Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization

Egg activation in cross-fertilization between Xenopus eggs and Cynops sperm may be caused by a protease activity against Boc-Gly-Arg-Arg-MCA in the sperm acrosome. To determine the role of the sperm protease in fertilization, the protease was purified from Cynops sperm using several chromatographic techniques. We found that purified sperm protease readily hydrolyzes Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that protease activity was inhibited by the trypsin inhibitors aprotinin and leupeptin, and that not only the purified protease, but also cathepsin B, induces activation in Xenopus eggs. We inseminated unfertilized Xenopus eggs with homologous sperm in the presence of various peptidyl MCA substrates or protease inhibitors and demonstrated that trypsin inhibitors or MCA substrates containing Arg-Arg-MCA reversibly inhibited fertilization of both fully jellied and denuded eggs. Sperm motility was not affected by the reagents. An extract obtained from Xenopus sperm showed hydrolytic activity against Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA. These results suggest that the tryptic protease in Xenopus sperm is involved in fertilization, most likely by participating in egg activation.