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BOR - Papers in Press, published online ahead of print February 1, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.046219
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BIOLOGY OF REPRODUCTION 74, 889–895 (2006)
DOI: 10.1095/biolreprod.105.046219
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Visualization of GM1 with Cholera Toxin B in Live Epididymal Versus Ejaculated Bull, Mouse, and Human Spermatozoa1

Danielle E. Buttke 3, Jacquelyn L. Nelson 3, Peter N. Schlegel 4, Gary R. Hunnicutt 5, and Alexander J. Travis 2 3

The James A. Baker Institute for Animal Health,3 College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Weill Medical College,4 Cornell University, New York, New York 10021 Population Council,5 The Rockefeller University, New York, New York 10021

ABSTRACT

The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside GM1. Using the pentameric B subunit of cholera toxin (CTB), we found GM1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in GM1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that GM1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access GM1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to GM1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing GM1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of GM1 in sperm of different species, and provide evidence for the segregation of GM1 to a stable subdomain in the plasma membrane overlying the acrosome.

epididymis, gamete biology, seminal vesicles, sperm

INTRODUCTION

The organization of colocalized receptor and effector complexes is an effective way to transduce signaling events into functional changes within a specific area of a cell [1]. Membrane subdomains enriched in sterols and sphingolipids such as gangliosides have been postulated to play an important role in a wide variety of cellular functions by acting as scaffolds or foci for the compartmentalization of signaling molecules to specific regions of membrane (see [2] for review). Such lipid raft subdomains have been found to anchor signaling molecules in somatic cells until stimulation allows for the release and activation of the molecule [3]. Dynamic changes in raft components and localization can also allow for the interaction of previously segregated signaling and effector molecules, as seen in the transduction of downstream events in B and T lymphocytes [4–6].

The presence of lipid raft subdomains in mammalian spermatozoa is of special interest because of the importance of plasma membrane alterations in sperm function. For example, the removal of sterols from the plasma membrane is a required stimulus for the process of capacitation, in which sperm acquire the ability to undergo acrosomal exocytosis [7] and a hyperactivated pattern of motility [8]. These changes render the sperm fertilization-competent. Because of their ability to transduce sterol efflux into functional changes, lipid rafts have been suggested to be involved in a number of processes of capacitation, including sterol efflux and acrosomal exocytosis [9–12]. Evidence of the presence of membrane subdomains in fixed/dried sperm has been provided by several methods, including direct labeling with membrane antibodies [13], fluorescence imaging of lipid-binding probes [14] and exogenous lipid probes [15, 16], freeze fracture, surface replica and freeze-etch electron microscopy [17–21], and atomic force microscopy [22]. For example, the polyene antibiotic filipin has been used to show that the distribution of sterols within the plasma membrane of fixed sperm is heterogeneous. Visualized by freeze-fracture electron microscopy or autofluorescence, filipin-sterol complexes delineate an area of extreme sterol enrichment in the plasma membrane overlying the acrosome, with a much lower sterol content found in the postacrosomal region [20, 23–25]. Caveolin-1, a sterol-binding protein associated with rafts [26], has since been shown to colocalize with the sterol-rich subdomain overlying the acrosome [10]. Caveolin-1 has been reported to scaffold signaling complexes and to participate in the movement of sterols across membranes (see [27] for review).

Despite the rapidly-growing literature regarding lipid raft membrane subdomains in both sperm and other cell types, legitimate controversy has arisen regarding whether lipid rafts are found in nature, or instead represent artifacts induced by the methods used to isolate them. For example, the use of fixatives/cross-linking reagents when trying to visualize lipid rafts can cause patching artifacts as different membrane components are brought into proximity. In addition, there is also controversy regarding the use of detergents during biochemical isolation, because they can cause artifactual coalescence of membrane components that might not interact under physiological conditions [28–30]. We have demonstrated that the same region of the plasma membrane that is enriched in sterols and caveolin-1 in fixed cells is enriched in the ganglioside GM1 in living, motile sperm [31]. This demonstrates that the membrane subdomain overlying the acrosome is not an artifact of fixation. In addition, we have been able to use a protocol without detergents to partition sperm membrane subdomains by means of their relative buoyancy alone [10]. Together, these data demonstrate the existence of membrane subdomains in sperm.

Much attention has been focused on GM1 because of its suggested association with lipid raft subdomains and the ease and specificity of its localization by means of fluorescence conjugates of the pentameric subunit B of cholera toxin (CTB). Previous work has also suggested that sperm-egg interactions in several nonmammalian species might be mediated in part by one or more gangliosides [32–34].

However, our experiments using murine sperm [31] have yielded results that contrast with other published results for the mouse [35] and rat [36], which themselves contrast with studies localizing GM1 in human [11] and boar sperm [12]. For example, we demonstrated localization to the plasma membrane overlying the acrosome in epididymal murine sperm, a region consistent in terms of size and stability [31], whereas other rodent studies localized GM1 to the postacrosomal plasma membrane [35, 36]. Conversely, GM1 localization in noncapacitated human and boar sperm was reported as nonexistent or patchy and inconsistent throughout the entirety of the sperm cell [11, 12]. One likely difference between the studies in rodents was the use of fixation conditions. We demonstrated that upon cessation of motility/cell death in unfixed or lightly fixed cells, GM1 moved rapidly from its position overlying the acrosome to the postacrosomal plasma membrane [31]. This finding underscores some of the difficulty inherent to visualization of lipid subdomains. One clear difference between the studies in rodents and those in other species was that the murine sperm were collected from the epididymis, whereas the human and boar sperm were collected from ejaculates. This raised the possibility of sperm source as an alternative to true species differences regarding the localization of GM1. In an attempt to resolve these dissimilar findings and improve understanding of the dynamics of membrane subdomains in mammalian sperm, we have compared the localization of GM1 in epididymal and ejaculated sperm of bull, mouse, and human.

MATERIALS AND METHODS

Reagents and Sources of Samples

All reagents were purchased from Sigma unless otherwise noted. CTB conjugated with Alexa-Fluor 488 and anti-rabbit IgG conjugated with Alexa-Fluor 647 were purchased from Molecular Probes. Purified PDC-109 (SPF1_BOVIN; UniProt/Swiss-Prot accession number P02784; also known as bovine seminal plasma [BSP] A1/A2) was a gift from the labs of Puttaswamy Manjunath and Susan Suarez. Rabbit polyclonal antiserum against PDC-109 was a gift from the lab of Susan Suarez. GVA mount was from Zymed. Semen was collected from proven high fertility bulls at Genex/CRI. Bull epididymides were collected from Genex, CRI, Cudlin's Meat Market, or Wyalusing Livestock Market. Male CD-1 mice were obtained from Charles River Laboratories. All animal work was conducted under the approval of Cornell University's Institutional Animal Care and Use Committee, in accordance with the Guide for Care and Use of Laboratory Animals. Human epididymal sperm were collected from the Center for Male Reproductive Medicine and Microsurgery at Cornell University's Weill Medical College from men with obstructive disorders undergoing epididymal aspiration for use in assisted reproduction. All procedures were performed as part of treatment of these patients and with institutional review board oversight at Weill Cornell Medical Center.

Collection and Handling of Sperm

Bull. Ejaculated bull semen was immediately diluted at a 1:4 ratio in sperm Hepes-buffered Tyrode-albumin lactate pyruvate (TALP H; 100 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 21.6 mM sodium lactate, 0.4 mM MgCl2, 40 mM Hepes, 0.4 mM EDTA, 10 mM NaHCO3, 2 mM CaCl2, 1 mM pyruvic acid, 50 µg/ml gentamycin, and 1 mg/ml poly-vinyl alcohol (PVA)) [37] and transported to the laboratory at 39°C. Sperm were washed at 39°C in TALP H. One milliliter of diluted semen was brought to 7 ml with TALP H and spun at 170 x g for 8 min. The sperm were transferred to a round-bottomed tube to repeat the spin twice more, resulting in a loose pellet of cells, which were resuspended in a final volume of 5 ml. Motility was assessed and sperm counted. No experiment was performed if motility was <50% immediately before fluorescent microscopy or fixation. Sperm (2 x 106) were then resuspended in 300 µl Tyrode-albumin lactate pyruvate (TALP; 100 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 21.6 mM sodium lactate, 0.4 mM MgCl2, 10 mM Hepes, 0.4 mM EDTA, 25 mM NaHCO3, 2 mM CaCl2, 1 mM pyruvic acid, 50 µg/ml gentamycin, and 1 mg/ml PVA) [37].

For experiments involving additional washes of ejaculated bull sperm, the sperm were washed under four separate media conditions: 1) TALP with 250 mM NaCl, 2) TALP with 500 mM NaCl, 3) TALP, pH 9, and 4) TALP, pH 11. Motility was assessed, and sperm were then incubated with CTB as described below.

Cauda epididymides of bulls were transported on ice and then warmed to room temperature for the dissection of the epididymides from the testis, blood vessels, and connective tissue. Epididymides were washed three times in PBS and blotted before being minced in TALP H. Sperm were allowed to swim out for 15 min before being washed in TALP H, resuspended in TALP, and incubated at 39°C and 5% CO2. Alternately, epididymal sperm were swum out in PBS, incubated at 39°C, and used for GM1 localization experiments as described below. There were no differences in results.

Mouse. A modified Whitten's medium (MW; 22 mM Hepes, 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, and 4.8 mM lactic acid hemicalcium salt, pH 7.35) [38] containing 5.5 mM glucose was used for all mouse sperm incubations. Mature sperm were collected from the cauda epididymides by a swim-out procedure as described previously [38] and washed at 37°C. All incubations of mouse sperm were conducted at 37°C in MW.

Human. Ejaculated human sperm were collected from healthy male donors, allowed to liquefy for 30 min at 37°C, and then diluted 1:4 in MW. The sperm were washed by centrifugation (200 x g) for 10 min at 37°C in MW. All incubations of human sperm were carried out at 37°C.

Microsurgical epididymal sperm aspiration was carried out as previously described [39]. Briefly, the epididymides of two patients with documented normal spermatogenesis and obstruction were explored, and the epididymal segment with optimal sperm motility was identified and aspirated using a micropuncture technique to avoid contamination with red blood cells. The presence of sperm and their motility were confirmed by evaluation during the operative procedure.

Fluorescence Localization in Live Sperm

All steps of localization experiments were carried out under dim lighting in a light-protected humidity chamber. Sperm (2 x 106) were incubated in 300 µl MW (mouse, human) or 300 µl TALP (bull) containing 10 µg/ml CTB for 10 min. For some experiments, different concentrations of epididymal bull sperm were preincubated with purified PDC-109 (0.4 mg/ml) for 30 min before CTB addition. For the mouse and bull, a 10 µl aliquot was transferred to a prewarmed slide and a coverslip was placed over the slide and viewed under a Nikon Eclipse TE 2000-U microscope equipped with a Photometrics Coolsnap HQ charge-coupled device (CCD) camera (Roper Scientific), and Openlab 3.1 (Improvision) automation and imaging software. Human sperm were visualized with an Olympus BX60 epifluorescent microscope equipped with a 37°C stage warmer and a Peltier-cooled CCD digital camera controlled by QCapture 2.68.6 software (Quantitative Image Corporation).

Fluorescence Localization in Fixed Cells

Bull sperm motility was assessed at the start and completion of washing, and after 1.5 and 3 h incubation. Mouse and bull samples were removed from the incubation tubes described above and settled on coverslips, and human sperm samples were placed onto Cel-Line HTC SuperCured 10-spot slides (Cel-Line Association Inc.) for 15 min to allow the sperm to attach before the supernatant was aspirated. Mouse and human sperm were fixed with 4% paraformaldehyde (PF), 0.1% glutaraldehyde, and 5 mM CaCl2 in PBS, whereas bull sperm were fixed with 1% PF and 12.5 mM CaCl2 in PBS. The sperm were then washed three times with PBS and incubated for 10 min in CTB (10 µg/ml) for GM1 localization.

Mouse Seminal Vesicle Fluid Collection and Incubation

The seminal vesicles (SV) were isolated from surrounding blood vessels and the coagulating glands by sharp dissection. Care was taken to avoid contamination with either blood or coagulating gland secretions, as these rapidly catalyze the precipitation of proteins within the SV fluid [40]. The glands were removed individually and the fluid contents were allowed to drip out and/or were manually expressed into microcentrifuge tubes. Using a large orifice pipette tip, 10 µl of SV fluid was aspirated and the tip and contents were placed in a humidity chamber at 37°C to await sperm addition. Sperm (4 x 106) were added to the SV fluid in the pipette tip, the tip was immersed in a 600-µl drop of PBS, and sperm were allowed to swim through the SV fluid, out of the tip, and into the PBS for 15 min. The tip and associated SV fluid were then carefully removed and CTB (10 µg/ml final concentration) was added to the coverslip and allowed to incubate for 10 min. The supernatant was aspirated before the coverslip was placed on a prewarmed slide and viewed for GM1 localization.

Protein assays were conducted on seminal vesicle fluid, and the concentrations (ranging from 167 to 350 mg/ml) were in accord with published levels, which range from 250 to 350 mg/ml [41]. As a control for exposure to an equivalent amount of protein to that found in the SV fluid, sperm (4 x 106) were incubated in 600 µl of PBS with casein that was varied to match the total amount of protein contained in the SV fluid (ranging from 1.67 to 3.5 mg) for 15 min before incubation with CTB. Casein was chosen as a control because we have found that it does not mediate sterol efflux as would BSA (data not shown).

Bull Seminal Plasma Isolation and Incubation

Ejaculated bull sperm were diluted 1:3 in TALP H and transported to the laboratory at 39°C. This volume was then spun at 800 x g for 10 min to pellet the sperm fraction. The top half of the supernatant was carefully aspirated without disturbing the sperm pellet and was snap-frozen in a dry ice/ethanol slurry for later use.

Aliquots of frozen, dilute seminal plasma collected as above were thawed at 39°C and examined with light microscopy to verify the absence of sperm before incubation. Epididymal bull sperm (4 x 106) were incubated in 600 µl dilute seminal plasma for 10 min before fluorescence localization of GM1 with 10 µg/ml CTB as described for live epididymal bovine sperm.

Immunofluorescence

Epididymal bull sperm incubated with and without purified PDC-109 were processed as previously described [42] with minor modifications. Briefly, aliquots of the sperm suspensions were added to slides on a 37°C warming stage and allowed to air dry. Slides were blocked in 0.1% BSA overnight before an additional overnight incubation with anti-PDC-109 (1:500) followed by three washes with PBS and incubation with secondary antibody. Slides were again washed three times in PBS before being mounted with GVA and visualized by epifluorescence.

RESULTS

Ejaculates from some bulls displayed a low percentage of sperm with faint CTB binding over the acrosome, but the vast majority of motile ejaculated bull spermatozoa failed to bind CTB. However, immotile bull sperm uniformly displayed a postacrosomal pattern of fluorescence (Fig. 1, A and B). These findings were remarkable in comparison with motile epididymal murine sperm, which show an acrosomal pattern of CTB in all motile cells (Fig. 1, C and D). We have shown previously that upon cessation of motility, epididymal murine sperm switch from having a pattern of CTB localization overlying the acrosome to a postacrosomal pattern ([31]; shown here in Fig. 1, E and F).


Figure 1
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  		FIG. 1. A comparison of motile and immotile ejaculated bull sperm incubated with CTB (n = 9 experiments; over 100 cells examined per treatment for this and subsequent experiments). Transmitted (A) and fluorescent (B) images of ejaculated bull sperm. Note the lack of signal in the motile cell. Arrows indicate the location of the live cell present next to an immotile cell displaying a postacrosomal pattern of fluorescence. Transmitted and fluorescent images of motile (C–D) and immotile (E–F) epididymal murine spermatozoa incubated with CTB.

During the process of ejaculation, sperm are exposed to several accessory sex gland secretions that interact with a sperm's plasma membrane [42, 43] and which could potentially interfere with the ability of exogenous reagents to bind to sites on this membrane. The differences in reported results between species, and between our findings in the mouse and the findings shown in Figure 1 in the bull, could therefore have been because of true species differences or a difference between sperm collected from the epididymis versus those from an ejaculate. To distinguish between these possibilities, we next localized GM1 in epididymal bull spermatozoa. All morphologically normal epididymal bull sperm that were motile (Fig. 2, A and B) displayed a pattern of GM1 localization identical to that seen in epididymal mouse sperm (Fig. 1, C and D), with fluorescence restricted to an area of the plasma membrane overlying the acrosome and also throughout the flagellum. Interestingly, live epididymal spermatozoa exhibiting abnormal morphology also displayed aberrant patterns of GM1 localization (Fig. 2C). For example, sperm with proximal droplets were frequently observed to have abnormal CTB binding over the area of the proximal droplet, connecting piece, and caudal portion of the postacrosomal subdomain of the head.


Figure 2
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  		FIG. 2. Epididymal bull sperm incubated with CTB (n = 4 experiments representing 12 individual bulls). Survey image of motile epididymal sperm displaying acrosomal patterns of fluorescence and a single immotile sperm displaying a postacrosomal pattern of fluorescence (A). Consistent with our data from the mouse (above, Fig. 1 and published [31]), sperm having a postacrosomal pattern of GM1 localization had a relatively brighter fluorescence intensity. Fluorescent image of a motile cell displaying an acrosomal pattern of fluorescence (B). Motile cell possessing a proximal cytoplasmic droplet and displaying abnormal CTB fluorescence (C).

Based on the results of GM1 localization in epididymal bull sperm, we hypothesized that exposure of murine epididymal sperm to accessory sex gland fluids would also result in a loss of CTB binding and/or fluorescence in the plasma membrane overlying the acrosome. The majority of both bull and mouse ejaculate volume originates from the SV [41, 44]. We therefore swam epididymal mouse sperm through SV secretions and observed the pattern of CTB fluorescence. As predicted, exposure to SV fluid significantly reduced the ability of CTB to bind GM1 as compared to normal epididymal murine sperm (Fig. 3; P < 0.05, n = 3). CTB fluorescence was not detected in the heads of the majority of cells (49%; Fig. 3, A and D), with remaining cells displaying a patchy, mottled pattern of fluorescence in the plasma membrane overlying the acrosome (47%; Fig. 3B). To demonstrate that nonspecific interactions with an equivalent amount of protein would not cause this decrease in binding, motile sperm were incubated in base medium alone (data not shown) or with casein (Fig. 3C). In both cases, motile sperm displayed an acrosomal pattern of fluorescence. In both of those treatment conditions, the vast majority of immotile cells displayed a postacrosomal pattern (n = 4 experiments; data not shown). Together, these results suggested an interaction of the SV fluid with the sperm plasma membrane overlying the acrosome that sterically and/or specifically prevented the binding and/or fluorescence of CTB to GM1. Similarly, epididymal bull sperm exposed to seminal plasma isolated from an ejaculate lost CTB binding over the acrosome and appeared identically as those collected from an ejaculate (n = 3 experiments; data not shown).


Figure 3
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  		FIG. 3. Effect of exposure to seminal vesicle fluid on CTB binding in murine spermatozoa (n = 3 experiments). Sperm incubated with SV fluid before incubation with CTB displayed little (A) or dim and mottled (B) fluorescence when compared to sperm incubated with an equivalent amount of casein before CTB incubation (C). Box whisker plot showing patterns of fluorescence after SV fluid exposure (D) showing no signal (NS), reduced signal (RS), and normal acrosomal fluorescence (APM). The boundaries of the boxes represent the 25th and 75th quantiles, the 50th quantile is displayed as a thin line within the box, and the mean as a thick line extending through the box. Whiskers extend to the 10th and 90th quantiles. In comparison, normal motile epididymal mouse sperm displayed 100% acrosomal fluorescence before SV fluid exposure (n = 3, data not shown). Statistically significant differences in the percentage of sperm showing the different patterns of localization were found between untreated epididymal sperm and epididymal sperm incubated with SV fluid using a Kruskal-Wallis analysis (P < 0.05).

The similarities between the pattern of GM1 localization reported for human sperm [11] and our results for epididymal mouse sperm exposed to SV secretions suggested that the localization in epididymal human sperm would also be in the plasma membrane overlying the acrosome. We found that CTB binding in ejaculated human sperm matched data previously reported for the human [11], with little to no fluorescence in motile cells (data not shown). We therefore examined GM1 localization in epididymal human sperm. One caveat regarding these experiments is that normal human epididymal sperm are difficult to procure. Typically, epididymal aspirations are performed in men who have obstructive pathologies, and the sperm collected tend to be abnormal. Accordingly, the epididymal human sperm we examined exhibited a very high percentage of abnormal morphologies, particularly large proximal droplets and irregular head shapes. However, the small population of motile cells with normal morphology seen did display an acrosomal pattern of fluorescence (Fig. 4, A and B). As found in the bull, a high percentage of cells possessing abnormal morphologies also exhibited atypical patterns of GM1 localization (Fig. 4, C–F).


Figure 4
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  		FIG. 4. GM1 localization in epididymal human sperm (n = 2 experiments, representing two individuals). Transmitted (A) and fluorescent images (B) of motile epididymal human sperm, showing normal morphology and an acrosomal pattern of fluorescence with CTB. Transmitted (C) and fluorescent images (D) of immotile human sperm displaying signal over the postacrosomal region and a proximal droplet. Other abnormal morphologies observed in brightfield, such as this misshapen head (E), were often accompanied by abnormal patterns of CTB binding and fluorescence (F).

In all three species examined, we observed a distinct and reproducible pattern of GM1 localization to the plasma membrane overlying the acrosome in motile epididymal sperm. Exposure of sperm of these three species to accessory sex gland secretion through either normal ejaculation (bull or human) or in vitro exposure (mouse and bull) resulted in a loss of signal. Two possible interpretations existed. One was that exposure to these secretions inhibited binding of CTB to the GM1, whereas the other was that exposure to seminal plasma removed GM1 from the plasma membrane. To rule out qualitatively the large-scale loss of GM1, we fixed ejaculated bull sperm and epididymal mouse sperm exposed to SV fluid before incubation with CTB. Sperm exposed to accessory sex gland secretions and subsequently fixed had a GM1 localization pattern that mirrored the pattern and intensity of live epididymal sperm and the pattern and intensity of CTB signal in epididymal sperm fixed without exposure to SV fluid (Fig. 5). This unmasking of GM1 with fixation suggested that the majority of GM1 remained in the sperm plasma membrane even after exposure to accessory sex gland secretions.


Figure 5
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  		FIG. 5. GM1 was not removed from sperm upon exposure to accessory sex gland secretions. GM1 localization in live epididymal bull sperm (A) or ejaculated bull sperm fixed with 1% paraformaldehyde and 12.5 mM CaCl2 before incubation with CTB (B; n = 6 experiments).

The majority of accessory sex gland secretions in the bull originate from the seminal vesicles, with the major constituent being PDC-109, which binds phospholipids in the sperm plasma membrane [42]. To determine whether PDC-109 binding to sperm was responsible for the loss of CTB binding to GM1, we incubated epididymal bull sperm with purified PDC-109 before incubation with CTB. We found no loss of CTB binding, with labeling of the plasma membrane overlying the acrosome as in untreated epididymal sperm (Fig. 6A; n = 6 experiments). As a control to demonstrate that the purified PDC-109 was binding these sperm, we performed indirect immunofluorescence (Fig. 6, B and C).


Figure 6
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  		FIG. 6. Incubation of epididymal bull sperm with purified PDC-109 did not prevent CTB binding to GM1. Fluorescent image of CTB bound to epididymal bull sperm after incubation with purified PDC-109 (A; n = 6 experiments). Indirect immunofluorescence localization of PDC-109 in epididymal bull sperm after this incubation, showing that the purified PDC-109 did bind to the plasma membrane overlying the acrosome (B; n = 6 experiments). Incubation of sperm with the secondary antibody alone served as a control for specificity of binding (C).

In a further attempt to remove and qualify the inhibition of CTB binding to GM1 in ejaculated sperm, ejaculated bull sperm were washed in TALP modified with either high salt (NaCl 250 mM and 500 mM) or high pH (9 and 11). Although binding was recovered in a low percentage (13.5%) of sperm washed in 500 mM NaCl, a treatment that also stopped motility, none of the conditions tested significantly altered the fluorescence pattern in ejaculated bull sperm from that of ejaculates washed in normal TALP H (pH 7.35, NaCl 100 mM; n = 3; data not shown). It should be noted that the highest pH condition clearly damaged the sperm, causing immotility and the appearance of a postacrosomal pattern. Therefore, it is impossible to discern what effect this wash condition might have had on CTB binding to GM1 in the plasma membrane overlying the acrosome, because the GM1 had redistributed to the postacrosomal plasma membrane subdomain.

DISCUSSION

Lipid rafts have been postulated to play a role in signaling and effector complex compartmentalization in sperm plasma membranes. Because of this, several studies have attempted to observe membrane subdomains in live and fixed sperm, but have reported widely differing results. The results obtained in this study suggest that exposure to secretions from accessory sex glands might mask the true localization of specific lipids—and therefore membrane subdomains—in mammalian sperm, accounting for some of the discrepancies within the literature.

Our previous work has described the segregation of the ganglioside GM1 to the plasma membrane overlying the acrosome in live epididymal murine spermatozoa [31]. This pattern was identical to the pattern of sterol localization seen in fixed sperm with filipin [20, 24, 25] and of the lipid-raft-associated protein caveolin-1 [10]. The segregation of a sphingolipid (GM1), sterols, and caveolin-1 to this region suggests the presence of a lipid raft subdomain extreme in terms of size and stability [31]. The biochemical partitioning of caveolin-1 to detergent-resistant membranes, as well as to fractions with light buoyant-density separated without the use of detergents [10], supports the raft-like nature of such a subdomain. Use of an exogenous lipid probe that partitions to liquid-ordered domains has recently confirmed the "raft" nature of this micron scale subdomain [45].

Other studies of CTB binding in epididymal rodent sperm had previously suggested a different pattern of localization, with GM1 being restricted to the plasma membrane of the postacrosomal subdomain in rat [36] and mouse [35]. Our recent discovery that CTB induces a redistribution of GM1 from the plasma membrane overlying the acrosome to the postacrosomal plasma membrane upon cell death and in lightly fixed sperm [31] can account for the differences observed in rodent studies. However, this report is the first to demonstrate that interaction with seminal plasma can account for many of the other reported differences among species.

For example, fluorescence localization of GM1 in ejaculated human spermatozoa was reported to show no large-scale segregation of GM1 [11]. Similar studies of ejaculated boar sperm reported a lack of GM1 localization in the majority of sperm heads, although this percentage decreased with time and incubation with reagents known to mediate sterol efflux [12]. The trend toward increased acrosomal fluorescence observed in that study was interpreted as sterol efflux-induced raft organization, which would be consistent with previous models suggesting that lipid rafts form during capacitation [9]. Both our published results and those herein contrast with such a model, and alternately suggest that lipid subdomain segregation exists on a micron scale in epididymal sperm before exposure to capacitating stimuli. Our results therefore help reconcile these conflicting reports, suggesting either a specific competitive inhibitor of binding to GM1 or a nonspecific steric masking of GM1 in the plasma membrane overlying the acrosome by substances within accessory sex gland secretions.

The binding of proteins secreted by the accessory sex glands to specific molecules of the sperm plasma membrane has been well characterized in several species, including the mouse and bull [42, 43, 46–50]. BSP proteins such as PDC-109 (SPF1_BOVIN), BSP A3 (SPF3_BOVIN), and BSP-30 kDa (SPF4_BOVIN) (UniProt/Swiss-Prot Accession numbers P02784, P04557, and P81019, respectively) preferentially bind to phospholipids enriched in the plasma membrane overlying the acrosome and promote membrane stabilization and subsequent destabilization upon their removal during capacitation [42, 43, 51, 52]. PDC-109 is the most abundant of these proteins, so we investigated whether its binding to plasma membrane phospholipids would inhibit CTB-GM1 binding. This was not the case, suggesting that either another protein or a seminal plasma component was responsible for this loss.

One additional finding of this work bears discussion for its possible clinical relevance. The aberrant patterns observed in human and bull sperm with proximal droplets and other abnormalities provide a correlation between morphological defects associated with reduced fertility and abnormal distribution of plasma membrane lipids. Interestingly, sperm with morphological defects in one region, such as proximal droplets, also showed abnormal GM1 distribution in surrounding regions that appeared morphologically normal at the level of light microscopy. This suggests that such defects might be more widespread than are immediately obvious at the level of light microscopy. Large proximal droplets have been associated with reduced fertility [53] (see [54] for review), although an exact cause for this impairment has not been described. It is intriguing to speculate that appropriate lipid compartmentalization and function might provide a molecular underpinning for such defects. The localization of lipids such as GM1 may therefore prove to be of value as a screening tool in evaluating male fertility.

The data presented in this paper provide evidence for the segregation of GM1 to the plasma membrane overlying the acrosome in three different families of mammals. These data suggest that the formation of large membrane subdomains in mammalian sperm has been conserved evolutionarily, and that these compartmentalized domains might have important roles in sperm function. The colocalization of GM1, sterols, and caveolin-1 to this lipid raft subdomain suggests possible mechanisms by which the process of sterol efflux might be transduced into the functional changes that allow a sperm to fertilize an egg. Because of this, studies into the dynamic responses of GM1 and this subdomain to stimuli associated with sperm capacitation, the acrosome reaction, and fertilization have begun.

ACKNOWLEDGMENTS

We would like to thank Puttaswamy Manjunath (Department of Medicine, University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada) for the generous gift of purified PDC-109, and Susan Suarez (College of Veterinary Medicine, Cornell University, Ithaca, NY) for the generous gifts of purified PDC-109 and PDC-109 antibody.

FOOTNOTES

1 Supported by National Institutes of Health grants R01-HD-045664, K01-RR00188 (A.J.T.), and R01-HD-038807 (G.R.H.), a Genex/CRI grant (A.J.T), and the Cornell College of Veterinary Medicine DVM/Ph.D. Dual Degree Program (D.E.B.). Back

2 Correspondence. FAX: 607 256 5608; ajt32{at}cornell.edu Back

Received: 29 July 2005.

First decision: 28 September 2005.

Accepted: 16 January 2006.

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