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Fish Physiol Biochem (2013) 39:1541–1554 DOI 10.1007/s10695-013-9807-5 Insulin-like growth factor binding protein-2 (IGFBP-2) in Japanese flounder, Paralichthys olivaceus: molecular cloning, expression patterns and hormonal regulation during metamorphosis Yuntong Zhang • Junling Zhang • Zhiyi Shi Wanying Zhai • Xiaozhu Wang • Received: 14 October 2012 / Accepted: 17 May 2013 / Published online: 22 August 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract In this study, we cloned and characterized cDNA sequences of two insulin-like growth factor binding protein-2 (IGFBP-2a and IGFBP-2b) from Japanese flounder, Paralichthys olivaceus. The fulllength cDNA of IGFBP-2a is 1,046 bp long and consists an open frame (ORF) of 876 bp, a 50 untranslated region (UTR) of 125 bp and a 30 -UTR of 45 bp. IGFBP-2b is 1,067 bp, including a 50 -UTR of 53 bp, a 30 -UTR of 198 bp and an ORF of 816 bp. Real-time quantitative PCR results revealed that IGFBP-2a -2b mRNA was expressed in all detected tissues. Interestingly, the levels of IGFBP-2a mRNA in all detected tissues were higher in female than male, but IGFBP-2b was precisely the opposite. At different embryonic stages, the levels of IGFBP-2a mRNA were typically higher than IGFBP-2b. After hatching, IGFBP-2a mRNA was gradually decreased to a relatively lower level. However, the expression of IGFBP-2b mRNA was increased after hatching, including 3, 7, 10, 14, 17, 20 and 23 days posthatching (dph), and it presents a higher level until 29 (metamorphic climax), 36 (post-climax) and 41 dph Yuntong Zhang and Junling Zhang have contributed equally to this work. Y. Zhang J. Zhang Z. Shi (&) W. Zhai X. Wang Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, People’s Republic of China e-mail: [email protected] (the end of metamorphosis). In levothyroxine sodium salt (T4, the main form of thyroid hormone in animals)-treated and thiourea (TU)-treated larvae, the expressions of IGFBP-2a had not visibly changed, except in T4-treated 17 dph larvae. The expressions of IGFBP-2b mRNA were distinctly increased from 17 to 23 dph, but suddenly dropped to a lower level in and after 29 dph. However, the levels of IGFBP-2b mRNA during metamorphosis were greatly down-regulated after TU treatment. These results provided basic information for further studies on the role of IGF system in flatfish development and metamorphosis. Keywords Cloning Gene expression IGFBP-2 Paralichthys olivaceus Thyroid hormone Introduction The insulin-like growth factor (IGF) system plays a pivotal role in vertebrate growth, development, proliferation and metabolic regulation. The IGF system is comprised of IGFs (IGF-I and IGF-II), the binding proteins (IGFBP-1 to -6) and the cell surface receptors (IGF-IR and IGF-IIR). Most circulating IGFs are bound to specific high affinity IGFBPs that protect them from degradation and modulate their actions (Duan and Clemmons 1998; Jones and Clemmons 1995; Rajaram et al. 1997). In vertebrates, six IGFBPs have been cloned and characterized (Duan and Xu 2005; Hwa et al. 1999; 123 1542 Jones and Clemmons 1995). Although the overall structure is conserved and related with high degree to each other, each IGFBP has unique properties and performs the functions of potentiating or inhibition of the biological activities of IGFs (Schneider et al. 2002). Among the six IGFBPs, IGFBP-2 is the second most abundant form in serum (Clemmons 1997). Recently, they only cloned one transcript of IGFBP-2 in chicken (Schoen et al. 1995), rat (Margot et al. 1989), rainbow trout (Oncorhynchus mykiss) (Kamangar et al. 2006), red seabream (Pagus major) (Funkenstein et al. 2002), goby (Gillichthys mirabilis) (Gracey et al. 2001), and yellowtail (Seriola quinqueradiata) (Pedroso et al. 2009). However, two subtypes of IGFBP-2s have been identified in many species, such as common carp (Cyprinus carpio) (Chen et al. 2009), zebrafish (Danio rerio) (Duan et al. 1999; Zhou et al. 2008), O. Latipes, E. coioides, S. aurata, F. obscurus and T. nigroviridis. Further studies reported that two zebrafish IGFBP-2s mRNA were expressed in liver, gonad, brain and many other tissues (Duan et al. 1999; Funkenstein et al. 2002; Kamangar et al. 2006), which suggest it has autocrine/ paracrine actions on regulating IGFs. However, IGFBP-2a was only found in liver and brain; IGFBP2b was detected in liver, intestine, kidney, ovary and muscle of female zebrafish, but only in liver of male zebrafish. In addition, the expressions of two IGFBP2s demonstrated different patterns in zebrafish embryonic and larval stages. These results suggest that two IGFBP-2s may have different physiological functions on growth and development of fish. While, analyzing the differential expression of two IGFBP-2s in staged embryos and larval, and tissues of adult fish are very conducive to illustrate the physiological significance of fish growth and development. The Japanese flounder (Paralichthys olivaceus) is an important marine economic flatfish in aquaculture. It undergoes a dramatic metamorphosis during larval development by changing its body form from a symmetrical bilateral pelagic larva to an asymmetrical benthic juvenile. This biological process is principally controlled by thyroid hormone (TH). Recently, it has been revealed that the IGF system could be important for metamorphic success in Atlantic halibut (Hippoglossus hippoglossus) (Hildahl et al. 2007a, b, 2008) and Japanese flounder (P. olivaceus) (Zhang et al. 2011a, c). Recent IGFBP-2 mRNA was reduced by growth hormone (GH) but increased by fasting in 123 Fish Physiol Biochem (2013) 39:1541–1554 zebrafish (Duan et al. 1999); 17b-estradiol could inhibit IGFBP-2 mRNA expression in orange-spotted ovary (Chen et al. 2010); IGFBP-2 mRNA was reduced by GH but increased by insulin in common carp (Chen et al. 2009). In hypothyroid mammals, TH treatment slightly increases IGFBP-2 mRNA expression (Clemmons 1997). We have demonstrated that TH affects the expression of IGF-I mRNA (Zhang et al. 2011a) and IGFBP-1 mRNA (Zhai et al. 2012) in P. olivaceus. But the relationship between IGFBP-2 and thyroid hormone has not been reported, and the molecular mechanism of IGFs system modulates flatfish metamorphosis is unknown. To understand the function of IGFBP-2 in the growth, development and metamorphosis of flatfish, we cloned and characterized the full-length cDNA of two P. olivaceus IGFBP-2 subtypes, examined their expression patterns during embryonic and larval development and in adult tissues and also investigated the effects of TH and thiourea (TU) on the levels of IGFBP-2 mRNA during metamorphosis, thus helps further illustrate the functional role of IGF system in flatfish development. Materials and methods Animals and sample collection Embryos, larvae and adult fish of P. olivaceus were obtained from Beidaihe Central Experimental Station (BCES), Chinese Academy of Fishery Sciences (CAFS), Hebei, China. All the embryos and fishes were maintained in circular culture tanks supplied with seawater at 16 ± 2 °C. Adult fish at same period were separated into two groups (n = 3) based on gender. Tissues containing heart, liver, spleen, stomach, kidney, brain, gill, gonad, muscle and intestine were promptly dissected, frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Embryos and larvae were maintained under intensive-culture conditions in 300 L tanks. They (n = 3 pools, 15–30 specimens/pool) were periodically collected at 0, 0.5 h post-fertilization (hpf, fertilized egg), 9 hpf (blastocyst stage), 26 hpf (gastrula stage), 42.5 hpf (neurula stage), 48 hpf (blastopore-closing stage), 71 hpf (heart-beating stage), 75 hpf (prehatching stage), 78.5 hpf (hatching stage) and 3 days post-hatching (dph), 10, 14, 17, 20, 23, 29, 36 and Fish Physiol Biochem (2013) 39:1541–1554 41 dph, respectively. The metamorphic stages of P. olivaceus were defined as Pre-metamorphosis, the stage prior to the start of eye migration (Stage D); the right eye just start to migrate (Stage E); Pro-metamorphosis, the right eye moved toward the dorsal margin, but still could not be seen from the left/ocular side (Stage F); Climax metamorphosis, the right eye became visible from the ocular side (Stage G); Postmetamorphosis, the right eye located on the dorsal margin (Stage H); both eyes were completely located on the left side of the head (Stage I) (Minami 1982; Miwa and Inui 1987; Miwa et al. 1988). According to this definition, larvae of 17, 20, 23, 29, 36 and 41 dph represented stage D, stage E, stage F, stage G, stage H and stage I, respectively. All the samples were frozen in liquid nitrogen immediately and stored at -80 °C until RNA extraction. To explain the effect of TH, larvae were sampled at 15 dph and divided into three groups (5,000 fish per group) (Inui and Miwa 1985). The three groups were reared in seawater containing either 0.1 mg/L concentration of T4 (the main form of TH in animals), 30 mg/L of TU, or none, with daily water exchange of one-third, keeping T4 and TU concentrations constant. We collected the larvae of each group after 2, 5, 8, 14, 21 and 26, respectively, and rapidly frozen in liquid nitrogen and stored at -80 °C until RNA extraction. All animal protocols have obtained the consent of the Review Committee for the Use of Animal Subjects of Shanghai Ocean University, Shanghai, China. 1543 manufacturers’ instructions. According to the IGFBP-2s cDNA sequences of P. olivaceus transcriptome, two pairs of primers (IGFBP-2a-f, IGFBP-2a-r and IGFBP2b-f, IGFBP-2b-r) were designed in non-conservative region for cloning and verifying the partial fragment of P. olivaceus IGFBP-2. In PCR amplification, 1 lL cDNA template, 2 lL of 109 Ex Taq buffer, 1.6 lL of dNTP (2.5 mM of each), 1 lL of the forward and reverse primers (10 lM), and 2.5 U of Ex TaqÒ DNA polymerase (TaKaRa, Japan) were combined and added up H2O to a total volume of 20 lL. After 4 min initial denaturation at 94 °C was carried out, 35 cycles of amplification were performed using a cycle profile of 94 °C for 30 s, a primer-dependent annealing temperature (Table 1) for 30 s, 72 °C for 30 s. Based on the two IGFBP-2s partial fragment sequences, four pairs of gene-specific primers were designed, respectively, for 30 -end and 50 -end cloning. Both 30 -end and 50 -end were amplified by two round PCR using the 30 and 50 -Full RACE Kit (TaKaRa, Japan). All the primers used were listed in Table 1. Amplification products were separated by agarose gel electrophoresis. The aimed PCR products were excised, and purified using a Gel Extraction Kit (Tiangen, China) and then ligated into pMDÒ19-T Vector (TaKaRa, Japan). The ligated products were transformed into Escherichia coli strain DH5a competent cells. Positive colonies were selected by color screening on LB plates containing X-gal and Amp. Five of the positive clones were sequenced on an ABI PRISM 3730 Automated Sequencer (ABI, USA). RNA extraction Total RNA from adult tissues, staged embryos, larvae and treated larvae was extracted by TrizolÒ Reagent (Invitrogen, USA) and treated with DNaseI (5 U/lL) (TaKaRa, Japan) for 1 h. The quality and integrity of RNAs were examined by agarose gel electrophoresis and spectrophotometer NANODROP 2000C (Thermo, USA). The samples with values of A260/A280 ratio ranged from 1.8 to 2.1 can be used in reverse transcription reactions. Cloning of IGFBP-2 cDNA Total RNA (2 lg) from liver of P. olivaceus, which treated with DNaseI (5 U/lL) (TaKaRa, Japan) for 1 h was reverse-transcribed using PrimeScriptTM 1st Strand cDNA Synthesis kit (TaKaRa, Japan), following the Sequence analysis The fragments were assembled together by DNAMAN software. The potential open-reading frame was analyzed using ORF Finder (http://www.ncbi.nlm.nih.gov/ gorf/gorf.html/). The cDNA sequences and the derived amino acid sequences were compared with the sequences in the GenBank database using BLAST program available for the NCBI website (http://www. ncbi.nlm.nih.gov/guide/). Alignment of amino acid sequences was achieved using DNAstar and CLUSTALW 1.8 program. The phylogenetic tree was constructed using MEGA 4.1 with Neighbor-Joining method (bootstrap 1000). The possible signal peptide was predicted by signal 4.0 Server (http://www.cbs.dtu. dk/services/SignalP/). 123 1544 Table 1 Nucleotide sequences of the primers used for partial fragment, 30 -RACE, 50 -RACE and real-time quantitative PCR of P. olivaceus IGFBP-2s cDNA Fish Physiol Biochem (2013) 39:1541–1554 Primers Sequences Tm (°C) Annealing temp (°C) Primers for partial fragment IGFBP-2a-f CGAGATGTGCGGCGTGTA 59.2 59.0 IGFBP-2a-r IGFBP-2b-f GGGATGATGGGATAGGTCGG TTCCGATGTCCAAGTTGTAC 61.2 51.4 51.0 IGFBP-2b-r CCACTTTTTG TCCACATC 45.0 Primers for 30 -RACE PCR 30 RACE outer primer TACCGTCGTTCCACTAGTGATTT 60.8 – 30 RACE inner primer CGCGGATCCTCCACTAGTGAT TTCACTATAGG 79.8 – 30 -IGFBP-2a CATATCCCCAACTGTGACAAGAG 58.8 56.0 30 -IGFBP-2a-nest GGCAGTATAACCTCAAACAGTGCA 54.9 54.0 3 -IGFBP-2b GTGCGGGGTCTATACACCGAGGT 66.2 62.0 30 -IGFBP-2b-nest CACAGGGGCTGAATTACCTTTGC 64.9 61.0 0 Primers for 50 -RACE PCR 50 RACE outer primer CATGGCTACATGCTGACAGCCTA 61.8 – 50 RACE inner primer CGCGGATCCACAGCCTACTGAT GATCAGTCGATG 84.7 – 50 -IGFBP-2a CTGCACCAGTTGCTTCAGGGGAA 68.2 63.0 50 -IGFBP-2a-nest GTTGCTTCAGGGGAAGCTCCGAG 68.3 63.0 50 -IGFBP-2b CTAAACCCTGGATGAGCTGCTGC 65.2 61.0 50 -IGFBP-2b-nest CCTCGGTGTATAGACCCCGCACAG 68.7 64.0 60.0 Primers for real-time quantitative PCR f forward primer, r reverse primer qIGFBP-2a-f TCTAAGATGCCCTTCAGAGATAAC 62.8 qIGFBP-2a-r GGGATGATGGGATAGGTCGG 66.2 qIGFBP-2b-f GTCCAGATACCGACGACGCCTAA 66.4 qIGFBP-2b-r ATCCTGACAGAGTTTTGAAGA 54.5 b-actin-f GGAAATCGTGCGTGACATTAAG 62.4 b-actin-r CCTCTGGACAACGGAACCTCT 61.2 Real-time quantitative PCR Total RNA (500 ng) from each sample was reversetranscribed using PrimeScriptTM RT reagent Kit (TaKaRa, Japan), according to the manufacturer’s instructions. Lack of genomic DNA contamination was confirmed by PCR amplification of RNA samples in the absence of cDNA synthesis. A 10 lL of each reverse-transcribed product was mixed with 90 lL sterile distilled water. This diluted cDNA was used as the template of each real-time quantitative PCR. The primers were designed using the Oligo 6.0 software and examined by electrophoresis on agarose gel, and PCR conditions were optimized by preliminary test to avoid from dimer formation and unspecific amplifications. 123 60.0 60.0 Real-time quantitative PCR was carried out on the CFX-96 (Bio-Rad). The 20 lL real-time PCR reactions contained 2 lL cDNA template, 0.2 lL of each of the gene-specific forward and reverse primers (qIGFBP-2af, qIGFBP-2a-r or qIGFBP-2b-f, qIGFBP-2b-r, 10 lM) and 10 lL of 2 9 iQTM SYBRGreen Supermix (BioRad). The PCR amplification procedure was used as follows: initial denaturation for 4 min at 95 °C, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. A melting curve was collected using two additional cycles by reading the fluorescence value from 65 °C to 95 °C, in order to assess the specificity of the PCR amplification. Negative controls contained non-template controls (NTC), and interplate calibrator (IPC) were performed. For normalization of the gene Fish Physiol Biochem (2013) 39:1541–1554 expression data, all samples were run in parallel with the internal control gene b-actin (primers: b-actin-f, b-actin-r). Each assay was repeated in triplicate. To estimate amplification efficiencies, a standard curve was generated for each gene based on fivefold serial dilutions of quantified larvae cDNA. All calibration curves exhibited correlation coefficients higher than 0.99, and the corresponding efficiencies (E) of PCR were from 0.95 to 0.99. Relative mRNA expressions for two IGFBP-2s gene were determined using the 2-DDCT method (Livak and Schmittgen 2001). Statistical analysis Quantitative data were expressed as mean ± Standard Error (SE) (n = 3). Comparisons among different stages and adult tissues were estimated by general linear model (GLM) method. A probability level of 0.05 or 0.01 was used to indicate significance. All statistics were performed using SPSS1 7.0 and SAS. Results Cloning and sequence analysis of P. olivaceus IGFBP-2 cDNA Using general-PCR and 30 - and 50 -RACE methods, we cloned and characterized two IGFBP-2 subtypes cDNA in P. olivaceus, and submitted them to Genbank (IGFBP-2a: KC914560; IGFBP-2b: KC914561). IGFBP-2a Paralichthys olivaceus IGFBP-2a full-length cDNA was cloned from the liver of adult P. olivaceus. The complete cDNA is 1,046 bp long and consists an open-reading frame (ORF) of 876 bp, encoding a predicted polypeptide of 291 amino acid residues with a putative signal peptide of 31 amino acids, a 125 bp 50 -untranslated region (UTR), and a 45 bp 30 -UTR. Like other vertebrate IGFBPs, the mature P. olivaceus IGFBP-2a has two highly conserved cysteine-rich domains: 12 cysteine residues in N-terminal and 6 cysteine residues in C-terminal. Typical IGFBPs motif (GCGCCXXC) was existed within the N-terminal domain of the peptide. And the characteristic Arg– Gly–Asp (RGD) sequence was observed at the C-terminal domain of P. olivaceus IGFBP-2a, which was 1545 practically present in all IGFBP-2s. The homology analysis based on the deduced amino acid sequences revealed that the predicted P. olivaceus IGFBP-2 exhibited significant identities to other vertebrates (see attached Fig. 1). The homology analysis based on the deduced amino acid sequences revealed that the predicted P. olivaceus IGFBP-2a exhibited significant identities to other vertebrates (Fig. 1). P. olivaceus IGFBP-2a protein displayed the highest homology of 73.8 % to S. salar IGFBP-2a, while it had lower identities to O. tshawytscha IGFBP-2a (72.5 %), O. mykiss (72.4 %), C. carpio (68.7 %), D. rerio IGFBP-2a (66.3 %), E. coioides (52.5 %), M. undulatus (50.5 %), S. quinqueradiata (50.1 %), D. labrax (48.9 %), S. maximus (48.6 %), G. gallus (44.3 %), M. musculus (44.0 %), O. aries (43.0 %), H. sapiens (42.7 %), respectively. IGFBP-2b Similarly, P. olivaceus IGFBP-2b full-length cDNA was obtained from liver of adult P. olivaceus. Its fulllength cDNA sequence is 1,067 bp long containing a 53 bp 50 -UTR, a 198 bp 30 -UTR and an open-reading frame of 816 bp, which encodes a predicted polypeptide of 271 amino acid residues with a putative signal peptide of 25 amino acids. Analogously, the mature P. olivaceus IGFBP-2b has two highly conserved cysteine-rich domains, respectively, are 12 cysteine residues in N-terminal and 6 cysteine residues in C-terminal, a typical IGFBPs motif (GCGCCXXC) existed within the N-terminal domain and the RGD sequence observed at the C-terminal domain. While, after the homology analysis, results display that P. olivaceus IGFBP-2b protein had identities to S. maximus (85.6 %), S. quinqueradiata (84.8 %), E. coiodes (84.1 %), D. labrax (81.1 %), M. undulatus (76.4 %), D. rerio IGFBP-2b (57.3 %), S. alpinus IGFBP-2b (56.3 %), O. tshawytscha IGFBP-2b (56.3 %), O. mykiss (55.2 %), C. carpio (54.2 %), G. gallus (39.8 %), M. musculus (37.6 %), O. aries (37.1 %) and H. sapiens (36.9 %), respectively. A phylogenetic tree of vertebrate IGFBPs was constructed to ascertain relationship of P. olivaceus IGFBP-2s and other vertebrate IGFBPs (see attached Fig. 2). The results indicate that P. olivaceus IGFBP2a is clustered with S. salar, O. mykiss, and 123 1546 123 Fish Physiol Biochem (2013) 39:1541–1554 Fish Physiol Biochem (2013) 39:1541–1554 b Fig. 1 Alignment of amino acid sequences of P. olivaceus IGFBP-2s and other species IGFBP-2s. The identical, highly and less conserved amino acid residues are indicated by different colors, respectively. (Color figure online) O. tshawytscha, while P. olivaceus IGFBP-2b is located in the same group with P. adspersus IGFBP-2. Differential expression of IGFBP-2 mRNAs in adult tissues The expression patterns of IGFBP-2 mRNAs in different tissues of both female and male adult P. olivaceus were examined by real-time quantitative PCR (Fig. 3). Both IGFBP-2 mRNAs were expressed in heart, liver, spleen, stomach, kidney, brain, gill, gonad, muscle and intestine, but the expression levels of IGFBP-2b in those detected tissues was relatively higher than IGFBP-2a, and largest gap distinctly displayed in liver. Interestingly, the levels of IGFBP-2a mRNA were higher in female than male, and yet, IGFBP-2b was just opposite, its levels in male were higher than in female (Fig. 3). Expression of IGFBP-2 mRNAs during embryonic and larval development The temporal expression of IGFBP-2 mRNA during early development was analyzed by real-time quantitative PCR. And the results were presented in Fig. 4. Two IGFBP-2 mRNAs were observed in all detected stages; but the transcripts of IGFBP-2b keep in a quite low level at embryonic stages, yet IGFBP-2a was higher. The levels of IGFBP-2a mRNA reached the peak at blastopore-closing stage and then began to decrease until the end of metamorphosis. The level of IGFBP-2 mRNA significantly increased at 3 dph, but it drop to a lower level at 7 dph. IGFBP-2 mRNA had a sharply increase in 10 dph, and gradually decreased and got to a nearly identical level with 7 dph at 23 dph, and it got to a higher level at 29 dph when the larvae were just at metamorphic climax, and slightly decreased at 36 dph, then, sharply increased to the highest level at the end of metamorphosis. Effect of thyroid hormone on the expression of IGFBP-2 mRNAs during metamorphosis In order to study the relationship between TH and the expression levels of IGFBP-2 mRNAs, larvae at 15 dph were treated by exogenous TH (0.1 mg/L) and TU (30 mg/L), respectively, and the levels of 1547 IGFBP-2 mRNAs were determined at 2, 5, 8, 14, 21 and 26 days after TH and TU treatment (Fig. 5). In THtreated larvae, IGFBP-2b mRNA showed dramatically increase from 17 to 23 dph, but suddenly drop to a lower level in and after 36 dph, compared with the same stage of untreated group. The levels of IGFBP-2b mRNA were visibly down-regulated in TU-treated larvae, especially at 29, 36 and 41 dph. Generally, IGFBP-2b mRNA exhibited higher expression levels from metamorphic climax to the completion of metamorphosis than in embryonic and early larval stages. Conversely, TH and TU have little effect on the levels of IGFBP-2a in metamorphic stages, except 17 dph. Discussion In this study, the full-length cDNAs of two IGFBP-2 genes from P. olivaceus were cloned and characterized. The deduced amino acid sequences of IGFBP-2a and IGFBP-2b, respectively, are 42.7–73.8 and 36.9–85.6 % homologous to the IGFBP-2s of mammal and other teleosts. Like other vertebrate IGFBPs, two P. olivaceus IGFBP-2s also have a cysteine-rich N- and cysteine-rich C-terminal domain. The two cysteine-rich domains, highly conserved among species, can specifically bind IGFs (Forbes et al. 1998; Hobba et al. 1998; Hwa et al. 1999). A typical IGFBP motif (GCGCCXXC) was existed in the N-domain of the P. olivaceus IGFBP-2s, which are necessary to IGFs binding (Kim et al. 1997). Residue Tyr-60 in the N-domain and a stretch of amino acids (K222HGLYNLKQCKMSLN236) in the C-domain of bovine IGFBP-2 has been shown to be important for IGF binding (Forbes et al. 1998; Hobba et al. 1998). These residues are well conserved in the P. olivaceus IGFBP-2s mature protein (IGFBP-2a: Tyr-61 and K200RGQYNLKQCKMSLH214; IGFBP-2b: Tyr-58 and R186HGLYNLKQCNMSTH200). A RGD motif was also present in the C-domain of the P. olivaceus IGFBP-2s. By the binding with a5b1 integrins in cell surface, the RGD motif in human IGFBP-1 has been shown to be responsible for the IGF-independent activity of IGFBP-1 on cell migration (Jones et al. 1993). This motif was also existed in IGFBP-2, which is essential for IGFBP-2 to mediate IGF action of proliferation. But, it reported that IGFBP-2 cell surface association was not dependent on the RGD motif in rat. The mutational sequence (RGD was replaced by RGE) of IGFBP-2 did not alter its growth inhibitory effect and affect its capacity 123 1548 Fish Physiol Biochem (2013) 39:1541–1554 b Fig. 2 Phylogenetic tree based upon the alignment of amino acid sequences of vertebrates IGFBPs and constructed using the Neighbor-Joining bootstrap method by Mega 4.1 with 1000 bootstrap replications. The full length of IGFBPs amino acid sequences used for analysis was extracted from GenBank. The number shown at each branch indicated the bootstrap values (%) of binding to cell surface in vivo (Hoeflich et al. 2002). Unlike mammals, heparin-binding site (PKKLRPP) was not perfectly conserved in fish IGFBP-2. The heparinbinding domain in IGFBPs binding to heparin or glycosaminoglycans changed the configuration of those IGFBPs, leading to significant lower affinity to IGF-I and enable the IGFBPs complex to type I receptors on the cell surface. The heparin-binding motif is also absent in P. olivaceus and D. rerio IGFBP-2 (Duan et al. 1999). It has proved that the mutants of D. rerio IGFBP-2 with altered RGD and heparin-binding site had similar effects in inhibiting growth and developmental rates, but altering the IGF binding site abolishes its biological activity (Zhou et al. 2008). Preliminary results demonstrated that D. rerio IGFBP-2 did not localize to the cell surface, but whether it has a growth inhibitory effect in P. olivaceus like D. rerio IGFBP-2 needs further studies. In addition, there were two IGFBP-2s in D. rerio, O. Latipes, E. coioides, C. carpio, S. aurata, F. obscurus and T. nigroviridis. In contrast to other species, there only found one transcript such as chicken (Schoen et al. 1995), rat (Margot et al. 1989). At present, there were two sequences of IGFBP-2 obtained in P. olivaceus, as the E. coioides (Chen et al., 2010), by rapid amplification of cDNA ends technique. P. olivaceus IGFBP-2a and IGFBP-2b share highly degree identity (51.1 %) in the amino acid level. In the present study, both IGFBP-2 mRNAs were ubiquitously expressed in various tissues, suggesting that IGFBP-2 is being synthesized in the liver, as well as in extrahepatic tissues locally. The IGFBP-2a mRNA in all detected tissues was rather lower, especially in male fish; however, the IGFBP-2b mRNA was extremely abundant in liver, but comparatively lower. The different spatial expression pattern between two IGFBP-2s in P. olivaceus revealed that IGFBP-2a and IGFBP-2b may have different functions on growth, development and even other physiological actions. The pervasive tissue distribution of IGFBP-2s in P. olivaceus is consistent with the 123 Fish Physiol Biochem (2013) 39:1541–1554 Fig. 3 Relative expression levels of IGFBP-2 mRNAs in different tissues of female and male P. olivaceus. Data were expressed as the mean fold difference (mean ± SEM, n = 3) from the calibrator group (heart of female P. olivaceus). a is relative expression levels of IGFBP-2a mRNA in different 1549 tissues of female and male P. olivaceus; b is relative expression levels of IGFBP-2b mRNA in different tissues of female and male P. olivaceus. The asterisk represented the statistical significant differences with the female fish (P \ 0.05) Fig. 4 Relative expression levels of IGFBP-2 mRNAs during P. olivaceus embryonic and larval development. Data were expressed as the mean fold difference (mean ± SEM, n = 3) from the calibrator group (the value of IGFBP2b mRNA in 0 h embryo). The asterisk represented the statistical significant differences between two IGFBP-2s subtypes (P \ 0.05) expression of IGFBP-2 found in mammalian and other fish species, whereas the extrahepatic tissue distributions of IGFBP-2 mRNA were practically different in vertebrates. Higher level of IGFBP-2 mRNA was found in the brain of D. rerio (Duan et al., 1999), O. mykiss (Kamangar et al., 2006) and C. carpio (Chen et al., 2009). However, in S. aurata (Funkenstein et al., 2002) and P. olivaceus, the level of IGFBP-2 mRNA in brain was relatively low. This phenomenon may cause by the discrepancies of physical conditions and life habits of different organisms. Interestingly, the spatial expression pattern of IGFBP-2 mRNA in P. olivaceus is not exactly consistent with that of IGF-I (Zhang et al. 2011a). It reported that the affinity of IGFBP-2 for IGF-II is fourfold higher than its affinity for IGF-I. (Clemmons 1997) partially suggests 123 1550 Fish Physiol Biochem (2013) 39:1541–1554 Fig. 5 Relative expression levels of IGFBP-2 mRNAs in TH and TU-treated larvae during P. olivaceus metamorphosis. Data were expressed as the mean fold difference (mean ± SEM, n = 3) from the calibrator group (untreated 17 dph larvae). Figure 1a is relative expression levels of IGFBP-2a mRNA in different tissues of female and male P. olivaceus; Fig. 1b is relative expression levels of IGFBP-2b mRNA in different tissues of female and male P. olivaceus. The asterisk represented the statistical significant differences with the untreated group (P \ 0.05). Control represented the untreated group; TH represented the thyroid hormone-treated group; TU represented the thiourea-treated group that IGFBP-2 not only modulate the actions of IGF-I but also IGF-II. Furthermore, IGFBP-2 also has its IGF-independent actions. To know the regulation and exact roles of IGFBP-2 in P. olivaceus still need further investigation. Interestingly, the levels of IGFBP-2b mRNA in all detected tissues were higher in male than female P. olivaceus; rather, the IGFBP-2a mRNA expression levels in male were lower than in female. As we all know, sexually dimorphic growth exists in many teleosts. In P. olivaceus, females grow significantly faster and larger than males. In vitro studies suggest that IGFBP-2 is primarily inhibitory to IGF actions (Clemmons 2001; Firth and Baxter 2002). Recent studies also indicate that IGFBP-2 could inhibit the growth of zebrafish by acting downstream in the GH– IGF-I axis (Duan et al. 1999). These data indicate that growth difference between male and female P. olivaceus may have much to do with IGFBP-2b. The decrease IGFBP-2b mRNA levels in female fish was accompanied by the IGFBP-2a mRNA levels increasing, which indicate that IGFBP-2a may cooperate with IGFBP-2b to exert other biological functions. Remarkably, the expression of IGFBP-2b mRNA in gonad, especially in ovary, is higher than other extrahepatic tissues. This phenomenon is also found in S. aurata (Funkenstein et al., 2002), O. mykiss (Kamangar et al., 2006), C. carpio (Chen et al., 2009), as well as in other animals. In S. aurata, a relatively high expression level of IGFBP-2 mRNA was detected in young gonads with a predominantly ovarian part, consisting mainly of pre-vitellogenic oocytes (Funkenstein et al. 2002). A higher level of IGFBP-2 mRNA was also observed in E. coioides ovary (Chen et al. 2010) and C. carpio gonad (Chen et al. 2009). In human, high levels of IGFBP-2 were found in follicular fluid from healthy, dominant follicles (Hughes et al. 1997). In addition, high level of IGFBP-2 mRNA in developing follicles was also reported in chicken (Onagbesan et al. 1999; Schams et al. 1999) and cattle (Armstrong et al. 1998; Yuan et al. 1998). These results indicate that IGFBP-2 may play an important role in animal gonadal development. In D. rerio (Zhou et al., 2008), S. aurata (Funkenstein et al., 2002), C. carpio (Chen et al., 2009) and E. coioides (Chen et al., 2010), IGFBP-2 mRNA was expressed during fish embryonic development and larvae at all the stages examined. Similarly, in our present study, two IGFBP-2 mRNAs were detected in all stages of embryonic development and sampled larval stages. While the levels of IGFBP-2a mRNA were obviously higher than IGFBP-2b at embryonic stages suggesting that IGFBP-2a may play a major role in embryonic development. It reported that 123 Fish Physiol Biochem (2013) 39:1541–1554 targeted knock-down of IGFBP-2 yielded embryos with reduced blood cell densities, disruptions to blood circulation, cardiac dysfunction and pronounced cerebral edema (Wood 2005). IGF-I, IGF-II, and IGF- IR mRNAs were previously reported in unfertilized D. rerio eggs (Maures et al. 2002), and Wood et al. authenticated that knock-down of IGFBP-2 significantly reduces IGF-I mRNA levels in D. rerio embryos (Wood 2005). Additionally, IGF-I and IGFIR were detected in embryonic development and larvae stages (Zhang et al. 2011a, b, c). These data suggest that IGFBP-2a may be involved in embryonic and larval development by modulating IGFs actions. With embryo hatching, the expression of IGFBP-2a decreased and maintained a lower level indicate that IGFBP-2b has gradually displaced the position of IGFBP-2a in growth and development. It indicated that IGFBP-2a plays a core role in embryonic development as well as IGFBP-2b in the larvae growth and development. In the present study, an abrupt increasing level of IGFBP-2b mRNA was detected at 3 dph and then dropped to a relative low level at 7 dph. Nutrition is a regulator of IGFBP-2 expression (Clemmons 1997). IGFBP-2 gene transcription was increased in starved rodents, and plasma concentrations were increased in fasted humans (Clemmons et al. 1991). In teleosts such as D. rerio, three-week fasting increased the IGFBP-2 mRNA tissue levels (Duan et al. 1999). The 3–4 dph is considered critical for the successful transition from endogenous nutrition to exogenous feeding, when the yolk sac is almost entirely absorbed (Bao et al. 1998). After 3 dph, larvae started feeding on nutritional supplements. Therefore, we speculate that the expression of IGFBP-2b mRNA increased at 3 dph may be due to poor endogenous nutrition and decreased at 7 dph may be owing to nutritional supplements after starting feeding. As we all know that the anlage of crow-like larval fin appeared and followed by the six fin rays of crow-like larval fin appeared one by one in pre-metamorphosis. IGFBP-2 is identified as a potent inhibitor of bone growth (Eckstein et al. 2002) and cell proliferation (Hoeflich et al. 1999). Research has shown that the P. olivaceus larvae underwent a faster growth in pre-metamorphosis (Liu 1996). Therefore, keeping a lower IGFBP-2b mRNA level and higher IGF-I mRNA level (Zhang et al. 2011a, b) could be imperative to stimulate premetamorphic larval faster growth and initiate 1551 metamorphosis. Considering these data, we conjecture that levels of IGFBP-2b mRNA observed from 10 to 23 dph put on a downward trend may be in relation to the fin rays appearance and extension and necessary to accelerate growth rate. At metamorphic climax, IGFBP-2b mRNA was increased to a higher level and maintained to the end of metamorphosis. Relatively higher expression of IGFBP-2b mRNA and lower IGF-I mRNA expression (Zhang et al. 2011a, b) during metamorphosis are consistent with the P. olivaceus larvae showing that the increasing weight rate evidently declined at this stage (Liu 1996). These results suggest that IGFBP-2 could play a vital role in modulating IGFs action during larval development and metamorphosis of the P. olivaceus. Previous studies have shown that TH treatment slightly increases IGFBP-2 mRNA expression in hypothyroid mammals (Clemmons 1997). Similarity with mammals, the expression and secretion of fish IGFBP-2 mRNA are also influenced by TH to some extent. In the present study, we used exogenous TH (0.1 mg/L) and TU (30 mg/L) treated the larvae at 15 dph to examine the effects of TH and TU on IGFBP-2 mRNA expression during P. olivaceus metamorphosis, found that IGFBP-2a mRNA level only distinctly raised in TH-treated 17 dph larvae; in TU-treated larvae and TH-treated larvae in other stage, have no visible change. However, IGFBP-2b mRNA level in TH-treated larvae showed obviously rise before 29 dph and in TU-treated larvae was markedly decreased during metamorphosis. This suggests that transcription of IGFBP-2b mRNA could be stimulated by TH in certain range of concentrations, while inhibited by high concentrations; TU could down-regulate IGFBP-2b mRNA expression level by inhibiting the secretion of endogenous TH; IGFBP-2b was the main subtype of IGFBP-2 participating in metamorphosis of Japanese flounder. Fu et al. (2012) indicate that TH-treated larvae were smaller in size than untreated and TU-treated larvae. Moreover, administration of TH to pre-metamorphic P. olivaceus larvae induced precocious metamorphosis, whereas a TU induced metamorphic stasis (Inui and Miwa 1985; Miwa and Inui 1987). Previous studies have shown that IGF-I and IGFBP-1 mRNA levels were significantly altered in TH-treated larvae compared with untreated larvae during the P. olivaceus metamorphosis (Zhai et al. 2012; Zhang et al. 2011a) indicating that the effect of TH on accelerating P. olivaceus 123 1552 metamorphosis may partly achieved by regulating IGFs system. In summary, the present study had cloned and characterized the full-length cDNA of two IGFBP-2 genes and expression patterns of P. olivaceus IGFBP2s. IGFBP-2a mRNA was more evenly expressed in all detected tissues, but IGFBP-2b mRNA was mainly synthesized in the liver, suggesting that IGFBP-2 has autocrine/paracrine actions on regulating IGFs system. Moreover, IGFBP-2a was mainly expressed in embryonic stages, and IGFBP-2b was mainly expressed in larval stages and adult fish. Furthermore, IGFBP-2b mRNA, rather than IGFBP-2a, was reduced by certain level of exogenous TH, while deduce by high level of TH in P. olivaceus larvae. The results showed that IGFBP-2b was the main subtype in regulating P. olivaceus metamorphosis. IGFBP-2a and IGFBP-2b may have different functions in IGFdependent and IGF-independent actions. Although the exact mechanism is necessary to be elucidated, these results suggest that the IGFBP-2 may be involved in the IGF system regulating developmental and metamorphic process in the P. olivaceus and provide molecular base for the further study of IGFs system and P. olivaceus metamorphosis. 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