Strategie di difesa attivate in risposta a stress, in embrioni di Paracentrotus lividus

Abstract

Sea urchin embryos are able to activate different defense strategies against stress. This model system allows to investigate numerous phenomena in multipotent cells, which interact among themselves, in their natural position, bypassing the disadvantages of isolated cells, deprived of their normal network. Cadmium (Cd) treatment triggers the accumulation of metal in embryonic cells and the activation of defense systems depending on concentration and exposure time, through the synthesis of heat shock proteins and/or the initiation of apoptosis. Here we show that Paracentrotus lividus embryos exposed to subacute/sublethal concentrations of Cd adopt autophagy as an additional stratagem to safeguard the developmental program and that a temporal/functional relationship between autophagy and apoptosis exists. In this work we employed Cd as a stressor for the induction of autophagy purely as a toxic insult and it in no way constitutes an environmental stressor, as the concentration of Cd is many orders of magnitude higher than would be found in a polluted situation. Using colorimetric and fluorimetric acidotropic dyes we found that embryos exposed to Cd display massive punctiform spots in the cytoplasm, indicative of acidic vesicular organelles (AVOs). Furthermore these data were validated through both protein gel blotting and immunofluorescence in situ analysis of LC3, a specific marker of autophagy. We found that in P. lividus embryos autophagic processes occur in greater amounts after Cd exposure and in specific phases of the physiological development. These results have been confirmed using bafilomycin A1 and 3-methyladenine, known inhibitors of this process. In particular we observed that embryos treated with sublethal Cd concentration activate a massive autophagic response after 18h, which decreases between 21 and 24 h, in the opposite of apoptotic process. In order to investigate a possible temporal relationship between autophagy and apoptosis, we tested apoptotic signals by TUNEL and immunofluorescence in situ assays of cleaved caspase-3. Quantitative analysis has shown that embryos activate a massive apoptosis after 24h of Cd-exposure. These results have been confirmed using Z-DEVD-FMK, a specific inhibitor of apoptosis. Therefore a functional relationship between autophagy and apoptosis was estimated evaluating apoptotic signals in Cd-exposed embryos, upon treatment with the autophagic inhibitor 3-methyladenine. We found that the inhibition of autophagy produced a contemporaneous reduction of apoptotic signals, suggesting that the two phenomena are functionally related. In effect using methylpyruvate, a cell-permeable substrate for ATP production, apoptotic signals were substantially restored. These data could be explained considering that autophagy could energetically contribute to apoptotic execution through its catabolic role.