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Biology of Reproduction 67, 1250-1258 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Major Proteins of Bovine Seminal Plasma Bind to the Low-Density Lipoprotein Fraction of Hen's Egg Yolk1

Puttaswamy Manjunath2,a, Veronica Nauca, Annick Bergerona, and Martin Ménarda

a Departments of Medicine and Biochemistry, University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada H1T 2M4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over the past 60 years, egg yolk (EY) has been routinely used in both liquid semen extenders and those used to cryopreserve sperm. However, the mechanism by which EY protects sperm during liquid storage or from freezing damage is unknown. Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane by inducing cholesterol efflux. Because cholesterol efflux is time and concentration dependent, continuous exposure to seminal plasma (SP) that contains BSP proteins may be detrimental to the sperm membrane, which may adversely affect the ability of sperm to be preserved. In this article, we show that the BSP proteins bind to the low-density fraction (LDF), a lipoprotein component of the EY extender. The binding is rapid, specific, saturable, and stable even after freeze-thawing of semen. Furthermore, LDF has a very high capacity for BSP protein binding. The binding of BSP proteins to LDF may prevent their detrimental effect on sperm membrane, and this may be crucial for sperm storage. Thus, we propose that the sequestration of BSP proteins of SP by LDF may represent the major mechanism of sperm protection by EY.

gamete biology, male reproductive tract, seminal vesicles, sperm, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cryopreservation of bull semen [1, 2] represents one of the most important achievements in dairy farming after the introduction of artificial insemination. These two approaches have enabled the worldwide distribution and use of desired genetic lines at a reasonable cost. Over the past 60 years, the cryoprotective media for sperm storage have been continuously revised, but the basic ingredients of the media remain unchanged. The egg yolk (EY) and glycerol represent the indispensable compounds of practically all media used for bull sperm preservation in liquid or frozen states. It is also clear that the interaction between the sperm and surrounding medium is a crucial factor affecting the preservation of sperm integrity and fertilizing ability.

The role of glycerol in cryopreservation is that it contributes to sperm integrity conservation [3, 4]; however, the protection afforded by EY is more complex. The EY has been shown to increase the sperm fertilizing ability when present in extenders for semen storage at ambient temperature [5–8] and appears to prevent sperm cell damage at cooling and freezing [9–11]. Various components of EY have been investigated to identify the most active component(s) responsible for the protective effect [11–19]. Evidence indicates that the low-density lipoprotein fraction (LDF), characterized biochemically by Banaszac et al. [20] and Kuksis [21], shows the highest protective ability; however, the mechanism by which this protection is provided to sperm remains elusive. It is speculated that the LDF associates with sperm membranes and provides protection against membrane damage, but there is contradictory evidence concerning the stability of this association [12, 22–24]. Vishwanath et al. [25] suggest that EY lipoproteins compete with detrimental seminal plasma (SP) cationic peptides (<5 kDa) in binding to the sperm membrane and thus protect the sperm.

Seminal plasma, which facilitates the transport of sperm in the female genital tract, also contains factors that influence sperm motility [26] and fertility [27, 28]. In addition, SP also appears to be detrimental for sperm storage [29–31]. Our studies indicate that the major protein fraction (50–70% or 35–50 mg/ml) of bovine seminal plasma is represented by a family of related proteins, designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins) [32–34]. The biochemical characteristics of BSP proteins have been well described [34, 35]. They bind to sperm membrane choline phospholipids at ejaculation [36, 37]. They also bind to capacitation factors such as high-density lipoprotein (HDL) and heparin [34, 38, 39], and the BSP proteins potentiate sperm capacitation induced by HDL and heparin [40, 41]. Thus, BSP proteins are beneficial for sperm function. In contrast, our recent studies also show that the BSP proteins induce changes in sperm plasma membrane by stimulating cholesterol and phospholipid efflux [42, 43]. This lipid efflux by BSP proteins is time and concentration dependent. Continuous exposure of sperm to SP that contains BSP proteins is detrimental to the sperm membrane, and this may render the membrane very sensitive to sperm storage in liquid or frozen states (cryopreservation). Therefore, BSP proteins in SP have the potential to act as both beneficial and detrimental factors to sperm depending on the concentration of SP and exposure time.

In the present study, we show that the BSP proteins, the major proteins of SP, interact with the LDF, the major component of EY extender. We discuss how this interaction could protect sperm and influence sperm storage.


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

BSA (fraction V), myoglobin, and lactoperoxidase were from Sigma (St. Louis, MO). Na125I was bought from Amersham (Oakville, ON, Canada). Anti-rabbit gamma globulin (goat anti-RGG) was from Medicorp Inc. (Montréal, PQ, Canada). Sephadex G-25, Sepharose CL-4B, lysozyme (egg white), and the low molecular weight (LMW) electrophoresis calibration kit were from Pharmacia Biotech Inc. (Baie d'Urfé, PQ, Canada). Acrylamide, bisacrylamide, SDS, and other electrophoresis products were obtained from Bio-Rad (Mississauga, ON, Canada). Polyethylene glycol (PEG) was from ICN Biomedicals, Inc. (Cleveland, OH). Centriflo CF-25 cones and PM-10 and YM-3 ultrafiltration membranes were from Millipore (Bedford, MA). The immobilon-P membrane and enhanced chemiluminescence (ECL) reagent kit were purchased from Mandel Scientific (Boston, MA). Tween-20 (enzyme grade) and EDTA were from Fisher Scientific (Nepean, ON, Canada). All other chemicals used were of analytical grade and were obtained from commercial suppliers.

Bull semen collected with an artificial vagina, EY Tris-glycerol (EYTG) extender (20% EY, 6% glycerol, 200 mM Tris, pH 6.7), and semen cryopreserved in EYTG extender were obtained from the Centre d'Insémination Artificielle du Québec Inc. (St. Hyacinthe, PQ, Canada). Crude seminal plasma proteins (cBSP) were prepared by ethanol precipitation of bovine seminal plasma followed by lyophilization. This preparation consists of 50–70% BSP proteins. The purification of the BSP-A1/-A2, -A3, and -30-kDa was done as described previously [33], and their purity was assessed by SDS-PAGE [44].

Preparation of BSP Protein-Depleted Seminal Plasma

To prepare the BSP protein-depleted bovine seminal plasma, 100 mg of cBSP was dissolved in 5 ml of 50 mM phosphate-buffered saline (PBS) and loaded onto a gelatin-agarose column (2.5 x 27 cm). The column was washed with the same buffer, and the unbound material (designated dBSP), consisting of all seminal plasma proteins but depleted with the BSP proteins, was dialyzed against ammonium bicarbonate and was lyophilized. This fraction generally contains less than 1% of the BSP proteins. The gelatin-agarose adsorbed proteins (designated GA-BSP) were eluted with 7 M urea, dialyzed against ammonium bicarbonate, and lyophilized. This fraction consists of a mixture of the BSP-A1/-A2, -A3, and -30-kDa proteins in the proportion (~8:1:1) that is generally present in the SP.

Preparation of Seminal Plasma

Fresh semen was diluted with nine volumes of 50 mM PBS, pH 7.4, and centrifuged in a microcentrifuge (Eppendorf, model 5415C) at low speed (5000 x g, 5 min). The supernatants were transferred into Eppendorf tubes, recentrifuged (10 000 x g, 10 min) to eliminate the remaining cells, and stored at -20°C until further analysis of SP proteins.

Isolation of Egg Yolk Lipoprotein

Egg yolk was separated from egg white and any adhering albumin was removed by blotting on filter paper. The yolk membrane was broken and the liquid yolk was collected. The liquid yolk was diluted 10-fold with 10 mM Tris-HCl (pH 7.4), the density was raised to 1.21 g/ml by adding solid potassium bromide, and the substance transferred into 11.5-ml Quick Seal tubes (Mandel Scientific Co., Guelph, ON, Canada). The tubes were centrifuged (Sorvall Ultracentrifuge; Rotor T-865) for 20 h at 60 000 rpm at 20°C. After centrifugation, the lipoproteins (designated low-density fraction, LDF) concentrated at the top of the tube were retrieved. The fraction was extensively dialyzed against 10 mM Tris-HCl (pH 7.4) and preserved at 4°C. The LDF was also isolated from 10x diluted (in 10 mM Tris-HCl buffer, pH 7.4) EYTG extender used routinely for cryopreservation of bull sperm by the same ultracentrifugation procedure. The protein concentration in LDF was determined by the modified Lowry procedure [45].

Agarose-Gel Electrophoresis

The interaction between LDF and BSP proteins or other proteins was studied using the Paragon electrophoresis kit (Beckman Instruments, Fullerton, CA). Lipo gels (0.5% agarose) and SPE (serum protein electrophoresis) gels (1% agarose) were used for lipoprotein and protein analysis, respectively. LDF was incubated in the presence or absence of cBSP or purified BSP proteins or other control proteins (BSA, myoglobin, lysozyme, heat-denatured BSP-A1/-A2, dBSP). After 3 min, 3–4 µl of incubation mixture were applied to each template slot. Agarose gels were subjected to electrophoresis for 30 min at 100 V, then immersed in fixative solution and dried. The LDF was visualized by lipid staining in Sudan Black B solution. The protein bands were revealed by staining in Paragon Blue solution.

Gel Filtration Chromatography

Gel filtration chromatography was carried out on a Sepharose CL-4B column (70 x 2.5 cm) equilibrated with PBS at a flow rate of 80 ml/h. After a 40-min wait, fractions of 3 ml were collected and the absorbance was determined at 280 nm. Elution profiles of SP proteins, purified BSP-A1/-A2, and dBSP were analyzed before and after incubation with LDF. The column was calibrated using blue dextran (Mr 2 x 106), thyroglobulin (Mr 669 000), BSA (Mr 66 000), and soy bean trypsin inhibitor (Mr 20 000).

Immunoblot Analysis of BSP Proteins

The chromatography fractions under elution profile corresponding to peaks a, b, and c (Fig. 5) were pooled separately and concentrated using Centriflo CF-25, PM-10, and YM-3 membranes, respectively. Aliquots of each concentrated fraction were delipidated using n-butanol/di-isopropyl ether (15:85 v/v) solvent. The proteins were separated on SDS polyacrylamide gels and were transferred onto Immobilon-P membrane according to the method of Towbin et al. [46]. Immunodetection was done with specific polyclonal antibodies against each BSP protein as described previously [35, 47] by using an ECL reagent kit.



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  		FIG. 5. Interaction of SP proteins with LDF. Solutions of 1.1 mg LDF protein, 2.2 mg total protein of SP, and mixture of LDF (1.1 mg) and SP (2.2 mg) in a final volume of l ml were prepared and subjected to chromatography on a Sepharose CL-4B column. The elution position of BD (blue dextran), TG (thyroglobulin), BSA (bovine serum albumin), and SBTI (soy bean trypsin inhibitor) are indicated. The data shown are representative of five experiments performed separately

Radioimmunoassay of BSP Proteins

The total content of each BSP protein in peaks a, b, and c (Fig. 5) were determined by radioimmunoassay (RIA) as described recently [47]. All reagents for RIA were diluted in the immunoassay buffer (50 mM phosphate buffer, pH 7.4, containing 5 mM EDTA, 0.45% NaCl, 0.25 mg/ml sodium azide, 0.5 ml/l Tween-20, and 0.1% BSA). The primary antibodies were used in the following dilutions: 1:1000 for anti-BSP-A1/-A2 and anti-BSP-30-kDa and 1:10 000 for anti-BSP-A3. After overnight (20-h) incubation, 50 µl of secondary antibodies (10%) were added, followed by another incubation for 12–16 h. At the end of the second incubation, 500 µl of 10% PEG-8000 were added to each tube (except total counts) and vortexed, and tubes were centrifuged at 2200 x g for 15 min. The supernatant was aspirated and the radioactivity associated with the pellet was determined in a gamma counter (1272 CliniGamma, Pharmacia Wallac, Finland).

Isolation of LDF-BSP Complex from Cryopreserved Semen

Cryopreserved semen in straws was subjected to a thawing procedure (40°C water bath, 1 min). The SP along with cryoprotective extender were separated from sperm by centrifugation at 3000 x g for 10 min and were then recentrifuged at 10 000 x g for 10 min to remove any remaining cell debris. The supernatant was subjected to ultracentrifugation as described previously to separate LDF-BSP protein complex (top 2 ml, yellow fraction) and the bottom opalescent fraction (~9–10 ml). Some white precipitates settled at the bottom of the tube were also recovered as a separate fraction. The same batch of EYTG extender (control) was also centrifuged to separate top (LDF) and bottom fractions. All fractions were extensively dialyzed against 10 mM Tris-HCl (pH 7.4) and were delipidated using n-butanol/di-isopropyl ether (15:85 v/v) solvent. The protein concentration was determined in each delipidated fraction by the modified Lowry procedure [45] and adjusted to 1 mg/ml for protein separation by SDS-PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Characterization of the LDF

The LDF from the EYTG extender and the EY were obtained as described in Materials and Methods. Two fractions were obtained from both sources. The top yellow fraction contained the LDF. The bottom fraction was slightly opalescent and corresponded probably to the water-soluble fraction described earlier [12, 48]. The LDF isolated either from the EYTG extender or EY had neutral surface charge as evaluated by agarose-gel electrophoresis (see later). The size-exclusion chromatography separated the LDF into two peaks: a minor peak, a (LDF-I), and a major peak, b (LDF-II; see later).

Interaction of cBSP Proteins with LDF

First, we investigated the interaction of cBSP proteins with the LDF. A constant amount of LDF was incubated (15 min) with increasing concentrations of cBSP and the LDF was reisolated along with the associated proteins. The addition of cBSP to LDF increased the total protein content of the reisolated fraction. Ten milligrams of LDF bound ~35 mg (70%) of proteins present in 50 mg of cBSP (Fig. 1). When 150 and 250 mg of cBSP was added to the same amount of LDF, ~65 mg (43%) and ~75 mg (30%) of cBSP proteins were associated with reisolated LDF.



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  		FIG. 1. Interaction between LDF and cBSP proteins. Increasing amounts of cBSP were added to 10 mg LDF protein. The mixtures were transferred into quick-seal tubes and subjected to ultracentrifugation as described under Materials and Methods. After ultracentrifugation, the top fraction, i.e., LDF-protein complex (•) and the bottom fraction ({triangledown}) were separated, dialyzed, and analyzed for total protein content by the modified Lowry procedure [45]. The data shown are representative of two experiments performed separately

Interaction of BSP Proteins with LDF

The binding of purified BSP-A1/-A2, -A3, and -30-kDa proteins to LDF was examined by electrophoresis. The LDF alone, when subjected to agarose-gel electrophoresis, remained at the point of application (Fig. 2A, lane 2), thus indicating a neutral surface charge. However, when LDF was mixed with either cBSP (Fig. 2A, lane 3) or each of the purified BSP proteins (Fig. 2A, lanes 4–9) and then subjected to the agarose-gel electrophoresis, a significant increase in migration of LDF particles was observed, indicating association of BSP proteins with LDF. The migration of the LDF-BSP protein complex was dependent on the ratio of LDF and BSP proteins. The complex migrated faster at a 1:4 LDF:BSP protein ratio than at a 1:1 ratio (lane 4 vs. 5, 6 vs. 7, and 8 vs. 9).



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  		FIG. 2. Specificity of interaction of BSP proteins with LDF. A) Interaction of LDF with BSP proteins. LDF (5.6 µg) and cBSP (22.4 µg) or purified BSP proteins (5.6 and 22.4 µg) were incubated in 15 µl of Tris-HCl buffer, and 4 µl of each sample was applied to the lipo gel slots. The electrophoresis and lipid staining were performed as described under Materials and Methods. Lanes 1 and 10: human serum (2 µl); lane 2: LDF (1.5 µg); lane 3: LDF (1.5 µg) + cBSP (6 µg); lanes 4 and 5: LDF (1.5 µg) + BSP-A1/-A2 (1.5 and 6 µg, respectively); lanes 6 and 7: LDF (1.5 µg) + BSP-A3 (1.5 and 6 µg, respectively); lanes 8 and 9: LDF (1.5 µg) + BSP-30-kDa (1.5 and 6 µg, respectively). The amount of each protein in a 4-µl sample is shown in parentheses. B) Interaction of LDF with control proteins. LDF (4 µg) and control proteins (16–48 µg) were incubated in 16 µl of Tris-HCl buffer, and 4 µl of each sample were applied to SPE gel slots. The electrophoresis and protein staining were performed as described under Materials and Methods. Lane 1: LDF (1 µg); lanes 2–4: LDF (1 µg) with myoglobin (6 µg), lysozyme (6 µg), and BSA (6 µg), respectively; lane 5: LDF (1 µg) + heat denatured BSP-A1/-A2 (4 µg); lane 6: heat denatured BSP-A1/-A2 (4 µg); lane 7: LDF (1 µg) + GA-BSP (4 µg); lane 8: GA-BSP (4 µg); lane 9: LDF (1 µg) + dBSP (12 µg); lane 10: dBSP (12 µg). BSP-A1/-A2 proteins were denatured by holding the tube containing protein solution (2 mg/ml in barbital buffer, pH 8.6) in boiling water for 20 min. The arrowheads indicate the point of sample application

Specificity of Interaction of BSP Proteins with LDF

In order to establish the specificity of the interaction between the BSP proteins and LDF, we studied the migration pattern of LDF with several control proteins (myoglobin, lysozyme, and BSA), heat denatured BSP-A1/-A2, dBSP (BSP depleted SP), and GA-BSP (gelatin-agarose adsorbed fraction, i.e., mixture of all BSP proteins). Incubation with control proteins did not change the surface charge of LDF particles, and the LDF band remained at the point of application (Fig. 2B, lanes 1–4). All control proteins migrated away from LDF. The heat denaturation of BSP-A1/-A2 inhibited its LDF-binding ability; thus, the denatured protein and LDF migrated separately (lane 5). The dBSP also did not interact with LDF (lane 9), whereas LDF with GA-BSP (lane 7) migrated as a single band and showed an increased migration compared with the GA-BSP alone (lane 8), indicating the formation of a complex between GA-BSP and LDF.

The specificity of binding of BSP proteins to LDF was further established by size-exclusion chromatography. The LDF was resolved into two lipoprotein peaks (Fig. 3; peak a, or LDF-I, 100–130 ml; and peak b, or LDF-II, 131–250 ml). BSP-A1/-A2 alone was eluted as a single peak (270–340 ml, designated peak c). When the mixture of LDF and BSP-A1/-A2 was subjected to size-exclusion chromatography, peak c corresponding to the BSP-A1/-A2 alone disappeared, whereas the absorbance under peaks a and b of the LDF showed a considerable increase, indicating an interaction between BSP-A1/-A2 proteins and LDF. In contrast, when the mixture of LDF and dBSP proteins was analyzed by size-exclusion chromatography (Fig. 3B), the elution profile did not differ from that of LDF alone in the area under peaks a and b, whereas peak c (270–340 ml), corresponding to the dBSP, was also present, indicating that the dBSP did not bind to LDF.



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  		FIG. 3. Gel-filtration analysis of LDF interaction with BSP-A1/-A2 and dBSP. A) Interaction of LDF with BSP-A1/-A2. Solutions of 1.1 mg LDF protein, 1.1 mg BSP-A1/-A2, and a mixture of LDF (1.1 mg) and BSP-A1/-A2 (2.2 mg) in a final volume of 1 ml were prepared and subjected to chromatography on a Sepharose CL-4B column. B) Interaction of LDF with dBSP. Solutions of 1.1 mg LDF protein, 1.1 mg dBSP proteins, and a mixture of LDF (1.1 mg) and dBSP proteins (2.2 mg) in a final volume of 1 ml were prepared and subjected to chromatography on a Sepharose CL-4B column. Chromatography was performed as described under Materials and Methods. The data shown are representative of five experiments performed separately

Saturation of the Binding of BSP Proteins to LDF

In order to establish the saturation of the binding sites on LDF, we incubated a constant amount of LDF with varying concentrations of BSP-A1/-A2 (Fig. 4) and analyzed their migration on agarose gel. At LDF and BSP-A1/-A2 ratios of 1:2.5 and 1:5, only the bands corresponding to LDF-BSP-A1/-A2 complex were visualized (lanes 2 and 3). Further increases in protein ratio (1:7.5, 1:10, 1:12.5, and 1:15) resulted in the appearance of the BSP-A1/-A2 band (lanes 4–7), whereas all LDF particles migrated with the complex. We chose BSP-A1/-A2 for this and the previous (Fig. 3A) experiments because it represents almost 80% of total BSP proteins [47]. Moreover, BSP-A1/-A2, BSP-A3, and BSP-30-kDa exhibit similar binding properties and biological activity.



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  		FIG. 4. Saturation of the binding of BSP proteins to LDF. LDF (4 µg) and BSP-A1/-A2 proteins (10–60 µg) were incubated in 12 µl of Tris-HCl buffer, and 3 µl of each sample was applied to SPE-gel slots. Electrophoresis and protein staining were performed as described under Materials and Methods. Lane 1: LDF (1 µg); lanes 2–7: LDF (1 µg) + BSP-A1/-A2 protein (2.5, 5, 7.5, 10, 12.5, and 15 µg, respectively); lane 8: BSP-A1/-A2 (10 µg). The amount of each protein present in 3-µl sample is shown in parentheses. The arrowhead indicates the point of sample application

Interaction of SP Proteins with the LDF

To verify whether the LDF and BSP proteins in SP form stable complexes, the LDF was incubated with SP and subjected to size-exclusion chromatography (Fig. 5). The chromatography of SP alone showed a minor peak (volume 100–130 ml, designated peak a) and a major peak (280–360 ml, designated peak c). The chromatography of the mixture of LDF and SP resulted in a considerable decrease in the area under the major protein peak of the SP (peak c) and a considerable increase in the area under peak a and b of the LDF, indicating association of large amounts of SP proteins with LDF.

Identification and Quantification of BSP Proteins in the Chromatography Fractions

In order to confirm the specific interaction of BSP proteins with LDF, fractions under peaks a, b, and c (Fig. 5) were pooled, concentrated, and subjected to immunoblotting using the specific antibodies against the BSP-A1/-A2 (Fig. 6A), BSP-A3 (Fig. 6B), and BSP-30-kDa (Fig. 6C). The results indicated the presence of BSP-A1/-A2, BSP-A3, and BSP-30-kDa in peaks a and b derived from the LDF-SP mixture. Because the polyclonal antibodies against BSP-A1/-A2, -A3, and -30-kDa are specific [47], the bands shown with the arrows in lane 5 of Figure 6 correspond to the degradation product of the respective BSP proteins.



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  		FIG. 6. Immunoblot analysis of chromatography peaks. Aliquots (200 ng total protein) corresponding to each pooled peak (Fig. 5) were reduced, denaturated, and then subjected to SDS-PAGE. The separated proteins were transferred to Immobilon-P and probed with antibodies directed against BSP proteins. A) Anti-BSP-A1/-A2. B) Anti-BSP-A3. C) Anti-BSP-30 kDa. Lanes 1–3: Correspond to peaks a–c derived from SP; lanes 4–6: correspond to peaks a–c derived from LDF and SP mixture; lanes 7–9: correspond to peaks a–c derived from LDF. The arrowheads in A, B, and C indicate the position of BSP-A1/A2, BSP-A3, and BSP-30-kDa, respectively. Arrows in A, B, and C correspond to degraded fragments of BSP proteins

BSP-A1/-A2, BSP-A3, and BSP-30-kDa protein content in pooled fractions under peaks a, b, and c of respective chromatography (SP, LDF, and SP+LDF) were further quantified by specific RIA and expressed in percent distribution in each peak. The results (Table 1) show that ~80% of BSP proteins were present in peak c when SP alone was chromatographed, whereas ~90% of these proteins were found in peak b upon SP preincubation with LDF and followed by chromatographic separation of the mixture.


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  		TABLE 1. The distribution of BSP proteins in pooled fractions (peaks a, b, and c) obtained by gel filtration chromatography of SP, LDF, and SP+LDF.a

Stability of LDF-BSP Protein Complex Following Freeze-Thaw of the Extended Semen

An experiment was carried out to verify whether the interaction between the BSP proteins and EY-LDF was stable during semen freezing and thawing. We isolated the LDF from frozen/thawed semen and analyzed the protein pattern by SDS-PAGE. Figure 7 shows the presence of the BSP proteins in the LDF (lane 5). The bottom fraction (lane 6) and the precipitates (lane 7) contained very insignificant amounts of BSP proteins. These results indicate that the EY-LDF-BSP protein complex remains stable during the freeze/thaw procedures.



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  		FIG. 7. Analysis of proteins in LDF isolated from frozen-thawed EYTG extended semen. Fractions obtained from EYTG extender (control) and from frozen/thawed extended semen by ultracentrifugation were dialyzed and delipidated. The proteins in these fractions were separated by SDS-PAGE and stained with Coomassie Blue. Lane 1: LMW standard; lane 2: whole EY proteins; lane 3: LDF from EYTG; lane 4: bottom fraction from EYTG; lanes 5 and 6, respectively: top (LDF) and bottom fractions from frozen/thawed semen; lane 7: precipitates from frozen/thawed semen; lane 8: cBSP proteins. a, b, and c indicate the positions of BSP-30-kDa, BSP-A1/-A2, and BSP-A3, respectively


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we have shown for the first time that the LDF isolated from EY interacts with the BSP proteins, factors in SP, which are detrimental to sperm. The binding of BSP proteins to LDF is rapid, specific, and saturable and the complexes formed (LDF-BSP proteins) are stable. This interaction may be critical for sperm protection and storage in the liquid or frozen state.

The isolated EY-LDF showed similarities with those reported by earlier workers [48–50]. The chromatographic separation of the isolated LDF resulted in two lipoprotein fractions (LDF-I and LDF-II; Figs. 3 and 5). Foulkes [12] reported the separation of LDF into three lipoprotein fractions, the second fraction (lipoprotein II) eluting as the shoulder in front of the third lipoprotein fraction (lipoprotein III). The lipoprotein fractions I, II, and III differed by their lipid:protein ratio (11.7, 6.6, and 2.8, respectively), and lipoprotein III was responsible for protecting bovine sperm against damage during freezing and thawing [12]. It is possible that our separation procedure did not resolve the lipoproteins II and III and that both these lipoproteins appeared in peak b (LDF-II; Figs. 3 and 5). In addition, differences were also noted from batch to batch in the proportion of LDF-I and LDF-II (Figs. 3 and 5). These differences may be attributed to LDF isolation techniques used or the EY sources.

We investigated first the interaction between the isolated LDF and cBSP proteins. Our results (Fig. 1) show a dose-dependent binding of cBSP proteins to LDF. At a LDF:cBSP ratio of 1:5, ~70% of cBSP proteins associated with reisolated LDF. Because ~70% of cBSP proteins correspond to BSP proteins, it is logical to assume that all the protein bound to LDF may be BSP proteins. Indeed, the SDS-PAGE indicated the presence of large amounts of the BSP proteins in the reisolated fraction (data not shown).

The specific binding of the BSP proteins to LDF was established by using purified BSP-A1/-A2, -A3, and -30-kDa proteins. The LDF-BSP protein complex was analyzed by agarose-gel electrophoresis and gel filtration chromatography. Agarose-gel electrophoresis (Fig. 2A) showed an increased electrophoretic mobility after LDF incubation with each purified BSP protein. This is due to the binding of the negatively charged BSP proteins [32, 34, 35] to the surface of LDF. In contrast, the control proteins (myoglobin, lysozyme, BSA) migrated either to cathode or anode, depending on their charge, and the LDF remained at the point of application (Fig. 2B). Similarly, the heat-denatured BSP-A1/-A2 or the dBSP (BSP protein-depleted fraction of seminal plasma) did not change the mobility of LDF, indicating no association of these proteins with the LDF (Fig. 2B). In addition, during gel filtration, the BSP-A1/-A2, but not dBSP, proteins eluted at the LDF region when coincubated (Fig. 3). The specificity of interaction between LDF and the BSP proteins was further confirmed by revealing the presence of the BSP proteins in peaks a and b derived from the LDF and SP chromatography (Fig. 6). The evaluation of BSP proteins and their percentages in each chromatographic peak (Table 1) of SP alone and LDF along with SP indicate that peak b of the LDF fraction bound most of the BSP proteins.

A higher mobility of particles noted in the presence of BSP-A1/-A2 may indicate a higher negative charge associated with BSP-A1/-A2 than with either BSP-A3 or BSP-30-kDa (Fig. 2A). Furthermore, the LDF-BSP protein complex had a higher negative charge than the purified BSP protein alone (Fig. 4, lane 8). This suggests changes in conformation of BSP proteins upon binding to LDF.

Our previous studies show that the BSP proteins bind specifically to choline phospholipid liposomes [36] and this binding occurs in less than a second [51]. In the current study, agarose-gel electrophoresis and gel filtration analysis were performed immediately after mixing BSP proteins with LDF, and in all instances, complex was observed. Therefore, it is reasonable to conclude that the LDF association with BSP proteins is rapid. Because the LDF-BSP complex is stable during ultracentrifugation, electrophoresis, and gel filtration, it is likely that the binding constant is in the µM range (high affinity binding).

The binding of the BSP proteins to the LDF is saturable. Based on the data shown in Figure 1, at saturation, 10 mg of LDF appeared to bind ~70 mg of cBSP proteins. The saturation of the BSP proteins binding to the LDF was also demonstrated by analyzing the complex at various ratios of LDF:BSP-A1/-A2 protein (Fig. 4). The saturation of LDF binding sites occurred between LDF:BSP-A1/-A2 at ratio of 1:5 and 1:7.5. This value is similar to that established by ultracentrifugation (i.e., 1:7 ratio).

The binding capacity of the LDF appears to be very high. LDF-I and LDF-II have an average Mr of 1.4 x 106 Da and 0.6 x 106 Da, respectively, as determined by gel filtration (calibrated with proteins of known molecular weight). BSP-A1/-A2 (Mr 16 500 Da), -A3 (Mr 15 500 Da), and -30-kDa (Mr 28 000 Da) proteins are present in an approximate ratio of 8:1:1 in SP [33, 47]. Assuming an average Mr of BSP proteins in SP to be 18 000, at saturation (1:7 ratio), nearly 555 and 243 moles of the BSP proteins can bind to a mole of LDF-I and LDF-II, respectively. The high capacity binding of LDF is important for sequestration of large amounts of BSP proteins present in semen. The average concentration of BSP proteins in semen is 35–50 mg/ml [47] and of LDF in 20% egg yolk is 10–15 mg/ml (current study). Semen is normally diluted 10 times and higher with 20% EY-containing medium (EYTG extender) prior to cryopreservation. At this dilution, most of the BSP proteins may be associated with LDF.

In the artificial insemination industry, semen is diluted with EYTG extender within minutes after collection, cooled to 4°C, packed in straws, frozen, and stored in liquid nitrogen. The LDF isolated from frozen-thawed semen also contained BSP proteins (Fig. 7). This indicates that the LDF-BSP protein complex remains stable even after semen cooling, freezing, and preservation in liquid nitrogen and after thawing.

In view of our discovery that the BSP proteins specifically bind to LDF and form a stable complex, we suggest a novel mechanism of sperm protection by EY lipoproteins (Fig. 8). Our previous studies have shown that the BSP proteins are added to sperm at ejaculation [34]. The BSP proteins coat the sperm membrane [36, 37] and induce cholesterol [42] and phospholipid efflux [43]. The lipid efflux is time and concentration dependent [42, 43]. At higher concentrations of the BSP proteins (i.e., SP) and/or at longer exposure (as in nondiluted semen), more cholesterol and phospholipids are removed. The removal of lipids, particularly cholesterol, results in sperm membrane destabilization. Evidence shows that the decrease in cholesterol content in plasma membrane also appears to decrease sperm resistance to cold shock and freezing [52, 53]. Therefore, prolonged exposure of sperm to SP that contains BSP proteins is deleterious to sperm. Because ejaculates are diluted with EY extenders within minutes after collection, the lipoproteins (LDF) may sequester most of the BSP proteins present in semen. This may result in a minimum modification of the sperm plasma membrane and allow better sperm storage. Thus, EY lipoproteins may offer protection to sperm by reducing the deleterious effect of SP proteins on sperm membranes. During natural mating, a mechanism may also exist to eliminate the detrimental effect of BSP proteins on sperm. After being ejaculated into the vagina, sperm swim through cervical mucus and enter the uterus within minutes. Cervical mucus acts as a barrier for SP. In the artificial insemination industry, the BSP proteins (i.e., SP proteins) are not removed from semen, but their effect is eliminated probably by the rapid formation of a stable complex with egg yolk lipoproteins.



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  		FIG. 8. The proposed mechanism of sperm protection by EY-LDF

Our model can provide an explanation for the various effects of SP. For instance, it is reported that sperm become more sensitive to cold shock and freeze-thaw following exposure to SP [7, 54, 55]. A recent report also indicates that the exposure of sperm to accessory sex gland fluid (AGF) is toxic and that rapid removal of sperm from SP or AGF is critical for maximal viability [56]. These deleterious effects on sperm could be essentially due to BSP proteins that modify the sperm membrane extensively by removing cholesterol. In ejaculates, the activity of BSP proteins on sperm membrane continues, whereas in extended semen, it is likely inhibited by EY-LDF. Consequently, extended sperm could better resist cold shock and/or freeze-thaw effects, as observed with epididymal sperm that are not exposed to SP [54].

The toxic effect of SP or AGF on sperm is concentration and time dependent [55, 56]. Studies have also suggested that the toxic effect is immediate and persists even after washing sperm [57]. Furthermore, the amount of EY required in semen extenders to provide protection against SP is proportional to the amount of SP in diluted semen [58]. The toxic effects of SP could be minimized by dilution with EY extenders within minutes of semen collection [9, 13]. All these effects can be explained with our model (Fig. 8). The modification of sperm surface (removal of cholesterol from sperm membrane or toxic effect) by BSP proteins is a concentration- and time-dependent phenomenon [42, 43]. Because of the magnitude of the BSP protein concentration (40–60 mg/ml) and the proteins' affinity to sperm membrane, the toxic effect could be caused within minutes and prolong even after washing the sperm. BSP proteins form a complex with LDF rapidly, and therefore their toxic effect on sperm could be minimized or inhibited within minutes. Because the LDF binding sites can be saturated by BSP proteins, the protection provided by the EY is proportional to its capacity to bind the BSP proteins.

The effect of egg yolk on the success of sperm storage and the mechanisms involved have been studied extensively [12, 22–25]. It is suggested that the EY lipoproteins associate with sperm membranes and shield against the toxic effect of SP. This hypothesis simply cannot explain all the effects of SP and why it requires large amounts of EY to protect sperm although a very small fraction of it may bind to sperm membrane. In fact, the specific binding of EY lipoproteins to the sperm membrane itself is controversial [22–25]. The current study suggests that the beneficial role of semen dilution with extenders containing EY is not limited to the direct binding of EY lipoproteins to the sperm plasma membrane but may involve interplay among BSP proteins (SP), EY-LDF, and the sperm membrane (i.e., how LDF and BSP association could effect the sperm membrane lipid composition). In this context, it is interesting to note that the extended washed sperm (unpublished data) and extended frozen-thawed washed sperm contain 80–85% less BSP proteins than washed ejaculated sperm [47]. This may result in less damage to sperm in extended semen than in unextended semen.

Our recent studies show that the BSP-like proteins present in stallion [59, 60], boar [60], ram, water buffalo, and human semen also bind to LDF (unpublished), suggesting that the mechanism of sperm protection by EY is similar in all these species. Furthermore, milk extender is also used for sperm storage, and it also contains lipoproteins and phospholipids. These milk components also interact with the BSP proteins (will be reported elsewhere), suggesting that the mechanism of sperm protection by milk is similar to that proposed for EY-LDF.

In summary, we have shown for the first time that the LDF isolated from EY and EYTG extenders interacts with the BSP proteins. By using a number of biochemical approaches, we conclude that this binding is rapid, specific, and saturable. In addition, LDF has a very high capacity for BSP proteins and the complex formed is stable even after freeze-thaw. BSP proteins destabilize the sperm membrane by removing cholesterol and phospholipids. However, it may be possible that this effect is abolished or minimized by the association of BSP proteins with EY lipoproteins, the major component of extenders used in sperm storage. Therefore, we suggest that the scavenging of the BSP proteins by EY lipoproteins may represent the major mechanisms of sperm protection by EY. Further studies on how LDF-BSP protein interaction affects a) BSP proteins binding to sperm membrane, b) sperm membrane lipid composition (cholesterol and phospholipid), and c) sperm functions (motility, viability, acrosomal integrity) will clarify the beneficial effects of LDF and BSP protein interaction on sperm storage. These studies may also aid in improving current protocols for sperm processing and developing better extenders for commercial application. In addition, the current discovery should aid in developing novel extenders for preservation of semen (liquid or frozen) from several farm animals, domestic animals, wild animals, and endangered species for which protocols do not exist presently.


   ACKNOWLEDGMENTS
 
We thank Dr. Gary Killian, The Pennsylvania State University, for critical review of the manuscript. We thank Dr. I. Thérien and Ms. M. Villemure for preparing figures. We appreciate the cooperation of Mr. Yves Brindle, Centre d'Insemination Artificielle du Québec Inc.


   FOOTNOTES
 
1 This work was supported by grants from the National Science and Engineering Research Council of Canada, Cattle Breeding Research Council of Canada, Boviteque Alliance, and Canadian Institute of Health Research. Back

2 Correspondence: P. Manjunath, Centre de recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 Boul. l'Assomption, Montréal, QC, Canada H1T 2M4. FAX: 514 252 3430; manjunap{at}medclin.umontreal.ca Back

Received: 8 February 2002.

First decision: 4 March 2002.

Accepted: 9 May 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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