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a Department of Medicine, University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada H1T 2M4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Capacitation and the acrosome reaction (AR) are two processes that sperm cells must undergo during their transit through the female genital tract in order to be able to fertilize the ovum [11–13]. The molecular events involved in sperm capacitation are not well understood, but capacitation probably involves several biochemical and ultrastructural changes in the sperm membrane (for review see [14]). Many studies have shown that one of the earliest steps in capacitation involves a change in the lipid composition of the sperm membrane. More specifically, capacitation is accompanied by an efflux of cholesterol, which appears to influence the fluidity and ionic permeability of the sperm membrane [15–19]. Capacitation is also associated with a loss of absorbed components originating from the seminal plasma, a redistribution of surface and intramembranous components, an increase in protein tyrosine phosphorylation, and sperm hyperactivation, which constitutes the last step of capacitation (for reviews, see [14, 20]). In the bovine female genital tract, two major capacitating factors have been identified, heparin-like glycosaminoglycans (GAG) [21–23] and high-density lipoprotein (HDL), which is a cholesterol acceptor [24, 25]. Our previous studies have shown that BSP proteins bind to heparin and to apolipoprotein A-I (apo A-I), which may or may not be associated with HDL [2, 26, 27]. Furthermore, we showed that the BSP proteins accelerate the capacitation of bovine epididymal sperm induced by heparin and HDL [7, 8]. We also showed that BSP proteins bind to choline phospholipids of the sperm membrane [6]. On the basis of these results, we previously postulated that after ejaculation, BSP proteins coat the sperm membrane and that sperm-bound BSP proteins interact with HDL, resulting in sequestration of phospholipids and cholesterol. This would result in the modification of the sperm membrane lipid composition or capacitation. In our recent work [9], we showed that the BSP proteins and HDL stimulate cholesterol efflux from bovine epididymal sperm independently. In view of this result, we proposed a modified mechanism for sperm capacitation. Accordingly, the BSP proteins stimulate a first cholesterol efflux at ejaculation, when sperm are exposed to seminal fluid for a brief period; and then HDL mediates a second efflux of cholesterol in the female genital tract, leading to capacitation. More recently, we also reported that the presence of both type II domains in BSP-A1/A2 protein is essential for the efflux and capacitation properties [28].
The mechanism by which the BSP proteins stimulate sperm cholesterol efflux remains to be elucidated. Since the BSP proteins do not directly interact with cholesterol but bind to choline phospholipids [6], we hypothesized that cholesterol removal by BSP proteins might require simultaneous phospholipid efflux. Studies using delipidated apo A-I have shown that the efflux of cellular cholesterol is accompanied by the release of phospholipids [29, 30]. In order to gain more insight into the mechanisms of BSP-mediated sperm capacitation, we studied whether or not BSP proteins induce phospholipid efflux from epididymal sperm membrane. We used two different methods: 1) direct determination of the amount of sperm choline phospholipids before and after incubation with BSP-A1/A2 protein, and 2) determination of radiolabeled choline phospholipids released from labeled sperm with [3H]palmitic acid, which specifically labels choline phospholipids. Our results showed, for the first time, that the BSP proteins stimulate sperm phospholipid efflux in addition to cholesterol efflux. This supports our view that the cholesterol efflux by BSP proteins requires phospholipid efflux.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BSA (fraction V fatty acid-free), taurine, L-epinephrine, erythrosin B, flavianic acid (naphthol), phosphatidylethanolamine (PE; purified from soybean), polyvinyl alcohol (PVA), and lysophosphatidylcholine (lyso-PC; purified from egg yolk) were from Sigma (St. Louis, MO). Penicillin G and streptomycin sulfate were from Gibco (Burlington, ON, Canada), and polyethylene glycol (PEG) was from Merck (Darmstadt, Germany). Immobilon-P membranes and the enhanced chemiluminescence reagent (ECL) kit were purchased from Mandel Scientific (Guelph, ON, Canada). Phosphatidylcholine (PC) and phosphatidic acid (PA) from egg yolk, phosphatidylinositol (PI) from pig liver, phosphatidylserine (PS) from bovine brain, cardiolipin from bovine heart, and sphingomyelin (SPM) from pig brain were obtained from Serdary Research Lab. (London, ON, Canada). [3H]Palmitic acid was from DuPont Canada (Mississauga, ON, Canada), and Sepharose CL-4B was purchased from Pharmacia Biotech (Uppsala, Sweden). Aluminum-backed silica gel thin-layer chromatography (TLC) plates were from Whatman (Maidstone, UK). The sulphopropyl (SP)-Sephadex (C-25) column was obtained from Amersham Pharmacia Biotech (Baie d'Urfé, QC, Canada). All other chemicals used were of analytical grade and obtained from commercial suppliers.
Bovine testes and epididymides were obtained from Abattoir Les Cèdres (St-Lazare, PQ, Canada). BSP-A1/A2, -A3, and -30-kDa were isolated using gelatin-agarose affinity chromatography [31]. The adsorbed fractions were eluted and resolved on Sephadex G-75 and G-200 columns under conditions described previously [32]. The purity of the BSP proteins was confirmed by SDS-PAGE.
Purification of Ribonuclease
Alcohol precipitates (4 g) of bovine seminal plasma were dissolved in 100 ml of 0.05 M PBS and centrifuged to separate any precipitate, and the supernatant was loaded onto an SP-Sephadex column (1.5 x 48 cm) previously equilibrated with 0.05 M PBS. The unadsorbed proteins were washed out with the same buffer, and the adsorbed proteins from the column were eluted with a linear gradient of 0.05 M PBS, pH 7.4 (500 ml), to 0.4 M NaCl in 0.05 M PBS, pH 8.1 (500 ml). The last protein peak eluted with this gradient was dialyzed against 0.05 ammonium bicarbonate and lyophilized (recovery ~110 mg). The molecular mass (14 kDa by SDS-PAGE), amino acid composition, and amino acid sequence confirmed that the purified protein was ribonuclease (RNase).
Determination of Sperm Choline Phospholipids
The medium used for washing and incubation of sperm was a modified Tyrode's albumin lactate pyruvate medium (mTALP) described previously [7], without albumin. Before use, 1 mg/ml PVA and 1 mg/ml PEG were added to the medium, and the pH was adjusted to 7.4. Caudal sperm were collected by retroperfusion of the epididymides obtained from four different bulls, pooled and washed 3 times (350 x g, 10 min) with 10 volumes of mTALP. Washed unlabeled epididymal sperm (final concentration of 5 x 107 cells/ml) were incubated for 8 h in the presence or absence of 120 µg/ml of BSP-A1/A2 proteins. After incubation, the sperm suspension was centrifuged (940 x g, 15 min) to remove the supernatant, which contained the efflux particles. Then, the sperm phospholipids were extracted by the method described by Bligh and Dyer [33]. After evaporation of the solvent with N2, the lipids were resuspended in isopropanol, and the amount of choline phospholipid was determined using the choline phospholipid determination kit (Boehringer Mannheim, Montreal, PQ, Canada; cat. no. 691844) according to the protocol described by the manufacturer.
Sperm Labeling and Phospholipid Efflux
The sperm were labeled using the method described by Roldan and Murase [34]. Labeling was carried out by incubating washed sperm (1.5 x 108/ml) for 60 min at 39°C in 5 ml of mTALP containing 5 µCi of [3H]palmitic acid/ml. Before phospholipid efflux studies, the sperm were rewashed through sucrose medium (mTALP with 222 mM of sucrose instead of NaCl, 1 mg/ml PVA, and 1 mg/ml of PEG, pH 7.4; 400 x g for 5 min and 1000 x g for 10 min), resuspended in mTALP, reincubated for another 15 min, centrifuged (350 x g, 10 min), and resuspended in medium to obtain 1 x 108 cells/ml. Approximately 50–60% of [3H]palmitic acid was incorporated into sperm and was < 0.01% of the sperm fatty acids mass. This amount of [3H]palmitic acid is unlikely to affect bulk properties of the sperm membrane. Studies of sperm phospholipid efflux were done by incubating 500 µl of the labeled sperm suspension with 500 µl of medium alone or 500 µl of medium containing different concentrations of purified BSP proteins (final concentration of 40–120 µg/ml). The sperm were then incubated for 8 h at 39°C under 95% air and 5% CO2, during which two samples of 50 µl were withdrawn at different times to determine the percentage of [3H]phospho-lipid present in the incubation medium. The percentage of label released into the medium was calculated from the radioactivity retrieved in the incubation medium divided by the total radioactivity associated with the sperm in the suspension [35]. The experiment was repeated under similar conditions in the presence of delipidated BSA (6 mg/ml) and RNase (120 µg/ml) as control proteins. The delipidation of BSA was carried out by charcoal treatment [36].
Lipid Extraction and TLC
After sperm labeling, the sperm suspension was resuspended in 0.8 ml of medium, and lipids were extracted with 3 ml of chloroform:methanol mixture (1:2 by volume), vortexed, and incubated for 40 min. Then, 1 ml of chloroform and 1 ml of water were added, and samples were vortexed and centrifuged (20 min at 3000 rpm). After centrifugation, the amount of radioactivity present in the chloroform phase was determined using a beta-counter. Fractions of the samples that corresponded to 100 000 cpm and 500 000 cpm were taken, and chloroform was evaporated under N2. With this extraction method, free [3H]palmitic acid (unincorporated into phospholipid pool) was mainly retrieved in the aqueous phase (85–90%), in contrast to the phospholipids, which were found in the chloroform phase. Lipids were then resuspended in chloroform and chromatographed on aluminum-backed silica gel TLC plates in chloroform:methanol:acetic acid:water (50:37.5:3.5:2 by volume). The plates were air-dried, and lipids were visualized either by iodine vapor and comparison with standards run on the same plate, or by autoradiography using Fuji RX film (Tokyo, Japan). The individual spots were cut, and the radioactivity in each spot was determined by liquid scintillation counting.
Chromatographic Analysis
After phospholipid efflux in the presence of 120 µg/ml BSP-A1/A2 or 6 mg/ml of BSA (8-h incubation), the medium containing the efflux particles was centrifuged twice (940 x g, 15 min) to remove the sperm. The supernatant (1 ml) was applied to a Sepharose CL-4B column (2.5 x 50 cm) previously washed with 0.050 M PBS, pH 7.4. The elution was carried out at a flow rate of 50 ml/h with the same buffer. Fractions of 2.3 ml were collected, and the radioactivity was determined using a beta-counter. When BSP-A1/A2 protein was used, 50 µl of each fraction was treated with trichloroacetic acid (TCA; final concentration of 15%) to precipitate the proteins, which were then analyzed by SDS-PAGE and immunoblotting.
SDS-PAGE Analysis and Immunoblotting
SDS-PAGE in 15% polyacrylamide gels was performed as described previously [37]. The proteins were then transferred to Immobilon-P membranes as described by Towbin [38], and immunodetection using affinity-purified polyclonal antibodies against BSP-A1/A2 protein was done as previously described [39] by using an ECL kit for detection.
Sperm Capacitation and AR
To determine the number of capacitated sperm after the efflux studies, lyso-PC was added at 100 µg/ml, and the sperm were reincubated for an additional 15 min. This concentration of lyso-PC was previously shown to induce the AR in capacitated sperm while having no effect on noncapacitated sperm [40]. The lyso-PC-induced AR is a well-characterized method that has been correlated with in vitro fertilization rates and validated by electron micrograph studies [40]. Before drying and staining, randomly selected slides were examined using light microscopy to verify sperm motility. The percentage of sperm that were acrosome-reacted was determined on air-dried sperm smears with a naphthol yellow-erythrosin B-staining procedure [22].
Protein Assay
The protein content of the samples was measured by weighing freeze-dried purified proteins on a Cahn microbalance (Model C-31; Fisher Scientific, Fairlawn, NJ).
Data Analysis
The data presented here were analyzed for significant differences by covariance analysis or by a Student's t-test on paired observations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first experiment, we studied the choline phospholipid efflux potential of BSP proteins by incubating washed epididymal sperm (not labeled, see below) for 8 h with or without BSP-A1/A2 protein. For this experiment, we used BSP-A1/A2 protein because it is the most abundant BSP protein in bovine seminal plasma (65% as determined by RIA). Before the incubation, a fraction of sperm was taken to determine the initial amount of choline phospholipid present in epididymal sperm. After incubation with or without BSP-A1/A2 protein, the sperm were centrifuged, and sperm choline phospholipids were extracted and quantified. After 8 h of incubation, the sperm that were incubated with BSP-A1/A2 protein (120 µg/ml) significantly lost 34.4% of their choline phospholipids (0.655 ± 0.177 mg/1 x 109 sperm versus 0.998 ± 0.262 mg/1 x 109 sperm, p < 0.05) compared to the sperm that were incubated with medium alone, which lost only 11.5% (0.883 ± 0.202 mg/1 x 109 sperm versus 0.998 ± 0.262 mg/1 x 109 sperm, not significant).
Labeling of Sperm Phospholipids
To study the kinetics of sperm phospholipid efflux, we labeled the bovine sperm phospholipids with [3H]palmitic acid. The incorporation of the labeled [3H]palmitic acid into the sperm was linear with time during 180 min. After 20 min, ~30% of the [3H]palmitic acid was incorporated; after 60 min, 50–60% of the [3H]palmitic acid was incorporated; and after 180 min, the majority of the label was associated with sperm. The incubation of bovine sperm with [3H]palmitic acid led to a small but a time-dependent incorporation of label into the phospholipid pool. After a 60-min incubation, only 7–10% of the label was present in the phospholipid pool. Figure 1A and Table 1 show that the majority of the label incorporated into phospholipid was associated with PC. However, when samples with higher levels of radioactivity were loaded onto the TLC plates (Fig. 1B), the presence of other phospholipids was revealed; ~80% of total phospholipids were associated with PC (including PC plasmalogen, which migrated at the same position on the chromatograms), ~12% was associated with PE, and a trace of the radioactivity was also associated with SPM and lyso-PC. PI and PS incorporated an insignificant amount of the label (0.6%) when the sperm were incubated with [3H]palmitic acid for up to 4 h. The pattern of incorporation was similar for each incubation time. The amount of radioactivity associated with PA could not be determined because the solvent used to develop the TLC plates does not separate PA and free [3H]palmitic acid or PA and [3H]palmitic acid incorporated into neutral lipids that migrated at the same position as PA. In bovine epididymal sperm, PA is present in trace amounts [41].
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Phospholipid Efflux from Bovine Epididymal Sperm in the Presence of the BSP Proteins
To study the sperm phospholipid efflux, labeled sperm were incubated in the presence or absence of the purified BSP proteins. As shown in Figure 2A, the medium alone and the BSP-A1/A2 protein enhanced the appearance of radiolabel in the medium. After an 8-h incubation, BSP-A1/A2 protein and the medium alone increased sperm radiolabel efflux to 54.3 ± 4.2% and 26.2 ± 1.9%, respectively. These percentages of radiolabel efflux appeared very high, and it seemed unlikely that the sperm could lose these amounts of phospholipids. Therefore, after 8 h of incubation, the sperm were centrifuged and the supernatant was analyzed by lipid extraction using chloroform:methanol (1:2 by volume). All the radioactivity in the supernatant of the control was recovered in the aqueous phase (see Materials and Methods). Therefore, all of the radiolabel efflux obtained for the control did not correspond to specific phospholipid efflux but corresponded to the release of [3H]palmitic acid not incorporated into the phospholipid pool (confirmed by TLC analysis). In contrast, when we analyzed the supernatant obtained after 8 h of incubation of the sperm with BSP-A1/A2 protein, we recovered the same amount of radioactivity in the aqueous phase that we found in the control supernatant, but a part of radioactivity was also recovered in the chloroform phase. The TLC analysis of the chloroform phase revealed that all of the radiolabel was associated with PC. These results were also confirmed by the analysis of the efflux particles obtained in the presence and absence of BSP-A1/A2 protein on a CL-4B column (see next section, Fig. 4). Therefore, the specific phospholipid efflux was obtained by subtracting at each time point the percentage of the efflux obtained in the control from the percentage of the efflux obtained in the presence of BSP proteins (Fig. 2B). The BSP-A1/A2 protein stimulated specific sperm phospholipid efflux in a dose-dependent manner, with a maximum stimulation of 28.1 ± 3.9% when 120 µg/ml of protein was used. The rate of sperm phospholipid efflux was very slow in the first hour and then rapidly increased up to 8 h. The specific efflux of phospholipids was significant when more than 40 µg/ml of BSP-A1/A2 protein was used. The efflux curves obtained in the presence of BSP-30-kDa and BSP-A3 were similar to that obtained with BSP-A1/A2 protein (Fig. 3). BSP-30-kDa protein (120 g/ml) stimulated a significant specific phospholipid efflux up to 29.5 ± 4.7%, similar to the efflux obtained in the presence of BSP-A1/A2 protein. In contrast to the other BSP proteins, BSP-A3 protein weakly stimulated the phospholipid efflux (8.3 ± 0.6%), but this efflux was significant (p < 0.005). To evaluate the specificity of efflux, we performed an experiment with bovine RNase as control protein. We used RNase as a control protein because this protein is present in high concentration (1–2 mg/ml) in bovine seminal plasma and has a molecular weight similar to that of BSP-A1/A2 or BSP-A3 proteins. RNase (120 µg/ml) failed to stimulate sperm phospholipid efflux (< 2.5%, Fig. 3). In contrast to RNase, a high concentration of BSA (6 mg/ml) stimulated a specific phospholipid efflux up to 22.6 ± 3.5% (Fig. 3). For all of the experiments, the epididymal sperm motility of treated sperm were the same as those observed in the control sample (medium alone). The motility of the sperm in the beginning was around 80–90% and decreased gradually during the long period of incubation (motility of 10–20% after 6–8-h incubation).
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Effect of BSP Proteins on the Capacitation of Epididymal Sperm in the Absence of BSA
Our previous experiments were always done in the presence of delipidated BSA (0.6%) [7–10]. In this study, BSA could not be used because previous results had demonstrated that BSA (0.5%) decreased the incorporation of label into phospholipids by 50% [42]. Therefore, all experiments described in this report were carried out by incubating the sperm in mTALP-PVA-PEG medium. We also investigated whether the absence of 0.6% of BSA in the medium affected the ability of the BSP proteins to promote sperm capacitation. After 8 h, an aliquot of labeled sperm suspension of the control sample as well as of that incubated with purified BSP proteins was taken to evaluate sperm capacitation and spontaneous AR (Table 2). Lyso-PC, which induces the AR only in capacitated sperm, was used to indirectly measure sperm capacitation (see Materials and Methods). Therefore, the AR in the present context is meant to reflect capacitation rather than the AR. After an 8-h incubation, the purified BSP proteins stimulated the AR (induced by lyso-PC) of labeled sperm (30.6–34.2%) compared to control (11.2 ± 1.3%) and stimulated the spontaneous AR (22.2–23.6%) compared to control (11.7 ± 0.9%).
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Chromatographic Analysis of the Efflux Particle
Chromatographic analysis, SDS-PAGE analysis, and immunoblotting were used to verify whether the efflux particles contained BSP proteins associated with [3H]phospholipid. After phospholipid efflux in the presence or absence of BSP-A1/A2 protein, the incubation media containing the efflux particles were chromatographed on a Sepharose CL-4B column (Fig. 4). Chromatographic analysis of the incubation medium in the absence of proteins (Fig. 4A) revealed a major peak of radioactivity (fractions 80–90). Lipid extraction of these fractions indicated that radioactivity corresponded to free [3H]palmitic acid. In contrast, chromatographic analysis of efflux particles produced in the presence of BSP-A1/A2 proteins on a Sepharose CL-4B column (Fig. 4B) revealed two peaks of radioactivity (peak 1, fractions 15–22 and peak 2, fractions 78–88). Lipid analysis of these fractions indicated that the radioactivity in peak 1 corresponded to [3H]phospholipid (mainly PC, verified by TLC plates) while the radioactivity in peak 2 corresponded to free [3H]palmitic acid. The same amounts of free [3H]palmitic acid were recovered in the supernatant of the control (fractions 80–90, Fig. 4A) and in the supernatant of sperm incubated with BSP-A1/A2 protein (peak 2, Fig. 4B; ~40 000 cpm). Furthermore, certain fractions shown in Figure 4B were analyzed by immunoblotting using antibody against BSP-A1/A2 protein (Fig. 4C). The major portion of the BSP-A1/A2 protein was detected in fractions 17–19, which corresponded to the first peak of radioactivity.
Our previous experiments were always done in medium containing BSA [7–10]. When we incubated labeled sperm with BSP proteins for 8 h in the presence of 0.6% of delipidated BSA and analyzed the supernatant on a CL-4B column under the same conditions as in the absence of BSA (Fig. 4), we obtained 3 peaks of radioactivity. The first peak (fractions 15–25, ~62 000 cpm) corresponded to labeled phospholipids associated with BSP proteins, the second peak corresponded to labeled phospholipids associated with BSA (fractions 65–80, ~300 000 cpm), and the third peak corresponded to [3H]palmitic acid unincorporated into phospholipids (fractions 80–94, ~55 000 cpm).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First, we evaluated the efflux potential of the BSP-A1/A2 protein, using direct determination of choline phospholipids in sperm. The amount of choline phospholipid in epididymal sperm before incubation was 0.998 ± 0.262 mg/1 x 109 sperm. On the assumption that choline phospholipids represent 55.3% of total epididymal sperm phospholipids [41], the total phospholipid content was ~1.805 mg/1 x 109 sperm, a value similar to that of 1.90 ± 0.07 mg/1 x 109 obtained previously by Lavon et al. [43]. The results indicated that the sperm incubated with BSP-A1/A2 protein lost a significant amount (34.4%) of choline phospholipids compared to the control sample (11.5%).
To study the kinetics of sperm phospholipid efflux, we used labeled epididymal sperm. This method allowed us to 1) use fewer sperm, 2) follow directly the kinetics of phospholipid efflux, 3) limit manipulation error and material loss that could occur during lipid extraction, and 4) evaluate the efflux of choline phospholipids and also of other phospholipids. Sperm phospholipids have saturated fatty acids only in position 1 and unsaturated fatty acids in position 2 [41, 44, 45]. Several investigators have attempted to label mammalian sperm with radioactive saturated and unsaturated fatty acids [34, 42, 45–47]. In these studies, only a small amount of phospholipid was labeled, and the majority of the label was incorporated into diglycerides. Vasquez and Roldan [48] showed that two pools of diglycerides exist in sperm and that only one of these pools (consisting of saturated fatty acids in position 1 and unsaturated fatty acids in position 2, the least labeled) contributes to phospholipid biosynthesis. We used [3H]palmitic acid, which constitutes the major fatty acid found in position 1 of epididymal sperm choline phospholipids (70–95% depending on the species of choline phospholipid) [41]. In addition, [14C]palmitic acid has been used to label phospholipids of bovine and boar sperm [48, 49]. Similar to the results mentioned above, when bovine epididymal sperm were labeled with [3H]palmitic acid, only a small amount (7–10%) of radiolabel was incorporated into the phospholipid pool. In contrast to other studies that were able to rapidly label PI and PS of bovine ejaculated sperm with [14C]palmitic acid [46, 49], in our studies using bovine epididymal sperm, these lipids did not incorporate [3H]palmitic acid for up to 4 h. PI and PS are minor components of sperm phospholipids, representing only 3.8% and 3.3%, respectively, of the total phospholipids of bovine epididymal sperm [41]. The majority of the phospholipids labeled were PC (~80%) and PE (~12%). After a 1-h incubation with 5 µCi of [3H]palmitic acid, 41 547 ± 3702 cpm/108 cells was associated with PC, which was 4 times that reported with ram sperm (10 349 ± 228 cpm/108 cells) [34]. Although the labeling was not very efficient, this amount of radioactivity associated with PC was enough to follow the movement of the phospholipids in the presence of BSP proteins.
BSP-A1/A2 and 30-kDa proteins stimulated a specific phospholipid efflux up to 29.5 ± 4.7%, and this value was similar to that obtained by direct determination of choline phospholipid (~23% when the value obtained for control was subtracted). In contrast to BSP-A1/A2 and BSP-30-kDa proteins, BSP-A3 protein only weakly stimulated phospholipid efflux, but all three proteins stimulated capacitation to the same level (Figs. 2 and 3, Table 2). In our previous study, we observed similar differences in cholesterol efflux [9]. BSP-A3 protein stimulated cholesterol efflux slowly at the beginning compared to BSP-A1/A2 or BSP-30-kDa proteins, but after 6–8 h, all three BSP proteins stimulated the cholesterol efflux (31.4 to 33.1%, control ~10%) and capacitation (33.5–38.5%, control 15 ± 0.4%) to the same levels. These results suggest that the higher cholesterol efflux from sperm is more important than the higher phospholipid efflux to enable sperm capacitation at the maximal level. It is not known why BSP-A3 protein stimulates phospholipid and cholesterol efflux differently from the other two BSP proteins. The three BSP proteins have two similar type-II domains arranged in tandem fashion, but they have a variable N-terminal portion. In addition, BSP-A3 protein is not glycosylated in contrast to BSP-A1/A2 and BSP-30-kDa proteins, and it has less affinity to choline than do the other two proteins. These differences in properties could influence the phospholipid efflux potential of BSP-A3 protein.
Seminal plasma contains albumin in addition to BSP proteins. BSA at 6 mg/ml stimulated phospholipid efflux (22.6 ± 3.5% after 8 h, Fig. 3) in a manner similar to that of BSP proteins. Although this is a significant stimulation, it occurs at high concentration of BSA and could be attributed to the hydrophobic property of this protein. Furthermore, the albumin concentration in semen is 0.32 mg/ml [50], and this low concentration is unlikely to stimulate a significant phospholipid efflux upon ejaculation. In addition, not all albumin is necessarily available for lipid efflux since it can interact with many other factors in seminal plasma. Moreover, the concentration of BSP proteins in seminal plasma is 20–40 mg/ml, about 100 times higher than the concentration of albumin, and the BSP proteins stimulate significant lipid efflux at a very low concentration (120 µg/ml) relative to BSA (> 6 mg/ml). Furthermore, the interaction of phospholipids with BSP proteins is choline-specific [39], and our recent results show that the ability of BSP protein-mediated lipid efflux from fibroblasts is inhibited by choline [51], which again affirms that the lipid efflux is a structure-dependent process. Consequently, BSP proteins are the factors in seminal fluid responsible for the lipid efflux.
Bovine seminal plasma contains ~198 µg/ml [52] of cholesterol, which may influence the lipid efflux properties of seminal plasma. This cholesterol is probably associated with a BSP protein-phospholipid complex. Our previous results show that bovine seminal plasma stimulates sperm cholesterol efflux in a dose-dependant manner similar to that of BSP proteins [9]. Therefore, BSP protein-lipid complexes and/or free BSP proteins in seminal fluid are not completely saturated with lipids and can accept more cholesterol and probably phospholipids.
Our previous study showed that the efflux particles obtained after incubation of sperm in the presence of BSP proteins are homogeneous particles with a density comparable to that of HDL. These particles were similar in size to very low-density lipoproteins and contained BSP proteins associated with [3H]cholesterol [9]. Our current results indicated similar particles containing BSP proteins associated with [3H]PC (Fig. 4). In contrast to the previous results [9], which indicated that only a small portion of BSP proteins participated in the formation of efflux particles, the majority of the BSP proteins in the current studies were associated with efflux particles (fractions 17–19, Fig. 4, B and C). This discrepancy could be attributed to the absence of BSA in the medium used for efflux studies. In the presence of 0.6% BSA, most of the phospholipids removed from sperm were sequestered by BSA, and, therefore, fewer phospholipids were available for the BSP proteins and many BSP proteins were free (eluted in the last fractions). In the absence of BSA, all the phospholipids were sequestered by the BSP proteins, and the majority seemed to be a part of the particles. Alternatively, lipids sequestered by BSP proteins may be transferred to BSA, which was present at a high concentration in the medium. The absence of BSA in the medium did not change the ability of BSP protein to promote sperm capacitation (Table 2). The percentages of AR induced by lyso-PC were similar to those obtained previously in the presence of 0.6% BSA (33.5–38.5%; control, 15.1 ± 0.4%) [9].
A continuous exposure of BSP proteins to sperm for 8 h stimulated a spontaneous AR (Table 2). It is possible that a high phospholipid efflux stimulated by BSP proteins after a long period could be due to the loss of membrane fragments as a result of a spontaneous AR. However, the incubation of sperm for a long period with BSP proteins is not a physiological situation, since seminal plasma that contains BSP proteins is gradually diluted and lost during sperm transit through the female genital tract [53]. Therefore, at the moment of the AR, only BSP proteins bound to sperm are present. Furthermore, these sperm-bound BSP proteins stimulate neither cholesterol efflux [9] nor the AR after incubation with heparin (5 h), and the presence of an acrosome inducer (lyso-PC) is necessary to stimulate the AR [7].
Our results show that bovine epididymal sperm incubated for a short period (20 min) with BSP-A1/A2 proteins, washed twice, and then incubated for 8 h with medium alone did not capacitate sperm [8]. During this period, the BSP-A1/A2 proteins stimulated a cholesterol efflux of 17.3 ± 4.0% (2.5-fold stimulation versus control) [9], but the phospholipid efflux remained low (Fig. 2B, current study). Therefore, the cholesterol efflux that occurred during the 20 min of incubation with BSP proteins is not sufficient to stimulate sperm capacitation. However, continuous exposure of sperm to BSP-A1/A2 proteins led to a 32.4 ± 1.3% cholesterol efflux [9] and a 28.1 ± 3.9% phospholipid efflux (Fig. 2B), and this resulted in a 2- to 3-fold higher capacitation versus control [9]. These results are in agreement with those reported by Zarintash and Cross [19], which showed that human sperm must lose cholesterol to respond to an acrosome reaction inducer. Their kinetics studies of the acrosome reaction as function of cholesterol efflux suggest that a threshold of cholesterol efflux is necessary before capacitation. Indeed, when the cholesterol efflux was less than 20%, the percentage of acrosome reactions was very low (< 5%), but when the cholesterol efflux was between 20% and 40%, the percentage of acrosome reactions was approximately 25–30%. Thus, it is possible that a threshold of cholesterol and phospholipid efflux is necessary to induce bovine sperm capacitation.
Davis suggested that capacitation involves a decrease in the membrane cholesterol:phospholipid (C:P) ratio [16]. Our present and previous results [9] showed that after ejaculation, the BSP proteins stimulate sperm phospholipid and cholesterol efflux. The phospholipid efflux was slow in the first hour and then rapidly increased up to 8 h. In contrast, the rate of cholesterol efflux was rapid in the first 2 h and then gradually reached a plateau after 4 h. Therefore, during the initial hours, BSP proteins seem to stimulate a preferential cholesterol-over-phospholipid efflux. It is plausible that the removal of cholesterol by BSP proteins might require a small initial phospholipid efflux. This could change the conformation of BSP proteins such that the phospholipid-BSP protein complex can sequester cholesterol. One explanation for the discrepancy in the kinetics of phospholipid and cholesterol efflux could be that the BSP proteins initially interact with cholesterol-rich microdomains on the membrane and consequently remove more cholesterol than phospholipids. Alternatively, it has been reported that the rate of desorption of cholesterol is 6 times more rapid than that of phospholipids [54]. That is, more cholesterol is removed than phospholipids during the short period (in vivo) of exposure to seminal fluid. As a result, there is a decrease in the sperm C:P ratio. This C:P ratio could decrease further because more cholesterol could be removed from sperm by HDL in the female reproductive tract. Therefore, our results support the current concept that there is a decrease in the C:P ratio during capacitation [15–19].
On the basis of our previous study [9] and the current study, we propose that the BSP proteins participate in membrane lipid modification events that occur during the early step of capacitation. After ejaculation, bovine sperm are exposed for a brief period (about 20–30 min) to the BSP proteins present in seminal fluid. During this brief exposure, a portion of the BSP proteins initiate a significant cholesterol efflux accompanied by the release of some phospholipids. This efflux of lipids (decrease in C:P ratio) may slightly destabilize the sperm membrane (capacitation-like state). At the same time, sperm are coated with BSP proteins. This binding of BSP proteins to the sperm membrane increases the number of heparin-binding sites on the sperm surface [26, 55]. The sperm then interact with capacitating factors (heparin-like GAG or HDL) in the oviduct and complete capacitation by two different mechanisms. Heparin induces capacitation by binding to BSP proteins present on ejaculated sperm and stimulates a series of intracellular events such as an increase in pH, Ca2+, cAMP, and tyrosine phosphorylation of a group of proteins (for review see [20]). On the other hand, HDL induces capacitation by stimulating a second efflux of sperm cholesterol in the female genital tract [9, 24, 35]. Since cholesterol is recognized to have a stabilizing effect on membranes [56], its efflux would be expected to provoke further reorganization or destabilization of the membrane and trigger some unknown signal transduction pathways. This could regulate the surface expression of sperm zona pellucida receptors [57]. The adhesion to the zona pellucida would then trigger the AR.
In summary, we show for the first time that the BSP proteins stimulate phospholipid efflux from epididymal sperm. These results together with our previous report [9] indicate that the BSP proteins concomitantly remove cholesterol and phospholipids. Since more cholesterol is removed than phospholipid, the C:P ratio decreases, which is favorable for capacitation. Furthermore, lipid efflux also results in the formation of BSP protein-phospholipid-cholesterol particles. Further characterization of these efflux particles is warranted to gain more insight into the membrane lipid modification events that occur during sperm capacitation.
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FOOTNOTES |
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2 Correspondence: P. Manjunath, Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 boul. de l'Assomption, Montréal, PQ, Canada H1T 2M4. FAX: 514 252 3430; manjunap{at}ere.umontreal.ca
Accepted: April 8, 1999.
Received: December 8, 1998.
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