A role for a TIMP-3-sensitive, Zn2+-dependent metalloprotease in mammaliangamete membrane fusion

During fertilization, sperm and egg plasma membranes adhere and then fuse b y a mechanism that is not well understood. Zinc metalloproteases are necess ary for some intercellular fusion events, for instance, cell-cell fusion in yeast. In this study we tested the effects of class-specific and family-sp ecific protease inhibitors on mouse gamete fusion. Capacitated, acrosome-re acted sperm and zona-free eggs were used in assays designed to define the e ffects of inhibitors on sperm-egg plasma membrane binding or fusion. Inhibi tors of the aspartic, cysteine, and serine protease classes had no effect o n sperm-egg binding or fusion. Both a synthetic metalloprotease substrate ( succinyl-Ala-Ala-Phe-amidomethylcoumarin) and the zinc chelator 1,10-phenan throline inhibited sperm-egg fusion but did not decrease sperm-egg binding. The fusion-inhibition effect of phenanthroline was reversible and activity of the inhibitable zinc metalloprotease was shown to be required during a short time window, the first 15 min after insemination. Tissue inhibitor of metalloprotease-3 and Ro 31-9790, specific inhibitors of zinc metalloprote ases in the matrixin and adamalysin families, also inhibited sperm-egg fusi on but not sperm-egg binding. These data indicate a role in gamete fusion f or one or more zinc metalloproteases of the matrixin and/or adamalysin fami lies that act after plasma membrane binding and before sperm-egg membrane f usion. (C) 2000 Academic Press.