INTRODUCTION In many mammalian species, sperm-egg interactions and mutual activation are mediated by the zona pellucida, the acellular gly- coprotein coat of the egg (reviewed by Saling et al., 1990). After the spermatozoon has penetrated the cumulus oophorous of the ovum, it binds to the zona pellucida with its plasma membrane intact. Sperm binding to the zona pellucida occurs via specific receptors to a zona pellucida glycoprotein (ZP3 in the mouse) which are localized over the anterior head region of the sperm. Zona pellucida binding stimulates the sperm to undergo the acrosome reaction. The latter is a stimulus-secretion coupled exocytotic event in which the exocytotic vesicle (the acrosome) fuses with the overlying plasma membrane (reviewed by Yanagamachi, 1994). The multiple fusions between the outer acrosomal membrane and the plasma membrane result in the release of hydrolytic enzymes (mostly acrosin) and in the exposure of new membrane domains, both of which are essential for fertilization to proceed further. The hydrolytic enzymes released from the acrosome digest the zona pellucida, allowing the sperm to approach the egg and fertilize it. Acrosome reaction will follow zona pellucida binding only if the sperm has previ- ously undergone a poorly defined process of maturation known as capacitation. Capacitation occurs in vivo upon exposure of the sperm to the female reproductive tract, or in vitro in the presence of various synthetic media (Yanagamachi, 1994). Acrosome reaction can be induced in vitro in capacitated sperm by incubation with solubilized zona pellucida. The ensuing signal transduction cascade invokes GTP-binding proteins (G-proteins), and receptor coupled tyrosine kinases (Naz and Ahmed, 1992; Damjanov et al., 1993; Lax et al., 1994). The activities of these lead to an increase in intracellu- lar calcium concentration followed by a rise in pH (reviewed by Florman et al., 1990) both of which are believed to be an essential component of the signal transduction cascade linking zona pellucida binding to acrosomal exocytosis. The elevation in Ca 2+ and pH have been implicated in activating phospholi- pases and protein kinase C (PKC). Acrosome reaction can also be induced in vitro by ionophores such as A23187 which exchange Ca 2+ for 2H + . In the case of ionophore induced acrosome reaction, the signal transduction cascade up to the elevation of intracellular calcium is bypassed, but the resulting acrosome reaction is morphologically indistinguishable from that induced by zona pellucida. The ability to induce acrosome reaction with solubilized zona pellucida as well as with calcium ionophores has permitted a partial temporal dissection of the acrosome reaction into two stages, namely upstream and downstream to Ca 2+ elevation. Thus, pertussis toxin (an inactivator of G i -like proteins) inhibits zona pellucida- but not ionophore-induced acrosome reaction (Endo et al., 1987), showing that G i -like proteins are involved only upstream to the Ca 2+ elevation. 2525 We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphopro- teins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and G i -like proteins, while involved in exo- cytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholi- pase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion. Key words: sperm, acrosome reaction, membrane fusion, exocytosis, phospholipase C, F-actin, cytoskeleton SUMMARY Sperm exocytosis reconstructed in a cell-free system: evidence for the involvement of phospholipase C and actin filaments in membrane fusion Ben Spungin*, Ilana Margalit and Haim Breitbart Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel *Author for correspondence Journal of Cell Science 108, 2525-2535 (1995) Printed in Great Britain © The Company of Biologists Limited 1995