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

5.1. Macrodomains and microdomains

When describing membranes of specialized cells with highly regionalized functions, macrodomains are defined; they correspond to morphologically distinguishable large membrane areas. Extending from the submicron level to the molecular scale, microdomains based on different lipid properties are also detected in cell membranes (61-64). Spermatozoa are a prime example of regionalized cells with specific functions localized in distinct regions: acrosome reaction takes place in the anterior region of the head, and sperm-oocyte plasma membrane fusion occurs in the equatorial region of the head. Motion energy from mitochondria occurs in the midpiece, while motility is exhibited in the tail. This regionalization affects the distribution of both plasma membrane lipids and proteins.

Lipid composition and lipid diffusibility in plasma membrane appears to be different for each of these distinct morphological regions of spermatozoa. Two macrodomains of different composition are observed in the head of spermatozoa from epididymis by means of freeze-fracture: the acrosomal cap and the post-acrosomal segment. The acrosomal cap portion of the plasma membrane is highly fusogenic and during acrosome reaction it fuses with the underlying acrosomal membrane. On the contrary, the post-acrosomal segment of the plasma membrane is non-fusogenic under the same conditions (8). Evidence of barriers to membrane protein and lipid lateral diffusion has been found at the equatorial region (65-67).

During capacitation and acrosome reaction, proteins are relocated. The migration of some proteins, such as PH-20, during acrosome reaction may partially result from an alteration of the barriers mentioned above (68, 69). SP-10 protein, a testis-specific acrosomal protein, is redistributed in capacitating sperm (70); SP-10 is detected in the equatorial segment and in the inner acrosomal membrane after acrosome reaction. Protein reorganization and clustering during capacitation has been shown in the acrosomal region of boar sperm by immunoelectron microscopy (71). A 78 kDa sperm antigen becomes clustered over the principal segment of the acrosome and it is localized in the postacrosomal and equatorial region after the acrosome reaction. During capacitation, a rat epididymal protein of 37 kD which is associated with sperm surface during maturation, migrates to the equatorial segment (72).

5.2. Biophysical, biochemical and cytochemical techniques for the analysis of lipid regionalization in the plasma membrane of sperm

In electron microscopy studies of freeze-fracture preparations, the polyene filipin has been widely employed as a probe of membrane structure because of its selective affinity for cholesterol and its sensitivity to the membrane-sidedness of sterol. Friend (8) has applied this technique to the study of guinea pig sperm membrane and he concluded that cholesterol is more abundant (4-fold) over the acrosomal cap region than over the post-acrosomal segment. Moreover, the inner fusogenic leaflet of the plasma membrane over the acrosome is relatively rich in free sterols (8).

Like the sterols, anionic phospholipids are demonstrable by polymyxin-B (50, 73) and by adriamycin (8). Different concentrations of acidic lipids are found in adjacent domains of plasma membrane of guinea pig sperm, with higher concentration localized over the fusogenic acrosomal cap; the concentration of these lipids increases as the membrane becomes fusionally competent prior to the acrosome reaction (8, 50). Since sulfo-galactolipid seminolipid is the most prominent anionic lipid in the outer leaflet of mammalian sperm plasma membranes, it is likely that polymyxin B detects the distribution of seminolipid in the surface of the membrane (67). Negative charge density of the exterior phospholipid-water interface at the acrosomal region has also been detected by using surface-directed spin labels (74, 75).

Inner and outer leaflets are considered by some authors as two large and almost independent domains, but with the possibility of trans-bilayer lipid redistribution (61). The distribution of phospholipid in sperm membrane is asymmetric. In the ram sperm choline containing phospholipids are situated mainly in the outer membrane monolayer, whereas cardiolipin (diphosphatidylglycerol) and phosphatidylserine are located predominantly in the inner layer (76).

Fluorescence recovery after photobleaching (FRP) measures the lateral diffusion of lipids in the plane of biological membranes. In most somatic cells, lipids are almost nearly free to diffuse laterally; in contrast, the plasma membrane of mammalian spermatozoa has large non-diffusing lipid fractions (77-79). These non-diffusing lipid areas increase as the spermatozoa differentiate during epididymal maturation (50% compared to 15% in most somatic cells) and also in capacitation. Immobile lipids are observed only in other polarized mammalian cells, such as epithelial cells, and may be due to lipid-lipid interactions (30).

At physiological temperature, most of the membrane lipids are organized in a fluid lamellar liquid-crystalline phase, (L_alpha phase). As the temperature decreases, bilayer lipids undergo a transition to the gel phase (L_ß phase), with more ordered fatty acids chains. In the gel-like areas, molecular motion is very slow and in the liquid-crystal-like microdomains, molecules move relatively freely. The phase transition temperature (T_m) depends on the lipidic composition of every membrane or even of each lipidic domain. Lipid phase transitions usually can be measured by differential scanning calorimetry (DSC) or in intact cells by fluorescence polarization and by Fourier transform infrared spectroscopy (FTIR). The phase behavior of egg yolk phosphatidylethanolamine as a function of temperature studied by DSC and FTIR is shown in Figure 3. Both curves show the phase transition L_alpha- L_ß around 20°C. A second transition (L_alpha - H_II), which will be discussed below, takes place around 50°C.

Figure 3 - Phase behavior of egg yolk phosphatidyl-ethanolamine as a function of temperature, studied by two techniques: a) Differential scanning calorimetry (DSC) and b) Fourier transform infrared spectroscopy (FTIR) (see the text for details). Adapted from Mantsch et al.. (80).

By using FTIR, Drobnis et al. (81) found a complex lipid phase behavior in the membranes of pig sperm that may represent multiple phase transitions, and which corresponds to various classes of lipids with different Tm. Alternatively, this may correspond to lipids located in the different membranes of the cell or in different domains.

Fluorescence polarization with lipid-like fluorescent probes gives a measure of membrane fluidity. Thus, a high fluorescence anisotropy corresponds to a more rigid membrane. By using this technique, when plasma membrane (PM) of sperm of rabbit was analyzed separately from the outer acrosomal membrane (OAM) and the inner acrosomal membrane (IAM), OAM was more rigid than the other two membranes.

Since sphingomyelin is known to condense the lipid bilayer by forming lipid-lipid hydrogen bonds in the hydrophylic-hydrophobic interface, this could be partially attributed to the higher sphingomyelin/phosphatidylcholine ratio in OAM (82).

In the sperm of the bull and rabbit, differences in the fluidity of the two leaflets of plasma membrane were observed by fluorescence anisotropy. The inner leaflets show a significantly higher fluidity than the outer leaflets. The same differences between the two leaflets were observed in OAM and in IAM (82). This can be attributed to the asymmetric distribution of cholesterol using filipin as a probe (8).

Membrane fluidity in biological membranes may be ascertained by using merocyanine 540, a fluorescent dye that preferentially binds to fluid-phase domains. This probe reveals that the fusogenic portion of guinea pig acrosome has a greater degree of fluidity than the post-acrosomal segment. It is interesting to note that after the cells become capacitated, possibly due to migration of proteins towards the equatorial segment, the fluorescence extends to this region (50).

5.3. Mechanisms accounting for the presence of distinct lipid domains

The intramembranous protein particles seem to play an important role in the formation of membrane microdomains. Nevertheless, in sperm plasma membrane, lipid-lipid interactions could be responsible for the immobilization of lipids and appear to contribute to the microdomain formation (83). This was demonstrated by FRP in liposomes formed exclusively with lipids extracted from the anterior region of head plasma membrane (10). The very unusual lipid composition of plasma membrane of sperm is very similar in its major components (one saturated fatty acid chain and one polyunsaturated 22:6 chain in the same phospholipid molecule) to the outer membrane of rods of the retina. Biophysical studies have provided evidence of lateral phase separation (formation of lipid domains) in the retinal membranes (84), as was found for sperm membranes. This lateral phase separation results from differences in lipid chain saturation, chain length, head group and charge and the concentration of cholesterol within the membrane (62). Wolf et al. (30) reported, at physiological temperature, two major phase transitions in the plasma membrane of ram sperm by using DSC, suggesting the co-existence of liquid-crystalline and gel phase lipids of different domains. Large amounts of polyunsaturated plasmalogens would segregate from other lipids and form fluid domains, whereas more saturated lipids would form more rigid domains at physiological temperature (30).

Cholesterol preferentially interacts with sphingomyelin or saturated phospholipids whereas it is unlikely to associate with highly unsaturated phospholipids, like 16:0-20:4 and 16:0-22:6 phosphatidylcholine. In systems with a highly unsaturated fatty acid content, there could be lateral separation into cholesterol-rich and cholesterol-depleted microdomains. Consequently, this would create highly saturated and unsaturated lipid domains (85, 86).

Lipid diffusion barriers might also be mantained by lipid structures such as glycolipids (sulfogalactosylglycerolipids or seminolipids) (65). Seminolipids have been localized in the equatorial segment of the acrosome reacted sperm (67). Lipid regionalization may also lead to protein regionalization by virtue of the preferential solubility of the proteins in different sites.