Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 μM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.