| In order to clarify the underlying growth regulation mechanisms of cultured starryflounder Platichthys stellatus, the full-length cDNAs of key endocrine factors alongGH/IGFs axis growth hormone (GH), insulin-like factors (IGF-I and IGF-II) wereisolated using RACE methods and their spatial and temporal expression patterns ofduring embryonic development, early growth stage and ovarian maturation cycle wereinvestigated by quantitative Real-time PCR (qPCR) method. The possible roles of GH,IGF-I and IGF-II in early life stages and ovarian maturation cycle were discussed bycombining qPCR analysis, radio immunoassay and histological observation.Furthermore, a in vitro prokaryotic expression systems of GH, IGF-I and IGF-II wereconstructed and biologically active recombinant proteins were obtained. These resultsare helpful for better understanding of the role of GH/IGFs axis in regulation of growthand reproduction in starry flounder. The main results are listed as follows:1. Molecular cloning and spatial expression patterns of growth hormone,insulin-like growth factor-I and-II in starry flounder Platichthys stellatusThe full-length cDNAs encoding GH, IGF-I and IGF-II were firstly isolated from starryflounder Platichthys stellatus pituitary and liver respectively using RACE methods. TheGH cDNA is957bp in length and encoding204amino acids. The predicted GH maturepeptide is180amino acids consisting of4conserved Cysteine residues and1N-linkedglycosylation site. The full-length cDNAs encoding IGF-I and IGF-II are1268bpencoding185amino acids and899bp encoding215amino acids, respectively. Themature peptides of IGF-I and IGF-II are141amino acids and168amino acidsrespectively containing B-C-A-D domains by cleaving the signal peptide and E domain,and consist of six highly conserved Cys residues which formed three disulfide bonds.The spatial expression patterns of GH, IGF-I and IGF-II mRNA in tissues of female and male starry flounder were determined by qPCR method. The results showed that GHmRNA exhibited highest expression level in pituitary from both sexes, and relativelylower expression levels were found in brain, gonad, stomach and muscle. The GHmRNA expression levels in female stomach and muscle were significantly higher thanmale(P＜0.05). IGF-I mRNA expression was detected in all the tissues examined, withliver being the highest expression organ for both sexes. The liver, gonad, gill, heart, andkidney IGF-I mRNA expression level in female was significantly higher than male, andvice versa in pituitary (P＜0.05). In contrast to IGF-I mRNA expression pattern, IGF-IImRNA showed high expression level in liver, brain and gill of males, whereas theexpression levels was lower in female liver and other tested organs. The IGF-II mRNAexpression level in pituitary, gonad, liver, gill, heart, head kidney, kidney, spleen,stomach and intestine was significantly higher in females than that of male, and viceversa in brain (P＜0.05). The spatial and sex-specific expression patterns of GH, IGF-Iand IGF-II mRNAs indicate that GH, IGF-I and IGF-II may be involved in the sexuallydimorphic growth pattern of starry flounder.2. Differential expression of GH, IGF-I and IGF-II mRNAs during early life stagesof starry flounderGH mRNA was not detected in unfertilized eggs and embryos, and it was firstlydetected in1d larvae and its expression level increased significantly with the growth（P＜0.05）. However, IGF-I and IGF-II mRNAs were detected in all the tested early lifestages including unfertilized egg, embryos, larvae and juveniles with differentexpression patterns. IGF-I mRNA showed high expression levels from unfertilized eggto low blastula stage which followed by a significant decrease at early gastrula stage（P＜0.05）, and then maintained lower levels from early gastrula stage to newly hatchedlarvae, but at post-embryonic stages it increased with growth and peaked at60d juvenilestage （P＜0.05）. IGF-II mRNA expression level exhibited low expression levels fromunfertilized egg to high blastula stage, and a surge at low blastula stage followed by agradually decrease （P＜0.05）from early gastrula stage to newly hatched larvae wasobserved. Then IGF-II mRNA expression level increased with larval developmentbefore18days（P＜0.05） and decreased significantly from18day-old larval stage to60day-old juvenile stage（P＜0.05）. These results showed that GH, IGF-I and IGF-IImight play important roles in starry flounder early growth and development. 3. The possible roles of GH, IGF-I and IGF-II during ovarian maturation cycle ofstarry flounderThe annual ovarian maturation cycle of starry flounder is divided into five stagesincluding Ⅱ、Ⅲ、Ⅳ、Ⅴand Ⅵ by using histological section observation. Plasmahormone expression levels of female brooders during annual maturation cycle weredetermined by radio immunoassay. Results showed that plasma GH leveland pituitaryGH mRNA expression level were lower at ovary maturation stage Ⅲ、Ⅳ、Ⅴ of starryflounder（P＜0.05）. However, plasma IGF-I level and liver IGF-I mRNA levelincreased（P＜0.05）with the ovary maturation course and peaked at stage Ⅴ followedby significant decline after ovulation（P＜0.05）. Moreover, IGF-I and IGF-II mRNAexpression levels both increased with ovary maturation（P＜0.05）and showed thehighest levels at stage Ⅵ. Plasma E2and T levels peaked at stage Ⅳ and Ⅴ,respectively. These results implied that GH, IGF-I and IGF-II were involved in ovarianmaturation through endocrine, paracrine and autocrine pathways.4. Prokaryotic expression and bioactivity analysis of recombinant GH, IGF-I andIGF-II from starry flounderThe mature peptide domains of GH, IGF-I and IGF-II genes were cloned from starryflounder. The recombinant plasmids constructed with the matured peptides fragmentsand the prokaryotic expression vector pET-28a were transformed into E. coli BL21(DE3) cells which could be induced by IPTG to produce special fusion polypeptidescontaining6×His tags at the N-terminus. The recombinant GH, IGF-I and IGF-IIproteins in form of inclusion bodies proximately account for45.2%,39.8%and42.7%of the whole bacterial protein respectively under the best induce conditions includingtime, temperature and IPTG concentration, which are confirmed by the SDS-PAGEanalysis and the SigmaScan software. Western blotting analysis indicated the threefusion proteins all had the antigenicity to6×His antibody with proper protein molecularweights. The IPTG-induced bacterial precipitation was denaturalize using6mol/Lguanidine HCl, purified using Ni-NTA affinity chromatography and annealed bygradient dialysis in urea, then the purified recombinant GH, IGF-I and IGF-II proteinwith molecular weight of24.9kD,12.1kD and11.4kD respectively were obtained.Moreover both the recombinant IGF-I and IGF-II can promote the proliferation of thehuman embryonic kidney cell HEK293T at low concentration levels and inhibit proliferation at high concentration levels. However, the recombinant GH only showedinhibition effect on proliferation of the human embryonic kidney cell HEK293T at highconcentration. The successful gain of starry flounder GH, IGF-I and IGF-II recombinantproteins in vitro will facilitate study on teleost GH/IGFs physiological functions.Furthermore, results would be helpful for better understanding of underlyingmechanisms of growth regulation and production of high effective growth promotionadditive used in starry flounder farming industry. |
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