Studies of molecular mechanisms for activation of amphibian eggs.

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In this study, we have purified a sperm agonist from sperm of the newt, Cynops pyrrhogaster to activate eggs of the frog, Xenopus laevis. By the purification with several chromatographies, we have found that the agonist is a sperm protease which shares properties of serine-proteases and have a molecular weight of 170 kDa. The sperm protease has a sugar moiety recognized by SBA-lectin and is localized at the acrosomal region of sperm heads. The inhibition of the protease activity by specific inhibitors or substrates caused the inhibition of egg activation, indicating its important role for activation of Xenopus eggs by Cynops sperm. Since we have purified it as a single band under SDS-PAGE,we are going to make a monoclonal antibody against it or to obtain c-DNA which encodes it to clarify the similar molecules in other vertebrates. On the other hand, the sperm agonist open Cl ion channels as well as Na ion channels at egg activation. We have demonstrated that Ca eflux through Ca channels on the egg plasm membrane is necessary for activation by the sperm agonist by detailed analysis of opening of ion channels by electrophysiological methods. Furthermore, increased Ca ions stimulate a propagative Ca-release dependent upon IP_3-receptors on endoplasmic reticulum.