During fertilization the sea urchin egg undergoes a global wave of secretion (the cortical reaction) followed by a period of endocytotic activity involved in membrane retrieval. Using a monoclonal antibody (MAb 1223) that recognizes a 130-kDa glycoprotein found in both the egg and embryo of Strongylocentrotus purpuratus, we recently suggested that glycoproteins bearing the 1223 epitope are stored in the cortical vesicles of the egg, secreted during fertilization, and subsequently endocytosed and routed to yolk platelets, a possible site of degradation.In the current study, to determine whether fertilized eggs could internalize an antibody probe directed at the cell surface, we coupled MAb 1223 to colloidal gold and presented it to eggs that had been stripped of fertilization envelopes and hyaline layers. Gold-conjugated mouse IgG (preimmune), bovine serum albumin (BSA), and goat-antimouse IgG served as controls. Colloidal gold (8 and 15 nm) was prepared and conjugated to BSA or antibody as previously described. For localization of the 1223 antigen in unfertilized eggs MAb 1223-gold was diluted 1:20 in buffer and applied to sections of eggs embedded in Lowicryl K4M. To remove the envelopes and hyaline layers, 20-30 seconds after addition of sperm the eggs were suspended in ice-cold artificial sea water containing 25 mM EGTA and passed through a nylon screen (64 μm mesh). Subsequently, 1 ml aliquots of the denuded eggs [10% suspension (vol/vol)] were gravity settled, removed from the Ca2+-chelator and resuspended in complete sea water. After a second wash, 20 μl aliquots of the eggs were transferred to fresh 1.0 ml volumes of sea water equilibrated at 14°C in the presence of the individual gold probes. The final dilution of the gold probes in sea water was 1:100. After gentle mixing of the eggs and gold probes, at time points 5,15,30 and 60 minutes specimens were fixed and embedded in Spurr resin as previously described.
The Fertilization Reaction in the Sea-Urchin Egg. The Effect of Nicotine