Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm.

Abstract

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation-a process preceding acrosome :plasma-membrane fusion-markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization . Both proteolytic digestion and capacitation failed to augment PXB-or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32p_ labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigràting with cardiolipin and phosphatidic acid . Vermiform protrusions elicited by PXB in the outer lamellae of card iolipin-phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane . We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane ; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function .That phospholipids mediate fusion in diverse biological or model membrane-systems has been advocated by a number of investigators. Particularly, workers in reproductive biology have shown that fertilization is not prevented by the treatment of eggs with proteolytic or carbohydrate-hydrolyzing enzymes which leave phospholipids intact (26) . Moreover, biophysicists have established that phospholipid vesicles can fuse, not only with each other (44) but with natural membranes (23,55) . Certain lipids have a greater potential for fusion than others. For example, liposomes composed solely of phosphatidylcholine (PC) resist merger for hours, yet vesicle membranes incorporating anionic lipids fuse readily upon the introduction of divalent cations (44) .The requirement for negatively charged lipids in the events of membrane :membrane fusion has been demonstrated in various systems-e.g., the entry of Semliki Forest virus into cells via the viral envelope-membrane union with the cell's lyso-604

Plasma-membrane diversity in a highly polarized cell.

“…But biochemical data suggest that, in addition, concurrent anionic-phospholipid synthesis takes place in sperm (30) . And comparing the information derived from PXB binding, which recognizes outer-leaflet lipids, and adriamycin, which does cross the bilayer, we can but conclude that the cytochemically detected differences in anionic-lipid concentration in adjacent domains of the membrane are also real (14,27,30) (Fig . 12).…”

“…In thin sections, the membranes of the two zones are similarly composed of a trilaminar unit-membrane, although the acrosomal-cap portion does exhibit a thick glycocalyx, itself separable into two layers (14) . Freeze-fracture preparations of sperm removed from the tail of the epididymis expose the typical feature of the fusigenic acrosomal cap of the membrane-the quiltlike pattern which mirrors the glycocalyx (Figs .…”

“…This probe reveals that the fusigenic portion has a greater degree of fluidity than the post-acrosomal segment: fluorescence, initially confined to the tip of the acrosomal cap, extends to the equatorial segment after the cells have been treated with a crude trypsin preparation or have become capacitated (14) .…”

“…Like the sterols, acidic phospholipids demonstrable by polymyxin-B (PXB) binding and membrane perturbation are also far more numerous over the anterior part of the acrosomal cap than over the post-acrosomal sector (14,30) (Fig . 12) .…”

Capacitation, acrosome function and chromatin structure in stallion sperm

“…The most important aspects of capacitation are the changes that enable a sperm to recognize the ZP and undergo the AR in response to ZP-binding; these changes include the facilitation of calcium entry into the cell (Baldi et al, 1991;Okamura et al, 1993;Parrish et al, 1999), increased intracellular calcium and cAMP levels (Visconti and Kopf, 1998;Florman et al, 1989;Visconti et al, 1990), increased membrane fluidity secondary to alterations in lipid composition and architecture (Bearer and Friend, 1982;Langlais and Roberts, 1985;Harrison et al, 1996;Flesch and Gadella, 2000;Harrison and Gadella, 2005) and changes in metabolic activity leading to the acquisition of hypermotility (Yanagimachi, 1994;Nassar et al, 1999). Once capacitated, a sperm cell can bind to the ZP and respond to this binding by undergoing the AR, an exocytotic event that is instrumental to ZP-penetration and subsequent fusion of the sperm plasma membrane with the oolemma.…”