Cellular localization of insulin-like growth factor-II protein in the sea bass (Dicentrarchus labrax) from hatching to adult.

The cellular localization of IGF-II protein was investigated during larval and postlarval developmental stages of sea bass (Dicentrarchus labrax) by immunohistochemistry using antisera raised against Sparus aurata IGF-II. At hatching, IGF-II immunoreactivity was already present in the skin, developing intestine and skeletal muscle. During larval life IGF-II protein was also observed in heart musculature, in kidney and gill epithelia as well as in liver. In fry skeletal muscle a moderate IGF-II immunostaining was detected in red fibres, whereas white muscle fibres exhibited a faint immunoreactivity. In adults, a marked IGF-II immunostaining was observed in red muscle fibres. A moderate immunoreactivity was also present in white fibres as well as in heart striated myocardial fibres. These results are in agreement with previous findings on the spatial localization of IGF-II and IGF type 1 receptor in S. aurata and Umbrina cirrosa, confirming the role of IGF system during development and growth of fish.

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress.

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.

Characterization and functional analysis of the 5' flanking region of myosin light chain-2 gene expressed in white muscle of the gilthead sea bream (Sparus aurata).

The promoter region ( approximately 1400 bp) of myosin light chain 2 gene of fast skeletal muscle from the marine fish Sparus aurata was cloned, sequenced and characterized. It contains a consensus sequence for TATA box, six perfect E-boxes known as binding sites to myogenic basic helix-loop-helix transcription factors and four putative MEF2-binding sites. Three genomic fragments (truncated at their upstream region) of 244, 650 and 1400 bp showed promoter activity evidenced by muscle-specific reporter gene activity using transient expression of green fluorescent protein in microinjected zebrafish embryos and in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA. The three genomic fragments also directed luciferase activity in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA showing a 60 to 150-fold higher luciferase activity compared to that obtained with pGL3-Basic. These experiments show that the three genomic fragments are functional muscle-specific promoters which will be useful for directing myostatin and follistatin expression in fish muscle in order to study their effect on fish muscle growth.

Long-term culture of muscle explants from Sparus aurata.

Although there are mammalian myoblast cell lines, no fish myoblast cell line has been developed so far. The aim of this study was to develop a culture system of muscle explants for fish, as explants provide an approximation of the in vivo conditions for cell proliferation and differentiation, and enable a close comparison with events in muscle regenerating in vivo. Here we describe the main features of a long-term in vitro culture system for muscle explants from Sparus aurata fry. At the time of sampling, the original fibres were damaged and subsequently degenerated as shown by the loss of parvalbumin (PV) and presence of apoptotic nuclei. This mechanical damage provoked a myogenic response by activation of myogenic precursor cells. After a few days, new mononucleate cells aligned with the original fibres were seen in the explants, some with proliferating cell nuclear antigen (PCNA-) and Myf-5-positive nuclei, indicating proliferation and their myogenic fate. By 1 week, multinucleate cells with desmin immunoreactivity but PCNA- and Myf5-negative nuclei were present, equivalent to differentiated, postmitotic myotubes. Some of these myotubes were also immunoreactive for PV and insulin-like growth factors (IGFs). By 11 days, many of the myotubes were also immunoreactive for myostatin (MSTN). By 23 days, many of the myotubes had increased in diameter, were packed with myofibrils, and were strongly PV-positive and immunoreactive for MSTN, IGF-I and IGF-I receptor. This study shows that a proliferative process occurs in the explants despite the death of the original muscle fibres, and new muscle fibres expressing growth regulators are formed by regeneration from myogenic precursors present in the explants at the time of sampling.

Hormonal control of the IGF system in the sea bream ovary.

Numerous studies have described the presence of an intragonadal IGF system involved in regulation of gametogenesis in teleost fish. In the present study, the in vivo effects of estradiol-17beta (E2) and growth hormone (GH) exposure on IGF-I, IGF-II, IGF1R, and IGFBP2 gene expression in sea bream ovary were monitored by RT-PCR during prereproductive and reproductive periods. The evidence demonstrates that both hormones investigated here affect the ovarian IGF system, showing that it is not only under GH control, but also can be regulated by sexual hormones; this hormonal modulation is related to reproductive phase.

Expression and cellular localization of insulin-like growth factor-II protein and mRNA in Sparus aurata during development.

The spatial localization of IGF-II protein and mRNA was investigated during larval and postlarval developmental stages of the gilthead sea bream (Sparus aurata) by immunohistochemistry and in situ hybridization, using specific antisera and riboprobes. Steady-state levels of IGF-II mRNA in larvae were determined by Northern blot analysis and were found to be increased. Immunoreactivity towards IGF-II was found in larval skin, muscle, gills, gut, olfactory epithelium and kidney. After metamorphosis, the strongest immunoreactivity was found in red skeletal muscle. Positive reaction with IGF-II antibodies was also found in the olfactory epithelium and in the epithelia of pharynx, oesophagus, stomach and kidney. In the adult, the most intense signal was observed in the red and pink musculature and in heart musculature. Immunostaining was also found in saccus vasculosus, thymus, spleen and ovary. IGF-II mRNA was detected by in situ hybridization in the brain, olfactory epithelium, eye, pharynx, skeletal musculature and liver. The spatial distribution of IGF-II shown in this study is consistent with previous findings on the cellular localization of IGF type 1 receptor in the sea bream and supports a role for IGF-II during development and growth of sea bream. Furthermore, these results suggest that IGF-II acts in an autocrine/paracrine manner.

Localization of IGF-I, IGF-I receptor, and IGFBP-2 in developing Umbrina cirrosa (Pisces: Osteichthyes).

In this study, the distribution of IGF-I, IGF type I receptor (IGF-IR), and IGF-binding protein 2 (IGFBP-2) was investigated during larval and post-larval developmental stages of the shi drum (Umbrina cirrosa) by immunohistochemistry using antisera raised against Sparus aurata IGF-I and IGF-IR, and against mouse IGFBP-2. Immunoreactivity of the mitogenic marker PCNA (proliferating cell nuclear antigen) was used for assessment of cellular proliferation. Distribution of IGF-I mRNA was studied by in situ hybridization. IGF-I immunoreactivity was detected in liver and developing intestine already in 1-5 day post-hatching larvae. From day 11, immunostaining in the intestine was evident in the enterocytes of the anterior intestine and in the apical zone of the epithelium of developing posterior intestine. Positive reaction with IGF-I antibody was also detected in chondrocytes, in the epithelium of the skin, gills and in the central nervous system (CNS), and lateral muscle. At hatching IGF-IR immunoreactivity was already detectable in developing CNS, notochord, and skin. From day 6 immunostaining was evident in the olfactory epithelium, in eyes and from day 11 in the developing olfactory bulbs and CNS. Positive reaction with IGF-IR antibody was also detected in chondrocytes, in the epithelium of the skin, gills, heart, and in the lateral muscle. Immunoreactive IGFBP-2, as detected by anti-mouse IGFBP-2 antiserum, exhibited generally a similar distribution pattern to that of IGF-I and IGF-IR. In situ hybridization, which has been performed by using riboprobes from S. aurata cDNA, revealed IGF-I mRNA in skeletal musculature, liver, and CNS. These data strongly suggest a role for the IGF system during development and growth of U. cirrosa.

Ontogeny, tissue distribution, and hormonal regulation of insulin-like growth factor binding protein-2 (IGFBP-2) in a marine fish, Sparus aurata.

In this study, we have cloned insulin-like growth factor binding protein (IGFBP)-2 from a marine hermaphroditic fish species, the gilthead sea bream (Sparus aurata), and determined its structure, ontogeny, tissue distribution, and hormonal regulation. The sea bream IGFBP-2 precursor consists of 286 amino acids, including a putative signal peptide of 22 residues and a mature protein of 264 residues. The overall sequence of sea bream preIGFBP-2 is 52-39% identical to that of zebrafish, chick, mouse, rat, pig, sheep, bovine, and human preIGFBP-2. The cysteine-rich N- and C-terminal domains, which are believed to be important for IGF binding, show a greater degree of conservation with a sequence identity of 73-56% and 53-50%, respectively. Sea bream IGFBP-2 contains 18 cysteine residues and their alignment is identical to those of other vertebrate IGFBP-2s. The Arg-Gly-Asp (RGD) sequence, present in the C-terminal domain of all known IGFBP-2, is also present in the sea bream. Northern blot analysis of RNA samples extracted from adult liver and developing larvae, revealed two transcripts of about 1.3 and 2.5 kb. RT-PCR analysis showed that IGFBP-2 was expressed in all adult tissues studied, with the highest levels found in liver and skin. IGFBP-2 transcripts were detected in gonad during reproductive cycle of S. aurata. Highest levels of IGFBP-2 mRNA were found in bisexual young gonads, and the levels decreased with gonad development and relatively high levels of IGFBP-2 mRNA were found in the ovary during spawning. By contrast, testicular IGFBP-2 mRNA levels were very low. RT-PCR detected IGFBP-2 mRNA throughout development in unfertilized eggs, embryos, and larvae, with highest levels observed after day 3 post-hatching, suggesting that this mRNA is the product of both the maternal and embryonic genomes. High variability in steady-state levels of hepatic IGFBP-2 mRNA was noted in adult fish, resulting in a statistically insignificant response to growth hormone treatment. These results suggest that the structure of IGFBP-2 is conserved in sea bream and that IGFBP-2 mRNA is expressed during early development and in gonad during the reproductive cycle, suggesting that it may play a role in gilthead sea bream development and reproduction.

Developmental expression, tissue distribution and hormonal regulation of fish (Sparus aurata) serum retinol-binding protein.

Retinol-binding protein (RBP) is the specific carrier of retinol in vertebrates and forms a 1:1 complex with transthyretin (TTR). A cDNA encoding serum RBP was cloned from liver and 7-day larvae of the marine fish Sparus aurata. The mature protein is 176 amino acids long and shows sequence identity of 77-78%, 56%, 63% and 62% with rainbow trout, Xenopus, chicken and human RBP, respectively. Northern blot analysis of hepatic RBP revealed two transcripts: a major one of approximately 1.4-1.5 kb and a minor of approximately 0.7 kb. Distribution of RBP mRNA in various tissues was studied by RT-PCR and showed high expression in liver and skin, and low expression in brain, kidney and gill filament (20-35% of the level in liver). RBP expression in intestine, pyloric caeca, muscle and pituitary was estimated to be approximately 7-14% of the level in liver. The ontogeny of RBP expression in S. aurata was examined in unfertilized eggs, embryos and larvae by using RT-PCR followed by hybridization with a specific probe. RBP transcript was found in all larval stages studied. Very low levels of RBP mRNA were detected in unfertilized eggs and in embryos 8 h after fertilization with a gradual increase at 12 h and 15-16 h post-fertilization. A single injection of estradiol-17beta to S. aurata immature, bisexual fish or to adult males reduced steady-state levels of hepatic RBP by 37 and 25%, respectively. The same treatment induced vitellogenin expression. The present data suggest that in fish, liver is the main site of RBP synthesis, but that RBP may have an important function in fish skin. RBP is expressed early in embryonic development and in fish its expression can be down regulated by estrogen.

Molecular characterization and high expression during oocyte development of a shrimp ovarian cortical rod protein homologous to insect intestinal peritrophins.

Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelline envelope and possess extracellular cortical rods (CRs) that extended into the cortical cytoplasm. These cortical specializations are precursors of the jelly layer (JL) of the egg. In searching for highly expressed mRNAs during oogenesis in the marine shrimp (Penaeus semisulcatus), two related cDNAs have been isolated that encode a mature protein of 250 amino acid residues. The deduced amino acid sequences revealed the presence of repeated cysteine-rich domains that are related to the chitin-binding domains of insect intestinal peritrophins. Similar cysteine-rich domains were reported in insect intestinal mucin, crustacean tachycitin, and invertebrate chitinases. The shrimp ovarian peritrophin (SOP) is glycosylated and can bind chitin when extracted from CRs. Its apparent molecular mass in SDS-PAGE is 29-35 kDa and 33-36 kDa, under nonreducing or reducing conditions, respectively. SOP is a major protein of CRs and the JL, and was immunodetected in ovaries; purified CRs; fertilized eggs that were surrounded by a JL matrix; and in the cloudy, whitish flocculent material appearing in sea water immediately after spawning. Immunolocalization in tissue sections determined that SOP was present in oocyte cytoplasm and in extraoocytic CRs. Shrimp expressed SOP mRNA in ovaries at all oocyte developmental stages, whereas expression in the hepatopancreas was restricted to vitellogenic stages. SOP mRNA was abundant in the shrimp ovary and was detected before the presence of the corresponding protein. This is the first demonstration that a protein with similar features to insect intestinal peritrophins is a component of CRs and is therefore a main precursor of the JL of spawned shrimp eggs.

Genomic structure and sequence of the gilthead seabream (Sparus aurata) growth hormone-encoding gene: identification of minisatellite polymorphism in intron I.

The growth hormone (GH) gene of the gilthead seabream (Sparus aurata) (saGH) has been cloned, sequenced, and characterized. The saGH gene spans approximately 4.3 kb and consists of six exons and five introns, as found for all cloned teleost GH genes with the exception of carps and catfish. The first and third introns contain long stretches of repetitive tandem repeats. The second intron, which is unusually long compared with that in other teleosts (and other vertebrates) spans 1747 nucleotides (nt) and contains several inverted repeats. Intron-targeted polymerase chain reaction (PCR) analysis identified length polymorphism of the first intron. Sequence analysis of four variants (405, 424, 636, and 720 nt) out of many variants found revealed that the variation in length is due to differences in the number of repeat monomers (17-mer or 15-mer) as well as minor changes in their length. This repeat unit contains the consensus half-site motif of the thyroid hormone response element (TRE) and estrogen response element (ERE). Polymorphism was found also in the third intron. This is the first report of such high polymorphism of the first intron of GH gene in a vertebrate.

Contrasting effects of estrogen on transthyretin and vitellogenin expression in males of the marine fish, Sparus aurata.

A partial cDNA encoding for the C-terminus of vitellogenin (VTG) was cloned from liver of Sparus aurata male treated with 17beta-estradiol (E(2)). E(2) treatment of S. aurata males resulted in increased synthesis and secretion of VTG protein into the plasma, determined by a specific enzyme-linked immunosorbent assay (ELISA) in a time-dependent manner. While VTG mRNA was induced by E(2) treatment, transthyretin (TTR) mRNA levels were reduced. These data provide the first demonstration that estrogen exhibits contrasting effect on VTG and on TTR gene expression in teleosts.

Insulin-like growth factor receptors and their ligands in gonads of a hermaphroditic species, the gilthead seabream (Sparus aurata): expression and cellular localization.

Expression of insulin-like growth factor (IGF)-I, IGF-II, and IGF type I receptor (IGF-1R) genes was studied in gonads at different developmental stages of the protandrous hermaphroditic species the gilthead seabream (Sparus aurata) by reverse transcription-polymerase chain reaction and Northern blot analysis. Both IGF-I and IGF-II mRNA levels were highest in bisexual gonads and decreased during gonadal development. Regardless of the stage of gametogenesis, IGF-II mRNA levels exceeded those of IGF-I. Transcripts for IGF-1R RNA were detected in gonads at all stages studied. A major transcript of 11 kb was found in gonads and in gill arch and brain, but it was not found in liver and muscle. Distribution of the two types of IGF-1R and IGF-I in gonads was studied by immunohistochemistry. Immunoreactive IGF-I was found in the granulosa and theca cells of follicles at different vitellogenic stages and in oocytes at the chromatin-nucleolus and perinucleolus stage. In the testis, immunoreactive IGF-I was found in somatic cells of the cyst wall, interstitial cells, and spermatogonia A. In addition, IGF-1R was detected in the membrane of previtellogenic oocytes and in the theca and granulosa cells of vitellogenic and late vitellogenic follicles. In the testis, a positive reaction was identified in spermatogonia A and spermatids for the germ cells and in somatic cells of the cyst walls and interstitial cells. Local expression and production of IGFs and their receptors in fish gonads support a role for the IGF system in fish gonadal physiology.

Ontogeny of the insulin-like growth factor system (IGF-I, IGF-II, and IGF-1R) in gilthead seabream (Sparus aurata): expression and cellular localization.

There is evidence for the presence of an insulin-like growth factor (IGF) system during fish development. The pattern of gene expression of IGF-I, IGF-II, and their cognate receptors during early development of gilthead seabream (Sparus aurata) was studied by reverse transcription-polymerase chain reaction (RT-PCR). Transcripts for IGF-I, IGF-II, and IGF-1R were detected throughout development in unfertilized eggs, embryos, and larvae, suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Analysis of IGF-1R mRNA in various adult tissues using RT-PCR revealed expression in all tissues studied, with the highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. The distribution of the two types of IGF-1R and IGF-I in gilthead seabream larvae was studied by immunohistochemistry and found to be tissue-specific and age-dependent. IGF-I and its receptors are widely distributed and appear in various tissues of seabream larvae. IGF-I immunoreactivity was highest in skeletal muscle and pancreas. The general distribution of the two types of IGF receptors in larval tissues appeared similar except for the muscle and the corpus cerebelli, in which IGF-1R was detected only by SpIR6 antisera. Both IGF-I and IGF-II may thus play a role during early development of teleosts, as in other vertebrates.

Preparation of recombinant gilthead seabream (Sparus aurata) growth hormone and its use for stimulation of larvae growth by oral administration.

Gilthead seabream (Sparus aurata) growth hormone (gsGH) cDNA coding for the mature protein was cloned in a pGEM-T vector and then transferred into prokaryotic expression vector pET-8 and expressed in E. coli BL21 (DE3) cells upon induction with IPTG. The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to over 98% purity, as evidenced by SDS-PAGE. Gel-filtration on a Superdex column under nondenaturing conditions and partial amino acid N-terminal sequence showed the purified protein to be a monomeric alanyl-gsGH. Over 90% pure bacterial beta-lactamase was copurified as a by-product. Binding assays of the [125I]gsGH to gs liver microsomal fraction resulted in high specific binding characterized by a Kd = 1.93 nM. Recombinant gsGH, like ovine placental lactogen, exhibited growth-stimulating activity when applied orally to S. aurata larvae or intraperitoneally to juvenile fish.

Cloning of putative piscine (Sparus aurata) transthyretin: developmental expression and tissue distribution.

cDNA encoding putative transthyretin (prealbumin, TTR) was cloned from liver of the marine fish Sparus aurata. The cDNA contains an open reading frame of 453 nt, encoding for a TTR precursor of 151 amino acids. The deduced amino acid sequence of S. aurata TTR shows identity of 54, 57.3 and 54.1% with lizard, chicken and rat TTR, respectively. Northern blot analysis revealed a TTR transcript of about 700 nt, highly expressed in liver, but also in skin. Low expression was detected in 12 other tissues by using RT-PCR. The ontogeny of TTR expression during early stages of larval development of S. aurata was examined by Northern blot analysis using poly(A+)RNA from larvae collected on different days after hatching. TTR mRNA was seen already on the first day after hatching and its steady-state levels increased from Day 15 onwards. Molecular cloning of a TTR-like cDNA from fish suggests that TTR evolved earlier in vertebrate development than previously thought. Furthermore, its expression in liver exceeds by several-fold that found in brain, yet high expression is also found in skin. These results suggest that in fish, liver is the main site of TTR synthesis, but that TTR may have an important function in fish skin.

Hyperglycaemic hormones inhibit protein and mRNA synthesis in in vitro-incubated ovarian fragments of the marine shrimp Penaeus semisulcatus.

The present work shows for the first time that peptides belonging to the Crustacean hyperglycaemic hormone family (CHH-family hormones) from Penaeus japonicus affect protein and mRNA synthesis in in vitro-incubated ovarian explant fragments removed from vitellogenic females of Penaeus semisulcatus. Reduced levels of protein synthesis, determined by TCA-precipitable 35S-labeled proteins, were found in the presence of crude sinus gland extracts from both P. semisulcatus and P. japonicus. A similar inhibitory effect compared to controls was found with each of the seven CHH-family peptides. Non-CHH-family peptides did not reduce protein synthesis. Crude sinus gland extracts prepared from P. semisulcatus were at least 20-fold more effective than sinus gland extracts of P. japonicus. The inhibition level was directly related to the concentration of the peptide in the incubation media, but its degree varied among the different tested peptides. The profile of proteins synthesized during in vitro incubation was analyzed using polyacrylamide gel electrophoresis under denatured and reduced conditions (SDS-PAGE), followed by autoradiography. Synthesis of several proteins was reduced, including proteins with electrophoretic mobility similar to that of vitellin. Immunoprecipitation with antiserum prepared against native ovarian vitellin confirmed the inhibitory effect of CHH-family peptides on vitellin synthesis. The crude sinus gland extract and CHH-family peptides also inhibited RNA synthesis, as determined by [3H]uridine incorporation into mRNA of ovarian fragments. It is concluded that in addition to their role in carbohydrate metabolism, CHH-family peptides may also influence ovarian physiology in crustaceans.

Piscine (Sparus aurata) alpha subunit of the G-protein transducin is homologous to mammalian cone and rod transducin.

A novel cDNA encoding alpha subunit of the GTP-binding protein, transducin, has been cloned from a marine fish, Sparus aurata. The cDNA contains an open reading frame of 1050 nt (encoding 350 amino acid residues). A high degree of identity was found with known mammalian transducin proteins of cones (Gt2 alpha) or rods (Gt1 alpha): human Gt2 alpha (80.2%), bovine Gt2 alpha (79.3%), mouse Tt1 alpha (78.2%), mouse Gt2 alpha (78%) and bovine Gt1 alpha (77.9%). Northern blot analysis of different tissues revealed a transcript of about 2.5 kb, which is expressed only in the fish eye and not in other tissues from adult fish, supporting its identification as transducin. Ontogeny of transducin mRNA expression during early development of Sparus aurata, determined by Northern blot analysis, showed very low levels in larvae 3 days after hatching but not earlier. Levels increased 3- and 6-fold on days 4 and 6 (respectively) compared with those on day 3 and remained essentially unchanged thereafter, until day 21 after hatching (the last day studied). Our results suggest that in fish only one alpha subunit of transducin is found, which shows similar identity with cone and rod alpha subunits of mammals.

Localization of IGF-I and IGF-I receptor mRNA in Sparus aurata larvae.

Studies of the ontogeny of IGF-I mRNA during embryonic and larval development of the gilthead sea bream Sparus aurata showed its expression during these early developmental stages. The present study applies in situ hybridization to localize IGF-I and IGF receptor mRNAs in 16-day larvae of S. aurata. Paraffin sections were hybridized with homologous RNA probes labeled by [35S]UTP. IGF-I mRNA expression was found mainly in chondrocytes, in both the gill arches and cranial cartilage, in skeletal muscle, in the brain, in the pancreas, in the retina, and in the epithelial cells surrounding the lens. A strong positive reaction for IGF receptor mRNA was found in skeletal muscle, in the pancreas, and in the lymphoid tissue found in the intertubular tissue of the kidney. Signals were less intense in brain and chondrocytes. It is suggested that in teleosts, as in higher vertebrates, IGF-I may be involved in the regulation of tissue growth and differentiation in an autocrine/paracrine manner.

Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization.

Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside. The expressed protein contained within the inclusion-body pellet was solubilized in 4.5 M urea, refolded for 24 h at pH 11.3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was approximately 200-fold lower than that of hIGF-1. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active.