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BOR - Papers in Press, published online ahead of print June 23, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030502
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BIOLOGY OF REPRODUCTION 71, 1367–1373 (2004)
DOI: 10.1095/biolreprod.104.030502
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Reorganization of Lipid Rafts During Capacitation of Human Sperm1

Nicholas L. Cross2

Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ejaculated mammalian sperm must complete a final maturation, termed capacitation, before they can undergo acrosomal exocytosis and fertilize an egg. In human sperm, loss of sperm sterol is an obligatory, early event in capacitation. How sterol loss leads to acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm sterol affects the organization of cold detergent-resistant membrane microdomains (lipid "rafts"). The GPI-linked protein CD59, the ganglioside GM1, and the protein flotillin-2 were used as markers for lipid rafts. In uncapacitated sperm, 51% of the CD59, 41% of the GM1, and 90% of the flotillin-2 were found in the raft fraction. During capacitation, sperm lost 67% of their 3ß-hydroxysterols, and the percentages of CD59 and GM1 in the raft fraction decreased to 34% and 31%, respectively. The distribution of flotillin-2 did not change. Preventing a net loss of sperm sterol prevented the loss of CD59 and GM1 from the raft fraction. Fluorescence microscopy showed CD59 and GM1 to be distributed over the entire sperm surface. Flotillin-2 was located mainly in the posterior head and midpiece. Patching using bivalent antibodies indicated that little of the GM1 and CD59 was stably associated in the same membrane rafts. Likewise, GM1 and flotillin-2 were not associated in the same membrane rafts. In summary, lipid rafts of heterogeneous composition were identified in human sperm and the two raft components, GM1 and CD59, showed a partial sterol loss-dependent shift to the nonraft domain during capacitation.

acrosome reaction, fertilization, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Freshly ejaculated mammalian sperm are not immediately capable of undergoing exocytosis (the acrosome reaction) and fertilizing an egg (reviewed in reference [1]). In the poorly understood process termed capacitation, they acquire these functions. Changes in the lipid composition and distribution within the plasma membrane are early events in capacitation (reviewed in references [2, 3]). Loss of sperm sterols begins soon after sperm are removed from seminal plasma and is obligatory for capacitation of human sperm [4]. Experimentally maintaining a high level of sterols in sperm inhibits progesterone- and calcium ionophore-induced acrosome reactions of human sperm [5], the zona pellucida-induced acrosome reaction of mouse sperm [6], and fertilization of rat, mouse, and rabbit eggs [7–9]. Sterol loss has been positioned upstream from the rise in intracellular pH [10] that is required for acrosomal responsiveness [11, 12], and it is also upstream from tyrosine phosphorylation of a set of sperm proteins [6] whose role in acrosomal exocytosis is not yet clear.

How sterols inhibit capacitation is unknown. In freshly ejaculated sperm, ß-OH sterols are abundant in the sperm plasma membrane [13, 14], and it has long been suggested that the critical results of sterol loss are increases in membrane fluidity and/or permeability [15]. We recently showed that the essential structural feature required for a sterol to inhibit capacitation is planarity of the fused ring structure [16], a feature that is required for maintaining high phospholipid order (reviewed in reference [17]). In fact, measurement of lipid order using steady-state fluorescence anisotropy revealed a sterol loss-dependent decrease in lipid order during capacitation [18], and binding of the dye merocyanine 540, which is believed to reflect disordered lipid packing, increases during capacitation [19]. However, application of agents that increase the bulk lipid fluidity did not accelerate capacitation, leading to the idea that lipid order in microdomains might be more important to sperm capacitation than bulk lipid fluidity [18].

There is considerable evidence that specific membrane lipids associate to form microdomains, too small to resolve with optical microscopy. The size, stability, and functional importance of the microdomains are not fully understood. Although not universally accepted, a strong case has been made that sterol-rich microdomains ("rafts") form organizing centers that affect the distribution of membrane proteins, activation of receptors, and triggering of signaling cascades [20–23]. Membrane fractions with the expected physical properties of rafts—higher order and lower fluidity than the sterol-poor lipid phase—can be isolated from cells by virtue of their insolubility and low buoyant density in cold Triton X-100 [24]. Raft fractions have been prepared from sperm of sea urchins, mice, guinea pigs, and boars [25–27].

Experimentally depleting cellular cholesterol typically disrupts rafts [23], raising the question whether sterol loss during sperm capacitation might alter the structure of sperm rafts. A recent study of boar sperm concluded that, at an amount of sterol loss sufficient to render the sperm acrosomally responsive to calcium ionophore, the lipid raft fraction did not decrease in abundance (measured by total protein content and light scattering). Only when cholesterol was depleted to the point of 100% sperm death was the raft fraction diminished [27].

The present experiments were designed to determine whether the loss of sperm sterols during capacitation of human sperm alters sperm lipid rafts. Three raft-associated markers were inspected. CD59 is a glycosylphosphatidylinositol (GPI)-anchored, externally exposed plasma membrane protein of 18–25 kDa with complement-inhibitory properties and possibly a role in sperm-egg interaction [28]. CD59 was expected to be raft associated, as it and many other GPI-linked proteins are in other cells [29, 30]. The ganglioside GM1 is concentrated in the external leaflet of the plasma membrane and can be detected with the B-subunit of cholera toxin (CTx), which binds to GM1 oligosaccharides [29, 31]. Flotillin-2 is a ubiquitous 42-kDa membrane protein that displays cold detergent resistance typical of rafts [32, 33]. Access of antibodies to flotillin-2 requires membrane permeabilization, suggesting a wholly intracellular location. The association of flotillin-2 with membrane rafts is not completely understood but is dependent on myristoylation, palmitoylation, and protein oligomerization [34].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

The following chemicals were used: methanol and chloroform (E M Science, Gibbstown, NJ), hexane and ethanol (Pharma Products, Brookfield, CT), BSA (Pentex Bovine Albumin, Fraction V, Reagent Grade, catalog number 81-066-7, lot 63; Serologicals Proteins Inc., Kankakee, IL), and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (Vector Laboratories, Burlingame, CA). Mouse monoclonal anti-CD59 (555761; BD Biosciences Pharmingen, San Diego, CA) was used at 5 µg/ml for immunofluorescence and 250 ng/ml for immunoblotting; anti-Na+/K+-ATPase {alpha}-1 chain (05-369; Upstate Biotechnology, Lake Placid, NY) was used at 1 µg/ml for immunoblotting; FITC-CTx (Sigma Chemical Co., St. Louis, MO) was used at 10 µg/ml; peroxidase-conjugated CTx (Sigma) was used at 1 µg/ml for dot blots; tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG (115-025-146; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 15 µg/ml; Cy3-conjugated Fab fragment of goat anti-mouse IgG (115-167-003; Jackson ImmunoResearch Laboratories) was used at 16 µg/ ml; and FITC-conjugated Fab fragment of goat anti-mouse IgG (115-097-003; Jackson ImmunoResearch Laboratories) was used at 17 µg/ml. Rabbit anti-CTx IgG (Fitzgerald Industries, International, Concord, MA) was used at 50 µg/ml. Anti-flotillin-2 (610383; BD Transduction Labs, San Diego, CA) was used at 5 µg/ml for immunofluorescence and 100 ng/ml for immunoblotting. For enhanced chemiluminescence, secondary antibodies and other reagents were obtained from Amersham Biosciences Corporation (Piscataway, NJ). All other chemicals were obtained from Sigma.

Sperm Preparation

Human sperm were treated as previously described [35, 36]. Informed consent was obtained from the semen donors, and an institutional review board approved this investigation. Briefly, semen was obtained by masturbation, and motile sperm were selected by centrifugation through a Percoll gradient, washed, and suspended in incubation medium modified from Suarez et al. [37] (117.6 mM NaCl, 0.36 mM NaH2PO4, 8.6 mM KCl, 2.4 mM CaCl2, 0.49 mM MgSO4·7H2O, 25 mM NaHCO3, 2 mM glucose, 0.25 mM Na pyruvate, 19 mM Na lactate, 0.05 mg/ml streptomycin sulfate, 0.075 mg/ml penicillin, and 26 mg BSA/ml). Sperm were incubated at a concentration of 2 x 106 sperm/ml at 37°C in a chamber containing a humidified atmosphere of 5% CO2/95% (v/v) air. In some experiments, sperm were incubated in medium containing 7.5 µM cholesterol, prepared by injecting an ethanolic solution of cholesterol (15 mM) into a 1000-fold volume of incubation medium while vortexing. The solution was agitated at room temperature for 15–60 min, passed through a 0.22-µm pore filter, and then combined with an equal volume of sperm suspension. Controls demonstrated that the solvent (0.05% [v/v] ethanol) did not affect sperm viability, acrosomal responsiveness, or sterol content (data not shown).

Capacitation cannot be assessed by fertilization of human eggs, so the ability of sperm to acrosome-react when exposed to progesterone (P4) was employed. Under the conditions used here, sperm are unresponsive to P4 for 4–6 h, and then responsiveness develops progressively and reaches a maximum at about 24 h (see following results and Zarintash and Cross [4]). Responsiveness to P4 correlates with capacitation in mouse sperm [38], and P4 and zonae pellucidae elicit similar responses in mouse and human sperm [38, 39]. Sperm viability and acrosomal status were assessed as previously described [36]. Briefly, sperm were incubated with Hoechst 33258 (H258, 0.5 µg/ml, 10 min) to label dead cells, then fixed and permeabilized in 95% (v/v) ethanol. The acrosomal contents were labeled with FITC-conjugated Pisum sativum agglutinin, and the sperm were examined by fluorescence microscopy. Spontaneously reacted sperm were defined as H258-negative, acrosome-reacted sperm in suspensions that had not been exposed to P4. P4-responsive sperm were defined as the number of H258-negative, reacted sperm following exposure to P4 (10 min, 1 µg/ ml), corrected for the number of spontaneously reacted sperm detected in matched aliquots of the same sperm suspension.

Preparation and Analysis of a Lipid Raft Fraction

A raft fraction was prepared as described [40]. Sperm were washed in PBS (138 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 1.0 mM CaCl2, 0.5 mM MgSO4), chilled on ice, and lysed on ice in TNE/ sucrose/Triton X-100 (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 10% w/v sucrose, 1% v/v Triton X-100, and a protease inhibitor mixture containing 16 µg/ml benzamidine HCl, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 0.42 mM 4-[2-aminoethyl]benzenesulfonyl fluoride) at a concentration of about 200 x 106 sperm/ml. The concentration of sperm (over the range 105 x 106 to 480 x 106 sperm/ml) did not have a significant effect on the percentage of CD59 in the raft versus nonraft fractions (data not shown). Sperm were sheared by 20–40 passages through a 22-g needle. After 30 min, stock Optiprep was added to bring the concentration to 40% (w/v), and the mixture was overlaid with aliquots of 35%, 30%, 25%, 20%, and 0% (w/v) Optiprep prepared in TNE/sucrose/Triton X-100. The tubes were centrifuged at 164 300 x gav in a SW55Ti rotor for 4 h, and then six fractions corresponding to the original layers were removed from the top of the tube.

After centrifugation, the distribution of GM1 was analyzed by drying 1-µl aliquots of the fractions onto nitrocellulose, blocking with blocking solution (5% w/v dry milk, 137 mM NaCl, 0.1% v/v Tween-20, 20 mM Tris HCl, pH 7.6), and incubating 1 h with peroxidase-conjugated CTx in blocking solution followed by enhanced chemiluminescence according to the manufacturer's directions. To analyze the distribution of Na+/K+-ATPase, flotillin-2, and CD59, gradient fractions were precipitated with trichloroacetic acid, separated on nonreducing 13% (w/v) acrylamide SDS-PAGE gels, and detected by immunoblotting and enhanced chemiluminescence. Samples to be analyzed for Na+/K+-ATPase were incubated in sample buffer for 1 h at 37°C to minimize aggregation that accompanies denaturation at higher temperatures; other samples were denatured at 90– 95°C for 3 min. Aliquots of each fraction equivalent to 2.5 x 106 or 5 x 106 sperm were used for each lane. The developed films were scanned using transmitted light, and the intensities were determined using the public domain ImageJ program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/ij/index.html). Brightness levels were converted to optical densities by scanning a set of calibrated density standards. The analyzed samples were within the linear range of a density-versus-concentration curve, as determined by inspection of serially diluted samples (data not shown).

Fluorescence Microscopy

Reagents to be applied to living cells were dialyzed against incubation medium. To determine the distribution of GM1 and CD59 on living cells, sperm were treated under conditions that minimized patching. GM1 was labeled with FITC-CTx (30 min, 37°C), and the cells were washed three times (800 g, 5 min) in incubation medium lacking BSA and containing 40 mM Hepes buffer, pH 7.4, with NaCl reduced by 40 mM (H-0BSA medium). Sperm were centrifuged onto coverslips (35 g, 5 min), fixed in 6% (w/v) paraformaldehyde in PBS for 10 min, rinsed in PBS, dried, and mounted in a solution of 100 mg/ml diazobicyclo-[2.2.2.]octane, 1 mg/ml sodium azide, 9:1 v/v glycerol:PBS, pH 9.0. To detect CD59, sperm were incubated with mouse anti-CD59 (30 min, 37°C), washed three times in H-0BSA medium (800 g, 5 min), incubated with FITC- or Cy3-conjugated Fab fragment of goat anti-mouse secondary antibody (30 min, 37°C), and then washed, fixed, and mounted as previously described. Because the epitope of flotillin-2 is not detectable on the surface of living cells, sperm were centrifuged onto coverslips, fixed in paraformaldehyde, and permeabilized in cold methanol (5 min, –20°C) before labeling with anti-flotillin-2 (2 h, room temperature) followed by Cy3-conjugated Fab fragment of goat anti-mouse secondary antibody (1 h, room temperature). Living sperm were labeled in a noncapacitating medium (incubation medium with BSA reduced to 3 mg/ml or replaced by 3 mg/ml ovalbumin). Fixed sperm were blocked with 4% (v/v) goat serum in PBS for 30 min and then labeled in the same solution.

To cross-link GM1 into patches and observe the effect on CD59, living sperm were incubated in FITC-CTx (30 min, 37°C), washed three times in H-0BSA medium (800 g, 5 min), and incubated in rabbit anti-CTx (10 min, 37°C). Sperm were then centrifuged onto coverslips, fixed in paraformaldehyde, blocked, and labeled with anti-CD59 (2 h, room temperature) followed by Cy3-conjugated Fab fragment of goat anti-mouse IgG (1 h, room temperature) as described previously. To cross-link CD59 and observe the effect on GM1, a similar procedure was used by treating sperm first with anti-CD59 followed by bivalent TRITC-conjugated goat anti-mouse IgG, fixing them, and finally labeling with FITC-CTx (30–45 min, room temperature). In some experiments, GM1 and CD59 were simultaneously patched by labeling with a mixture of FITC-CTx and anti-CD59 (30 min, 37°C), washing, and then a mixture of anti-CTx and TRITC-conjugated goat anti-mouse IgG (10 min, 37°C). The sperm were then centrifuged onto coverslips, fixed in paraformaldehyde, dried, and mounted as described previously.

Sperm were inspected with an Olympus BH-2S fluorescence microscope. To observe FITC, BP-490 + EY-455 exciter filters, a DM-500 dichroic mirror, and a O-515 emission filter were used. To observe Cy3 or TRITC, a BP-545 exciter filter, DM-580 dichroic mirror, and a O-590 long-pass emission filter were used. For doubly labeled cells, accessory filters were added to prevent bleed-through: a 535 ± 20-nm band-pass emission filter for observing FITC and a O-515 long-pass excitation filter for observing TRITC or Cy3. Images were recorded with an Olympus C-4040 digital camera, and Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA) was used to crop, align, and superimpose pairs of images. In merged images, the color level was adjusted to increase visibility. Grayscale images were produced by discarding color information and adjusting the intensity levels to optimize contrast.

Sterol Assays

Sperm sterol content was determined either immediately after motile sperm were prepared from semen or after 24 h of incubation. Sperm were collected by centrifugation (10 min, 800 x g) and washed and suspended in PBS. The sperm concentration after washing was determined with CyQuant, a fluorescent DNA-binding dye (Molecular Probes, Eugene, OR), according to the manufacturer's directions. The internal standard {alpha}-cholestane was added to each tube of washed sperm, and lipids were extracted with chloroform and methanol as previously described [16]. The extracted material was dissolved in hexane and analyzed by gas chromatography using a Perkin-Elmer Autosystem XL with Turbochrom 4.1 for control and analysis and a DB-17 column (J&W Scientific, Folsom, CA; 0.53 mm i.d. x 30 m long). The carrier was helium (18 ml/min), and the flame ionization detector was supplied with hydrogen (45 ml/min) and air (450 ml/min). Preliminary experiments determined that the ratios of sterols to {alpha}-cholestane were not altered by the extraction procedure. To ensure that the washing procedure removed soluble extracellular sterol from the sperm suspension, blank samples were prepared with sterol in incubation medium but lacking sperm. This protocol assays free, unesterified sterols; in this report, all references to sterols mean the unesterified form.

Statistics

Means were compared by analysis of variance with Tukey posttests using InStat (GraphPad, Inc., San Diego, CA) with P < 0.05 indicating significance. Percentage data were transformed before analysis (arcsine [%/100]1/2;) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Raft Markers

When sperm were extracted in ice-cold 1% (v/v) Triton X-100 and centrifuged on an Optiprep-sucrose density gradient, the markers CD59, flotillin-2, and GM1 were found mainly in four fractions: two at the top of the gradient (0% and 20% Optiprep) and two at the bottom (35% and 40% Optiprep) (Fig. 1). Na+/K+-ATPase is a nonraft protein [41]; it was found only in the 35% and 40% fractions. When sperm were extracted in Triton X-100 at 37°C, CD59 was found only in the 35% and 40% Optiprep fractions (not shown). These results indicate that the detergent-resistant membrane raft components were found in the 0% and 20% fractions, and nonraft, detergent-soluble components were found in the 35% and 40% fractions. The relative amounts in the 35% versus 40% fractions or in the 20% versus 0% fractions varied among experiments, probably because a substantial amount was found at the interface of the adjacent fractions and was not divided to the same degree during collection of the gradient fractions. In the text that follows, "raft" means the sum of materials found in the 20% and 0% fractions, and "nonraft" means the sum of materials found in the remaining fractions.



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  		FIG. 1. Distribution of markers in density gradient fractions. Sperm were lysed in ice-cold 1% v/v Triton X-100, separated on an Optiprep-sucrose flotation step gradient, and analyzed by immunoblot using anti-Na+/K+-ATPase {alpha}-1 chain, anti-CD59, or anti-flotillin-2. GM1 was detected by dot blot using peroxidase-conjugated CTx. Flotillin-2, CD59, and GM1 were partly located in a Triton X-100-insoluble fraction that floated to the interface of 20% and 0% Optiprep. The nonraft marker Na+/K+-ATPase {alpha}-1 chain was found only in the 35% and 40% Optiprep fractions

Changes in the Density Distribution of Raft Markers During Capacitation

During 24-h incubation in vitro, sperm lose 67% of their 3ß-hydroxysterols, cholesterol, and desmosterol and become acrosomally responsive [4, 18] (Fig. 2). During this period, the percentage of CD59 and GM1 in the raft fraction decreased significantly (Fig. 3). The distribution of flotillin-2 did not change. The total amount of these three markers recovered from the gradients did not change significantly during the 24-h incubation (data not shown). To determine if the redistribution of CD59 and GM1 from raft to nonraft fractions was sterol loss dependent, sperm were incubated in medium supplemented with cholesterol. Under these conditions the sperm cholesterol content remains high, and sperm do not become acrosomally responsive [4, 16, 42] (Fig. 2). A concentration of cholesterol was used that replaced both cholesterol and desmosterol in sperm because desmosterol has about the same ability as cholesterol to create order in model phospholipids [16]. When sperm sterol loss was prevented in this fashion, CD59 and GM1 did not shift out of the raft fraction (Fig. 3). Flotillin-2 was not affected.



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  		FIG. 2. A) Changes in the incidence of dead sperm, spontaneously acrosome-reacted (AR) sperm, and sperm that acrosome-react on exposure to progesterone (P4-AR). Sperm were assayed when they were uncapacitated (U; shortly after ejaculation), after capacitation by incubation in vitro for 24 h (C), and after incubation 24 h with 7.5 µM cholesterol (Ch). Data are shown as the mean ± SEM (n = 4). B) Sperm content of cholesterol (white bars) and desmosterol (black bars) during incubation in vitro. Data are shown as the mean ± SEM (n = 3). The negative-going bar is for cholesterol, and the positive-going bar is for desmosterol



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  		FIG. 3. Changes in the distribution of components of lipid raft fractions isolated by density gradient centrifugation. Fractions were prepared from sperm that were uncapacitated (U; analyzed shortly after ejaculation), after capacitation by incubation in vitro for 24 h (C), and after incubation 24 h with 7.5 µM cholesterol (Ch). A) CD59. B) GM1. C) Flotillin-2. Data are shown as the mean ± SEM (n = 4–14). Asterisks mark groups that are significantly different from both of the other two treatment groups in the same set (P < 0.05)

Fluorescent Labeling of Raft Markers

GM1 and CD59 on the sperm surface were detected by labeling living sperm with FITC-CTx and anti-CD59, respectively. GM1 and CD59 were detected on all regions of all sperm (Fig. 4, A and B). The labeling was generally uniform (when a monovalent second antibody was used to detect anti-CD59), although small areas of variations in intensity occurred, perhaps because of the multivalent nature of FITC-CTx and anti-CD59. The posterior head was somewhat variable in labeling with FITC-CTx. It usually had the same intensity as the anterior head, as in Figure 4A, but on some sperm it was either more or less intense than the anterior head (not shown). Flotillin-2 was not detectable on living sperm, but after sperm were fixed and permeabilized, it was detected mainly in the posterior head and midpiece (Fig. 4C). A small amount of labeling was sometimes present on the flagellum and anterior head. Much of the flotillin-2 was in small patches, but the posterior head of some sperm contained a uniform band of label (Fig. 4C). When control mouse IgG was used in place of the first antibody, no labeling was seen with any of the secondary antibodies. There were no discernible differences in the labeling patterns of GM1, CD59, and flotillin-2 in uncapacitated sperm compared to 24-h capacitated sperm (not shown).



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  		FIG. 4. Distribution of GM1 (labeled with FITC-CTx), and CD59, and flotillin-2 (labeled with specific antibodies). A) GM1. B) CD59. C) Flotillin-2. Left, phase contrast; right, fluorescence. D–F) GM1 was patched using FITC-CTx and rabbit anti-CTX, and then CD59 was localized using anti-CD59 and Fab Cy3-anti-mouse IgG. D) Phase contrast. E) GM1. F) CD59. G–J) GM1 and CD59 were patched simultaneously. A flagellum is shown. G) Phase contrast. H) GM1. I) CD59. J) Merged images. Areas of overlap are yellow (arrows). K–L) GM1 was patched using FITC-CTx and rabbit anti-CTx, and then flotillin-2 was inspected using anti-flotillin-2 and Fab Cy3-anti-mouse IgG. K) Phase contrast. L) GM1. M) Flotillin-2. N) Merged images. Areas of overlap are yellow (arrows). Bars = 2 µm. The bar in A applies to all figures except G–J

The association of GM1 and CD59 on uncapacitated sperm was tested by causing one to gather into patches and observing whether the other also patched. GM1 was patched by incubating living sperm with FITC-CTx followed by rabbit anti-CTx. The sperm were then fixed and labeled with mouse anti-CD59 followed by monovalent Cy3-conjugated anti-mouse IgG to reveal the distribution of CD59 (Fig. 4, D–F). Controls indicated that there was no visible bleed-through of the two fluorescent emissions and that the anti-mouse antibody was species specific. The treatment caused GM1 to form patches on all regions of the sperm surface (Fig. 4E). In contrast, CD59 was unperturbed (Fig. 4F), suggesting that the two molecules were not stably associated. A similar result was observed when CD59 was caused to patch with bivalent TRITC-conjugated anti-mouse, and then the distribution of FITC-CTx was inspected (not shown). In both cases, essentially all the nontargeted molecule remained unpatched.

Finally, GM1 and CD59 were simultaneously patched on living, uncapacitated sperm using a mixture of FITC-CTx and mouse anti-CD59, followed by a mixture of rabbit anti-CTx and TRITC-conjugated anti-mouse IgG (Fig. 4, G–J). In two experiments, 1959 patches on 51 sperm were inspected. Of the FITC-CTx-labeled patches, 89% had no overlap with CD59-containing patches. Of the CD59-containing patches, 72% had no overlap with CTx-containing patches.

The association of GM1 and flotillin-2 was also inspected in uncapacitated sperm. Because flotillin-2 can be detected only on fixed, permeabilized sperm, it could not be patched in living sperm. GM1 was patched using FITC-CTx followed by rabbit anti-CTx, and then the sperm were fixed, permeabilized with methanol, and labeled with mouse anti-flotillin-2 followed by monovalent Cy3-conjugated anti-mouse IgG. The distribution of flotillin-2 was not perceptibly altered by this treatment, and only minor areas of overlap were detected between GM1 and flotillin-2 (Fig. 4, K–N).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The lipids of mammalian sperm undergo remarkable remodeling as the sperm prepare for fertilization. Two thirds of the ß-OH sterols are lost, causing a decrease in bulk lipid order [4, 18, 19, 43]. The phospholipid composition changes, and the transverse asymmetry of the phospholipids in the plasma membrane decreases [44–46]. Some lipids redistribute horizontally [47]. This report adds the reorganization of raft domains to that list.

Because cholesterol helps stabilize lipid rafts, one might expect that loss of sperm cholesterol during capacitation would affect the amount or composition of lipid rafts. The role of desmosterol in raft stability has not been studied, but experiments utilizing model systems suggest that the lipid-ordering property of a sterol determines its ability to stabilize rafts [48, 49]. In this regard, desmosterol is about equivalent to cholesterol [16], so desmosterol may also play an important role in organizing sperm lipid rafts.

Rafts are operationally defined entities, and their composition depends on the conditions used for cell fractionation [50]. The isolation procedure used here—solubilization in ice-cold Triton X-100 followed by density gradient centrifugation—is the most widely used protocol. Three raft markers were studied. Two of these, GM1 and CD59, behaved similarly. About 40%–50% of these molecules were in a raft fraction isolated by density gradient ultracentrifugation. It is likely that CD59 is restricted to the external leaflet of the plasma membrane because it can be completely removed from sperm by extracellular phosphatidyl inositol-specific phospholipase C [28]. The location of GM1 has not been determined in human sperm, but in neuroblastoma cells most is externally exposed on the cell surface [51]. In the present experiments, when fluorescent labels were applied to living sperm, the patterns reported here were present on most or all of the sperm, so these patterns reflect the distribution of externally exposed, plasma membrane GM1 and CD59.

In uncapacitated sperm, GM1 and CD59 were present more or less uniformly over the whole sperm surface. This is a bit surprising because reports using the probe filipin have shown that the ß-OH sterol content of the human sperm plasma membrane is concentrated in the anterior head, and one might expect some preference for that environment [14]. Whether rafts are in a higher concentration in the head remains to be determined. Because 50%–60% of GM1 and CD59 were not in the raft fraction, one cannot equate their distribution as revealed by fluorescence microscopy with the distribution of lipid rafts.

It is also interesting that not all GPI-linked proteins have the same distribution on human sperm: CD59 and CD52 are found on the entire sperm surface, while plasma membrane GPI-linked hyaluronidase occurs only in the membrane of the head [28, 52, 53]. Clearly, the GPI linkage does not solely determine where on the sperm surface a protein resides, and it does not restrict all GPI-linked molecules to raft domains.

GM1 has been reported to change its location on the surface of rat and boar sperm as the sperm become capacitated, moving either rostrad (from a dispersed distribution to the head in boar sperm [27]) or caudad (from postacrosomal/head cap to the equatorial region, acrosome, and tail in rat sperm [54]). Whether GM1 relocates during capacitation of mouse sperm is controversial (compare references [55] and [27]). In the present study, however, neither GM1 nor the proteins CD59 and flotillin-2 changed their distributions on the human sperm surface during capacitation.

During incubation in vitro for sperm to become acrosomally responsive, raft GM1 and CD59 partly shifted to the nonraft fraction. Because the shift could be blocked by preventing a net loss of sperm sterol, it is likely that the capacitation-associated loss of sterols modifies the structure and/or abundance of rafts. The simplest explanation is that loss of sterols causes some rafts to disperse, releasing all their components to the less ordered nonraft domain. It is premature to conclude that dispersal is so nonselective, however. In porcine sperm, a 70% reduction in sperm cholesterol by 5 mM methyl-ß-cyclodextrin caused sperm to become acrosomally responsive to calcium ionophore but did not decrease the abundance of rafts (measured by total protein content and light scattering) [27]. The alternative explanation is that sterol loss causes selected raft components to leave the rafts, which otherwise remain intact. Another unresolved issue is whether all sperm exhibit the same effects. About 25% of the sperm become acrosomally responsive, and it is not yet known whether sterol loss and raft reorganization is confined to these cells or experienced by all cells.

Although CD59 and GM1 had similar surface locations and sterol loss-dependent shifts out of the raft fraction, it appears that in the plasma membrane they are not stably associated in the same raft domains. When one of the molecules was clustered using a bivalent antibody, the other was hardly affected. When the markers were clustered simultaneously, the patches showed minor overlap. If there is an association between these two molecules, it is not strong enough to cause coincident patching, suggesting that little if any of these two markers coexist in the same type of lipid raft. GM1 and CD59 are also in different microenvironments in the membrane of epithelial cells [56], while contradictory results have been reported for T cells [57, 58].

The third raft marker studied was flotillin-2. Its distribution in sperm was different from CD59 and GM1, as it was concentrated in the posterior head, neck, and midpiece. Labeling was generally punctate, as reported in other types of cells [59], even though the sperm were fixed in paraformaldehyde and methanol, and a monovalent secondary antibody was used to reduce cross-linking. Copatching experiments did not reveal an association between GM1 and flotillin-2. Flotillin-2 was more fully associated with the raft fraction (about 90%) compared to GM1 or CD59, and this did not change significantly when sperm were capacitated. As in hemopoietic cells, flotillin-2 apparently does not depend on cholesterol for association with rafts [60]. It is likely that most of the flotillin-2 resides in a third type of lipid raft.

In summary, this report demonstrated that human sperm contain a heterogeneous collection of lipid rafts, some of which are altered in composition and/or abundance because of sterol loss during capacitation. Could these changes play an essential role in capacitation? Although the functional importance of rafts is not fully understood, rafts are typically considered to be foci that concentrate interacting molecules to initiate signaling cascades [23]. According to this model, raft dispersal might terminate a set of reactions that suppress capacitation, or exit of an inhibitor might activate the remaining raft-associated molecules to trigger capacitation. Alternatively, one might consider that heterogeneous rafts could segregate potential partners and that releasing them to the more fluid phase would facilitate their interaction. These possibilities merit further attention.


   ACKNOWLEDGMENTS
 
I thank Dr. Lin Liu for many helpful discussions.


   FOOTNOTES
 
1 Supported by NIH grant HD30763. Back

2 Correspondence: Nicholas L. Cross, Department of Physiological Sciences, 264 McElroy Hall, Oklahoma State University, Stillwater, OK 74078. FAX: 405-744-8263; ncross{at}okstate.edu Back

Received: 1 April 2004.

First decision: 21 April 2004.

Accepted: 10 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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