A possible role of autophagy for the execution of apoptosis in cadmium-exposed sea urchin embryos

The sea urchin embryo is a suitable model system that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium treatment provokes the accumulation of metal in dose- and time-dependent manner in embryonic cells and the activation of defence systems, such as the synthesis of HSPs and/or the initiation of apoptosis. Analysing autophagy, by neutral red, acridine orange and LC3-detection, we demonstrated that Cd-exposed embryos adopt this process as an additional stratagem to safeguard the developmental program. We observed that embryos treated with subletal Cd concentration activate a massive autophagic response after 18h of treatment. In addition, autophagy decreases between 21 and 24h, in the opposite of apoptotic process. In order to investigate a possible temporal relationship between autophagy and apoptosis, we tested apoptosis by immunodetection in situ of cleaved caspase-3 and TUNEL assays. We showed that embryos activate a massive apoptosis after 24h of Cd exposure. In addition, a functional relationship between autophagy and apoptosis was estimated evaluating apoptosis in Cd-exposed embryos with inibited autophagy, by treatment with 3-methyladenine (3-MA). We found that the inhibition of autophagy produced a reduction of apoptotic signals, suggesting that the two phenomena are functionally related. Considering the catabolic role of autophagy, an energetic hypothesis to explain this relationship was evaluated; in this case autophagy could contribute to apoptotic process providing ATP, necessary for the execution of the apoptotic programme. In effect, using methylpyruvate (MP), a substrate for ATP production, in embryos with inhibited autophagy, apoptosis was substantially restored. In this context, autophagy could play a crucial role in stress response of this suitable model system.