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  • Author: B Funkenstein x
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GROWTH HORMONE INCREASES PLASMA LEVELS OF INSULIN-LIKE GROWTH FACTOR (IGF-I) IN A TELEOST, THE GILTHEAD SEABREAM (SPARUS AURATA)

B. Funkenstein, A. Silbergeld, B. Cavari, and Z. Laron

ABSTRACT

A heterologous radioimmunoassay (RIA) was applied for the determination of immunoreactive (IR)insulin-like growth factor (IGF-I) in a teleost, the gilthead seabream (Sparus aurata). Serial dilutions of the fish plasma gave a linear curve when added to constant amounts of 125I-labelled human IGF-I(53-70) and antiserum prepared against this fragment. The RIA was used to study the effect of GH on plasma levels of IR-IGF-I in S. aurata. A single injection of human recombinant GH (1 μg/g) resulted in a significant increase in IR-IGF-I at 29, 48 and 72 h, when compared with saline-injected fish. This novel observation suggests that in fish, as in mammals, circulating IGF-I levels are modulated by GH.

DOI:
https://doi.org/10.1677/joe.0.120R019
Volume 120: Issue 2,
Pages:
R19–R21
Free access

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

G Radaelli, M Patruno, L Maccatrozzo, and B Funkenstein

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.

DOI:
https://doi.org/10.1677/joe.0.1780285
Volume 178: Issue 2,
Pages:
285–299 (15 total)
Restricted access

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

M Fine, R Amuly, Y Sandowski, T A Marchant, S J Chan, A Gertler, and B Funkenstein

Abstract

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 ∼200-fold lower than that of hIGF-I. 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.

Journal of Endocrinology (1997) 153, 139–150

DOI:
https://doi.org/10.1677/joe.0.1530139
Volume 153: Issue 1,
Pages:
139–150 (12 total)