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Abstract: In bony fish, IGF-I released from the liver under the control of pituitary GH is the main endocrine regulator of growth, maintenance and development, and the amount of circulating IGF-I regulates synthesis and release of GH. In mammals and amphibia, evidence indicates that anterior pituitary endocrine cells also contain IGF-I. However, only preliminary and conflicting data exist on IGF-I gene expression in bony fish pituitary. Thus, we investigated the presence of IGF-I in the tilapia (Oreochromis niloticus) pituitary by quantitative real-time RT-PCR, in situ hybridisation and immunohistochemistry. The absolute amount of IGF-I mRNA in the whole pituitary (7.4±3.3×10−3 pg/μg total RNA) was 1000-times lower than in liver (7.5±3.1pg/μg total RNA). IGF-I peptide occurred in both neuro- and adenohypophysis but IGF-I gene expression was mainly restricted to the adenohypophysis. In the neurohypophysis, only few cells, probably pituicytes, contained IGF-I mRNA whereas IGF-I peptide was found also in numerous axons in the pars nervosa. In the adenohypophysis, both IGF-I mRNA and peptide were present in the majority of ACTH cells in all individuals investigated. In α-MSH cells, only IGF-I mRNA but no IGF-I peptide was detected likely suggesting an immediate release of IGF-I after synthesis. IGF-I mRNA and peptide were further observed in GH cells but their presence showed pronounced inter-individual differences likely due to the physiological, e.g., nutritional, status of the individual. IGF-I released from the GH cells may serve as auto/paracrine mediator of a negative feedback mechanism in addition to liver-derived endocrine IGF-I. Generally, the constitutive synthesis of IGF-I in ACTH cells and the varying content in GH and α-MSH cells suggest particular roles for IGF-I. Local IGF-I may regulate synthesis and release of pituitary hormones in an autocrine and/or paracrine manner as well as prevent apoptosis and stimulate proliferation of endocrine cells. [Copyright &y& Elsevier]

Abstract: Recently, in addition to IGF-1 and IGF-2 the existence of a third form of IGF, termed IGF-3, limited to fishes, to be present only in the gonads and encoded by a separate gene has been reported. However, no further data have been presented on IGF-3. The present study on tilapia (Oreochromis niloticus) uses quantitative real-time PCR specific for tilapia IGF-1 and IGF-3. The organ distribution of IGF-3 mRNA in adult fish and the early ontogeny of IGF-3 in male and female gonads were studied. The potential sensitivity of IGF-3 to GH was revealed by intraperitoneal injections of bream GH using IGF-1 as control gene. The effects of 17α-ethinylestradiol (EE2) exerted after feeding of high EE2 doses and exposure to low environmentally relevant EE2 doses on IGF-3 expression in testis and ovary during early development were determined. Low IGF-3 mRNA expression levels were detected in most organs studied, with the highest extra-gonadal amount in the pituitary. During development, the IGF-3 gene was significantly upregulated in male but downregulated in female gonad. Injections of GH elevated IGF-1 mRNA in male and female liver and ovary. IGF-3 did not respond to GH treatment neither in ovary nor in testis. Both EE2 treatments resulted in significant downregulations of IGF-3 mRNA in testis while ovarian IGF-3 mRNA did not respond. Thus, IGF-3 may be involved in reproduction of fishes most likely in the male gonad only. Whether IGF-3 also has some physiological significance in ovary or other organs should be the topic of further studies. [Copyright &y& Elsevier]

Intensified aquaculture has strong impact on fish health by stress and infectious diseases and has stimulated the interest in the orchestration of cytokines and growth factors, particularly their influence by environmental factors, however, only scarce data are available on the GH/IGF-system, central physiological system for development and tissue shaping. Most recently, the capability of the host to cope with tissue damage has been postulated as critical for survival. Thus, the present study assessed the combined impacts of estrogens and bacterial infection on the insulin-like growth factors (IGF) and tumor-necrosis factor (TNF)-α. Juvenile rainbow trout were exposed to 2 different concentrations of 17β-estradiol (E2) and infected with Yersinia ruckeri . Gene expressions of IGF-I, IGF-II and TNF-α were measured in liver, head kidney and spleen and all 4 estrogen receptors (ERα1, ERα2, ERβ1 and ERβ2) known in rainbow trout were measured in liver. After 5 weeks of E2 treatment, hepatic up-regulation of ERα1 and ERα2, but down-regulation of ERß1 and ERß2 were observed in those groups receiving E2-enriched food. In liver, the results further indicate a suppressive effect of Yersinia -infection regardless of E2-treatment on day 3, but not of E2-treatment on IGF-I whilst TNF-α gene expression was not influenced by Yersinia -infection but was reduced after 5 weeks of E2-treatment. In spleen, the results show a stimulatory effect of Yersinia -infection, but not of E2-treatment on both, IGF-I and TNF-α gene expressions. In head kidney, E2 strongly suppressed both, IGF-I and TNF-α. To summarise, the treatment effects were tissue- and treatment-specific and point to a relevant role of IGF-I in infection. [ABSTRACT FROM AUTHOR]

Oestrogens and insulin-like growth factors (Igfs) play both a central role in the regulation of reproduction and growth and can interact especially in species showing a clear-cut sex-linked growth dimorphism (SGD) like in tilapia. Aromatase is essential in ovarian differentiation and oogenesis since it controls oestrogen synthesis. During tilapia sex differentiation, aromatase cyp19a1a expression increases from 9 days post-fertilization (dpf), resulting in high oestradiol level. High temperature, exogenous androgens or aromatase inhibitors override genetic sex differentiation inducing testes development through the suppression of cyp19a1a gene expression and aromatase activity. Supplementation with 17ß-oestradiol (E2) of gonadectomized juveniles induced a sustained and higher E2 plasma level than in intact or gonadectomized controls and both sexes showed reduced growth. Juvenile and mature females treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione had 19% lower E2 plasma level compared to controls and they showed a 32% increased growth after 28 days of treatment. Altogether, these data suggest that E2 inhibits female growth leading to the SGD. Regarding Igf-1, mRNA and peptide appeared in liver at ∼4 dpf and then in organs involved in growth and metabolism, indicating a role in early growth, metabolism and organogenesis. Gonad igf-1 showed an early expression and the peptide could be detected at ∼7 dpf in somatic cells. It appeared in germ cells at the onset of ovarian (29 dpf) and testicular (52 dpf) meiosis. In testis, Igf-1 together with steroids may regulate spermatogenesis whereas in ovary it participates in steroidogenesis regulation. Igf-1 and Igf-2 promote proliferation of follicular cells and oocyte maturation. Igf-3 expression is gonad specific and localized in the ovarian granulosa or testicular interstitial cells. In developing gonads igf-3 is up-regulated in males but down-regulated in females. In contrast, bream Gh injections increased igf-1 mRNA in male and female liver and ovaries but gonadal igf-3 was not affected. Thus, local Igf-1 and Igf-2 may play crucial roles in the formation, development and function of gonads while Igf-3 depending on the species is involved in male and female reproduction. Furthermore, precocious ethynylestradiol (EE) exposure induced lasting effects on growth, through pituitary gh inhibition, local suppression of igf-1 expression and in testis only down-regulation of igf-3 mRNA. In conclusion, SGD in tilapia may be driven through an inhibitory effect due to E2 synthesis in female and involving Igfs regulation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Diabetes mellitus is on the rise in domestic cats and resembles T2D in humans. [•] We experimentally induced insulin suppression in serum and β-cells of healthy cats. [•] We observed plasma and urinary ketones related to the induced insulin suppression. [•] We for the first time localize IGF-I in distinct feline islet cells of Langerhans. [•] We propose a protective effect of IGF-I within the β-cells against hyperglycaemia. [ABSTRACT FROM AUTHOR]

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 fish, 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 5′end 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 verified (IGF-I: 91 nt; IGF-II: 94 nt). The PCR products were used to create IGF-I and IGF-II cRNAs which were quantified in dot blot by comparison with defined amounts of standardised kanamycin mRNA. Standardised threshold cycle (Ct) 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 β-actin as internal calibrator (template: 86 nt), and validation experiments were performed demonstrating similar amplification efficiencies 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/μg total RNA, IGF-II: 3.59 ± 0.98 pg/μg total RNA) and extrahepatic sites, such as heart, kidney, intestine, spleen, gills, gonad, and brain considering the different lengths of cRNAs and mRNAs by correction factors. The reliability of the method was confirmed in additional experiments. The amplification of descending dilutions of cRNA and total liver RNA resulted in parallel slopes of the amplification curves. Furthermore, amplification plots of the standard cRNA and the IGF-I and IGF-II mRNAs showed signals starting at the expected Ct values. Thus, the one-tube RT-PCR described here is highly sensitive (detection level ∼2 pg/μg total RNA) and allows precise absolute quantification. The method is rapid as there are neither separate reverse transcriptions nor post-amplification 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. [Copyright &y& Elsevier]