Molecular identification of Igf3 and its roles in grass carp (Ctenopharyngodon idella)

Abstract

As a new member of the Insulin-like growth factors (IGFs) family of proteins, Igf3 has been found to be involved in fish reproduction. To evaluate the functions of Igf3 in grass carp, the igf3 gene was isolated from the gonads via RT-PCR (reverse transcription polymerase chain reaction). The ORF of grass carp igf3 was found to be 606 bp long, encoding 201 amino acids. The grass carp Igf3 precursor contains five domains, termed B-C-A-D-E. Tissues distribution analysis showed that igf3 was widely expressed in multiple tissues, and is highly expressed in the gonads, cerebellum and hypothalamus of grass carp. The oral glucose tolerance test (OGTT) showed that igf3 expression levels increased in the hypothalamus, hepatopancreas and muscle after glucose treatment. The fasting/refeeding showed that igf3 expression in hepatopancreas and muscle were markedly inhibited in the fasting group and were significantly increased in refeeding group. A recombinant grass carp Igf3 protein was obtained using a prokaryotic expression system. The function of this recombinant Igf3 were assessed in vitro and in vivo experiments. The expression of igf1, igf2b, and ghr was significantly increased, and igf2a expression was markedly decreased by Igf3 treatment in vitro and in vivo. The expression of igf3 and igf1r was significantly increased by Igf3 treatment in vitro. The expression of igfbp1 decreased and the expression of igfbp2, igfbp3, akt and pi3k increased after Igf3 treatment in vivo and in vitro. The expression of igfbp5a and igfbp5b were also increased by Igf3 treatment in vitro. The effects of Igf3 treatment on the expression of igf1, igf2, and ghr were relieved after treatment with the PI3K inhibitor wortmannin in grass carp primary hepatocytes. To our knowledge, our study was the first to reveal that Igf3 regulates the expression of growth-related genes in teleosts.