraperitoneal injection of E2, igfals and igfbp3 mRNA expression were similiarly changed. Levels of liver igfals and igfbp3 mRNA were significantly lower than that of control at early days, and then significantly higher than that of control. At last, their expressions went to normal levels. However, E2 influenced igfl mRNA expression in a different manner.Not only in gene and amino acid sequence, but in response to hormones, zebrafish igfals is quite different from mammals, which suggested that the function of igfals in zebrafish may be different from mammals. IGFALS could play more complicated roles than our predictation in the development, growth and reproduction.