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BOR - Papers in Press, published online ahead of print January 30, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.066514
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BIOLOGY OF REPRODUCTION 78, 921–931 (2008)
DOI: 10.1095/biolreprod.107.066514
© 2008 by the Society for the Study of Reproduction, Inc.

Gamete Biology; 

Seminal Plasma Proteins Regulate the Association of Lipids and Proteins Within Detergent-Resistant Membrane Domains of Bovine Spermatozoa1

Julie Girouard , Gilles Frenette , and Robert Sullivan 2

Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Quebec City, Quebec, Canada G1V 4G2

ABSTRACT

Maturing spermatozoa acquire full fertilization competence by undergoing major changes in membrane fluidity and protein composition and localization. In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched detergent-resistant membrane (DRM) domains. These proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Since seminal plasma regulates sperm cell membrane fluidity, we hypothesized that seminal plasma factors could be involved in DRM disruption and redistribution of DRM-associated proteins. Our results indicate that: 1) the sperm-associated proteins, P25b and adenylate kinase 1, are linked to DRM of epididymal spermatozoa, but were exclusively associated with detergent-soluble material in ejaculated spermatozoa; 2) seminal plasma treatment of cauda epididymal spermatozoa significantly lowered the content of cholesterol and the ganglioside, GM1, in DRM; and 3), seminal plasma dissociates P25b from DRM in epididymal spermatozoa. We found that the seminal plasma protein, Niemann-Pick C2 protein, is involved in cholesterol and GM1 depletion within DRM, then leading to membrane redistribution of P25b that occurs in a very rapid and capacitation-independent manner. Together, these data suggest that DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival and attachment within the female reproductive tract and to potentiate recognition, binding, and penetration of the oocyte.

epididymis, gamete biology, male reproductive tract, sperm maturation

INTRODUCTION

Leaving the testis, mammalian spermatozoa are unable to bind to the zona pellucida and fertilize the ovum. In order to progressively acquire motility and fertilizing ability, extensive remodeling of the sperm plasma membrane occurs during the epididymal transit and in the female reproductive tract by processes generally referred to as epididymal maturation and capacitation, respectively. Through these two sperm maturation processes, certain components, such as antigens and enzymes [1, 2], membrane lipids [3], and cholesterol [4], are topographically reorganized into specific regions of the sperm cell surface [5].

Generally, membrane remodeling or lipid modifications are caused by exchanges between cell membranes and lipid donors or acceptors. In this context, it was demonstrated that sperm membrane lipid modification could be mediated in a capacitation-dependent manner by seminal plasma phospholipid-binding proteins [6–9]. These proteins, termed bovine seminal plasma (PDC-109) proteins, are well characterized in bovines, and the presence of similar proteins in human seminal plasma and that of other species has been reported [10]. PDC-109, which is secreted by the seminal vesicles, stimulates cholesterol efflux in ejaculated spermatozoa upon the association with the plasma membrane. Another high-affinity cholesterol-binding protein, Niemann-Pick C2 (NPC2), is also found in the epididymal fluid as well as in the seminal plasma of various species [11, 12]. While the precise biological role of NPC2 is not well understood, this protein is thought to be implicated in cholesterol transport during sperm maturation. Other seminal plasma proteins, such as phospholipid transfer protein, albumin, and glycodelin-S [13–15], are also involved in the regulation of lipid remodeling of the sperm plasma membrane. Thus, cholesterol efflux seems to be an important step in sperm membrane remodeling and, therefore, in sperm maturation. However, the impact of cholesterol efflux and the role of cholesterol-binding proteins in regulating the reorganization of integral and peripheral sperm membrane proteins are poorly understood and documented. This is of physiological relevance, because perturbation of membrane restructuring could result in impaired sperm survival or attachment within the female reproductive tract and reduced oocyte recognition, binding, and/or penetration [16–18].

Recent studies have identified the existence of sphingolipid-containing membrane clusters enriched in cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins [19, 20] in the plasma membrane of spermatozoa from various mammalian species [21–23]. It is proposed that these membrane domains, generally termed lipid rafts or detergent-resistant membranes (DRMs) [24, 25], form organizing centers that regulate the distribution of membrane proteins, activation of receptors, and triggering of intracellular signaling cascades (reviewed in [24, 26]). Proteomic analysis of DRM established that several sperm-associated proteins—such as hexokinase-1, testis serine proteases-1 and-2, hyaluronidase (PH20/SPAM1), and Izumo—are included in these membrane domains [27]. During in vitro capacitation, most of these proteins dissociate from DRM, suggesting that cholesterol efflux, which characterizes this process, may be directly linked to this effect [27, 28]. These results are consistent with the fact that sperm capacitation results in an extensive membrane reorganization involving changes in membrane fluidity and in composition, structure, and localization of membrane proteins [23]. They also suggest that functional lipid rafts are present in mature spermatozoa and could play a regulating role in the reorganization of sperm membrane proteins during the sperm maturation process.

We previously demonstrated that P25b, a member of a family of xylulose reductases identified in our laboratory [29–31], appears and accumulates at the bovine sperm surface during the epididymal transit of testicular spermatozoa [32, 33]. Other members of this family, such as P26h and P34H (DCXR) proteins, are acquired in the epididymis and are known to cover the acrosome region of hamster and human spermatozoa, respectively [34, 35]. These proteins are GPI-anchored [36] to the sperm membrane, and recent evidence indicates that they are important in sperm-zona pellucida interactions [29–31]. Our preliminary two-dimensional SDS-PAGE and Western blot analysis performed on DRM from cauda epididymal spermatozoa have determined that the sperm proteins, P25b and adenylate kinase (AK) 1 are associated with these membrane domains. Further analysis of DRM from caput and cauda epididymal spermatozoa indicates that the epididymal protein, P25b, is accumulated and concentrated in DRM during the epididymal transit, but is dissociated from DRM in ejaculated spermatozoa. Since seminal plasma regulates sperm cell membrane fluidity, we plan to investigate whether seminal plasma factors could be involved in DRM disruption and redistribution of P25b and AK1 proteins. Our results demonstrate that loss of cholesterol and the ganglioside GM1, a lipid raft marker, rapidly occurs in DRM of ejaculated sperm, indicating that lipid rafts may undergo major changes in a capacitation-independent manner. DRM analysis of cauda epididymal spermatozoa incubated with seminal plasma and repleted with cholesterol show that a seminal plasma-mediated cholesterol efflux is associated with membrane redistribution of P25b and AK1 proteins. Further studies indicate that the seminal plasma proteins, PDC-109 and NPC2, are implicated in the dissociation of P25b proteins with DRM domains in a sequential manner. Considering the role of P25b in fertilization, this process could be physiologically relevant by allowing sperm cell progression within the female reproductive tract and by improving sperm fertilizing ability.

MATERIALS AND METHODS

Bovine Sperm Preparation

Epididymides from mature bulls were obtained from a commercial slaughterhouse. The epididymides were dissected and kept on ice until used. Fluid from the caput epididymidis was recovered by neatly cutting tubules with a razor blade, applying light pressure to the proximal portion of the dissected caput epididymidis. Fluid from the cauda epididymidis was obtained through retrograde flushing by applying air pressure with a syringe in the proximal scrotal segment of the vas deferens. Spermatozoa were isolated from the caput and cauda epididymal fluids and extensively washed in saline solution by centrifugation at 500 x g for 5 min. Freshly ejaculated bull semen was obtained from Centre d'insémination artificielle du Québec (St.-Hyacinthe, PQ, Canada) and washed in saline within 1 h of ejaculation.

Cell Fractionation and DRM Domain Isolation

Washed spermatozoa (500 x 106) were treated with ice-cold TNE (10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA), containing 1% (v/v) Triton X-100, for 30 min on ice. To remove whole sperm and sperm particulates, a precentrifugation step at 2000 x g for 5 min was performed on the Triton X-100-treated sperm. Supernatant was separated from the pellet and submitted to ultrasons treatment to ensure the release of DRM from the Triton-insoluble material remnant and prior to be subjected to a second step of centrifugation at 200 000 x g in a three-step (42.5%, 30%, 5%) sucrose gradient as previously described [37–40]. Briefly, Triton X-100- and ultrasons-treated sperm extracts were mixed with an equal volume of 85% (w/v) sucrose and overlaid with 2.4 ml of 35% (w/v) sucrose and 1 ml of 5% (w/v) sucrose. Centrifugation was performed at 200 000 x g in a SW60Ti rotor at 4°C for 16 h. Eleven fractions (numbered 1–11) were collected from the tube starting from the top: the low-density fractions (3–5) containing DRM or rafts, the intermediate-density fractions (6–8), and the high-density fractions (9–11) containing the Triton-soluble (T-S) proteins (Fig. 1A). DRM and T-S material recovered from spermatozoa were initially characterized by measuring the light scattering of these fractions at 400 nm and by evaluating the lipid:protein ratio, as previously described [21, 41] (see Supplemental Figure S1 available online at www.biolreprod.org).


Figure 01
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  		FIG. 1. P25b and AK1, but not MIF or AKR1B1, are associated with lipid rafts of bovine spermatozoa along the epididymis. A) Schematic representation of sperm subcellular compartment distribution following ultracentrifugation of cold Triton X-100 sperm extracts on a discontinuous sucrose gradient. Caput (B) and cauda (C) epididymal spermatozoa were treated with 1% Triton X-100 and subjected to fractionation in discontinuous density sucrose gradient. Western blot detection of P25b molecules (28 kDa), as well as AK1 (25–26 kDa), MIF (12 kDa), and AKR1B1 (36 kDa) were performed in reducing conditions; 25 x 106 spermatozoa per sample for each fraction were loaded. Results are representative of three independent experiments. DRM, Detergent-resistant membranes; T-S, Triton-soluble fraction.

Alternatively, DRM fractions were isolated from spermatozoa treated with ice-cold TNE buffer containing 1% (v/v) Triton X-100. The supernatant from a low-speed centrifugation step (2000 x g for 5 min at 4°C) containing DRM was pelleted by a straightforward centrifugation step at 16 000 x g for 30 min, and then completely separated from the Triton-soluble fractions [40]. For this purpose, the insoluble pellet was washed twice, and homogenously dispersed by ultrasound treatment in a volume of TNE buffer equivalent to the Triton-soluble fraction. Dot blot analysis determined that the lipid raft marker, GM1, isolated from Triton X-100-treated cauda epididymal spermatozoa, is detected in the 16 000 x g pellet, but not in the Triton X-100-soluble material, confirming that DRM was efficiently pelleted down at 16 000 x g (see Supplemental Figure S2 available online at www.biolreprod.org).

GM1 Detection

The ganglioside GM1 is known to be specifically targeted by the cholera toxin B (CTB) subunit and to be exclusively associated with lipid rafts of somatic and germinal cells [28, 42]. Sucrose gradient fractions or total sperm extracts were dotted onto nitrocellulose membranes by means of a dot spot apparatus. Membranes were blocked in 5% (v/v) skim milk in PBS containing 0.05% v/v Tween-20 and then incubated with horseradish peroxidase (HRP)-conjugated CTB (Sigma, St. Louis, MO) in PBS containing 3% (w/v) BSA. CTB-probed GM1 was visualized using the ECL detection kit (Roche Diagnostics, Quebec City, PQ, Canada).

Cholesterol Extraction and Quantification

Sperm lipids were extracted by the Folch method adapted for spermatozoa [43]. Briefly, an aliquot of 50 x 106 epididymal or ejaculated spermatozoa was mixed with 8 ml chloroform:methanol (2:1, v/v), and then 1.5 ml distilled water was added, mixed again, and allowed to stand at room temperature for 1 h after centrifugation at 500 x g for 10 min at 4°C. The upper layer was mixed with 8 ml of chloroform:methanol (2:1, v/v) and centrifuged at 500 x g for 10 min at 4°C, after which chloroform extracts were pooled. Finally, the organic layers were evaporated under nitrogen flow. The dried material was dissolved in chloroform:methanol (2:1, v/v) before cholesterol quantification. Cholesterol was assayed using Lieberman-Burchard reagent [44]. In order to extract and quantify cholesterol in different sucrose fractions of epididymal and ejaculated sperm extracts (150 x 106 spermatozoa), aliquots of different fractions were pooled as follows: low-density fractions 3–5, intermediate-density fractions 6–8, and heavy-density fractions 9–11. The concentrations of total cholesterol (nmoles cholesterol per 50 x 106 spermatozoa) were expressed as mean of triplicates ± SEM. Significant difference between the type of sperm cells or treatments was determined by one-way ANOVA using both the Fisher protected least-squares difference (PLSD) test and the Scheffé test.

Western Immunoblotting

Sperm proteins fractionated in sucrose gradient fractions or from Triton-insoluble (DRM) and Triton-soluble (T-S) fractions were precipitated with MeOH/CHCl3 [45] and resuspended in SDS-PAGE sample buffer (2% [v/v] SDS, 2.5% [v/v] β-mercaptoethanol, 50 mM Tris, pH 6.8). Protein fractionation was resolved by SDS-PAGE [46] and transferred onto nitrocellulose membrane. In order to detect P25b protein, AK1, macrophage migration inhibitor factor (MIF), and the aldose reductase, AKR1B1, membranes were incubated with a rabbit anti-P26h/P25b antiserum from our laboratory [33], an anti-MIF (generously provided by Dr. M. Nishobori from the Department of Pharmacology, Okayama University, Okayama, Japan) and an anti-AKR1B1 (a gift from Dr. M.A. Fortier, Université Laval, PQ, Canada) [47]. Membranes were then incubated with HRP-conjugated anti-rabbit IgG antisera (1:10 000) for 1 h at room temperature. Parallel studies were conducted in which membranes were also probed with anti-AK1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), then incubated with anti-goat IgG antisera (1:10 000) for 1 h at room temperature. Immunological complexes were visualized using the ECL detection kit.

Whole and Fractionated Seminal Plasma Incubation

In a first set of experiments, we investigated the effects of seminal plasma on membrane integrity of cauda epididymal spermatozoa by evaluating the content of cholesterol and GM1 and the membrane localization of P25b. Whole seminal plasma was obtained from freshly ejaculated bull semen. Cauda epididymidal spermatozoa were then incubated in whole seminal plasma at a final concentration of 500 x 106 spermatozoa/ml for 60 min at 37°C and extensively washed and incubated in isotonic saline solution. To investigate the effects of cholesterol repletion following seminal plasma incubation on P25b membrane localization, seminal plasma-treated spermatoza were extensively washed and then incubated at 37°C for 3 h in saline solution or in saline solution containing 200 µM cholesterol-loaded methyl-β-cyclodextrin (Sigma). To examine the effects of seminal plasma components on P25b membrane localization, free-spermatozoa seminal plasma was centrifuged twice (120 000 x g for 2 h) to eliminate prostasome vesicles, or dialysed (cut-off, 6–8 kDa) in saline solution (150 mM NaCl). Prostasomes are small membranous vesicles originating from prostatic secretion and found in seminal plasma [48, 49]. Equivalent volumes of intact, centrifuged or dialysed seminal plasma were incubated with 250 x 106 cauda epididymal spermatozoa for 60 min at 37°C. The pH of incubating solution was adjusted to a pH range of 6–7 by the addition of 10 mM 2-(4-morpholino)-ethane-sulfonic acid/PIPES buffer. Spermatozoa were then extensively washed in saline solution. Proteins from seminal plasma were also isolated by ammonium sulfate precipitation (0.603 g/ml) and then dialysed in saline solution. Seminal plasma proteins were incubated with 250 x 106 cauda epididymal spermatozoa.

Purification and Analysis of PCD-109 and NPC2 Proteins

Bovine seminal plasma proteins, termed PDC-109 (BSP-A1/A2), were purified from bovine seminal plasma as described by Manjunath et al. [50, 51]. Cauda epididymal spermatozoa (250 x 106) were incubated with BSA (4 mg/ml) (fraction V fatty acid-free; Sigma) or PDC-109 (400 µg/ml, as previously described [8]) in isotonic NaCl solution for 1 h and 4 h at 37°C. NPC2 was purified from bovine cauda epididymal fluid as previously described [11]. The purity of NPC2 was verified by Coomassie-stained gel and by Western blot using a rabbit anti-NPC2 [12] (data not shown). The 250 x 106 cauda epididymal spermatozoa were incubated for 1 h with NPC2 at a final concentration of 1 mg/ml.

Densitometry

Densitometric quantitation of proteins probed with secondary HRP-conjugated antibodies was carried out with the use of an image analyzer system (Alpha Innotech Corp., San Leandro, CA). Area density for each band of the Western blots was expressed as percent of total distribution:


Formula 78

Results from GM1 detection were expressed as the ratio of the area density of GM1 on the area density of GM1 detected in cauda epididymal spermatozoa. These values were expressed in percent of GM1. The values presented in the Results section correspond to the mean ± SEM for each similar experiment.

RESULTS

Specific Sperm-Associated Proteins Are Linked to DRM in Epididymal Spermatozoa

As depicted in the Figure 1A, DRM domains were isolated from sperm cells based on their insolubility in cold Triton X-100, a nonionic detergent, and their low buoyant density in sucrose gradient [25, 37–40]. To initially characterize DRM and T-S material recovered from caput and cauda epididymal spermatozoa, the light scattering of these fractions was assessed by measuring the optical density at 400 nm (Supplemental Figure S1 available online at www.biolreprod.org). As expected, the optical density of DRM-containing fractions (fractions 3–5; Fig. 1A) was significantly higher than that of the other fractions (intermediate-density and high-density fractions). As reported for other species [21, 52], this increased ability of DRM fractions to disperse the light beam reflects a higher content of insoluble particulates (vesicles) present in these fractions. In addition, the lipid:protein ratio is also higher in DRM-containing fractions than in T-S fractions (Supplemental Table S1 available online at www.biolreprod.org), as previously determined for the DRM in different cell models [21, 53].

In a first attempt to determine which sperm proteins are associated with DRM in epididymal spermatozoa, proteomic analysis was performed on Triton X-100-insoluble fractions from cauda epididymal spermatozoa (data not shown). Two-dimensional SDS-PAGE and Western blot analysis performed on these fractions confirmed proteomic data showing that the P25b and AK1 proteins are associated with the low-density sucrose gradient fractions. These observations prompted us to examine whether P25b and AK1 were differently associated with spermatozoal membrane domains according to their maturation stage in the epididymis. Caput and cauda epididymal spermatozoa were treated with cold Triton X-100, fractionated on a sucrose density gradient, and analyzed by Western blot using highly specific antibodies. As shown in Figure 1B, P25b and AK1 in caput epididymal spermatozoa were predominantly detected in the low-density sucrose fractions (DRM fractions 3–5), but were barely detected or absent in the high-density sucrose fractions (T-S fractions 9–11). Immunoblotting analysis of proteins extracted from cauda epididymal spermatozoa indicated that almost all the P25b proteins were detected in DRM fractions, while AK1 proteins were equally dispersed in the DRM and the T-S fractions (Fig. 1C). These results clearly indicate that, in contrast to AK1, the epididymal protein, P25b, is mainly accumulated in DRM domains during epididymal transit.

In contrast to P25b and AK1, other sperm-associated proteins, such as MIFand aldose reductase (AKR1B1), are totally excluded from DRM, but are associated with the T-S fractions (9–11) from caput as well as from cauda spermatozoa (Fig. 1, B and C). MIF and AKR1B1 were previously examined in our laboratory. MIF is particularly known as a T-cell cytokine acting as proinflammatory mediator, but is also extensively expressed in different tissues where it is multifunctional. Recent data demonstrated that MIF is found associated with the dense fibers of bovine and human spermatozoa [54], and is involved in flagellar beating and motility. We also found that AKR1B1 may modulate sperm motility, since aldose reductase activity was detected along the epididymis [55, 56]. Aldose reductase is an enzyme of the polyol pathway that transforms glucose to sorbitol, which is used as an energy source by cells [57].

The Proteins P25b and AK1 Completely Dissociate from DRM in Ejaculated Spermatozoa

Given that P25b and AK1 proteins are associated with DRM in caput as well as in cauda epididymal spermatozoa, we next investigated the membrane localization of these proteins in ejaculated sperm. Spermatozoa recovered from 60-min postejaculated semen were washed, treated with cold Triton X-100, fractionated on a sucrose density gradient, and analyzed by Western blot. As shown in Figure 2A, P25b and AK1 proteins are both excluded from the DRM fractions and are, instead, found in the T-S fractions. To further determine the kinetic changes in the membrane localization of P25b and AK1 after ejaculation, DRM and T-S fractions were isolated from ejaculated sperm at different times after ejaculation. To this end, aliquots of the same ejaculate were incubated in a shaking bath at 37°C immediately after ejaculation and separated from seminal plasma by centrifugation after 15, 30, 60, and 180 min. Seminal plasma-free spermatozoa were treated with Triton X-100 on ice and the sperm extracts were fractionated by a straightforward centrifugation step at 16 000 x g for 30 min at 4°C. Since GM1 were almost exclusively detected in the 16 000 x g pellet (Supplemental Figure S2 available online at www.biolreprod.org), this method was appropriated to isolate DRM from the T-S fractions. Using this simple and rapid method, we found that, 15 min after ejaculation, P25b is mainly in DRM fractions, but some P25b dissociates from DRM and is found in the T-S fraction. Within 30 min after ejaculation, a large portion of P25b dissociated from DRM and was localized in the T-S fraction. In contrast to P25b, most of the AK1 was found associated with the T-S fraction as early as 15 min after ejaculation (Fig. 3B). Together, these results suggest that the association of P25b and AK1 depends on the duration of sperm contact with seminal plasma, and that DRM-AK1 association is more sensitive to the effects of seminal plasma components.


Figure 02
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  		FIG. 2. Time-dependent membrane localization of P25b and AK1 proteins in ejaculated spermatozoa. A) Freshly ejaculated spermatozoa (60 min after ejaculation) were treated with 1% Triton X-100 and subjected to fractionation in discontinuous density sucrose gradient. B) Freshly ejaculated spermatozoa were incubated in seminal plasma for 15, 30, 60, and 180 min, then washed and treated with 1% Triton X-100. The Triton-insoluble fractions containing DRM were pelleted by centrifugation and then separated from the corresponding Triton-soluble fractions; 25 x 106 cells per sample for each fraction of sucrose gradient and for the DRM and T-S fractions were loaded. Detection of P25b (28 kDa) and AK1 (25–26 kDa) protein was performed. Results are representative of three independent experiments. DRM, detergent-resistant membranes; T-S, Triton-soluble fraction.


Figure 03
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  		FIG. 3. Contents of cholesterol and GM1 decrease from cauda epididymal spermatozoa to ejaculated spermatozoa. A) Cholesterol levels in total lipid extract of cauda epididymal sperm cells and of ejaculated spermatozoa were assayed using Lieberman-Burchard reagent. Spermatozoa were also treated with 1% Triton X-100 and subjected to fractionation by the sucrose gradient method. Different fractions collected along the sucrose gradient were pooled to obtain the L-D (fractions 3–5), the I-D (fractions 6–8), and the H-D (fractions 9–11) fractions. Cholesterol levels were quantified in each fraction. Results are expressed as mean ± SEM for three independent experiments. Different letters indicate significant difference of cholesterol levels within and between each sperm type and density fractions (P < 0.05). B) Total extract and the fractions of sucrose gradient of cauda epididymal and of ejaculated spermatozoa were dotted on membranes, and GM1 levels were detected with HRP-conjugated CTB. Values are expressed as the percent decrease of the GM1 content (% GM1) in ejaculated spermatozoa. Data presented are mean ± SEM for three independent experiments. Cd, Cauda epididymidis; Ej, ejaculated; H-D, high-density sucrose fraction; I-D, intermediate-density sucrose fraction; L-D, low-density sucrose fraction.

Cholesterol and GM1 Levels in DRM Are Decreased in Ejaculated Spermatozoa Compared with Cauda Epididymal Spermatozoa

Since the association of P25b proteins with DRM domains depends on the duration of sperm contact with seminal plasma, we propose that seminal plasma components affect the stability of DRM. This hypothesis is supported by the fact that seminal plasma induces cholesterol efflux in membrane spermatozoa [8], and that DRM stability is known to be altered by cholesterol efflux [22, 37, 58]. In order to confirm that lipid composition of DRM is modified in ejaculated spermatozoa, the total content of cholesterol and the ganglioside, GM1, two major structural components of DRM domains, were first examined in cauda epididymal spermatozoa as well as in ejaculated spermatozoa. As shown in Figure 3A, the analysis of the total organic extract from 50 million cauda epididymal spermatozoa had a cholesterol content of 43 nmol. This value significantly decreased in ejaculated spermatozoa to a value of 28 nmol. To determine whether DRM domains and T-S fractions were both affected by cholesterol efflux, DRM and T-S fractions of ejaculated sperm cells were separated by centrifugation on a discontinuous sucrose gradient, as described above. The cholesterol level was quantified separately in different pools of low- (L-D; fractions 3–5), intermediate- (I-D; fractions 6–8), and high- (H-D; fractions 9–11) sucrose density fractions (Fig. 1A). As reported in Figure 3A, the cholesterol level in the DRM-containing L-D fraction decreased significantly from cauda epididymal spermatozoa (15 nmol) to ejaculated sperm (7 nmol). Furthermore, in the I-D and H-D fractions, the cholesterol levels from cauda epididymal to ejaculated sperm remained unchanged.

Recent studies demonstrated the presence of the ganglioside, GM1, a well-characterized lipid raft marker, on the sperm head surface of different species [59]. Using HRP-conjugated CTB subunit, which specifically binds GM1, we compared by dot blot analysis the GM1 level in epididymal versus ejaculated spermatozoa. Similar to cholesterol content, the levels of GM1 in total organic extracts were lowered in ejaculated spermatozoa (25.8% below the levels of GM1 detected in cauda epididymal spermatozoa) compared with cauda epididymal spermatozoa (Figure 3B). Detection of GM1 in separated DRM and T-S fractions clearly showed that GM1 was exclusively detected in DRM fractions in both cauda epididymal and ejaculated spermatozoa. These results correlate well with the observation that GM1 was mainly detected in DRM. However, as expected, the level of GM1 was lowered in DRM of ejaculated spermatozoa compared with DRM of cauda epididymal spermatozoa. Together, these data indicate that DRM domains are depleted in cholesterol content after ejaculation, and suggest that a structural reorganization of DRM takes place when sperm cells are mixed with seminal plasma.

Seminal Plasma Dissociates P25b from DRM in Cholesterol-Depleted Cauda Epididymal Spermatozoa

To determine if seminal plasma directly affects DRM structure, cholesterol and GM1 levels were measured in total organic extracts from cauda epididymal spermatozoa incubated with or without whole seminal plasma for 60 min. As shown in Figure 4A, incubation of cauda epididymal spermatozoa with seminal plasma significantly decreases the cholesterol content of 50 million sperm cells, from 43 to 24 nmol. Similarly, the level of GM1 decreases in cauda epididymal spermatozoa after incubation in seminal plasma (Fig. 4B). As evaluated by densitometric analysis, the level of GM1 decreased by 33.8% when compared with untreated cauda epididymal spermatozoa.


Figure 04
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  		FIG. 4. Involvement of seminal plasma in the cholesterol and GM1 efflux and in the P25b dissociation of DRM fractions. A) Cauda epididymal sperm cells were left untreated (control) or treated with seminal plasma (SP) for 60 min, and their respective total cholesterol levels were assayed using Lieberman-Burchard reagent. Results are expressed as mean ± SEM for three independent experiments. Different letters indicate significant difference of cholesterol levels within and between each sperm type (P < 0.05). B) Detection of GM1 levels from total sperm extract of cauda epididymal sperm cells were left untreated (control) or treated with SP for 60 min. Values are expressed as the percent decrease of the GM1 content in cauda epididymal spermatozoa incubated with SP. C) Sperm cells from cauda epididymidis were incubated without (control) or with SP for 60 min. SP-treated spermatozoa were then incubated with or without cholesterol-loaded MβCD (200 µM). Spermatozoa were treated with 1% Triton X-100. The Triton-insoluble fractions containing lipid rafts were pelleted by centrifugation and then separated from the corresponding Triton-soluble fractions; 25 x 106 spermatozoa per sample for each fraction were loaded. P25b protein (28 kDa) was detected by Western blot. Densitometric values indicated the percent of distribution of P25b in each fraction for the different treated spermatozoa. Results are representative of three independent experiments. Cd, Cauda epididymidis; Chol, Cholesterol; DRM, detergent-resistant membranes; SP, Seminal plasma; T-S, Triton-soluble fraction.

To further explore the possibility that a structural reorganization of DRM takes place when cauda epididymal spermatozoa are bathed in seminal plasma, the presence of P25b proteins was assessed in DRM and T-S fractions of seminal plasma-treated cauda epididymal spermatozoa. Our results indicated that the association of P25b proteins with DRM was highly reduced after a 60-min incubation period with seminal plasma (Fig. 4C). In order to corroborate that cholesterol depletion induced by seminal plasma is responsible for DRM disruption and dissociation of P25b, cholesterol-loaded methyl-ß-cyclodextrin was added immediately after seminal plasma exposure for 60 min at a final concentration of 200 µM. This approach was previously used in other studies [37, 60] to reconstitute DRM after a treatment of cells with a cholesterol-depleting agent. After cholesterol addition, P25b association to DRM and the intensity of the DRM-associated P25b band of the blot were similar to those observed in DRM of control cauda epididymal spermatozoa (Fig. 4C). Similar results were also observed with AK1 molecules (data not shown). Together, these results strongly suggest that seminal plasma dissociated P25b proteins from DRM in cauda epididymal spermatozoa by a mechanism involving cholesterol depletion and disruption of DRM integrity.

Seminal Plasma Proteins Are Involved in P25b Dissociation with DRM

To determine the nature of seminal plasma constituent involved in the disruption of DRM-P25b association, bovine seminal plasma was centrifuged to remove prostasome vesicles, or dialysed in an isotonic saline solution to remove low-molecular weight molecules. Cauda epididymal spermatozoa were then incubated for 60 min with or without untreated, centrifuged (prostasome-free) or dialysed (metabolite-free) seminal plasma. Untreated or treated cauda epididymal spermatozoa were treated with 1% Triton X-100, and the Triton-insoluble fractions containing DRM were pelleted by centrifugation and separated from the corresponding T-S fractions. As shown in Figure 5A, P25b proteins were dissociated from DRM domains following incubation with complete seminal plasma, as well as with centrifuged or dialysed seminal plasma (Fig. 5). Given that this result suggests that prostasomes and small molecules are not involved in DRM-P25b dissociation, we explored the possibility that seminal plasma proteins are responsible for this effect. Total proteins from seminal plasma were isolated by ammonium precipitation, washed with saline, and incubated with cauda epididymal spermatozoa for 60 min. The analysis of DRM and T-S fractions demonstrated that incubation of cauda epididymal spermatozoa with proteins from seminal plasma results in the dispersion of P25b from DRM to the T-S fractions (Fig. 5). These results indicate that a protein or a group of proteins from seminal plasma is able to dissociate P25b proteins from DRM in ejaculated and seminal plasma-treated cauda spermatozoa.


Figure 05
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  		FIG. 5. Proteins, but not prostasomes or small molecules, from seminal plasma are involved in the dispersion of P25b from DRM to the Triton-soluble fraction. Seminal plasma isolated from bovine ejaculated spermatozoa were centrifuged to remove prostasomes or dialysed against isotonic NaCl to remove small molecules. Proteins were prepared from seminal plasma fluid by ammonium sulfate precipitation. Cauda epididymal spermatozoa were then left untreated (control) or treated for 60 min with intact, centrifuged, or dialysed seminal plasma, as well as seminal plasma proteins isolated by ammonium sulfate precipitation. After incubation, spermatozoa were treated with 1% Triton X-100. The Triton-insoluble fractions containing lipid rafts were pelleted by centrifugation and then separated from the corresponding Triton-soluble fractions; 25 x 106 spermatozoa per sample for each fraction were loaded. P25b protein (28 kDa) was detected by Western blot. Densitometric values indicated the percent of distribution of P25b in each fraction for the different treated spermatozoa. DRM, detergent-resistant membranes; SP, seminal plasma; T-S, Triton-soluble fraction.

The Seminal Plasma Proteins, PDC-109 and NPC2, Dissociate P25b Proteins from DRM

PDC-109 is the major protein found in bovine seminal plasma, and is known to interact with, and bind to, ejaculated spermatozoa. Given that these proteins induced a cholesterol efflux to efficiently initiate capacitation of ejaculated spermatozoa [8], it is possible that PDC-109 proteins may be involved in the regulation of P25b membrane localization. According to Therien et al. [8], optimal cholesterol efflux occurred after 4–6 h incubation of cauda epididymal spermatozoa with highly purified PDC-109 proteins. Moreover, albumin is another protein known to induce cholesterol efflux by acting as cholesterol acceptor, and is frequently used to induce sperm capacitation in vitro [14]. In the present study, 250 x 106 cauda epididymal spermatozoa were incubated for 1 h and 4 h with or without PDC-109 proteins or albumin (both at 400 µg/ml), respectively, then washed and treated with cold Triton X-100 detergent. The mixture was centrifuged in order to isolate DRM from T-S fractions. P25b immunodetection demonstrated that incubation for 1 h with PDC-109 proteins or albumin did not dissociate P25b from DRM (Fig. 6A). However, P25b molecules were completely excluded from DRM after a 4-h incubation period with PDC-109 proteins, but not with albumin (Fig. 6A). These results are consistent with the fact that PDC-109 proteins induce an optimal cholesterol efflux in cauda epididymal spermatozoa after an incubation period of 4–6 h. The fact that PDC-109 or albumin proteins are unable to rapidly dissociate P25b from DRM as observed with complete, centrifuged or dialysed seminal plasma suggests that mechanisms independent of capacitation are involved in this process.


Figure 06
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  		FIG. 6. Seminal plasma proteins, PDC-109 and NPC2, regulate in P25b membrane localization. A) Cauda epididymal spermatozoa were incubated for 1 h or 4 h in the absence (control) or the presence of PDC-109 and albumin. B) Cauda epididymal spermatozoa were left untreated (control) or treated with 1 mg/ml NPC2 protein for 1 h. After incubation, spermatozoa were treated with 1% Triton X-100. The Triton-insoluble fractions containing lipid rafts were pelleted by centrifugation and then separated from the corresponding Triton-soluble fractions. P25b protein (28 kDa) was detected by Western blot. Densitometric values indicated the percent distribution of P25b in each fraction for the different treated spermatozoa. C) Cholesterol levels of total sperm extract from cauda epididymal sperm cells (control) or from cauda epididymal sperm exposed to NPC2 were assayed using Lieberman-Burchard reagent. GM1 from total sperm extract was also detected. Different letters indicate significant difference of cholesterol levels within and between each sperm type and density fractions (P < 0.05). Values are expressed as percent decrease of the GM1 content in cauda epididymal spermatozoa incubated with NPC2. SP, seminal plasma; T-S, Triton-soluble fraction.

To further identify the protein responsible for P25b-DRM dissociation, we examined the effects of NPC2 on P25b membrane localization and cholesterol efflux. NPC2 protein was first characterized as a major secretory protein in the human epididymis, and was also detected in bovine epididymal fluid and seminal plasma. The NPC2 protein binds cholesterol with submicromolar affinity at neutral and acidic pH [11]. Notably, this protein is involved in cellular transport and transfer of cholesterol to membrane [61–63]. In order to determine whether NPC2 molecules induce P25b-DRM dissociation, NPC2 proteins were purified from bovine epididymal fluid. Cauda epididymal spermatozoa were then treated with purified NPC2 for 60 min at 37°C. As shown in Figure 6B, the incubation of spermatozoa with NPC2 protein for 60 min dissociates a portion of P25b protein from DRM. To confirm whether NPC2 affects the cholesterol and GM1 levels in spermatozoa, the cholesterol content and the GM1 level were examined in NPC2-treated spermatozoa. As shown in Figure 6C, the cholesterol level decreased from 43 nmol in cauda epididymal spermatozoa to a value of 32 nmol in NPC2-treated cauda epididymal spermatozoa. Similar to cholesterol content, GM1 level decreases in cauda epididymal spermatozoa following NPC2 incubation. As evaluated by densitometric analysis, the level of GM1 decreased to 26.9% below that detected in untreated cauda epididymal spermatozoa. These data suggest that within 1 h after ejaculation, DRM are reorganized by specific seminal plasma proteins that induce cholesterol and GM1 efflux, as well as dissociation of the GPI-anchored P25b proteins from DRM.

DISCUSSION

In the plasma membrane of somatic cells, DRMs, also known as lipid rafts, play a key role in the compartmentalization of specific lipids and proteins, as well as in cell signal transduction and vesicular trafficking (reviewed in [24, 26]). Structurally, DRMs are defined as cholesterol- and sphingolipid-enriched membrane microdomains. Acylated and lipid-modified proteins [64, 65], GPI-anchored proteins [19, 20] as well as lipidified signaling molecules [66] are found sequestered in these domains at the surface of somatic cells. DRM are also present within the membranes of sperm cells [21–23, 28, 67], whereas their physiological roles in sperm biology and functions are poorly understood and documented. However, it has recently been reported that lipid rafts must undergo a structural reorganization during sperm capacitation, an effect directly linked to a loss of membrane sterols [22, 23, 28]. Moreover, proteomic studies have demonstrated that DRM isolated from cauda epididymal mouse sperm are associated with several functional proteins [27]. On the other hand, when the protein content of DRM isolated from capacitated versus noncapacitated spermatozoa were analyzed by one-dimensional SDS-PAGE, a marked decrease in DRM-associated proteins was observed in the capacitated sperm cell population [27]. It is proposed that such protein redistribution might take place to regulate signaling pathways leading to capacitation [27]. While it remains to be confirmed convincingly, this concept is in accordance with the proposed role played by lipid raft domains as a platform for cell signal transduction in the plasma membrane of somatic cells [58]. Additionally, these findings indicate that functional lipid rafts are present in mature spermatozoa, and could play an essential role during the fertilization process.

The experimental data we present here support the hypothesis that lipid rafts may be involved in the localization of specific sperm-associated proteins, and provide the basis for a mechanism explaining why P25b and AK1 are mainly associated to DRM in caput and cauda epididymal spermatozoa. This is first supported by the observations that P25b and AK1 were detected in GM1-enriched fractions of the sucrose gradient. It is well documented that some GPI-anchored proteins are found clustered in DRM and contribute to the formation of these domains through the interaction with phospho- and sphingolipids, including GM1 [68, 69]. Our previous data showed that P25b protein is mainly associated with the sperm plasma membrane covering the acrosomal region, and is GPI-anchored to the sperm surface of epididymal as well as ejaculated spermatozoa [33, 70]. In contrast to P25b, our present results demonstrate that AK1 is in part associated with DRM in bovine epididymal spermatozoa, and that DRM-AK1 association is more sensitive to the effects of seminal plasma components. This could be explained by the fact that AK1 may be differently anchored to the sperm membrane. Accumulating evidence indicates the existence of two structurally distinct AK1 proteins in mouse and human cells: a typical mitochondrial form, and a membrane-bound isoform (AK1beta) [71–73]. The extra amino acid residues at the N-terminal end of AK1beta provides a consensus signal for N-terminal myristoylation [71]. Moreover, myristoylated proteins are well known to have affinity for lipid rafts [74, 75].

Our previous study indicated that MIF and AKR1B1 are acquired by bovine spermatozoa along the epididymis [54–56]. Eickhoff et al. [76] clearly demonstrated that MIF is associated with the outer dense fibers of mid and principal piece of spermatozoa flagellum. These structures are involved in sperm flagellar beating and motility, suggesting that MIF has a potential role in modulating sperm motility of the maturing spermatozoa. Although cellular distribution of aldose reductase in spermatozoa remains to be determined, this enzyme modulates sperm motility as well. Since aldose reductase activity is high along the epididymis except in the distal cauda epididymidis, it is suggested that this enzyme contributes to maintaining sperm motility in a repressive state by depriving the intracellular compartment of sperm cells from energy sources generated by the polyol pathway [77]. While the interaction and binding mechanisms of MIF and AKR1B1 with the spermatozoa are still unknown, our results demonstrate that these epididymal proteins are completely excluded from DRM (Fig. 1, B and C). These results are in accordance with the subcellular localization of these molecules.

In contrast to the membrane localization observed in epididymal spermatozoa, both P25b and AK1 proteins dissociate from DRM, and are completely redistributed in the Triton-soluble material in ejaculated spermatozoa (Fig. 2). P25b dispersion in the Triton-soluble fraction is reached progressively, since this event is dependent on the time of contact with seminal plasma (Fig. 2B). Our data also indicate that immediately after ejaculation, spermatozoa undergo a lipid remodeling, since the levels of total cholesterol and GM1 are lower in freshly ejaculated sperm than in cauda epididymal spermatozoa. Moreover, the cholesterol content is decreased in DRM of ejaculated spermatozoa. However, the cholesterol content of DRM fractions that we have isolated by sucrose gradient centrifugation are similar or lower than Triton-soluble fractions in cauda and ejaculated spermatozoa, respectively. Similar data were reported in epithelial cells [78] and suggest that low cholesterol content in DRM is a physiological state and probably dependent on cell type. As reported for epididymal mouse spermatozoa [79], our recent preliminary data similarly indicated that bovine caput epididymal spermatozoa have a higher cholesterol level than mature spermatozoa. Moreover, these results indicate that the cholesterol content of DRM in caput epididymal sperm cells is high compared with that measured in Triton-soluble fractions. This strongly suggests that the low cholesterol content in DRM is in fact a normal physiological state of cauda and ejaculated bovine spermatozoa. This loss of cholesterol is probably required to regulate the membrane reorganization of sperm cells after ejaculation.

Given that seminal plasma plays a regulatory role in membrane fluidity [6], we propose that redistribution of raft-associated proteins occurs as a result of cholesterol removal and subsequent lipid raft dissociation. We thus planned to test the hypothesis that seminal plasma factors could be involved in lipid raft disruption and cellular redistribution of lipid raft-associated proteins. In order to test this hypothesis, cauda epididymal spermatozoa were incubated with seminal plasma. Our data indicate that seminal plasma rapidly dissociates DRM domains by inducing cholesterol efflux and GM1 loss (Fig. 4A). Seminal plasma treatment of cauda epididymal spermatozoa significantly dissociates P25b from DRM domains (Fig. 4B). Similar observations were also made for AK1 protein (data not shown). Together, these results establish that seminal plasma regulates the membrane compartmentalization of P25b and AK1 proteins—a process too rapid to be associated with capacitation.

Seminal plasma is composed of mixed secretions of the male accessory reproductive glands [80]. Upon ejaculation, seminal plasma components, such as prostasomes, inorganic salts, metabolites, and proteins, interact with the sperm plasma membrane. This event is required to prevent spontaneous acrosome reaction, to induce sperm capacitation, and to potentiate zona pellucida-induced acrosome reaction [6, 81, 82]. However, we demonstrated that neither prostasomes nor small molecules, such as bicarbonate, which is involved in cholesterol efflux occurring during sperm capacitation [83], are implicated in the dispersion of P25b proteins from DRM to the Triton-soluble fractions (Fig. 5). Moreover, the incubation of epididymal sperm with the total extract of proteins contained in seminal plasma suggests that specific proteins might regulate the membrane localization of P25b (Fig. 5). PDC-109 proteins, the major proteins found in seminal plasma, bind spermatozoa and stimulate cholesterol and phospholipid efflux from epididymal sperm, suggesting that these proteins participate in membrane lipid modification [8, 9]. In addition, cholesterol efflux results in a decrease in the cholesterol:phospholipids ratio and promotes capacitation, which is a necessary event before the acrosome reaction and, finally, fertilization. However, PDC-109 must interact with heparin or high-density lipoprotein found in the female genital tract in order to induce capacitation [6]. Since PDC-109 may provoke membrane reorganization or destabilization, we set out to examine the effects of this protein on the localization of the sperm-associated protein, P25b. As shown in Figure 6A, P25b is excluded from DRM when cholesterol efflux is induced by a 4-h exposure to PDC-109 proteins. On the other hand, P25b association with DRM is not abrogated after a 1-h exposure to PDC-109 proteins. These results are in agreement with the fact that 25% of cholesterol removal is accomplished only after a 4-h exposure to PDC-109 proteins [6]. The optimal cholesterol efflux induced in bovine spermatozoa following PDC-109 incubation takes about 4–6 h to occur [8]. Furthermore, albumin, which also promotes cholesterol loss from sperm membrane, does not dissociate P25b proteins from lipid rafts after exposure periods of 1 or 4 h. This result indicates that P25b-DRM dissociation could be accomplished after cholesterol removal, but could involve a more complex mechanism induced by specific seminal plasma proteins. Given that rapid dissociation of P25b with DRM cannot be reproduced after a 1-h incubation period with purified PDC-109 or albumin (Fig. 5B), it appears that other unknown protein(s) of seminal plasma disrupts P25b-DRM association before the sperm enter the female reproductive tract.

Recently, NPC2, a protein secreted by the epididymis, was shown to specifically bind free cholesterol with high affinity, and to function as a cholesterol transfer protein in vitro [50]. In order to determine its involvement in the membrane localization of the P25b and the regulation of the lipid composition of the sperm membrane, NPC2 was purified in our laboratory from the epididymal fluid. We initially performed biochemical and immunological studies to confirm that NPC2 preparations were free of other protein contaminants. Highly purified NPC2 is able to dissociate a large part of P25b molecules from DRM domains and lower the content of cholesterol and GM1 of the spermatozoa (Fig. 6B). Our results strongly suggest that NPC2 is involved in sperm membrane reorganization in a capacitation-independent process.

Moreover, cholesterol repletion after DRM disruption induced by seminal plasma allows a complete recovery of P25b association with DRM (Fig. 4C). Since P25b protein is compartmentalized in the acrosomal region of spermatozoa [33], and is proposed to be involved in the zona pellucida binding, it is tempting to speculate that lipid rafts are directly involved in the acquisition and localization of specific epididymal proteins, such as P25b, to the acrosomal region of spermatozoa, allowing optimal sperm-egg interactions. This hypothesis is supported by our results revealing that P25b accumulates and then concentrates in DRM domains during epididymal maturation (Fig. 1, B and C). Interestingly, while AK1 is positioned mainly in DRM of caput spermatozoa, it was found to be partitioned in DRM as well as in the Triton-soluble fractions in cauda epididymal sperm cells (Fig. 1, B and C), and to be completely excluded by DRM in ejaculated spermatozoa (Fig. 2). These data are in accord with previous studies showing that AK activity is important for biosynthesis of ATP and then for flagellar motility [84]. In bovine ejaculated spermatozoa, AK activity is mostly detected in flagella [84]. Moreover, it was found that AK1 molecules are associated with the outer microtubular doublets and outer dense fibers of ejaculated spermatozoa [85]. Since motility is acquired progressively during epididymal transit, these data strongly suggest that the compartmentalization of AK1 in DRM could be essential to modulate AK1 enzymatic activity during sperm maturation.

Together, these findings indicate that an important intermediate maturation process, independent of capacitation, occurs rapidly after sperm ejaculation and results in membrane relocalization of the sperm-associated proteins, P25b and AK1. NPC2 in the bovine seminal plasma acts as a membrane reorganization factor by regulating the association of lipid and proteins within DRM in epididymal spermatozoa. Consequently, protein-DRM dissociation may be central to modulate the function of proteins, such as P25b or AK1, which play a crucial role in the fertilization process. This study also suggests that lipid rafts may be essential for the compartmentalization and or distribution of some proteins within specific region of the spermatozoa.

ACKNOWLEDGMENTS

We wish to acknowledge Centre d'Insémination Artificiel du Québec for the generous gift of bovine semen samples. Dr. P. Manjunath (Université de Montréal, Montreal, PQ, Canada) and Dr J.L. Gatti (INRA-Université de Tours, Nouzilly, France) are acknowledged for the generous gifts of purified BSP proteins and HE1/NPC2, respectively. Drs. M. Nishibori (Okayama University, Okayama, Japan) and M.A. Fortier (Université Laval, Laval, PQ, Canada) are both acknowledged for generous gifts of anti-MIF and anti-AKR1B1 antibodies, respectively. We wish to thank Dr. M.K. Holland from the Monash Institute for Medical Research (Melbourne, VC, Australia), Dr. C. Reyes-Moreno (Université du Québec à Trois-Rivières, Trois-Rivières, PQ, Canada), and Dr. P. Leclerc (Université Laval) for critical reading of the manuscript.

FOOTNOTES

1Supported by Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council-Canada grants (to R.S). J.G. is supported by a Ph.D. scholarship from the CIHR. Back

Correspondence: 2Robert Sullivan, Unité d'Ontogénie-Reproduction, Centre de Recherche du Centre Hospitalier de l'Université Laval, 2705 boulevard Laurier, T1-49, Quebec City, PQ, Canada G1V 4G2. FAX: 418 654 2765; e-mail: robert.sullivan{at}crchul.ulaval.ca

Received: 5 November 2007.

First decision: 26 November 2007.

Accepted: 14 January 2008.

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