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

4.1. Cholesterol efflux from sperm membrane

Plasma membranes of ejaculated sperm are considered to be destabilized by loss of cholesterol from the membrane during the course of capacitation (12,43). Cholesterol is known to regulate the fluidity of membrane lipid bilayers and the permeability of membrane (44, 45) and to modulate the lateral mobility of integral proteins and functional receptors within the membrane. The "flip-flop" of phospholipids, that is to say, their passage from one leaflet of the bilayer to the other, is markedly restricted by the increasing rigidity and order conferred on lipid bilayers by cholesterol (11). Davis was the first to report that removal of membrane cholesterol, with a consequent decrease in the cholesterol/phospholipid ratio, constitutes an important step in sperm capacitation (46). Hoshi et al.. reported the existence of a close relationship between human sperm capacitation and the cholesterol/phospholipid ratio: lower ratios correspond to faster sperm capacitation, as indicated by the sperm penetration assay (43).

Cross reported that human spermatozoa in the continued presence of seminal plasma do not become acrosomally responsive. The purified inhibitory activity in seminal plasma was identified as that of cholesterol on the basis of its solubility in organic solvent, its chromatographic behavior and its mass spectrum (47). This cholesterol from plasma membrane is thought to be transferred to high density lipoproteins present in the oviductal fluid in the female genital tract, thus promoting capacitation.

Part of the cholesterol in the plasma membrane is present in the form of sulfoconjugates.

The cholesterol sulfate accounts for only 2% of the total sterol present in human sperm; however, as it is concentrated in the membranes overlying the acrosome, it probably accounts for as much as 20% of the sperm head surface and is likely to contribute to membrane stability in this region (37, 40).

Cholesterol sulfates inhibit the ability of capacitated rabbit sperm to penetrate the zona pellucida, because of their membrane-stabilizing properties (48). The difference between cholesterol sulfate and unesterified cholesterol may be due in part to the different polar headgroups of these two molecules. Cholesterol has a relatively small polar group: a poorly hydrated hydroxyl group; in contrast, cholesterol sulfate has a large charged and hydrated polar sulfate group. Consequently, cholesterol sulfate stabilizes the membrane bilayer, thus avoiding disruption to a non-bilayer organization (37, 49). The action of the cumulus steroid sulfatases on steroid or cholesterol sulfate has been postulated to be part of the mechanism of capacitation (11). Cholesterol removal, after desulfation, is thought to increase membrane fluidity and to allow greater lateral movements of integral membrane proteins. This is associated with a greater permeability to calcium, which is apparently the key trigger in the acrosome reaction (48). Alternatively, factors that serve to neutralize the charge of sulfate groups, such as high ionic strength or binding of divalent cations appear to reduce the bilayer stabilizing capacity of cholesterol sulfate (49).

A question arises concerning the regions of sperm membrane from which cholesterol is released. Electron microscopic evaluation of freeze-fractured filipin-treated guinea pig spermatozoa cultured under capacitating conditions revealed a loss of cholesterol from fusogenic areas of the plasma membrane overlying the acrosome (8, 50). Cholesterol in both the anterior and the equatorial regions of human sperm head was also detected by Tesarík and Fléchon by examinig filipin-cholesterol complexes. Acrosomal plasma membrane is probably prevented from fusion with the acrosomal outer membrane by its high concentration in anti-fusogenic sterols. During in vitro capacitation, cholesterol-free patches are developed, these patches becoming the only sites favorable to the initiation of membrane fusion (51). The periacrosomal plasma membrane, which is rich in cholesterol (51, 52), is the region where membrane fusion takes place during the acrosome reaction. It is from this region that cholesterol is preferentially removed (53).

4.2. Cholesterol acceptors

Albumin and high-density lipoproteins present in uterine fluid, follicular fluid, or an in-vitro medium containing serum albumin act as sterol acceptors and induce cholesterol efflux from sperm membrane (46, 54). Human sperm cholesterol efflux after 4 hours of capacitation in human follicular fluid or serum is about 50% of the total sperm-bound cholesterol (54). On the other hand, cholesterol-enriched albumins, whose sterol binding sites are saturated, inhibit fertilization in a sterol concentration-dependent manner (55, 56). Following a 60 minute incubation with bovine serum albumin (BSA), mouse spermatozoa become depleted (29%-50%) of sterol in a time-dependent manner (55).

For in-vitro capacitation, cholesterol-free liposomes have also been used. Ehrenwald et al. (53) showed 28%-31% cholesterol removal from bovine spermatozoa by incubating them for 90 minutes in the presence of cholesterol-free liposomes. Incubation of those spermatozoa with lysophosphatidylcholine resulted in acrosome reaction in 40% of cells as compared with a 14% acrosome reaction in the control spermatozoa whose cholesterol was not removed. Ehrenwald et al.(57) also demonstrated that the capacity of bovine sperm to penetrate both zona-free hamster oocytes and in vitro matured bovine oocytes was markedly increased by a reduction in cholesterol of sperm membrane. These results suggested that cholesterol efflux may be an early step in bovine sperm capacitation.

Although it is well established that albumin itself can function as a sterol acceptor from living mammalian cells (58), Ravnick et al. (59) presented evidence that the lipid transfer protein-1 (LTP-1) is responsible for this activity. They suggested that the variable ability of albumin preparations to support in-vitro sperm capacitation is largely attributable to the presence of the contaminating LTP-1. They proposed that this protein, which is present in human follicular fluid, may contribute to the capacitation of sperm (60).