In the present study, we cloned IGFBP-2 cDNA from common carp (Cyprinus carpio) liver. The 1879 bp full-length cDNA encodes 274 amino acid residues containing a putative signal peptide of 22 residues. Two IGFBP-2 transcripts with estimated sizes of 2.2 and 1.5 kb have been detected with Northern blot analysis in liver. Relatively high levels of IGFBP-2 mRNA were observed in all regions of brain, liver, pituitary, ovary and testis. Intermediate levels were observed in white muscle, thymus gland and head kidney, while in retina, heart and other tissues IGFBP-2 mRNA levels were very low. A significant level of IGFBP-2 mRNA was firstly detected at lens formation stage, and it continued to increase to the highest level at blood cycling stage, and fell to a relatively high level until hatching. The expression pattern of IGFBP-2 mRNA was similar during different stages of testis and ovary. At recrudescing stage the expression level was extremely low, but it sharply increased to a high level at matured stage, and finally brought back to the very low level at regressed stage. Hepatocytes IGFBP-2 mRNA was greatly reduced by growth hormone but increased by insulin. PD-98059 and LY-294002, the specific inhibitor of MEK and PI3K, increased IGFBP-2 mRNA expression level and completely blocked the inhibitory effect of GH. It is suggested that the MAPK and PI3 kinase-ignaling pathways were involved in the decrease of IGFBP-2 mRNA expression induced by GH in primary cultured hepatocytes.

GH secretagogue receptor (GHSR) is the receptor of ghrelin, a circulating GH-releasing and appetite-inducing hormone. In this paper, two Ghsr cDNAs, gpGhsr1a and gpGhsr1b, were identified and characterized in a teleost, the orange-spotted grouper (Epinephelus coioides). The gpGHSR1a is 1512 bp in length with an open reading frame (ORF) that encodes a protein of 383 amino acids with seven transmembrane (TM) domains, while the 1703 bp gpGHSR1b contains an ORF encoding for 303 amino acids with five TM domains. Comparison between cDNA and gene sequences showed that the two transcripts are two alternative splicing forms of a single gpGhsr gene. Tissue distribution and ontogeny of two gpGhsr mRNAs were examined by RT-PCR. The gpGHSR1a is mainly expressed in brain and pituitary gland, when compared with a more widespread expression of gpGHSR1b. During embryonic and larval development, the gpGhsr1b mRNA appears before the gpGhsr1a mRNA. Furthermore, quantitative real-time PCR performed on brain showed that both transcripts have the highest expression level in the pituitary gland. The expression level of gpGHSR1a was generally higher than that of gpGHSR1b. GHSR expressing cells were also detected widely in grouper brain by in situ hybridization, with a broader distribution than previous reports in mammals. Finally, an in vitro study showed that expression of both gpGHSR transcripts in pituitary and hypothalamus is downregulated by GH and ghrelin but not by des-acyl ghrelin, and this suggests that feedback regulation of GHSR also exists in teleostean fishes.

Giant grouper (Epinephelus lanceolatus) and orange-spotted grouper (Epinephelus coioides), which belong to Pereiformes, Serranidae, Epinephelinae, Epinephelus, are important seawater breeding fishes. But the growth ratio of Giant grouper and orange-spotted grouper are different. Studies about orange-spotted grouper on the development and reproduction had been well reported, but little on giant grouper. In this study, we used reverse transcription and rapid amplification of cDNA ends (RACE) to obtain the three full-length cDNA sequences encoding for GH and two forms of IGF-I. The GH precursor cDNA consists of 916 bp in size with an open-reading frame encoding 204 amino acid (aa), a 65 bp 5'-untranslated region and a 236 bp 3'-untranslated regions. The sequence of giant grouper GH shared 100% aa sequence homology with GH reported in orange-spotted grouper (Epinephelu coioides). Two forms of IGF-I precursor cDNA were cloned from giant grouper, one consisting of 186aa, IGF-Ia, and the other of 159aa, IGF-Ib. They shared 98.4% and 98.7% amino acid identity with IGF-I reported in orange-spotted grouper, respectively. Giant grouper IGF-I a and b had the same signal peptide and B, C, A and D domains but the different E domain. Using quantitative real-time reverse transcription-PCR strategies, Tissue distribution profile showed that GH, IGF-I mRNA signals were totally detected in pituitary, brain, liver and ovary . Interestingly, IGF-Ia mRNA level in liver was found to be more abundant than IGF-Ib. These findings will contribute to the understanding of the evolution of the GH, IGF-I and provide insight into the roles of GH and two forms of IGF-I in the growth, development and metabolism of giant grouper.

One pepsinogen C gene was cloned from the gastric mucosa of orange-spotted grouper (Epinephelus coioides) for the first time. This gene consisted of nine exons interrupted by eight introns. The nucleotide sequence with the coding region and 3-flanking region was also determined. The deduced polypeptide sequence was composed of a prosegment of 55 residues and a pepsin moiety of 332 residues. The putative substrate-binding subsites were well conserved among fish with the exception of the residue Thr292 in the S2 subsite and displayed a significantly low value of hydropathy and high flexibility. These differences may affect the catalytic function and substrate specificity. RT-PCR assay followed by Southern blot revealed that pepsinogen C was primarily distributed in the stomach, but also expressed in various tissues, including gill, intestine, pyloric ceca, esophagus and ovary. This is the first observation of pepsinogen C expression in various tissues of one species of fish. Pepsinogen C transcript was first detected at 41 dph and continuously expressed through to adult fish, coinciding with the pepsin-like enzymes activity during developmental stages. Pepsin-like enzymes activity was present in the early larva stage, increased significantly at the end of juvenile stage and remained at similar levels in young fish and adult. Northern blot analysis suggested that three forms of transcripts were expressed differently during experimental periods. Our results suggest that pepsin C possesses any other functions besides digestion.