Proteases stimulate fertilization-like responses in starfish eggs.

Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (congruent to 30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodeoxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 nM) such that Ca influx would be reduce by > 10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.