FUSION OF ARTIFICIAL MEMBRANES WITH MAMMALIAN SPERMATOZOA - SPECIFIC INVOLVEMENT OF THE EQUATORIAL SEGMENT AFTER ACROSOME REACTION

The fusogenic properties of bovine and human spermatozoa membranes wer e investigated, using phospholipid bilayers (liposomes) as target memb ranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was v isualized by fluorescence microscopy, using fluorescent lipid vesicles . Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all s permatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as t riggered by calcium ionophore A23187 or zonae pellucidae (proteins), w hile the fluorescence distribution was mainly restricted to the equato rial segment (ES). However, with spermatozoa that had undergone a free ze/thawing cycle, domains other than ES also became labeled. Hence, th e redistribution of the lipid probes over the entire membrane occurrin g during lipid mixing with cryopreserved bovine sperm is probably rela ted to membrane perturbations caused by long-term cryopreservation. Fu sion with liposomes was governed by spermatozoa factors and required t he presence of acidic phospholipids like cardiolipin and phosphatidyls erine in the liposomal bilayer. Incorporation of the zwitterionic lipi d phosphatidylcholine in the vesicles inhibited the fusion reaction. F usion was pH dependent. The results indicate that the ES is the primar y domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds ) pp. 135-185, Raven Press, New York] to represent the putative, initi al fusion site for the oocyte.