Cellular localisation of insulin-like growth factor binding protein-2 (IGFBP-2) during development of the marine fish, Sparus aurata

Abstract

The spatial localisation of insulin-like growth-factor-binding protein-2 (IGFBP-2) and its mRNA was investigated during larval and post-larval developmental stages of the gilthead seabream ( Sparus aurata) by immunohistochemistry and in situ hybridisation with specific antisera and riboprobes. During larval development, immunoreactivity was found in skin, muscle, gills, pharynx, intestine, liver and olfactory epithelium. After metamorphosis, immunoreactivity was found in the oesophageal epithelium (the strongest reaction) and in red skeletal muscle, heart muscle, the thymus and the epithelium of renal tubules. In the adult, immunostaining with IGFBP-2 antibody was also found in the saccus vasculosus, ovary and testis. IGFBP-2 mRNA was detected by in situ hybridisation mainly in the intestine, skeletal musculature and ovary. These results show that IGFBP-2 protein and mRNA are expressed in a variety of seabream tissues, suggesting that IGFBP-2 regulates the actions of IGFs on these tissues during development and growth.

Real-time polymerase chain reaction, in situ hybridization and immunohistochemical localization of insulin-like growth factor-I and myostatin during development of Dicentrarchus labrax (Pisces: Osteichthyes)

“…Following hydration in graded methanol, larvae were processed as described earlier (Radaelli et al 2005) with slight modifications. Briefly, after incubation in 6% H 2 O 2 for 15 min, larvae were permeabilized by treatment with 10 μg/ml proteinase K (Roche) in PBS containing 0.1% Tween 20 (PBT) for about 5 min (duration of treatment depended on the size of the sample), washed in PBT and fixed again in 4% paraformaldehyde plus 0.2% glutaraldehyde for 20 min before the hybridization step.…”

“…Frozen sections Sections of larvae (as above) and fry (80 dph) were processed for ISH experiments as described elsewhere (Radaelli et al 2005) with the following modifications. The riboprobes (500 ng/ml final concentration) were resuspended in the following hybridization buffer: 50% (deionized) formamide, 1× SSC, 10% dextran sulfate, 1× Denhardt's solution, 0.67 M NaCl, 0.1 μg/μl yeast tRNA, and 0.1 μg/μl herring sperm DNA.…”

Immunocytochemical Tools Reveal a New Research Field Between the Boundaries of Immunology and Reproductive Biology in Teleosts

“…Especially in the gilthead seabream, numerous antibodies have been obtained in the recent years, particularly those specific for studying the endocrine regulation of multiple biological processes such as reproduction, growth, metabolism, etc. (Abad et al, 1992;García-Ayala et al, 2003;Modig et al, 2008;Morgado et al, 2007;Picchietti et al, 2007;Pinto et al, 2009;Pirone et al, 2008;Radaelli et al, 2003Radaelli et al, , 2005Santos et al, 2001;Villaplana et al, 1997). Regarding the gilthead seabream immune response, an array of monoclonal and polyclonal antibodies have been developed for leukocyte markers (Sepulcre et al, 2002), immune molecules ( Corrales et al, 2010;García-Castillo et al, 2004;Pelegrín et al, 2004) or immunoglobulins (Picchietti et al, 2006).…”

Applications of Immunocytochemistry

“…Especially in the gilthead seabream, numerous antibodies have been obtained in the recent years, particularly those specific for studying the endocrine regulation of multiple biological processes such as reproduction, growth, metabolism, etc. (Abad et al, 1992;García-Ayala et al, 2003;Modig et al, 2008;Morgado et al, 2007;Picchietti et al, 2007;Pinto et al, 2009;Pirone et al, 2008;Radaelli et al, 2003Radaelli et al, , 2005Santos et al, 2001;Villaplana et al, 1997). Regarding the gilthead seabream immune response, an array of monoclonal and polyclonal antibodies have been developed for leukocyte markers (Sepulcre et al, 2002), immune molecules ( Corrales et al, 2010;García-Castillo et al, 2004;Pelegrín et al, 2004) or immunoglobulins (Picchietti et al, 2006).…”