PROTEASES STIMULATE FERTILIZATION-LIKE RESPONSES IN STARFISH EGGS

Application of proteases to eggs of the starfish, Asterina miniata, ca used several responses like those seen at fertilization. Cortical gran ule exocytosis and fertilization envelope elevation occurred within ab out 1 min after exposure to trypsin, chymotrypsin, or pronase; proteas e inhibitors prevented these responses. Kallikrein caused cortical gra nule exocytosis and fertilization envelope elevation, but this respons e required more time (similar 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, chymotryp sin, or pronase caused an increase in intracellular free calcium, dete cted by calcium green dextran, and stimulation of DNA synthesis, detec ted by incorporation of bromodeoxyuridine. Exocytosis also occurred wh en trypsin or chymotrypsin was applied in artificial sea water in whic h the free calcium was reduced to a low level (40-70 nM) such that Ca influx would be reduced by > 10,000-fold; this indicated that the prot eases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extrace llularly exposed protein that when proteolyzed can induce fertilizatio n-like responses; this protein may be a receptor that transduces a sig nal from the sperm to initiate egg activation at fertilization. (C) 19 95 Academic Press, Inc.