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a Department of Food Biophysics, Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom
b The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The plasma membrane of mammalian spermatozoa has a pronounced domain organization with many glycoprotein antigens segregated into the acrosome, postacrosome, midpiece, and principal piece regions [8]. It is not clear whether this situation also applies to component lipids. Unlike most somatic cells, mammalian sperm lipids tend to be very unsaturated (in the ram, bull, and boar, ~60% of phospholipid-bound fatty acids are 20:4, 22:5, and 22:6, respectively) and contain a high proportion (20–40%) of plasmalogen phospholipids [9]. Theoretically, their membranes should be very "fluid," a prediction supported by recent work, using generalized polarization spectroscopy with Laurdan, which showed that lipids in the plasma membrane of human spermatozoa are in a liquid-crystalline (fluid) phase throughout the cell [10]. Other biophysical techniques, however, such as differential scanning calorimetry (DSC), electron spin resonance (esr), and Fourier Transform Infrared Spectroscopy (FTIR), have detected thermotropic phase transitions in membrane vesicles and lipid extracts from bull, ram, boar, and guinea pig spermatozoa [11–14]. Multiple transition temperatures are consistent with the sperm plasma membrane's being a mosaic of gel and fluid phases, and it has been suggested that this is caused by the preferential affinity of certain lipids for transmembrane glycoproteins, the modulating effects of cholesterol, or the presence of intramembranous barriers that impede free diffusion [15].
Although informative, DSC, esr, and FTIR do not have the necessary spatial resolution to analyze "fluidity" in specific regions of the sperm plasma membrane. This information is important for understanding processes such as antigen migration and membrane fusion that are, for the most part, confined to specific domains of the plasma membrane. One technique with the necessary resolution is fluorescence recovery after photobleaching (FRAP), in which a fluorescent reporter probe is inserted into the bilayer and its diffusion calculated after laser-induced bleaching and recovery. Previous FRAP analysis of ram and mouse spermatozoa with the fluorescent lipid analogue DiIC16 suggested that sperm membranes contain a large immobile phase (40–50%) indicative of extensive gel phase domains [6]. Recent work in the bull, however, has shown that DiIC16 stains only dead or permeabilized spermatozoa and that lipid diffusion in these cells is highly compromised relative to that in live motile ones [16]. It is not known whether this is generally true for other species, and in this investigation we have compared lipid diffusion in various surface membrane domains of bull, boar, ram, mouse, and guinea pig spermatozoa using the lipophilic reporter probe 5-(N-octadecanoyl)aminofluorescein (ODAF). The diffusion of ODAF has been measured by FRAP analysis in both live and dead spermatozoa. In addition, we have investigated the effects of temperature on ODAF diffusion as well as cross-linking membrane proteins with paraformaldehyde.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals and reagents were of the highest purity available commercially and were purchased from Sigma Chemical Co. (Poole, UK) or BDH-Merck (Lutterworth, UK). ODAF was obtained from Molecular Probes (Eugene, OR); its chemical structure is shown in Figure 1.
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Collection and Labeling of Spermatozoa with ODAF
Bull Ejaculated bull semen was collected from 3 Holstein Friesian bulls maintained at an artificial insemination center (Genus Breeding, Milton Keynes, UK). The bulls were of proven fertility, and the semen was transported to the laboratory within 3 h of collection. Ten microliters of whole semen was diluted with 40 µl of bovine sperm washing medium (BSWM; [16]) and mixed with 50 µl of BSWM containing 12.5 µM ODAF in 2% ethanol. (ODAF is made up as a stock solution of 5 mM in 100% ethanol and stored in the dark at -20°C. The probe is not completely soluble in ethanol at 25°C, and the concentration is best calculated from its extinction coefficient EM = 85 x 10-3/cm.) Spermatozoa were incubated for 15 min at 25°C, washed twice by centrifugation at 400 x g for 10 min in 1 ml of glucose-free BSWM, and resuspended in a final volume of 0.5 ml with BSWM containing 0.1% sodium azide. Samples of spermatozoa were immediately analyzed by FRAP.
Ram Ejaculated semen was collected from 4 Suffolk rams using an artificial vagina and an aliquot diluted 1:10 with neat ram seminal plasma. Thereafter, the labeling protocol was the same as described above for bull spermatozoa except that a modified Krebs-Ringer phosphate glucose medium (KRPG; [17]) was substituted for BSWM.
Boar The sperm-rich fraction of ejaculated semen was collected from 3 fertile boars (Large White variety) and filtered through 4 layers of surgical gauze to remove seminal gel. The labeling protocol thereafter was the same as described above for bull spermatozoa except that KRPG was used for dilution and washing instead of BSWM.
Mouse Male mice (CFLP strain; Harlan Olac Ltd., Bicester, Oxon, UK) were killed by CO2 asphyxiation, and the cauda epididymidis was dissected free of fat and connective tissue. Spermatozoa were released by gentle mincing of the cauda in 0.5 ml of Whittingham's medium (WhM; [18]) containing 0.3% BSA at room temperature. For loading with ODAF dye, 20 µl of sperm suspension was mixed with 30 µl of WhM, followed by 50 µl of 3.5 µM ODAF in WhM/1% ethanol, and incubated for 15 min at 25°C. Spermatozoa were washed twice by centrifugation at 600 x g for 10 min in 1 ml of glucose-free WhM and finally resuspended in 0.5 ml of the same medium.
Guinea pig Three male guinea pigs were killed and spermatozoa collected from the cauda epididymidis as described above except that they were diluted into modified Tyrode's medium (TALP; [19]) containing 0.3% BSA and 5.6 mM glucose. An aliquot (100 µl) was diluted with 100 µl of TALP/BSA/glucose, containing 3.5 µM ODAF and 1% ethanol, and incubated for 15 min at 25°C. Labeled spermatozoa were washed twice by centrifugation at 300 x g for 5 min in 5 ml TALP (without glucose or BSA) and resuspended in a final volume of 0.5 ml of the same medium.
Fluorescence Microscopy
Sperm motility was assessed by phase-contrast microscopy at room temperature. Uptake of ODAF was checked by epifluorescence microscopy on a Zeiss (Carl Zeiss, Thornwood, NY) Axiophot photomicroscope fitted with a 100 W mercury vapor lamp using excitation filters at 485 nm and emission filters at 530 nm. Color photographs were taken on Kodak (Eastman Kodak, Rochester, NY) Ektar 1000 film.
FRAP Analysis
The principle of the FRAP technique is shown in Figure 2. Briefly, the membrane is loaded with a nonperturbing amount of fluorescent reporter probe to give a measurable signal when excited at the appropriate wavelength. A laser beam (~2.5-µm diameter) is focused on the membrane, and the initial fluorescence intensity is measured (pre-bleach intensity, Fig. 2a). The strength of the laser is then increased 1000-fold for ~5 msec to irreversibly bleach the fluorophore within the monitoring area of the beam (Fig. 2b). Due to random diffusion, unbleached molecules from outside the monitoring beam mix with bleached molecules inside the beam with the result that the fluorescence signal recovers with time (Fig. 2, c and d). The kinetics of fluorescence recovery reflects the rate of diffusion (D) of the reporter probe within the plane of the membrane, while the extent of recovery (%R) indicates the proportion of reporter molecules that are freely diffusing. Typically, membrane lipids have high diffusion coefficients (between 10-7 to 10-8 cm2/sec) whereas membrane proteins, especially those attached to the cytoskeleton, are several orders of magnitude slower (10-10 to 10-11 cm2/sec). A significant feature of the instrumentation system used in these experiments is the presence of 2 epi-illumination attachments on the microscope that allow spermatozoa to be viewed before photobleaching and to be classified as "live-pattern" or "dead-pattern" (see later). As described below, this distinction is important for interpretation of results. Protocols describing the FRAP instrumentation system, data collection, and correction formulae for calculating the diffusion coefficient (D) and percentage recovery (%R) of fluorescence have been published [16, 20].
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Statistical Analysis
Results were analyzed for statistical significance by one-factor ANOVA and Microsoft (Redmond, WA) Excel 97.SR-1 "t-test" assuming samples of unequal variances.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Motile spermatozoa from all 5 species investigated labeled uniformly over the whole surface membrane with ODAF reporter probe using the protocols described (Fig. 3). These spermatozoa also excluded DNA-binding dyes such as propidium iodide; this staining is henceforth referred to as live-pattern staining. Preliminary experiments showed that the amount of ODAF incorporated into the membrane increased with time of incubation and concentration of probe as judged by the intensity of fluorescence (results not shown). However, to reduce to a minimum the possible perturbing effects of the reporter probe itself on membrane structure, spermatozoa were loaded with the lowest amount of ODAF necessary to generate a signal for observation and FRAP analysis. Consequently, live-pattern spermatozoa always appear weakly stained. By contrast, dead and permeabilized spermatozoa absorbed more of the probe, with typically stronger fluorescence over the acrosome and midpiece regions than on the postacrosome or principal piece of the tail (Fig. 3). These spermatozoa were immotile, and since they stained strongly with propidium iodide, they are referred to as dead-pattern spermatozoa.
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ODAF Diffusion in Various Regions of Live and Dead Bull, Boar, Ram, Mouse, and Guinea Pig Spermatozoa
The measured diffusion coefficients and percentage recoveries for ODAF in various surface domains of live bull, boar, ram, mouse, and guinea pig spermatozoa are shown in Table 1. Typical recovery curves for bull, ram, and guinea pig spermatozoa are illustrated in Figure 4. The D values obtained were typical of membrane lipids and with one exception were significantly higher over the sperm head than on the tail; differences between these domains varied from 2-fold for the bull to 3-fold for the ram and mouse to 5-fold for the boar. The exception was guinea pig spermatozoa, for which D values were not significantly different between the acrosome and midpiece. In all species, however, the %R was high on the sperm head (78–94%) but variable on the tail—from as low as 25.1% on the midpiece of guinea pig spermatozoa to as high as 80% and 90% on mouse and bull spermatozoa, respectively.
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In keeping with the differential staining patterns observed by fluorescence microscopy, ODAF diffusion in the plasma membrane of dead-pattern bull, ram, boar, and mouse spermatozoa was substantially different from that measured in live-pattern cells (Table 1; Fig. 4). The most extreme case was found in the bull, in which dead-pattern spermatozoa developed a large immobile phase throughout the plasma membrane to the extent that %R was < 15% in all regions and calculations of D became too variable to be reliable. In dead-pattern ram spermatozoa, however, %R was sufficiently large to be measurable although it decreased substantially relative to that for live cells on the postacrosome (from 82% to 42%), midpiece (from 54% to 24%), and principal piece (from 77% to 37%). On the acrosome of dead-pattern spermatozoa, %R remained relatively unaffected (from 85% to 73%, respectively), although D was ~3 times lower than on the acrosome of live-pattern cells (Table 1). Similarly, there was a ~4-fold decrease in D on the postacrosome. In contrast, the lower %R in the midpiece and principal piece of dead-pattern ram spermatozoa had little effect on ODAF diffusion in the remaining mobile phase (Table 1). Thus, on ram spermatozoa, different surface domains appear to behave independently of one another as they respond in different directions to loss of membrane integrity.
This unpredictability in the direction of the response to loss of membrane integrity was also seen in mouse and boar spermatozoa. Dead-pattern mouse spermatozoa showed substantially lower %R on the postacrosome and midpiece regions than live-pattern spermatozoa, whereas on the acrosome the %R was largely unaffected (Table 1). D values on the acrosome and postacrosome of dead-pattern spermatozoa, however, were significantly lower than on live-pattern spermatozoa (Table 1), whereas those on the midpiece were essentially the same. Similarly, on dead-pattern boar spermatozoa, D values were 4–5 times lower on the acrosome and postacrosome than on the equivalent regions of live-pattern cells while %R values were not significantly different (Table 1). Once again, the exception was guinea pig spermatozoa; D values on all regions of dead-pattern guinea pig spermatozoa were not significantly different from the equivalent areas on live-pattern cells (Table 1).
ODAF Diffusion in Sperm Membranes Was Temperature Sensitive
In addition to the degree of unsaturation of fatty acids, phospholipid:cholesterol ratios, etc., temperature has a strong influence on the phase organization of bilayer lipids that should be reflected in FRAP data, provided the reporter probe intercalates into the membrane. To verify that ODAF was in contact with the lipid bilayer and not simply bound electrostatically to the surface, we investigated the effects of temperature on its diffusion coefficient in various surface domains of bull, mouse, boar, and guinea pig spermatozoa. As shown in Table 2, D values in all regions of bull and mouse spermatozoa were significantly lower at 5°C than at 23°C, whereas at 37°C, ODAF diffusion was 2–6 times faster than at 23°C (the extent depended on the membrane domain). The situation was similar for the acrosomal domain of boar spermatozoa. ODAF diffusion on guinea pig spermatozoa, on the other hand, was poorly responsive to temperature changes, showing only a doubling of D between 8°C and 37°C. Overall, however, the direction of these responses to changing temperature is consistent with the known behavior of lipid bilayers and suggests that ODAF was in direct contact with the hydrophobic environment of the sperm plasma membrane.
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Effects of Paraformaldehyde Fixation on Regionalized Diffusion of ODAF in Live-Pattern Bull Spermatozoa
To find an explanation for the large immobile fraction of ODAF that develops in the plasma membrane of bull spermatozoa after permeabilization, attempts were made to mimic this effect by cross-linking membrane proteins with paraformaldehyde, while accepting that ODAF is essentially a reporter for lipids. For this purpose, bull spermatozoa, which show development of extensive immobile phases after permeabilization, were prelabeled with ODAF followed by fixation in 4% paraformaldehyde for 30 min at room temperature. Spermatozoa were then washed twice in BSWM and analyzed by FRAP as before. Although immotile, more than 85% of spermatozoa still retained the live pattern of labeling. FRAP analysis of these live-pattern spermatozoa gave D values that were significantly lower than values for nonfixed controls on the acrosome, postacrosome, and midpiece domains (Table 3). Paradoxically, D on the principal piece increased slightly after fixation. Percentage recoveries after fixation were also significantly less than for controls on the postacrosome and principal piece domains, but, overall, there was no formation of a large immobile phase throughout the membrane similar to that found on dead-pattern spermatozoa. Thus, although the dynamics of ODAF within the plasma membrane of bull spermatozoa can be affected by cross-linking protein components with paraformaldehyde, it is not possible to reproduce exactly by this means the extensive rigidification found after cell death.
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Lateral Diffusion of ODAF on the Inner Acrosomal Membrane of Guinea Pig Spermatozoa
The inner and outer acrosomal membranes are formed by fusion of Golgi cisternae in the early round spermatid [21], whereas the surface plasma membrane is derived initially from the spermatogonium and represents the culmination of all the various testicular and posttesticular developmental processes (e.g., maturation changes in phospholipids, endoproteolysis of membrane proteins, etc.) that lead to the formation of a fertile spermatozoon [22]. It is possible that the different cellular origins of these membranes are reflected in the biophysical behavior of their component lipids. To investigate this hypothesis, guinea pig spermatozoa that were either acrosome intact or had lost their acrosomes completely were labeled with ODAF and analyzed by FRAP. In this species, these two cell types are easily distinguished from each other because of the size of the acrosome (Fig. 3, e and e'). As shown in Table 4, there were no significant differences in ODAF diffusion between the plasma membrane overlying the acrosome of intact cells and the membrane exposed on the head of spermatozoa that had lost their acrosomes. We presume that the latter membrane represents the inner acrosomal membrane. The data also indicate that lipid diffusion on the anterior part of the acrosomal plasma membrane of live spermatozoa is not significantly different from that on, or close to, the equatorial segment. In keeping with the earlier observations on dead-pattern guinea pig spermatozoa, ODAF diffusion on spermatozoa permeabilized by cold shock was not significantly different from that for live cells in any of the surface domains investigated (Table 4). These cold-shocked spermatozoa characteristically stained strongly on the midpiece and inner acrosomal membrane, the latter structure appearing as a "ghost" inside the acrosome (Fig. 3e'; compare with the typical live pattern in Fig. 3e).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The validity of ODAF as a reporter probe for membrane lipids has been discussed in detail in an earlier publication [16]. Its principal advantages are that 1) it is incorporated homogenously into all regions of the plasma membrane of live spermatozoa; 2) it labels live and dead spermatozoa differentially, thereby enabling them to be distinguished from each other before FRAP analysis—as shown in Table 1, the ability to classify spermatozoa in this way is crucial since dead cells show significant and unpredictable differences from live cells in terms of ODAF diffusion and %R; 3) D values for ODAF are temperature sensitive consistent with the probe being incorporated into the membrane [20]. Therefore, it is reasonable to presume that its diffusion characteristics are an acceptable reflection of real-time lipid dynamics within the bilayer.
In all species investigated, dead spermatozoa characteristically incorporated more ODAF into the acrosome and midpiece regions than into the postacrosome and principal piece. An obvious explanation for this pattern is that ODAF is gaining access to internal membranes in the head and midpiece (e.g., the outer acrosomal and mitochondrial membranes) through holes in the overlying plasma membrane. Alternatively, it may reflect a generalized loss of membrane organization leading to a change in the density or "packing" of lipids in these regions with a subsequent increase in uptake of ODAF. A third possibility is that proton displacement from uncoupled ion pumps could cause a localized increase in surface pH (over, say, the midpiece) to which ODAF would respond with a stronger fluorescent signal. Wolf and coworkers [23, 24] did not distinguish between live and dead cells in their studies on DiIC16 diffusion in ram and mouse spermatozoa, and the fact that they observed more intense staining over the acrosomal and midpiece domains suggests that they may well have been measuring permeabilized spermatozoa. Whatever the reason, it is clear from the data presented in Table 1 that it is important to distinguish between live and dead spermatozoa prior to FRAP analysis.
Two major questions arising from the observations presented above are 1) what maintains differences in lipid diffusion between the sperm head and tail and 2) what causes the immobile fraction in certain regions (mostly the tail) of the plasma membrane, particularly after cell death.
Lipid Domains in Sperm Plasma Membranes
There is much evidence from work on whole cells and subcellular organelles that biological membranes have an in-plane heterogeneity that ranges in size from a few nanometers to several microns. In unfertilized Xenopus eggs (diameter ~1 mm), for example, the diffusion coefficient for hexadecanoylaminofluorescein (HEDAF) in the animal pole was 1.5 x 10-8 cm2/sec whereas in the vegetal pole it was 7.6 x 10-8 cm2/sec [25]. After fertilization, this difference increased 100-fold. At the micrometer scale, significant disparities have been found between apical and basolateral plasma membranes of epithelial cells [26] and in focal versus nonfocal contact points of cultured chicken fibroblasts [27]. Microheterogeneity is also present at the nanometer level as found for the so-called collar lipids surrounding transmembrane proteins [28, 29]. Thus, the 2- to 5-fold higher diffusion coefficients found for ODAF on the acrosomal domain relative to the midpiece/principal piece domains of bull, ram, boar, and mouse spermatozoa are consistent with the concept of lateral asymmetry.
The high %R observed on all regions of live-pattern bull, ram, boar, mouse, and guinea pig spermatozoa, together with generally fast diffusion coefficients, suggests that fluid phase lipids predominate in the plasma membrane of these species. This is in agreement with the high proportion of polyunsaturated fatty acids in sperm phospholipids that, theoretically, would favor fast diffusion because of their low transition temperatures. It is also consistent with the work of Palleschi and Silvestroni [10], who found only liquid crystalline (fluid) phase lipids in live human spermatozoa using generalized fluorescence spectroscopy with Laurdan probe. However, FTIR and DSC techniques have revealed multiple phase transition temperatures in phospholipid extracts and membrane vesicles prepared from ram, boar, and rat spermatozoa [11–14, 30]. These results suggest the coexistence of fluid and gel phase lipid domains in sperm plasma membranes, induced by either localized variations in lipid composition or differential interactions with component glycoproteins, or both. The complexity of the situation would obviously be exacerbated during cooling of spermatozoa, when some lipids would reach their transition temperatures well before others, leading to a rapid spreading of gel phases. Edidin [7] has suggested that much of the confusion surrounding lipid domains in membranes stems from the difficulties of estimating their size and duration. Small-scale lipid-lipid interactions are unlikely to last more than a few microseconds and probably involve only tens of molecules. Larger domains containing hundreds or thousands of molecules, such as those that form around transmembrane proteins and reach 30–40 nm in diameter, may persist for much longer. These collar domains could grow larger still if the proteins in question reached sufficient density. It is not inconceivable, therefore, that a lipid domain approaching the size of the sperm acrosome or midpiece could persist in a dynamic state, especially if it was constrained between diffusion barriers such as the posterior ring in the neck region and the annulus at the end of the midpiece [31]. The functionality of these structures in spermatozoa is still an open question. Some glycoprotein antigens appear constrained between diffusion barriers whereas others (e.g., PH20, 2B1, CE9, and fertilin) are able to migrate to new cellular locations during maturation and capacitation. Since they move against large-concentration gradients, an active transport mechanism across the barrier is implied [32–36]. It is not inconceivable, therefore, that the posterior ring and annulus could segregate lipid domains of different composition and/or phase organization. This is supported by the observations of Arts et al. [37], who found that liposomes containing anionic phospholipids (PS, PE) bound specifically to the equatorial segment of acrosome-reacted human spermatozoa and were retained therein for up to 40 min. A PE-binding protein has also been described in rat spermatozoa [38, 39], but its subcellular distribution and effects on PE diffusion in the membrane are not known.
Origins of Immobile Phase Lipids in the Plasma Membrane of Spermatozoa
A significant immobile fraction is present in the midpiece region of live ram, boar, and guinea pig spermatozoa. These immobile lipids increase substantially after cell death, reaching an extreme situation in the bull. In the latter case, the apparent "rigidification" of the membrane has been found to be irreversible and stable up to 42°C [16]. The problem of poor recovery after photobleaching is well known from work on membrane glycoproteins, which frequently show large immobile fractions that can be readily explained on the basis of their cross-linking to underlying cytoskeletal elements, to each other, or to so-called `post proteins in the ectodomain of the membrane [40]. However, in our experiments we presume that ODAF intercalates only into the outer leaflet of the lipid bilayer and over the time course of our experiments does not flip-flop to the inner leaflet to any appreciable extent. Its low %R, therefore, is more difficult to explain. One possibility is that the probe may equilibrate into nanometer-size gel phase domains that, being numerous and much smaller than the diameter of the laser beam, would recover only very slowly after photobleaching. Metcalf et al. [41] found only 35–65% recovery in soybean protoplasts labeled with nitrobenzoxadiazole glycerophosphoethanolamine (NBD-PE) or DiIC18 and interpreted this as evidence for a mosaic of immiscible gel and fluid phase domains of varying dimensions in their membranes. Probably the situation closest to the large immobile fraction in dead bull spermatozoa is found in Xenopus eggs. Before fertilization, HEDAF in the animal pole diffuses very rapidly (D = 15 x 10-9 cm2/sec), but after fertilization it becomes completely immobile [25]. In early FRAP work, Wolf and colleagues [23, 24], using DiIC16 as a reporter probe, also described a large immobile fraction in ram and mouse spermatozoa that was similar in magnitude to that found here for dead cells. It is noticeable that in all species it is the tail domain that shows the lowest %R, emphasizing once again possible compositional and/or organizational differences from the sperm head.
There are a number of other possible explanations for formation of the large immobile fraction. One is cross-linking of ODAF to negatively charged phospholipids and sulphogalactosylglycerolipids by Ca2+ ions leading to microaggregation [42, 43]. These so-called lipid blocks, however, are temperature sensitive, whereas no such response could be detected in the plasma membrane of dead spermatozoa [16]. While cross-linking protein within the membrane with paraformaldehyde had some effect on ODAF diffusion, it did not cause widespread immobilization, suggesting that the probe does not bind directly or strongly to transmembrane proteins. A second possible explanation is entrapment within hexagonal II-type lipids [44]. Some phospholipids (e.g., cardiolipin) are known to form non-bilayer structures depending on the pH, salt concentration, and their acyl chain length [45]. A notable feature of these non-bilayer arrangements is their stability, something that would explain the persistence of immobile fractions in spermatozoa over time. A third possibility is interaction with the submembranous cytoskeleton. Actin, thymosin-ß-10, and annexins have been demonstrated in spermatozoa [46–48] and could conceivably influence lipid order directly or indirectly via membrane proteins. A fourth possible explanation is localized variations in cholesterol concentrations. Cholesterol-rich and cholesterol-poor regions have been described in sperm membrane from studies on filipin binding [49, 50] and are likely to influence lipid diffusion through interactions with unsaturated fatty acyl chains. This would obviously enhance any in-plane compositional heterogeneity. Lastly, there could be loss of transverse asymmetry caused by depletion of ATP and inactivation of flippases [3].
The reasons ODAF diffusion in guinea pig spermatozoa does not conform to the general pattern found in the bull, boar, ram, and mouse are not clear. The lipid composition of plasma membranes from guinea pig spermatozoa is similar to other species [51, 52] and unlikely, therefore, to account for the different behavior of ODAF following FRAP. In addition, the D values for ODAF obtained in this study compare favorably with those reported for DiIC14 on intact and acrosome-reacted guinea pig spermatozoa by Cowan et al. [53, 54]. These workers recorded diffusion coefficients of 8.9 x 10-9 cm2/sec and 10.5 x 10-9 cm2/sec on the acrosome and postacrosome, respectively, of intact spermatozoa and 5.4 x 10-9 cm2/sec on the inner acrosomal membrane of acrosome-reacted cells. Thus, the fact that two different probes give similar D values after FRAP indicates that the unusual diffusional properties of lipids in guinea pig sperm membranes are more likely to have an organizational than a compositional basis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: July 30, 1998.
Received: May 28, 1998.
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