Insulin-like growth factor-I cDNA cloning, gene expression and potential use as a growth rate indicator in Nile tilapia, Oreochromis niloticus (original) (raw)
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Insulin-like Growth Factor-I (IGF-I) is a physiological mediator and a potentially important growth indicator candidate in teleost fishes. In this study, the effects of increased temperature on the growth and hepatic IGF-I gene expression in Oreochromis niloticus were evaluated. Twenty all-male fish were reared separately at temperatures below 24°C for 12 days and then water temperature in 15 aquaria was gradually raised to 30°C within a day. Growth and hepatic IGF-I gene expression in five fish were obtained before the temperature change and after 2, 5 and 7 days of increasing the water temperature. The growth rate of the fish reared in the warmer temperature for 2, 5 and 7 days was significantly increased in a time dependent manner (r = 0.93). Mean hepatic IGF-I mRNA levels in fish reared at warm temperature for 2, 5 and 7 days were elevated 1.6-, 2.5-, and 3.6-fold, respectively compared to that of fish reared at cold temperature (<24°C). The IGF-I levels were significantly elevated after at least 5 days of exposure to warm temperature, which is consistent with the idea that hepatic IGF-I gene expression can be used as a short-term growth rate indicator for O. niloticus. A significant positive correlation was observed between days of rearing at warm temperature and hepatic IGF-I levels (r = 0.92); between specific growth rate (length) and IGF-I levels (r =0.92); and between condition factor and IGF-I levels (r = 0.55). The high positive association between IGF-I mRNA and growth rate validated the assertion that hepatic IGF-I levels are sufficiently sensitive to be used as instantaneous growth rate indicator in this species of fish.
Journal of Endocrinology, 2000
In spite of the importance of IGF-I for growth and development, knowledge about regulation of its production in submammalian species is rather limited. In order to create a tool for investigation of direct regulatory effects on the expression of IGF-I in bony fish liver, a primary cell culture of hepatocytes from Oreochromis mossambicus, the tilapia, was established. The cells were viable for up to 3 days and IGF-I mRNA synthesis was detected by northern blot and semiquantitative reverse transcriptase (RT)-PCR. Northern blot analysis of the primary cultured hepatocytes revealed four different IGF-I transcripts, 0.5, 1.9, 3.9 and 6.0 kb in size, which were identical to those in liver tissue. However, the expression rate was weaker than that in liver. The direct effects of recombinant tilapia (rt) growth hormone (GH) and salmon (s) IGF-I on the expression of IGF-I in primary cultured hepatocytes were investigated in time-course and dose-response experiments. In untreated cultures, IGF...
General and Comparative Endocrinology, 2004
We developed a one-tube two-temperature real-time RT-PCR that allows to absolutely quantify the gene expression of hormones using the standard curve method. As our research focuses on the expression of the insulin-like growth factors (IGFs) in bony Wsh, we established the technique for IGF-I and IGF-II using the tilapia (Oreochromis niloticus) as model species. As approach, we used primer extension adding a T7 phage polymerase promoter (21 nt) to the 5Јend of the antisense primers. This procedure avoids the disadvantages arising from plasmids. Total RNA extracted from liver was subjected to conventional RT-PCR to create templates for in vitro transcription of IGF-I and IGF-II cRNA. Correct template sizes including the T7 promoter were veriWed (IGF-I: 91 nt; IGF-II: 94 nt). The PCR products were used to create IGF-I and IGF-II cRNAs which were quantiWed in dot blot by comparison with deWned amounts of standardised kanamycin mRNA. Standardised threshold cycle (C t) values for IGF-I and IGF-II mRNA were achieved by real-time RT-PCR and used to create standard curves. To allow sample normalisation the standard curve was also established for-actin as internal calibrator (template: 86 nt), and validation experiments were performed demonstrating similar ampliWcation eYciencies for target and reference genes. Based on the standard curves, the absolute amounts of IGF-I and IGF-II mRNA were determined for liver (IGF-I: 8.90 § 1.90 pg/ g total RNA, IGF-II: 3.59 § 0.98 pg/ g total RNA) and extrahepatic sites, such as heart, kidney, intestine, spleen, gills, gonad, and brain considering the diVerent lengths of cRNAs and mRNAs by correction factors. The reliability of the method was conWrmed in additional experiments. The ampliWcation of descending dilutions of cRNA and total liver RNA resulted in parallel slopes of the ampliWcation curves. Furthermore, ampliWcation plots of the standard cRNA and the IGF-I and IGF-II mRNAs showed signals starting at the expected C t values. Thus, the one-tube RT-PCR described here is highly sensitive (detection level »2 pg/ g total RNA) and allows precise absolute quantiWcation. The method is rapid as there are neither separate reverse transcriptions nor post-ampliWcation steps, and can be executed with low risk of contamination. Therefore, it will be helpful when investigating gene expression in any species and tissue whenever absolute levels are of concern.
Transgenic Research, 2007
Several lines of growth hormone (GH)-overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 ± 0.75 ng/ml) in transgenic than in wild-type (15.01 ± 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2-times higher than in wild-type (16.0 ± 2.21 vs. 3.83 ± 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 ± 0.82 vs. 7.3 ± 0.49 pg/μg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 ± 0.02 vs. 0.16 ± 0.01 pg/μg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.
Molecular and Cellular Endocrinology, 2010
Contradictory studies suggest IGF-I in fish liver and gills is involved in osmoregulation, but nothing is known about the kidney and intestine's role nor about IGF-II's role in any organ. Tilapia were transferred from freshwater (FW) to seawater (SW) for 1 week (wk) and retransferred to FW for another week. At 4 h, 1 d, 2 d, 3 d and 1 wk after SW-transfer and FW-retransfer IGF-I, IGF-II and growth hormone receptor (GHR1) mRNA were measured by real-time PCR. Hepatic IGF-I, IGF-II and GHR1 mRNA were downregulated in parallel after SW-transfer, recovered and were again downregulated after FWretransfer. In gills, IGF-I, IGF-II and GHR1 were upregulated synchronously after SW-transfer and, partially also after FW-retransfer. The renal genes were downregulated after SW-transfer and partially upregulated after FW-retransfer. Persisting upregulation in intestinal IGF-I mRNA occurred after FW-retransfer. Thus, endocrine and auto/paracrine IGF-I and IGF-II seem to be involved in fish osmoregulation in an organ-specific manner.
Cell and Tissue Research, 2006
The cellular sites of insulin-like growth factor I (IGF-I) synthesis in the early developing tilapia (0-140 days post fertilization, DPF) were investigated. IGF-I mRNA and peptide appeared in liver as early as 4 DPF and in gastro-intestinal epithelial cells between 5-9 DPF. In exocrine pancreas, the expression of IGF-I started at 4 DPF and continued until 90 DPF. IGF-I production was detected in islets at 6 DPF in non-insulin cells and occurred throughout life. In renal tubules and ducts, IGF-I production started at 8 DPF. IGF-I production in chondrocytes had its onset at 4 DPF, was more pronounced in growing regions and was also found in adults. IGF-I mRNA and peptide appeared in the cytoplasm of skeletal muscle cells at 4 DPF. In gill chloride cells, IGF-I production started at 6 DPF. At 13 DPF, IGF-I was detected in cardiac myocytes. IGF-I-producing epidermal cells appeared at 5 DPF. In brain and ganglia, IGF-I was expressed in virtually all neurones from 6 to 29 DPF, their number decreasing with age. Neurosecretory IGF-I-immunoreactive axons were first seen in the neurohypophysis around 17 DPF. Endocrine cells of the adenohypophysis exhibited IGF-I mRNA at 28 DPF and IGF-I immunoreactivity at 40 DPF. Thus, IGF-I appeared early (4-5 DPF), first in liver, the main source of endocrine IGF-I, and then in organs involved in growth or metabolism. The expression of IGF-I was more pronounced during development than in juvenile and adult life. Local IGF-I therefore seems to have a high functional impact in early growth, metabolism and organogenesis.
General and …, 2007
Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identiWcation and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR ampliWcation, using gene speciWc oligonucleotides as ampliWcation primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated Wsh also were substantially induced over controls at 12 h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.