Autophagy induction in sea urchin embryos exposed to gadolinium ions

Abstract

Sea urchin embryos are highly sensitive to several kinds of stressors, and able to activate different defense strategies. Gadolinium (Gd) is a metal of the lanthanide series of the elements: its chelates are employed as contrast agents for magnetic resonance imaging since the 1980s. Gd complexes are released in the aquatic environment, making Gd an emergent environmental pollutant. In this study we focused on the effects of Gd ions on sea urchin embryos development. The study was conducted looking at three different processes: general development, apoptosis and autophagy. At the whole morphological level, Paracentrotus lividus sea urchin embryos continuously exposed to Gd ions displayed morphological abnormalities and a significant inhibition of skeleton elongation and patterning. The study of apoptosis performed by immunofluorescence (IF) staining using an anti-cleaved-caspase-3 antibody on whole-mount embryos after 24h (gastrula) and 48h (pluteus) exposure showed no apoptotic induction. Autophagic processes were investigated by Western blot analysis of total lysates and IF staining (autophagosomes) on whole-mount embryos to detect LC3 protein and acridine orange (AO) vital staining to highlight the presence of acidic vesicular organelles (autophagolysosomes) in whole embryos. In particular, Western blots of embryos exposed to Gd showed a 2.6-fold increase relative to controls at 24h and a 4-fold increase at 48h, suggesting that the autophagic process is acting as a cell survival strategy to defend the developmental program. In agreement, AO vital staining and LC3 IF confirmed an increased number of autophagosomes and autophagolysosomes. Taken together, results show that autophagy is a molecular process activated in sea urchin embryos exposed to Gd ions. Work supported by University of Palermo FFR to MCR and partially by PO-FESR 2007-2013 DeCroMed Project to VM. Authors thank Mr. M. Biondo for technical assistance.