Paz Martínez¹ and Antoni Morros²

¹ Instituto de Biología Fundamental. Unidad de Inmunología, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) Spain.

² Unitat de Biofísica. Departament de Bioquímica i de Biologia Molecular,Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) Spain

Received 05/06/96; Accepted 06/18/96; On-line 07/01/96

7.1. Cholesterol efflux

A mechanism for free (un-esterified) cholesterol efflux from liposomes has been widely demonstrated, in which cholesterol molecules desorb from the donor lipid-water interface and diffuse throughout the aqueous phase until they collide with an acceptor particle (for reviews, see references 92, 93). Cholesterol may desorb very rapidly (half time of 30 minutes) from small unilamellar vesicles (SUV). The kinetics of cholesterol release from biological membranes has been described to be slower and cell-dependent (half time 1-50 hours).

The efflux of cholesterol from several mammalian biological membranes can be explained by the above mechanism. However, this process, due to the presence of an "unstirred" or immobile water layer around the cells, which is not present around vesicles, is more complex. This layer acts as a diffusion barrier between the cell surface and the "bulk" water; then, cholesterol concentrates in the surrounding layer until an acceptor enters this area and encounters it. Therefore, cholesterol efflux from membranes involves both the entrance of the cholesterol acceptor into the immobile water layer and collision with the desorbed cholesterol molecules (92-95). The high density of charged hydrophilic oligosacharide side-chains of glycoproteins of the glycocalyx region retains a layer of immobile water. The plasma membrane of sperm head exhibits a thick glycocalyx, consisting of glycoproteins anchored to the bilayer, extending 100-150 Å from the phospholipid-water interface. The morphological features of this glycocalyx are different in the acrosomal cap from those of the postacrosome (8).

Ejaculated non-capacitated sperm adsorb proteins in their surface, mainly of seminal plasma origin. The amount of one kind of coating protein that belongs to the family of spermadhesins is sufficient to cover one-third of the entire sperm head surface. Spermadhesins are acidic proteins of low molecular weight (15-17 kDa); these heparin binding proteins are carbohydrate- and zona pellucida-binding proteins and are thought to be adsorbed to this glycocalyx. Alternatively, spermadhesins and other coating proteins may interact and bind to phospholipids (96). They are involved in at least two important aspects of fertilization: sperm capacitation and sperm-egg interaction (97, 98). Spermadhesins seem to be related to the major proteins from bull seminal plasma, designated BSP (bovine seminal plasma), since they also have an affinity to heparin (99, 100).

It has been established both in vivo and in vitro that heparin or glycosaminoglycans capacitates sperm (101, 102). First and Parrish have suggested that capacitation of bovine sperm by glycosaminoglycans includes removal of decapacitation proteins from seminal plasma (15, 103, 104).

Removal of these proteins appears to be a prerequisite for the acrosome reaction. Ehrenwald et al. established that the rate of cholesterol exchange between lamellar liposomes and epididymal sperm is three to four times greater than for the ejaculated sperm (53). Taking all these data into consideration, the early events of sperm capacitation may be explained by the following (Figure 5): the layer of coating proteins covers the glycocalix zone, prevents the entrance of cholesterol acceptors and interferes with the removal of the cholesterol that might have migrated from sperm plasm membrane to the underlying viscous glycocalix.

Figure 5 - Hypothetical model for the mechanism of cholesterol efflux in human sperm capacitation. This model shows the phospholipid-water interface to which cholesterol migrates and the glycocalyx zone consisting of glycoproteins anchored to the plasma membrane. Ejaculated non-capacitated sperm adsorb several coating proteins on the external charged surface. These proteins progressively dissociate by binding to the glycosaminoglycans in the female genital tract or by in-vitro incubation with heparin. Then, some cholesterol acceptors (93), such as albumin, may bind cholesterol and the lipidic bilayer becomes more fusogenic. Some receptors for hormones (progesterone) or for oligosaccharides (mannose-ligand) become accessible after capacitation, and acrosome reaction may be induced by progesterone or by zona pellucida.

Once the coating proteins are removed from the surface of sperm, cholesterol acceptors are free to interact with the cholesterol which has already been removed from the sperm membrane, thus facilitating cholesterol efflux.

7.2. Effects of cholesterol efflux

Cholesterol is not randomly distributed within biological membranes. Some areas of membrane are cholesterol-rich whereas other domains are cholesterol-poor (36). After cholesterol efflux in capacitation, changes in the physical state of the sperm plasma membrane are observed: the lipid diffusibility becomes regionalized. A lipid re-distribution after capacitation would not be surprising, as cholesterol is known to alter dramatically the lateral domain organization of lipidic bilayers. Such reorganizations also affect the function of membrane proteins.

The anterior acrosome region of the human sperm plasma membrane, due to its high concentration of anti-fusogenic sterols, seems to be resistant to immediate fusion. It is the formation of sterol-depleted patches in the anterior acrosomal region that renders it susceptible to membrane fusion (51). Lipid re-distribution during capacitation appear to provide the fusogenic domains required for membrane fusion in the acrosome reaction (50).

One of the important consequences of cholesterol efflux is the massive influx of extracellular Ca²⁺, this is considered a prerequisite for the acrosome reaction to occur. The entrance of calcium may be a consequence of the changes in the fluidity of the membrane that renders the membrane more permeable to Ca²⁺. Alternatively, there is an opening of voltage-dependent Ca²⁺ channels, probably by stimulation of phospholipase C (105).

Increased intracellular Ca²⁺ concentration can trigger different pathways involved in the acrosome reaction: generation of diacylglycerol (DAG) through phospho-inositide breakdown; DAG stimulation of Ca²⁺ dependent phospholipase A2 and participation in the membranefusion itself. Phospholipase A2 action on phospholipids gives rise to lysophospholipids and arachidonic acid or other fatty acids, which are known to be highly fusogenic. (106, 107).

Ca²⁺ could also act directly on negatively charged membrane lipids, by neutralizing anionic phospholipids or cholesterol sulfate. This may induce membrane destabilization and fusogenic intermediates that are thought to induce a massive acrosome reaction in the vicinity of oocyte envelopes (49).