Abstract

Bovine seminal plasma (BSP) contains a family of novel phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa; collectively called BSP proteins) that potentiate sperm capacitation induced by heparin or by serum high-density lipoprotein (HDL). BSP proteins stimulate lipid efflux from sperm that may occur during the early events of capacitation. Here, we investigated the role of BSP proteins, bovine follicular fluid (FF), and bovine follicular fluid HDL (FF-HDL) in sperm capacitation. FF and FF-HDL alone stimulated epididymal sperm capacitation (19.5% ± 0.8% and 18.2% ± 2.8%, respectively, control, 9.0% ± 1.9%) that was increased by preincubation with BSP-A1/-A2 proteins (30.2% ± 0.4% and 30.9% ± 1.5%, respectively). In contrast, lipoprotein-depleted follicular fluid (LD-FF) alone was ineffective, and a preincubation with BSP-A1/-A2 proteins was necessary before sperm capacitation was stimulated (up to 22.8% ± 1.4%). The interaction of BSP proteins with FF components was analyzed using ultracentrifugation, Lipo-Gel electrophoresis, SDS-PAGE, and gel filtration. We established that the BSP proteins interact with factors present in FF including FF-HDL. Additionally, we obtained evidence that BSP proteins, found associated with FF-HDL, were released from the sperm membrane during capacitation. These results confirm that the BSP proteins and the FF-HDL play a role in sperm capacitation.

Introduction

In mammals, sperm emerging from the male reproductive tract are incapable of fertilizing eggs, but they acquire this ability during their transit in the female reproductive tract [1, 2]. This required period of conditioning is called capacitation [3]. Sperm capacitation is a multistep process that is not well understood and involves several biochemical and ultrastructural changes in the sperm membrane. These changes include the loss of adsorbed components originating from the seminal plasma, the modification of membrane lipid composition, and an increased permeability to ions [412]. The process of capacitation renders the spermatozoa capable of interaction with the oocyte and thereby induce the acrosome reaction (AR).

Many years ago, Davis [13, 14], based on several studies, proposed that the loss of cholesterol from the sperm membrane and the resulting decrease in the cholesterol/phospholipid (C/P) molar ratio is an important step in the process of sperm capacitation. This concept is supported by other studies [1522]. In the bovine species, Erhenwald’s as well as our studies have shown that the efflux of membrane cholesterol leads to sperm capacitation [20, 21, 23].

Because capacitation occurs during the transit of spermatozoa through the female reproductive tract, a number of investigators tried to identify those factors in the oviduct that may be responsible for reducing the sterol content of the sperm membrane. Many studies have shown that albumin [7, 17, 24] and high-density lipoprotein (HDL) [25, 26] are acceptors of cholesterol. HDL, probably derived from plasma, is the only class of lipoprotein present in follicular and oviductal fluids (FF and OF, respectively) [25, 27, 28]. The apolipoprotein associated with OF-HDL is apolipoprotein A-I (apoA-I) and has a molecular mass of 28 kDa [25]. Albumin is the major protein present in OF (>90%), in the bovine and human species; however, the HDL appears to be a more efficient acceptor of cholesterol than albumin [23, 25, 26]. Our previous results showed that, in vitro, purified human serum HDL induces capacitation of bovine epididymal sperm slightly (1.4-fold) [29]. However, when bovine ejaculated sperm were used, human serum HDL increased capacitation up to 2.5-fold [30]. Therefore, factors from seminal plasma accelerate the sperm capacitation process triggered by HDL, and one of these factors is the bovine seminal plasma (BSP) family of proteins [29].

The BSP family of proteins are phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa) that are secreted by the seminal vesicles [3133]. Biochemical properties and structure of these proteins have been studied in detail [3137]. The BSP proteins bind to epididymal spermatozoa via their interaction with choline phospholipids [38], interact with the bovine sperm capacitating factors, heparin [39], and bind to purified human plasma apoA-I and apoA-I associated with HDL [40, 41]. Furthermore, the BSP proteins accelerate the capacitation of bovine epididymal sperm induced by heparin and serum HDL [29, 42]. In our further work, we showed that the exposure of epididymal sperm to BSP proteins alone for a brief period results in the stimulation of a significant sperm membrane cholesterol efflux accompanied by some phospholipid efflux [23, 43].

Our previous work was done with HDL isolated from human serum. However, bovine FF-HDL appear to be better particles to stimulate sterol efflux and to capacitate sperm. These HDLs are cholesterol-poor particles compared with serum HDL and contain a higher amount of phospholipid [44]. Moreover, biochemical characterization of FF-HDL showed the presence of a high proportion of pre-β1-HDL [45], a subclass of HDL recognized to play a major role as an initial cholesterol acceptor in the reverse cholesterol transport (RCT) [46, 47]. In this study, we attempted to gain further insight into the molecular mechanism of sperm capacitation by BSP proteins and FF capacitation factor (FF-HDL).

Materials and Methods

Materials

BSA (fraction V fatty acid-free), taurine, l-epinephrine, erythrosin B, flavianic acid (naphthol), and lysophosphatidylcholine (lyso-PC; purified from egg yolk) were from Sigma (St. Louis, MO). Penicillin G, streptomycin sulfate, and antibiotic-antimycotic (100×) were from Gibco BRL (Burlington, ON, Canada); eosin B from Fisher Scientific (Fair Lawn, NJ); and nigrosine from Kodak (Rochester, NY). Immobilon-P membranes and enhanced chemiluminescence reagent (ECL) kit were purchased from Mandel Scientific (Guelph, ON, Canada). Sepharose CL-4B was purchased from Amersham Pharmacia Biotech (Baie d’Urfé, PQ, 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 was obtained by the Centre d’Insémination Artificielle du Québec (St-Hyacinthe, PQ, Canada). Crude seminal plasma proteins (cBSP) were prepared by ethanol precipitation of BSP followed by dialysis of the precipitates against 50 mM ammonium bicarbonate and lyophilization. This preparation consists of 60–70% BSP proteins. The purification of BSP-A1/-A2, -A3, and -30-kDa proteins is described previously [32, 33, 42].

Isolation of FF and FF-HDL

Bovine FF was obtained from postmortem ovaries. Briefly, within 20 min after slaughter, ovaries were isolated from the genital tract and placed in warm (33°C) saline (0.9% NaCl) with antibiotic-antimycotic agents (100 units/ml of penicillin, 100 μg/ml of streptomycin and 0.25 μg/ml of amphotericin B) and transported to the laboratory. Between 3 and 4 h of slaughter, the contents of follicles (5–10 mm) were aspirated with a syringe and transferred to a sterile conical tube. Then, the FF was centrifuged (10 min, at 10 000 × g) to remove cellular debris. FF-HDL was isolated from bovine FF by ultracentrifugation as described previously for the isolation of HDL from human serum [29]. After centrifugation of FF, EDTA (1 mM) was added, and the density of the FF was raised to 1.21 kg/L by the addition of KBr and the samples were centrifuged for 20 h at 60 000 rpm at 20°C. After centrifugation, the FF-HDL (only lipoprotein present) was concentrated in a layer at the top of the tube and the bottom fraction contained lipoprotein-depleted FF (LD-FF). The fractions obtained were dialyzed (10 mM Tris-HCl buffer, pH 7.4) to remove salt. The concentration of protein in each fraction and in FF was measured by the modified Lowry procedure [48]. The purity of the FF-HDL was assessed by the Lipo-Gel kit following the manufacturer’s instructions (Beckman Instruments, Fullerton, CA) and by SDS-PAGE after precipitation of apolipoproteins with trichloroacetic acid (TCA, final concentration of 15%).

Sperm Capacitation and AR

The medium used for washing and incubation of sperm was a modified Tyrode medium (mTALP) described previously [42]. Caudal sperm were collected from the epididymides obtained from four different bulls, pooled, and washed twice (300 × g, 10 min) with 10 volumes of mTALP. Washed epididymal sperm (final concentration of 5 × 107 cells/ml) were preincubated in a humidified atmosphere (5% CO2, 37°C) for 20 min in the presence or absence of BSP-A1/-A2 proteins (40 or 120 μg/ml). BSP-A1/-A2 proteins were chosen because they are the most abundant BSP proteins in bovine seminal plasma (∼65%). The cells were then washed three times (300 × g, 10 min), resuspended in mTALP containing different concentrations of FF-HDL (final concentration 40–160 μg/ml), or FF (30% or 18–30 mg protein/ml), or LD-FF (30% or 25–40 mg protein/ml). At the end of the incubation (37°C, 5% CO2 atmosphere), two samples of 50 μl were withdrawn and lyso-PC was added (final concentration of 100 μg/ml), and the sperm were reincubated for an additional 15 min. This concentration of lyso-PC induces the AR in capacitated sperm while having no effect on noncapacitated sperm [49]. 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 [49]. Prior to 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 [50], and viability was estimated by the staining protocol of Dott and Foster [51].

Agarose-Gel Electrophoresis

The binding of BSP proteins to FF-HDL was demonstrated by agarose-gel electrophoresis using the Lipo-Gel kit. Different amounts of cBSP proteins (7.5–150 μg in 10 μl of 50 mM PBS buffer, pH 7.4) were incubated at room temperature (23°C) with purified FF-HDL (7.5 μg in 10 μl of 50 mM PBS buffer, pH 7.4) or FF (572.5 μg protein which contained ∼6.25 μg of FF-HDL in 10 μl of 50 mM PBS buffer, pH 7.4). After 30 min, 4 μl of incubation mixture was applied to each template slot of the agarose gel (0.6%), and the gel was subjected to electrophoresis for 30 min at 100 V. After electrophoresis, the gel was immersed in fixative solution and dried. The particles on dried Lipo-Gel were visualized by staining with Sudan black B solution (lipid stain) or with 8-amino-7-(3-nitrophenylazo)-2-(phenylazo)-1-naphthol-3,6-disulfonic acid disodium salt (protein stain).

Ultracentrifugation of FF-HDL and cBSP Proteins

To determine the binding of BSP proteins to FF-HDL, 5 mg of cBSP proteins (in 50 mM PBS, pH 7.4) was incubated 30 min with 1 mg of FF-HDL or 1.6 ml of FF (which contained ∼1 mg of FF-HDL). After incubation, PBS was added and the density of the incubation mixture was raised to 1.210 kg/L by the addition of KBr. Then, each mixture was centrifuged for 20 h at 60 000 rpm at 20°C. After centrifugation, three distinct fractions were obtained: top, middle, and bottom fractions. The concentration of protein in each fraction was measured by a modified Lowry procedure [48]. Then, each fraction was analyzed by agarose gel electrophoresis. The fractions were also subjected to SDS-PAGE and immunoblotting after precipitation of proteins with TCA (15%).

Chromatographic Analysis

For chromatographic analysis, 5 mg of cBSP proteins (in 50 mM PBS buffer, pH 7.4) was incubated for 30–40 min with 1 mg of FF-HDL or 1.6 ml of FF (that contained ∼1 mg of FF-HDL). The incubation mixtures were then applied to a Sepharose CL-4B column (2.5 × 73 cm) previously washed with 50 mM PBS buffer, pH 7.4. The elution was carried out at a flow rate of 80 ml/h with the same buffer. After 60 min, fractions of 3 ml were collected. After chromatography, each fraction was treated with TCA (15%) to precipitate the proteins that were then analyzed by SDS-PAGE and immunoblotting. Chromatographic analysis of cBSP alone (5 mg), FF-HDL alone (1 mg), and FF alone (1.6 ml) on a Sepharose CL-4B column was also performed under similar conditions.

Analysis of BSP Proteins in Incubation Media and on Sperm

To determine the fate of BSP proteins during capacitation by FF and FF-HDL, at different time intervals, an aliquot of each sperm suspension was taken and centrifuged (5 min, 10 000 rpm) to remove sperm from incubation media. The sperm pellet (washed twice with 50 mM PBS) and the supernatant (incubation media) were frozen for subsequent analysis of BSP proteins. To analyze the presence of BSP proteins, the fractions were thawed, dried, and delipidated using chloroform-methanol (2:1 by volume) [52]. The protein component of each fraction was then analyzed by SDS-PAGE and immunoblotting.

SDS-PAGE Analysis and Immunoblotting

SDS-PAGE in 15% polyacrylamide gels was performed as described previously [53]. The proteins were then transferred to Immobilon-P membranes as described by Towbin et al. [54]. Immunodetections using affinity-purified polyclonal antibodies against purified BSP proteins were done as previously described [55] by using an ECL kit for detection. Apparent molecular weights of the protein bands were determined using the low-molecular weight calibration kit (Amersham Pharmacia Biotech) containing phosphorylase b (Mr 94 000), BSA (Mr 67 000), ovalbumin (Mr 43 000), carbonic anhydrase (Mr 30 000), trypsin inhibitor (Mr 20 100), and α-lactalbumin (Mr 14 400).

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) or by the modified Lowry procedure [48].

Data Analysis

The data presented here were analyzed for significant differences by covariance analysis or by a Student t-test on paired observations.

Results

Isolation and Characterization of FF-HDL

The FF-HDL fraction, isolated by ultracentrifugation in KBr solution, contained only one class of detectable lipoproteins that correspond to HDL when visualized with a lipid stain (Fig. 1A) or protein stain (Fig. 1B). The SDS-PAGE revealed a major protein with a molecular mass around 28 kDa that correspond to apoA-I (Fig. 1C) [56].

Fig. 1

Characterization of FF-HDL. The purity of the FF-HDL isolated by ultracentrifugation was analyzed by Lipo-Gel electrophoresis. The particles were visualized by staining the gel with Sudan black B solution (lipid stain, A) or with 8-amino-7-(3-nitrophenylazo)-2-(phenylazo)-1-naphthol-3,6-disulfonic acid disodium salt (protein stain, B). Lane 1, human serum; lane 2, human serum HDL; lane 3, FF-HDL; lane 4, LD-FF; lane 5, FF. • C represents the point of application of samples. C) SDS-PAGE of isolated FF-HDL after precipitation of proteins with TCA (15%). std, Low molecular weight calibration kit (LMW standard, Pharmacia); lane 1, human serum (15 μg); lane 2, human serum HDL (5 μg); lane 3, FF-HDL (5 μg); lane 4, FF-HDL (10 μg); lane 5, FF (15 μg); lane 6, LD-FF (15 μg)

Fig. 1

Characterization of FF-HDL. The purity of the FF-HDL isolated by ultracentrifugation was analyzed by Lipo-Gel electrophoresis. The particles were visualized by staining the gel with Sudan black B solution (lipid stain, A) or with 8-amino-7-(3-nitrophenylazo)-2-(phenylazo)-1-naphthol-3,6-disulfonic acid disodium salt (protein stain, B). Lane 1, human serum; lane 2, human serum HDL; lane 3, FF-HDL; lane 4, LD-FF; lane 5, FF. • C represents the point of application of samples. C) SDS-PAGE of isolated FF-HDL after precipitation of proteins with TCA (15%). std, Low molecular weight calibration kit (LMW standard, Pharmacia); lane 1, human serum (15 μg); lane 2, human serum HDL (5 μg); lane 3, FF-HDL (5 μg); lane 4, FF-HDL (10 μg); lane 5, FF (15 μg); lane 6, LD-FF (15 μg)

Effect of FF, FF-HDL, and LD-FF on Bovine Sperm Capacitation in the Presence of the BSP-A1/-A2 Proteins

The capacitation potential of FF, FF-HDL, and LD-FF was determined in the presence or the absence of BSP-A1/-A2 proteins (Figs. 2 and 3). After 2 h of incubation (Figs. 2A and 3A), no stimulation of the AR (induced by lyso-PC) was observed with any treatment except when the combined presence of BSP-A1/-A2 proteins and 30% FF was used which stimulated the AR up to 2.2-fold (10.2 ± 0.5 compared with control, i.e., media alone, 4.7% ± 0.4%). After a longer incubation period (5 h), FF alone stimulated the AR. The percentage of AR at this time was low but represented an increase of 2.0-fold (12.7% ± 1.1% compared with control, i.e., without FF, 6.3% ± 0.6%). The preincubation of sperm with BSP-A1/-A2 proteins (120 μg/ml) increased the incidence of the AR with a maximum level of 3.1-fold (19.7% ± 0.1%; compared with control, i.e., medium alone, 6.3% ± 0.6%). After 8 h (Fig. 2C) with 30% FF, the percentage of AR was much higher, i.e., 2.2-fold (19.5% ± 0.8%, compared with control, i.e., medium alone, 8.8% ± 0.4%). However, preincubation with 120 μg/ml of BSP-A1/-A2 proteins followed by incubation with 30% FF caused the incidence of AR to increase further to 3.4-fold (30.2% ± 0.4% compared with control, medium alone).

Fig. 2

Effect of FF and LD-FF on the AR of epididymal sperm incubated with BSP-A1/-A2 proteins. Sperm were preincubated 20 min with different concentrations of BSP-A1/-A2 proteins, washed, and incubated with FF (30% or 18–30 mg protein/ml) or LD-FF (30% or 25–40 mg protein/ml) (see Materials and Methods). After a 2-h (A), 5-h (B), or 8-h (C) incubation, lyso-PC (100 μg/ml) was added to induce the AR. Results represent the mean ± SEM of three independent experiments, with duplicate samples for each experiment and 200 sperm assayed per sample. Significant difference versus control (without FF or LD-FF): *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 2

Effect of FF and LD-FF on the AR of epididymal sperm incubated with BSP-A1/-A2 proteins. Sperm were preincubated 20 min with different concentrations of BSP-A1/-A2 proteins, washed, and incubated with FF (30% or 18–30 mg protein/ml) or LD-FF (30% or 25–40 mg protein/ml) (see Materials and Methods). After a 2-h (A), 5-h (B), or 8-h (C) incubation, lyso-PC (100 μg/ml) was added to induce the AR. Results represent the mean ± SEM of three independent experiments, with duplicate samples for each experiment and 200 sperm assayed per sample. Significant difference versus control (without FF or LD-FF): *P < 0.05; **P < 0.01; ***P < 0.005

When isolated FF-HDL was used instead of FF, we obtained similar results (Fig. 3). FF-HDL alone stimulated sperm AR induced by lyso-PC after 5–8 h of incubation (up to 2.0-fold after 8 h compared to control, medium alone), but the preincubation of sperm with BSP-A1/-A2 proteins (120 μg/ml) increased the AR to a higher level (up to 3.4-fold after 8 h compared to control, medium alone). The effect of BSP-A1/-A2 proteins or FF-HDL was dose dependent; when the concentrations of BSP-A1/-A2 proteins or FF-HDL were increased, we observed an increased incidence in the AR.

Fig. 3

Effect of FF-HDL on the AR of epididymal sperm incubated with BSP-A1/-A2 proteins. The same protocol as in Figure 2 but using different concentrations of FF-HDL instead of FF or LD-FF. After a 2-h (A), 5-h (B), or 8-h (C) incubation, lyso-PC (100 μg/ml) was added to induce the AR. Results represent the mean ± SEM of three independent experiments, with duplicate samples for each experiment and 200 sperm assayed per sample. After 5 h and 8 h of incubation, the percentage of AR in the presence of more than 80 μg/ml of FF-HDL (in the presence or the absence of BSP-A1/-A2 proteins) was significantly different from control (without FF-HDL; P < 0.05)

Fig. 3

Effect of FF-HDL on the AR of epididymal sperm incubated with BSP-A1/-A2 proteins. The same protocol as in Figure 2 but using different concentrations of FF-HDL instead of FF or LD-FF. After a 2-h (A), 5-h (B), or 8-h (C) incubation, lyso-PC (100 μg/ml) was added to induce the AR. Results represent the mean ± SEM of three independent experiments, with duplicate samples for each experiment and 200 sperm assayed per sample. After 5 h and 8 h of incubation, the percentage of AR in the presence of more than 80 μg/ml of FF-HDL (in the presence or the absence of BSP-A1/-A2 proteins) was significantly different from control (without FF-HDL; P < 0.05)

In contrast with FF and FF-HDL, the LD-FF fraction alone did not stimulate the AR, and the preincubation of sperm with the BSP-A1/-A2 proteins was essential to stimulate the AR (up to 2.4–2.5-fold compared to control, medium alone, Fig. 2). In any case, BSP-A1/-A2 proteins alone could not stimulate significantly the AR of epididymal sperm (Figs. 2 and 3). Note that the FF, LD-FF, and HDL concentrations were based on protein and not lipid content.

The viability of sperm was affected by the period of incubation and by some treatments. The viability of the sperm in the beginning was around 69–71% for all samples and decreased gradually during the long period of incubation (data not shown). After 8 h, the viability of the samples incubated with FF-HDL (40–160 μg/ml) was not significantly different from that observed in the control (40–50%). When FF (30%) or LD-FF (25–40 mg protein/ml) were used, the viability of sperm decreased a little further and reached 27–34% after 8 h of incubation. The preincubation of sperm with BSP-A1/-A2 proteins did not affect significantly the viability of sperm.

Interaction of FF Components and Isolated FF-HDL with the BSP Proteins

A series of experiments evaluated the interation of BSP proteins and FF-HDL in which varying amounts of cBSP proteins were incubated with known amounts of FF or FF-HDL, and the resultant mixture was analyzed on Lipo-Gel electrophoresis (Fig. 4). An apparent change in the mobility (increased charge) of FF-HDL (isolated or not) when the cBSP proteins were present (Fig. 4A, lanes 3–9) is consistent with complex formation between one or more components of the cBSP proteins and FF-HDL.

Fig. 4

Analysis of the interaction of cBSP proteins and FF or FF-HDL by Lipo-Gel electrophoresis and after ultracentrifugation. A) Different amounts of cBSP proteins (1.5–30 μg) were incubated with FF (2 μl that contained ∼1.25 μg of FF-HDL) or purified FF-HDL (1.5 μg) and the mixtures were analyzed on Lipo-Gel. Lane 1, human serum (2 μl or ∼90 μg); lane 2, FF-HDL (1 μl or ∼3 μg); lanes 3–6, FF-HDL (1.5 μg) and cBSP proteins (lane 3, 1.5 μg; lane 4, 7.5 μg; lane 5, 15 μg; lane 6, 30 μg); lanes 7–9, FF (contained ∼1.25 μg FF-HDL) and cBSP proteins (lane 7, 1.5 μg; lane 8, 15 μg; lane 9, 30 μg); lane 10, FF (4 μl). In another experiment, cBSP proteins (5 mg) were incubated with FF (1.6 ml that contained ∼1 mg of FF-HDL) or purified FF-HDL (1 mg), and the mixtures were analyzed by density gradient ultracentrifugation following Lipo-Gel analysis of the fractions obtained (B), and by SDS-PAGE and immunoblotting with purified antibodies against BSP proteins (C, anti-A1/-A2; D, anti-A3; E, anti-30-kDa). B) Lane 1, human serum (2 μl or ∼90 μg); lane 2, FF-HDL (1 μl, ∼3 μg); lane 3, FF (4 μl, ∼230 μg); lane 4, top fraction of FF-HDL and cBSP proteins (8 μl, ∼5 μg); lane 5, bottom fraction of FF-HDL and cBSP proteins (8 μl, ∼3 μg); lane 6, top fraction of FF and cBSP proteins (8 μl, ∼5 μg); lane 7, middle fraction of FF and cBSP proteins (8 μl, ∼5 μg); lane 8, bottom fraction of FF and cBSP proteins (8 μl, ∼330 μg). • C represents the point of application of samples. CE) Lane 1, LMW standard; lane 2, cBSP proteins (60 ng); lane 3 FF-HDL (10 μg); lane 4, top fraction of FF-HDL and cBSP proteins (10 μg); lane 5, bottom fraction of FF-HDL and cBSP proteins (10 μg); lane 6, top fraction of FF and cBSP proteins (10 μg); lane 7, middle fraction of FF and cBSP proteins (10 μg); lane 8, bottom fraction of FF (10 μg) and cBSP proteins; lane 9, FF (10 μg).

Fig. 4

Analysis of the interaction of cBSP proteins and FF or FF-HDL by Lipo-Gel electrophoresis and after ultracentrifugation. A) Different amounts of cBSP proteins (1.5–30 μg) were incubated with FF (2 μl that contained ∼1.25 μg of FF-HDL) or purified FF-HDL (1.5 μg) and the mixtures were analyzed on Lipo-Gel. Lane 1, human serum (2 μl or ∼90 μg); lane 2, FF-HDL (1 μl or ∼3 μg); lanes 3–6, FF-HDL (1.5 μg) and cBSP proteins (lane 3, 1.5 μg; lane 4, 7.5 μg; lane 5, 15 μg; lane 6, 30 μg); lanes 7–9, FF (contained ∼1.25 μg FF-HDL) and cBSP proteins (lane 7, 1.5 μg; lane 8, 15 μg; lane 9, 30 μg); lane 10, FF (4 μl). In another experiment, cBSP proteins (5 mg) were incubated with FF (1.6 ml that contained ∼1 mg of FF-HDL) or purified FF-HDL (1 mg), and the mixtures were analyzed by density gradient ultracentrifugation following Lipo-Gel analysis of the fractions obtained (B), and by SDS-PAGE and immunoblotting with purified antibodies against BSP proteins (C, anti-A1/-A2; D, anti-A3; E, anti-30-kDa). B) Lane 1, human serum (2 μl or ∼90 μg); lane 2, FF-HDL (1 μl, ∼3 μg); lane 3, FF (4 μl, ∼230 μg); lane 4, top fraction of FF-HDL and cBSP proteins (8 μl, ∼5 μg); lane 5, bottom fraction of FF-HDL and cBSP proteins (8 μl, ∼3 μg); lane 6, top fraction of FF and cBSP proteins (8 μl, ∼5 μg); lane 7, middle fraction of FF and cBSP proteins (8 μl, ∼5 μg); lane 8, bottom fraction of FF and cBSP proteins (8 μl, ∼330 μg). • C represents the point of application of samples. CE) Lane 1, LMW standard; lane 2, cBSP proteins (60 ng); lane 3 FF-HDL (10 μg); lane 4, top fraction of FF-HDL and cBSP proteins (10 μg); lane 5, bottom fraction of FF-HDL and cBSP proteins (10 μg); lane 6, top fraction of FF and cBSP proteins (10 μg); lane 7, middle fraction of FF and cBSP proteins (10 μg); lane 8, bottom fraction of FF (10 μg) and cBSP proteins; lane 9, FF (10 μg).

When the incubation mixture was subjected to ultracentrifugation in KBr solution to isolate the complex cBSP-FF-HDL, three fractions were obtained that were analyzed by Lipo-Gel (Fig. 4B), by SDS-PAGE, and by immunoblot (Fig. 4, C–E). The Lipo-Gel analysis confirmed our previous results that components of the cBSP proteins became associated with FF-HDL. SDS-PAGE and immunoblot analysis of each fraction obtained by ultracentrifugation revealed the presence of the major portion of each BSP protein in the upper fraction (Fig. 4, C–E, lanes 4 and 6), the fraction that contained FF-HDL. When BSP proteins alone were analyzed on ultracentrifugation in KBr solution as above, they were recovered in the bottom fractions (data not shown).

Then, cBSP proteins, FF, FF-HDL, and the mixture of cBSP proteins and FF or FF-HDL were analyzed by chromatography (Figs. 5 and 6). When cBSP proteins alone were applied on the column (Figs. 5A and 6A), two detectable protein peaks were obtained; a small peak of proteins (fractions 14–18) and a major peak (fractions 72–97). Immunoblot of selected fractions with antibodies against BSP proteins indicated that the BSP proteins were present in the second peak (fractions 72–97, data not shown). With FF alone (Fig. 5A), three distinct protein peaks were obtained (fractions 14–18, 48–84, and 86–104). The mixture of FF and cBSP proteins revealed a similar elution pattern (Fig. 5A) as found with FF alone except for the appearance of a new shoulder at fractions 44–60 that was rich in BSP proteins (Fig. 5, B–E). When isolated FF-HDL was applied on the column, a single peak was obtained (fractions 52–75, Fig. 6A). When FF-HDL and cBSP proteins were incubated together (Fig. 6A), the peak that contained FF-HDL was shifted to the left (fractions 40–68), and we observed an increase of the absorbance value compared to that obtained with FF-HDL alone. Also, the absorbance value obtained for the peak of cBSP proteins (fractions 73–95) was smaller compared to that obtained with cBSP proteins alone (fractions 72–97). SDS-PAGE and immunoblot analysis revealed the presence of the major portion of the BSP proteins in fractions 46–63 of FF-HDL and cBSP chromatography (Fig. 6, B–E).

Fig. 5

The analysis of the interaction of cBSP proteins and FF by chromatography. A) The cBSP proteins (5 mg) were incubated with FF (1.6 ml), and the mixture was applied to the column. After waiting 60 min, fractions of 3 ml were collected and the absorbance (280 nm) was determined. The chromatography of cBSP proteins (5 mg) alone and FF (1.6 ml) alone on a Sepharose CL-4B column was also performed. Then, selected fractions were treated with 15% TCA to precipitate the proteins and analyzed by SDS-PAGE (B) and immunoblotting using antibodies against purified BSP proteins (C, anti-A1/-A2; D, anti-A3; E, anti-30-kDa). std, LMW standard

Fig. 5

The analysis of the interaction of cBSP proteins and FF by chromatography. A) The cBSP proteins (5 mg) were incubated with FF (1.6 ml), and the mixture was applied to the column. After waiting 60 min, fractions of 3 ml were collected and the absorbance (280 nm) was determined. The chromatography of cBSP proteins (5 mg) alone and FF (1.6 ml) alone on a Sepharose CL-4B column was also performed. Then, selected fractions were treated with 15% TCA to precipitate the proteins and analyzed by SDS-PAGE (B) and immunoblotting using antibodies against purified BSP proteins (C, anti-A1/-A2; D, anti-A3; E, anti-30-kDa). std, LMW standard

Fig. 6

Analysis of the interaction of cBSP proteins and FF-HDL by chromatography. The same protocol as in Figure 5 but using purified FF-HDL (1 mg) instead of FF. A) Chromatography patterns. B) SDS-PAGE pattern. CE) Immunoblotting using polyclonal antibodies against purified BSP proteins (C, anti-A1/-A2; D, anti-A3; E, anti-30-kDa). std, LMW standard

Fig. 6

Analysis of the interaction of cBSP proteins and FF-HDL by chromatography. The same protocol as in Figure 5 but using purified FF-HDL (1 mg) instead of FF. A) Chromatography patterns. B) SDS-PAGE pattern. CE) Immunoblotting using polyclonal antibodies against purified BSP proteins (C, anti-A1/-A2; D, anti-A3; E, anti-30-kDa). std, LMW standard

Fate of BSP Proteins Following Capacitation Induced by FF and FF-HDL

To determine the fate of BSP proteins following capacitation induced by FF, FF-HDL, or LD-FF, an aliquot of each sperm suspension, during the capacitation studies, at different time intervals, was taken and centrifuged. The sperm pellet and the supernatant (incubation media) were separated and analyzed for the presence of BSP proteins by SDS-PAGE and immunoblotting (Fig. 7). After 2–5 h, the majority of the BSP-A1/-A2 proteins were present in the supernatant when sperm were incubated with FF, FF-HDL, or LD-FF but were associated with sperm when sperm were incubated (0–8 h) with the medium alone.

Fig. 7

The fate of BSP proteins after capacitation. Analysis of the presence of BSP-A1/-A2 proteins in sperm pellet (S) and in the incubation media (M) after incubation of sperm in the presence (+) or the absence (−) of BSP-A1/-A2 proteins (40 μg/ml) for 20 min, washed, and reincubated with FF (30%) or LD-FF (30%) (A) or FF-HDL (80 μg/ml) (B). std, LMW standard; cBSP (100 ng A and 60 ng B).

Fig. 7

The fate of BSP proteins after capacitation. Analysis of the presence of BSP-A1/-A2 proteins in sperm pellet (S) and in the incubation media (M) after incubation of sperm in the presence (+) or the absence (−) of BSP-A1/-A2 proteins (40 μg/ml) for 20 min, washed, and reincubated with FF (30%) or LD-FF (30%) (A) or FF-HDL (80 μg/ml) (B). std, LMW standard; cBSP (100 ng A and 60 ng B).

To determine whether BSP-A1/-A2 proteins found in the media were associated with FF components, at the end of the incubation under capacitation conditions, each sample was centrifuged and the supernatant was analyzed by ultracentrifugation, SDS-PAGE, and immunoblotting (Fig. 8). The BSP-A1/-A2 proteins were present only in the top fraction in those samples incubated with BSP-A1/-A2 proteins and FF-HDL or FF.

Fig. 8

The ultracentrifugation analysis of the incubation medium after capacitation in the presence of BSP-A1/-A2 proteins (120 μg/ml) and FF-HDL (160 μg/ml) or FF (30%). After capacitation studies, an aliquot of each sperm suspension was taken and centrifuged (5–10 min, 10 000 rpm) to separate sperm cells. The incubation media (supernatant) were then subjected to ultracentrifugation in KBr solution (60 000 rpm, 20 h), and three fractions (top, T; middle, M; bottom, B) were recovered. Then, each fraction was treated with 15% TCA and analyzed by SDS-PAGE and immunoblotting with antibodies against BSP-A1/-A2 proteins. 1, Sperm alone; 2, sperm incubated with FF-HDL; 3, sperm incubated with FF; 4, sperm preincubated with BSP-A1/-A2; 5, sperm preincubated with BSP-A1/-A2 and FF-HDL; 6, sperm preincubated with BSP-A1/-A2 and FF. cBSP (60 ng).

Fig. 8

The ultracentrifugation analysis of the incubation medium after capacitation in the presence of BSP-A1/-A2 proteins (120 μg/ml) and FF-HDL (160 μg/ml) or FF (30%). After capacitation studies, an aliquot of each sperm suspension was taken and centrifuged (5–10 min, 10 000 rpm) to separate sperm cells. The incubation media (supernatant) were then subjected to ultracentrifugation in KBr solution (60 000 rpm, 20 h), and three fractions (top, T; middle, M; bottom, B) were recovered. Then, each fraction was treated with 15% TCA and analyzed by SDS-PAGE and immunoblotting with antibodies against BSP-A1/-A2 proteins. 1, Sperm alone; 2, sperm incubated with FF-HDL; 3, sperm incubated with FF; 4, sperm preincubated with BSP-A1/-A2; 5, sperm preincubated with BSP-A1/-A2 and FF-HDL; 6, sperm preincubated with BSP-A1/-A2 and FF. cBSP (60 ng).

Discussion

In mammals, sperm are always in a process of modification and complete their maturation only in the female genital tract where they are exposed to various fluids including FF and OF [57]. In vivo, sperm cells require the interaction with OF and FF to be able to capacitate and fertilize the oocyte. At the time of ovulation, most of the FF passes into the oviduct where fertilization occurs. In the current study, bovine HDL was isolated from FF and characterized. As observed by other groups [27, 28, 58], only one class of lipoprotein was present in the FF (Fig. 1), which corresponds to HDL. The FF-HDL particles were similar to the HDL particles isolated from bovine oviductal fluid by Ehrenwald et al. [25]. Surprisingly, purified FF-HDL migration was slightly higher than HDL migration in complete FF. The reasons for this difference are not clear but the isolation of bovine FF-HDL was done in Tris-HCl buffer (pH 7.4) after ultracentrifugation and dialysis, in contrast to FF that was loaded onto Lipo-Gel without any treatment. Alternatively, it is possible that FF-HDL was modified slightly following ultracentrifugation in KBr solution.

The present results indicate that FF and FF-HDL alone stimulated the capacitation of epididymal bovine sperm but, interestingly, this stimulation was increased by a preincubation with BSP-A1/-A2 proteins (Figs. 2 and 3). The ability of FF to capacitate mammalian sperm has been demonstrated previously by other groups [24, 5962]. In bovine animals, McNutt and Killian [59] reported that in vitro, FF was capable of capacitating sperm (determined indirectly by inducing the AR with lyso-PC) within 2 h of incubation when more than 60% of FF was used and within 4 h if only 20% of FF was used. After 5 h, 20% and 40% of FF stimulated the AR (induced by lyso-PC) by approximately 46% and 56% (control, medium alone; approximately 18%). Therefore, the number of acrosome-reacted sperm induced by lyso-PC was higher in their samples than in our samples using 30% FF. The differences observed may be due to the ejaculated sperm they used versus epididymal sperm exposed to BSP-A1/-A2 proteins in the current study. Ejaculated sperm are often exposed to seminal plasma proteins (BSP proteins; 30–50 mg/ml) for a certain period of time before treatment. Because incubation of sperm with a high concentration of BSP proteins accelerates sperm capacitation [23], the prolonged contact of sperm with seminal plasma could influence the percentage of capacitated sperm obtained.

A few groups [23, 25, 63] have also determined the effect of FF and female genital tract HDL on sperm cholesterol efflux. Ehrenwald [20, 21], using liposomes with and without cholesterol, showed that removing cholesterol from the sperm membrane predisposed the sperm to undergo the AR induced by lyso-PC and facilitated oocyte penetration. They also reported that bovine oviductal HDL stimulates the efflux of sperm cholesterol [25]. Our recent results show that human serum HDL stimulates bovine sperm cholesterol efflux and sperm capacitation [23, 29]. However, to our knowledge, this is the first study that demonstrates the direct effect of isolated FF-HDL on sperm capacitation. FF-HDL alone stimulated sperm AR induced by lyso-PC, but preexposure to the BSP-A1/-A2 proteins increased the incidence of the AR. In contrast to human serum HDL that requires 8 h of incubation to stimulate the lyso-PC-induced AR [29], the FF-HDL required only 5 h of incubation to stimulate the same phenomenon (Fig. 3). Moreover, the stimulation induced by FF-HDL compared with the control was always higher than the stimulation obtained with serum HDL in the presence or in the absence of the BSP-A1/-A2 proteins. Therefore, the FF-HDL appears to be a better capacitating factor than human serum HDL.

In contrast to FF and FF-HDL, the LD-FF fraction stimulated capacitation only when the BSP-A1/-A2 proteins were present on the sperm membrane. The LD-FF fraction contained all of the constituents of FF except the HDL particles. Bovine FF is a rich source of glycosaminoglycans (GAGs: dermatan sulfate and heparan sulfate) [64]. In bovine animals, GAGs possess the ability to capacitate sperm [49]. Our previous results indicated that heparin stimulates sperm capacitation only when the sperm are preincubated in the presence of BSP proteins [42], which is very similar to the phenomenon observed with the LD-FF fraction. Therefore, the effect observed with the LD-FF fraction could be due to the presence of heparin-like GAGs in the bovine FF. When sperm were incubated with BSP-A1/-A2 proteins and LD-FF fractions, the BSP-A1/-A2 were recovered in the supernatant. The LD-FF fraction may contain several factors (GAGs and apoA-I not complexed with HDL) that could remove BSP proteins from sperm.

We observed that the viability and the motility (data not shown) of sperm incubated with the FF and the LD-FF fraction decreased more rapidly than the control. Langlais et al. [24] observed the same phenomenon; when they incubated human sperm with 10% of FF, the sperm viability decreased from 64% to 49% after only 2 h. They could abolish the toxicity of FF by heating the fluid (30 min, 56°C). However, the factor(s) from FF that affected the sperm viability was not identified. McNutt and coworkers [59, 60] also observed a significant decrease in sperm motility when more than 20% of FF was used. They hypothesized that the observed decrease in motility is due to sperm agglutination that occurred to a greater extent in FF.

Analysis of sperm pellet and supernatant (media) following capacitation experiments revealed that BSP proteins interact with FF-HDL to form a complex. In this study, we showed for the first time that the BSP proteins were released from sperm membrane during capacitation and were recovered with FF-HDL. Ehrenwald and coworkers [25] incubated bovine ejaculated sperm with oviductal fluid and found that two proteins of approximately 16 and 18 kDa were transferred from sperm to the HDL fractions. These proteins could correspond to the BSP proteins. Currently, we do not know the significance of the removal of the BSP proteins from sperm and their association with FF-HDL. Many groups have postulated that the removal of sperm-bound seminal plasma proteins that act as decapacitating factors is essential for the AR in some species [9, 65]. Thus, the binding of BSP proteins to sperm could ensure that the sperm cells remain uncapacitated until they reach the appropriate time and place, and that the sperm undergo capacitation only in the female genital tract upon interaction with HDL or GAGs.

Many studies report that BSP contains factors that have a positive regulatory role in capacitation [42, 66, 67]. Indeed, bovine ejaculated sperm always capacitated faster than epididymal sperm in response to capacitating factors (GAGs or HDL) and a preexposure of epididymal sperm to seminal plasma or the purified BSP proteins reduced the time required for capacitation [29, 42]. Because the BSP proteins are the most abundant proteins of seminal plasma, it is reasonable to conclude that these proteins are the physiological molecules in BSP responsible for acrosomal stabilization and destabilization (see below).

Based on our systematic studies with epididymal sperm and BSP proteins, we propose the following mechanism of sperm capacitation (Fig. 9). At ejaculation, sperm are exposed to seminal fluid (BSP proteins, contributed by seminal vesicles). During this brief exposure, BSP proteins remove a significant amount of cholesterol (first cholesterol efflux) accompanied by the release of some phospholipids. This lipid efflux (decrease in C/P ratio) may slightly destabilize the sperm membrane (priming). At the same time, BSP proteins coat the sperm surface via their interaction with choline phospholipids. This coating of BSP proteins prevents free movement of phospholipids and thereby stabilizes the sperm membrane (arrested state). In the second-step, the oviduct/follicular fluid HDL removes BSP proteins from the sperm membrane. As a result, sperm membrane lipids are free to move. In addition, HDL may induce a second efflux of cholesterol. Because cholesterol is recognized to have an important stabilizing effect on membranes, its efflux would be expected to provoke further destabilization of the membrane and trigger certain unknown signal transduction pathways. This could regulate the surface expression of sperm zona pellucida (ZP) receptors. The adhesion to the ZP would then trigger the AR. Alternatively, BSP proteins may play a role via their interaction with heparin-like GAGs in the uptake of Ca2+, in the intracellular alkalinization and in tyrosine-phosphorylations that occur during heparin-induced capacitation.

Fig. 9

Mechanism of sperm capacitation by the BSP proteins and HDL.

Fig. 9

Mechanism of sperm capacitation by the BSP proteins and HDL.

In summary, the current studies indicate that FF-HDL stimulates bovine sperm capacitation and that the BSP proteins accelerate capacitation by physiological inducers (FF, FF-HDL, and components of the LD-FF fraction). Furthermore, we showed for the first time that the BSP proteins are released from sperm membrane during capacitation and are sequestered by FF-HDL. Our results confirm that the BSP proteins and factors in FF play an important role in sperm capacitation. Further studies are necessary to determine whether or not the removal of the BSP proteins from the sperm membrane during capacitation leads to signal transduction downstream to induce the AR.

Acknowledgments

We are grateful to Ms. Nathalie Morin for collecting bovine FF and to Dr. Kenneth D. Roberts for proofreading the manuscript.

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Author notes

1
This work was supported by a grant from the Medical Research Council of Canada. I.T. is a Fellow of the Lalor Foundation.