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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022996
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BIOLOGY OF REPRODUCTION 70, 708–717 (2004)
DOI: 10.1095/biolreprod.103.022996
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Low-Density Lipoprotein Fraction from Hen's Egg Yolk Decreases the Binding of the Major Proteins of Bovine Seminal Plasma to Sperm and Prevents Lipid Efflux from the Sperm Membrane1

Annick Bergeron3, Marie-Hélène Crête3, Yves Brindle4, and Puttaswamy Manjunath2,3

Departement of Medicine,3 University of Montreal, and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Québec, Canada H1T 2M4, Centre d'Insémination Artificielle du Québec,4 Ste-Hyacinthe, Québec, Canada J2S 7B8


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For sperm preservation, semen is generally diluted with extender containing egg yolk (EY), but the mechanisms of sperm protection by EY are unclear. The major proteins of bull seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) bind to sperm surface at ejaculation and stimulate cholesterol and phospholipid efflux from the sperm membrane. Since EY low-density lipoprotein fraction (LDF) interacts specifically with BSP proteins, it is proposed that the sequestration of BSP proteins in seminal plasma by EY-LDF represents the major mechanism of sperm protection by EY. In order to gain further insight into this mechanism, we investigated the effect of seminal plasma, EY, and EY-LDF on the binding of BSP proteins to sperm and the lipid efflux from the sperm membrane. As shown by immunodetection, radioimmunoassays, and lipid analysis, when semen was incubated undiluted or diluted with control extender (without EY or EY-LDF), BSP proteins bound to sperm in a time-dependent manner, and there is a continuous cholesterol and phospholipid efflux from the sperm membrane. In contrast, when semen was diluted with extender containing EY or EY-LDF, there was 50%–80% fewer BSP proteins associated with sperm and a significant amount of lipid added to sperm membrane during incubation. In addition, sperm function analysis showed that the presence of EY or EY-LDF in the extender preserved sperm motility. These results show that LDF is the constituent of EY that prevents binding of the BSP proteins to sperm and lipid efflux from the sperm membrane and is beneficial to sperm functions during sperm preservation.

assisted reproductive technology, gamete biology, male reproductive tract, seminal vesicles, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Seminal plasma, which is mixed with epididymal sperm at ejaculation, serves as the carrier of sperm to the female genital tract and has been described as both beneficial and detrimental for sperm. More precisely, several workers have described seminal plasma factors that support sperm functions, such as motility [1] and viability and fertility [2, 3], and that facilitate capacitation [4]. However, seminal plasma is also known to contain factors that are detrimental for sperm fertilizing ability [5], such as decapacitation factor [6–8] and motility inhibiting factor [9]. In addition, seminal plasma is detrimental to sperm storage [10] because it contains factors that negatively affect sperm viability [11–13].

The major protein fraction of bovine seminal plasma is represented by a family of closely related proteins designated BSP-A1/A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins) [14, 15]. The BSP proteins represent ~65% of seminal plasma total protein, and their biochemical characteristics have been extensively described (for a review, see [16]). BSP proteins bind to sperm membrane choline phospholipids at sperm ejaculation and potentiate sperm capacitation induced by high-density lipoprotein and heparin [17, 18] by stimulating cholesterol and phospholipid efflux from the sperm membrane. Therefore, BSP proteins are beneficial to sperm. In contrast, the same BSP proteins may be deleterious for the sperm membrane in vitro. The lipid efflux stimulated by BSP proteins is time and concentration dependent [19, 20], and therefore a continuous exposure of sperm to seminal plasma that contains BSP proteins may damage the sperm membrane [16].

Hen's egg yolk (EY) is the most effective agent to protect sperm against cold shock and has been shown to improve sperm functions and preserve sperm fertility after storage in liquid [21–24] or frozen state [25–28]. Despite the use of extender containing EY for more then 60 yr, the mechanisms involved in sperm protection by EY against storage, cooling, and freezing damages remain unclear. The low-density lipoprotein fraction (LDF) of EY appears to be the constituent responsible for sperm protection against cold shock and freezing damages [29–32]. Several mechanisms of sperm protection by EY-LDF have been proposed. It is suggested that EY-LDF provides protection by associating with sperm membrane [28, 30, 31, 33, 34]. Another hypothesis is that EY-LDF prevents the loss of membrane phospholipids, thus increasing the sperm tolerance to the cold shock [35], and previous studies that indicate that EY phospholipids protect sperm from cold shock support this speculation [31, 36]. Studies from our laboratory indicate that the EY-LDF interacts specifically with the BSP proteins [37]. The binding of the BSP proteins to LDF is rapid and saturable, and the binding capacity of the LDF is very high. Furthermore, this interaction is stable even after freeze-thawing. In view of this, we have proposed that the scavenging of the BSP proteins by EY lipoproteins on dilution of semen with extender containing EY protects sperm from deleterious effects of BSP proteins present in seminal plasma.

In order to gain further insight into the mechanisms of sperm protection by EY, it is essential to study further the interplay among BSP proteins, extender constituents, and sperm. In the present study, it is hypothesized that dilution of semen with EY containing extender prevents the binding of BSP proteins to sperm and prevents a lipid efflux from sperm membrane. First, we investigated the effect of semen dilution in EY Tris-glycerol (EY-TG) extender on 1) the binding of BSP proteins to the sperm membrane, 2) cholesterol and phospholipid efflux from the sperm membrane, and 3) sperm functions (motility, viability, acrosomal integrity). The second objective of this study was to test the hypothesis that the EY-LDF is the extender constituent responsible for preventing BSP proteins from binding to sperm and stimulating sperm lipid gain during storage.


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

BSA (fraction V), lactoperoxidase, leupeptin, erythrosin B, and flavianic acid (naphthol) were from Sigma (St. Louis, MO). Anti-Rabbit immunoglobulin G (H + L), acrylamide, bisacrylamide, SDS, and other electrophoresis products were obtained from Bio-Rad (Mississauga, ON, Canada). Low-molecular-weight (LMW) electrophoresis calibration kit was from Pharmacia Biotech Inc. (Baie d'Urfé, PQ, Canada). Polyethylene glycol (PEG) was obtained from ICN Biomedicals Inc. (Cleveland, OH). Immobilon-P membrane and enhanced chemiluminescence (ECL) reagent kit were purchased from Mandel Scientific (Boston, MA). 125I was purchased from Perkin-Elmer Life Science (Boston, MA). Goat anti-rabbit gamma globulin (RGG) was from Medicorp Inc. (Montréal, PQ, Canada). All other chemicals used were analytical grade and obtained from commercial suppliers.

Freshly ejaculated semen was collected with an artificial vagina from bulls at the Centre d'Insémination Artificielle du Québec (St-Hyacinthe, PQ, Canada). Bulls were handled by qualified technicians according to the Guide for the Care and Use of Agricultural Animals established by the Ministry of Agriculture and Fisheries, Québec. EY-TG extender (200 mM Tris, pH 6.7; 20% EY, 5.6% glycerol) was added to the semen. The extender was prepared by adding fresh egg yolk to Tris-glycerol base.

Isolation of EY-LDF Lipoprotein Fraction

EY used to isolate EY-LDF was from the same batch used to prepare 20% EY extender. EY was diluted three times with 10 mM Tris-HCl (pH 7.4), and the density was raised to 1.21 mg/ml by adding solid potassium bromide and centrifuged for 20 h at 366 257 x g at 20°C as described previously [37]. The floating lipoproteins (designated low-density fraction [LDF]) were retrieved and extensively dialyzed against 10 mM Tris-HCl (pH 7.4). The volume of dialyzed LDF was adjusted with 10 mM Tris-HCl to original volume (EY used for isolation).

Preparation of Semen Samples

The same four bulls were used to conduct this study. In each experiment, three separate ejaculates from four different bulls were used. In the first experiment, one ejaculate from each bull was undiluted or immediately diluted after collection with TG (200 mM Tris, 5.6% glycerol, pH 6.7) or EY-TG (TG, 20% EY) extender to reach a sperm concentration of 80 x 106/ml as routinely done in the artificial insemination center and incubated at 37°C for 24 h. After dilution, final concentration of seminal plasma in diluted ejaculates was between 4.2% and 9.8% (v/v). In the second experiment, one ejaculate from each bull was diluted right after collection with TG, EY-TG, or LDF-TG extender to reach a sperm concentration of 80 x 106/ml and were incubated at 4°C to mimic the cooling procedure in the artificial insemination center. After dilution, final concentration of seminal plasma in diluted ejaculates was between 4.7% and 10.0% (v/v).

Final pH of the extenders was 6.7, and extenders were kept at 37°C prior to semen dilution. At 0, 1, 2, 4, 6, 8, and 24 h of incubation, samples of semen were taken of which one aliquot was used to assess sperm functions and two aliquots were used for sperm protein and lipid analysis. Time "zero" is the time at which the semen was mixed with EYTG or LDF or TG extender, and an aliquot was removed immediately and diluted with phosphate-buffered saline (PBS) for sperm washing. All samples were treated in the same manner at time zero in both experiment 1 and experiment 2. When the sample was undiluted, the time zero is considered to be the start of the incubation, which is usually within 5–6 min (time normally elapsed before mixing with extender) of semen collection. For protein and lipid analysis, semen samples were diluted (1:20) with 50 mM PBS in 15-ml plastic tubes and centrifuged at 1840 x g for 10 min. This washing procedure was repeated five times to remove extender or seminal plasma from sperm. Then the pellets were resuspended in 900 µl PBS, transferred into 1.5-ml tubes, and centrifuged at 15 800 x g for 10 min. The supernatants were discarded, and the sperm pellets were stored at -20°C until used for protein solubilization or lipid extraction.

Preparation of Protein Extracts from Sperm Membrane

The protein extracts from sperm membrane were prepared as described previously [38] and were stored at -20°C until used for sperm protein analysis. The protein contents of seminal plasma or sperm extracts were determined by the modified Lowry procedure [39].

SDS-PAGE and Immunoblot Analyses of BSP Proteins

Seminal plasma proteins or sperm membrane proteins were reduced, denatured, and separated in 15% polyacrylamide gels. For immunoblotting, the proteins in the gel were transferred on to Immobilon-P membrane as described by Towbin et al. [40], and the immunodetection was done with specific polyclonal antibodies against each BSP protein as described previously [38, 41].

Quantification of BSP Proteins on Sperm by Radioimmunoassays

Iodination of the BSP proteins was performed by the lactoperoxidase method as described previously [42]. Radioimmunoassays (RIAs) for each BSP protein were performed on sperm protein samples as described in Nauc and Manjunath [38]. Briefly, the assay tubes containing the 125I-labeled and unlabeled antigen, the primary antibodies (anti-BSP-A1/A2, anti-BSP-A3, or anti-BSP-30-kDa), and normal rabbit serum (1.5% v/v) were incubated. After 20 h, 50 µl of 10% goat anti-RGG were added, and assay tubes were incubated for 16 h. Then 500 µl of 10% polyethylene glycol were added, the antibody-antigen complex was separated by centrifugation (2200 x g, 20 min), and the radioactivity associated with the pellet was determined in a gamma counter (1272 CliniGamma, Pharmacia Wallac, Finland).

Sperm Cholesterol and Choline Phospholipid Determination

Sperm lipids were extracted from pellets kept at -20°C using chloroform/methanol as described previously [19]. After solvent evaporation under N2, the lipids were resuspended in isopropanol. The amount of cholesterol and choline phospholipids was determined following the protocol described in the cholesterol determination kit (catalog no. 139 050, Boehringer Mannheim, Roche Applied Science, Indianapolis, IN) and the choline phospholipid determination kit (catalog no. 691 844, Boehringer Mannheim), respectively.

Sperm Function Analysis

Sperm motility in each sample was assessed subjectively in duplicate by estimating the percentage of motile sperm in a drop of Tris-citrate buffer (200 mM Tris, 73 mM citric acid) on a warm slide using light microscopy. Three fields per drop were examined under 100x magnification. The viability was assessed by staining of sperm according to the protocol of Dott and Foster [43]. Acrosomal integrity was assessed by determining the percentage of sperm acrosome-intact on air-dried sperm smears stained according to a naphthol yellow-erythrosin B-staining procedure [44]. For viability and acrosomal integrity, two samples by treatments and 200 sperm by sample were assayed.

Statistical Analysis

The data for sperm functions, RIAs data, and sperm cholesterol and phospholipid loss or gain were analyzed for significant difference by ANOVA. The significant differences among treatments were determined with the protected Fisher least significant difference (LSD) test within each incubation time. A value of P < 0.05 was considered statistically significant. The data for sperm function analysis were transformed as a percentage of motility, viability, or acrosomal integrity at time 0 h (time 0 h being 100%) before analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SDS-PAGE Patterns of Sperm Protein

SDS-PAGE analyses were performed to verify whether modifications occurred in the sperm protein pattern during incubation of semen. In undiluted semen and semen diluted with TG extender and incubated at 37°C, electrophoresis of sperm protein samples corresponding to each incubation time (0, 1, 2, 4, 6, 8, and 24 h) revealed changes in protein pattern (Fig. 1, A and B, respectively; lanes 2–8). The intensity of 15–16.5-kDa and 28-kDa bands (corresponding to BSP proteins) increased during incubation of sperm to reach a maximal intensity at 24 h incubation (lane 8). In contrast, in sperm protein extracts from semen diluted with EY-TG extender (Fig. 1C), intensity of the bands corresponding to BSP proteins decreased significantly.



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  		FIG. 1. SDS-PAGE pattern of sperm proteins from semen incubated undiluted or diluted with TG or EY-TG extender and incubated at 37°C. Aliquots of sperm proteins (15 µg) from semen incubated for 0 to 24 h were reduced, denatured, and separated on 15% polyacrylamide gel and stained with Coomassie Blue R-250 dye. A) Sperm proteins from undiluted semen. B) Sperm proteins from semen diluted with TG extender. C) Sperm proteins from semen diluted with EY-TG extender. In A–C, lane 1 corresponds to LMW standard (phosphorylase b, 94 kDa; BSA, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; and {alpha}-lactalbumin, 14.4 kDa) and lanes 2–8 correspond to samples at 0, 1, 2, 4, 6, 8, and 24 h of incubation at 37°C. a, b, and c indicate the position of BSP-30-kDa, BSP-A1/A2, and BSP-A3, respectively. The experiment was performed on four ejaculates, and a representative experiment is shown

In a similar manner, in the second experiment, electrophoresis of sperm protein extracts from semen diluted with TG extender and incubated at 4°C revealed that the intensity of 15–16.5-kDa and 28-kDa bands (corresponding to BSP proteins) increased during the incubation to reach a maximal intensity at 24 h incubation (Fig. 2A, lanes 2–8). In sperm protein extracts from semen diluted with EY-TG or LDF-TG extender (Fig. 2, B and C, respectively), the intensity of the bands corresponding to BSP proteins decreased significantly.



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  		FIG. 2. SDS-PAGE pattern of sperm proteins from semen diluted with TG, EY-TG, or LDF-TG extender and incubated at 4°C. Aliquots of sperm proteins (15 µg) from semen incubated for 0 to 24 h were reduced, denatured, and separated on 15% polyacrylamide gel and stained with Coomassie Blue R-250 dye. A) Sperm proteins from semen diluted with TG extender. B) Sperm proteins from semen diluted with EY-TG extender. C) Sperm proteins from semen diluted with LDF-TG extender. In A–C, lane 1 corresponds to LMW standard (see Fig. 1). Lanes 2–8 correspond to samples at 0, 1, 2, 4, 6, 8, and 24 h of incubation, respectively. a, b, and c indicate the positions of BSP-30-kDa, BSP-A1/A2, and BSP-A3, respectively. The experiment was performed on four ejaculates, and a representative experiment is shown

Immunoblot Analysis of BSP Proteins in Sperm Protein Extract

In order to confirm SDS-PAGE results, we subjected sperm protein extracts from each incubation time (0, 1, 2, 4, 6, 8, and 24 h) to immunoblotting using antibodies against BSP-A1/A2-, BSP-A3-, and BSP-30-kDa proteins. In the first experiment, in semen incubated undiluted or diluted with TG extender, intensity of the bands corresponding to BSP-A1/A2, BSP-A3, and BSP-30-kDa (Fig. 3, A–C, respectively) at each incubation time (0, 1, 2, 4, 6, 8, and 24 h) increased in a time-dependent manner to reach maximal intensity at 24 h of incubation. In contrast, when the semen was diluted with EY-TG extender and subjected to immunoblotting, it revealed that the intensity of the bands corresponding to BSP-A1/A2 and BSP-A3 proteins at each incubation time remained the same, and the intensity of the bands corresponding to BSP-30-kDa increased slightly (Fig. 3C, lanes 6 and 7).



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  		FIG. 3. Immunoblot analysis of BSP proteins associated with sperm during incubation at 37°C of semen undiluted or diluted with TG or EY-TG extender. Aliquots of sperm protein samples (100 ng) corresponding to each incubation time (0, 1, 2, 4, 6, 8, and 24 h) in undiluted semen or semen diluted with TG or EY-TG extender were separated by SDS-PAGE. The separated proteins were transferred to Immobilon-P and probed with purified antibodies directed against BSP proteins. A) Anti-BSP-A1/A2. B) Anti-BSPA3. C) Anti-BSP-30-kDa. Lanes 1–7 correspond to 0, 1, 2, 4, 6, 8, and 24 h of incubation at 37°C. The arrowheads in A–C correspond to the position of BSP-A1/A2, BSP-A3, and BSP-30-kDa, respectively. Arrows in C correspond to BSP-30-kDa immunoreactive proteins. The experiment was performed on four ejaculates, and a representative experiment is shown.

In a similar manner, in the second experiment, intensity of the bands corresponding to BSP-A1/A2, BSP-A3, and BSP-30-kDa (Fig. 4, A–C, respectively) at each incubation time (0, 1, 2, 4, 6, 8, and 24 h) increased in a time-dependent manner to reach maximal intensity at 24 h of incubation in semen diluted with TG extender and incubated at 4°C. Moreover, when the semen was diluted with EY-TG or LDF-TG extender and subjected to immunoblotting, it revealed that the intensity of the bands corresponding to BSP-A1/A2 and BSP-A3 proteins at each incubation time seemed to be the same or slightly decreased. Immunoblotting using antibodies against BSP-30-kDa revealed that the intensity of the bands corresponding to each incubation time increased slightly (Fig. 4C).



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  		FIG. 4. Immunoblot analysis of BSP proteins associated with sperm during incubation at 4°C of semen diluted with TG, EY-TG or LDF-TG extender. Aliquots of sperm protein samples (100 ng) corresponding to each incubation time (0, 1, 2, 4, 6, 8, and 24 h) in semen diluted with TG, EY-TG, and LDF-TG extender were separated by SDS-PAGE. The separated proteins were transferred to Immobilon-P and probed with purified antibodies directed against BSP proteins. A) Anti-BSP-A1/A2. B) Anti-BSPA3. C) Anti-BSP-30-kDa. Lanes 1–7 correspond to 0, 1, 2, 4, 6, 8, and 24 h of incubation at 4°C. The arrowheads in A–C correspond to the position of BSP-A1/A2, BSP-A3, and BSP-30-kDa, respectively. Arrows in C correspond to BSP-30-kDa immunoreactive proteins. The experiment was performed on four ejaculates, and a representative experiment is shown

Amount of BSP Proteins Associated with Sperm During Incubation of Semen

To confirm the immunoblot results, the concentration of BSP proteins in sperm protein extracts was assessed by RIAs. In the first experiment, during the incubation, the quantity of BSP-A1/A2, BSP-A3, and BSP-30-kDa associated with sperm increased in a time-dependent manner in undiluted semen or semen diluted with TG extender and remained the same in semen diluted with EY-TG extender (Fig. 5, A–C, respectively). Furthermore, in semen incubated undiluted or diluted with TG extender, there is no significant difference between the amounts of each BSP protein associated with sperm. However, at the start of the incubation, a decrease in the quantity of BSP proteins associated with sperm was evident in semen diluted with EY-TG extender as compared to undiluted semen or semen diluted with TG extender, and it represented an average of 79% for BSP-A1/A2 and BSP-A3 and 55% for BSP-30-kDa. In the presence of EY, the average amount of BSP-A1/A2, BSP-A3, and BSP-30-kDa associated with sperm at the start of the incubation was 29.4 ± 7.3, 8.7 ± 3.0, and 47.1 ± 7.5 ng/106 sperm, respectively.



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  		FIG. 5. Amount of BSP proteins associated with sperm during incubation at 37°C of semen undiluted or diluted with TG or EY-TG extender. Each BSP protein was quantified by RIAs in sperm protein extracts from semen incubated 24 h at 37°C undiluted (circles) or diluted with TG (squares) or EY-TG extender (triangles). A) BSP-A1/A2. B) BSP-A3. C) BSP-30-kDa. Results represent the mean ± SEM of four ejaculates performed in triplicate. Significant differences vs. control (0 h): * P < 0.01; ** P < 0.001. Within each incubation time, amount of BSP proteins associated with sperm in undiluted semen and semen diluted with TG extender is significantly different as compared to semen diluted with EY-TG extender according to the protected least significant difference (LSD) test (P < 0.05).

In the second experiment, the quantity of BSP-A1/A2, BSP-A3, and BSP-30-kDa associated with sperm increased in a time-dependent manner during the incubation of semen diluted with TG extender and remained the same during the incubation of semen diluted with EY-TG or LDF-TG extender (Fig. 6, A–C, respectively). Moreover, there is no significant difference in the amount of BSP proteins associated with sperm in semen diluted with EY-TG or LDF-TG extender. Furthermore, at the start of the incubation, 70% less BSP-A1/A2 and 50% less BSP-A3 and BSP-30-kDa was bound to sperm in semen diluted with EY-TG or LDF-TG extender as compared to sperm incubated in TG extender. In semen diluted with TG extender, the average amount of BSP-A1/A2, BSP-A3, and BSP-30-kDa associated with sperm at the start of incubation was 102.5 ± 10.2, 11.9 ± 1.0, and 36.7 ± 4.1 ng/106 sperm, respectively.



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  		FIG. 6. Amount of BSP proteins associated with sperm during incubation at 4°C of semen diluted with TG, EY-TG or LDF-TG extender. Each BSP protein was quantified by RIAs in sperm protein extracts from semen incubated 24 h at 4°C diluted with TG (solid triangles), EY-TG (solid circles), or LDF-TG (solid squares) extender. A) BSP-A1/A. B) BSP-A3. C) BSP-30-kDa. Results represent the mean ± SEM of four ejaculates performed in duplicate. Significant differences vs. control (0 h): * P < 0.01; ** P < 0.001. Amount of BSP proteins associated with sperm diluted with TG extender is significantly different as compared to semen diluted with EY-TG and LDF-TG extender within each incubation time according to the protected least significant difference (LSD) test (P < 0.05).

Sperm Cholesterol and Choline Phospholipid Analysis

Lipid analyses were used to verify if there is a continuous cholesterol and choline phospholipid efflux from the sperm membrane caused by BSP proteins present in seminal plasma during incubation of undiluted semen or semen diluted with TG extender and if the presence of EY in the extender protects sperm against cholesterol and choline phospholipid loss. In the first experiment, the average amount of cholesterol and choline phospholipid associated with sperm at the start of the incubation was 213.1 ± 10.4 µg/109 sperm and 816.2 ± 27.8 µg/109 sperm, respectively. Right after dilution of semen with EY-TG extender, no differences were observed in the amount of cholesterol and choline phospholipids associated with sperm as compared to undiluted semen or semen diluted with TG extender. As shown in Figure 7, a gradual loss of sperm cholesterol and choline phospholipids was observed during incubation of undiluted semen and semen diluted with TG extender. After 24 h of incubation, sperm from semen incubated undiluted or diluted with TG extender lost 51.0% ± 2.1% and 35.1% ± 7.5% of their cholesterol, respectively (P < 0.001; Fig. 7A), and 42.2% ± 4.0% and 40.2% ± 7.1% of their choline phospholipids, respectively (P < 0.001; Fig. 7B). However, after 24 h of incubation, sperm diluted with EY-TG extender gained 47.9% ± 10.3% of cholesterol (P < 0.001; Fig. 7A) and 61.1% ± 9.7% of choline phospholipids (P < 0.001; Fig. 7B).



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  		FIG. 7. Sperm cholesterol and choline phospholipids loss or gain in undiluted semen or semen diluted with TG or EY-TG extender and incubated at 37°C. Fresh semen samples were collected and incubated undiluted (circles) or diluted with TG (squares) or EY-TG (triangles) extender. At 0, 1, 2, 4, 6, 8, and 24 h of incubation at 37°C, semen samples were taken, and the cholesterol and choline phospholipid amounts associated with sperm were determined as described in Materials and Methods and expressed as the loss or gain in sperm cholesterol and choline phospholipids. A) Cholesterol. B) Choline phospholipids. Results represent the mean ± SEM of four ejaculates performed in duplicate. Significant differences vs. control (0 h): * P < 0.05; ** P < 0.01; *** P < 0.001. Different letters within each incubation time denote significant differences between treatments according to the protected least significant difference (LSD) test (P < 0.05)

In the second experiment, the amount of cholesterol and choline phospholipids associated with sperm at the start of incubation was 185.9 ± 11.6 µg/109 sperm and 733.9 ± 60.7 µg/109 sperm, respectively. No significant differences were observed in the average amount of cholesterol and choline phospholipids associated with sperm in semen diluted with EY-TG or LDF-TG extender as compared to semen diluted with TG extender (control). As shown in Figure 8, a gradual loss of sperm cholesterol and choline phospholipids was observed over 8 h of incubation, and then it reached a plateau in semen diluted with TG extender. After 24 h of incubation, sperm from semen diluted with TG extender lost 15.6% ± 4.2% of their cholesterol (P < 0.01; Fig. 8A) and 12.4% ± 3.5% of their choline phospholipids (P < 0.001; Fig. 8B). However, during incubation of semen diluted with EY-TG or LDF-TG, a gradual gain of cholesterol and choline phospholipids was observed during 8 h, and then it reached a plateau. After 24 h of incubation, sperm diluted with EY-TG or LDF-TG extender gained 35.7% ± 17.1% or 21.6% ± 6.1% of cholesterol respectively (P < 0.01; Fig. 8A), and 48.1% ± 17.8% and 33.6% ± 2.6% of choline phospholipids, respectively (P < 0.01; Fig. 8B).



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  		FIG. 8. Sperm cholesterol and choline phospholipids loss or gain in semen diluted with TG, EY-TG, or LDF-TG extender and incubated at 4°C. Fresh semen samples were collected and diluted with TG (solid triangles), EY-TG (solid circles), or LDF-TG (solid squares) extender. At 0, 1, 2, 4, 6, 8, and 24 h of incubation at 4°C, semen samples were taken, and the cholesterol and choline phospholipid amounts associated with sperm were determined as described in Materials and Methods and expressed as the loss or gain of sperm cholesterol and choline phospholipids. A) Cholesterol. B) Choline phospholipids. Results represent the mean ± SEM of four ejaculates performed in duplicate. Significant differences vs. control (0 h): * P < 0.05; ** P < 0.01; *** P < 0.001. Different letters within each incubation time denote significant differences between treatments according to the protected least significant difference (LSD) test (P < 0.05)

Seminal Plasma and EY Effect on Sperm Functions

The sperm function analyses were performed in order to determine the deleterious effects of seminal plasma in undiluted semen or semen diluted with TG extender and the protective effect of the presence of EY or EY-LDF in the extender on sperm viability, motility, and acrosomal integrity during semen incubation. Table 1 shows that during the 24 h of incubation at 37°C, sperm in undiluted semen and semen diluted with TG or EY-TG extender underwent a time-related decrease of viability as compared to control (0 h) (P < 0.01). After 24 h of incubation, more sperm were alive in semen diluted with EY-TG extender as compared to undiluted semen or semen diluted with TG extender (P < 0.01). However, after 24 h of incubation, sperm from undiluted semen and semen diluted with TG or EY-TG extender were immotile. A gradual decrease in sperm motility was observed during the first 8 h of incubation of undiluted semen and semen diluted with TG or EY-TG extenders (data not shown). The acrosomal integrity remained the same during 24 h of incubation in undiluted semen or in semen diluted with TG or EY-TG extender.


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  		TABLE 1. Effect of seminal plasma, TG, and EY-TG extenders on sperm functions during 24-h of incubation at 37°C

Effect of the Presence of LDF or EY in the Extender on Sperm Functions During Incubation at 4°C

In the second experiment (Table 2), percentage of live sperm remained the same, and sperm acrosome remained intact during incubation of semen in the absence or presence of EY or LDF in the extender. However, there was a gradual decrease in motility during the first 8 h of incubation, reaching a plateau during 24 h when semen was diluted with TG or EY-TG extender (data not shown), while motility in semen diluted with LDF-TG extender remained the same. After 24 h of incubation, percentage motility was better in semen diluted with LDF-TG extender as compared to semen diluted with EY-TG extender, which was better than semen diluted with TG extender (P < 0.05).


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  		TABLE 2. Effect of TG, EY-TG, and LDF-TG extenders on sperm functions during 24 h of incubation at 4°C


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
For sperm preservation, the semen of several species is generally diluted with extender containing EY, and on dilution, sperm have fewer interactions with seminal plasma and more interactions with the surrounding extender. A previous study shows that EY-LDF binds BSP proteins, the major proteins of seminal plasma (35–50 mg/ml), and it is suggested that this interaction plays a key role in sperm protection by EY lipoproteins [37]. At ejaculation, the BSP proteins bind to sperm and stimulate cholesterol and choline phospholipid efflux from the sperm membrane. The lipid efflux is time and concentration dependent. If the ejaculate is undiluted, sperm are exposed to a high concentration of seminal plasma (BSP proteins), and there is a continuous lipid efflux that could be detrimental to sperm membrane. Since ejaculates are diluted with extender containing EY for sperm preservation, the EY-LDF may sequestrate BSP proteins and prevent them from binding to sperm and cause extensive modifications to the sperm membrane. The current study is focused on gaining more insight into this proposed mechanism of sperm protection by EY.

BSP proteins bind to choline phospholipids of the sperm membrane right after ejaculation [45, 46], but the current data show that there is a time-related increase in the BSP protein binding to sperm during the incubation of undiluted semen (Figs. 1, 3, and 5) or semen diluted (>10 times) with TG extender (Figs. 1– 6). Thus, during a continuous contact of sperm with seminal plasma (diluted or not), there is a continuous binding of BSP proteins to sperm. In contrast, the presence of EY in the extender prevented the increased binding of BSP proteins to sperm during the incubation of semen (Fig. 5), and it is EY-LDF that is responsible for this effect (Fig. 6). In addition, on dilution of semen with EY-TG or LDF-TG extenders, there is 50%–80% less BSP proteins bound to the sperm surface (Figs. 5 and 6). In a previous study, it was shown that sperm diluted with EY-TG extender and frozen-thawed contained almost 80% fewer BSP proteins than sperm from fresh ejaculates [38]. EY-LDF is the only component of EY that binds specifically the BSP proteins [37]. Thus, EY-LDF is responsible for preventing the binding of BSP proteins to sperm on dilution in extender containing EY. Since the polyclonal antibodies against BSP-A1/A2, BSP-A3, and BSP-30-kDa are specific [38], the arrows in Figures 3C and 4C may correspond to the proteolytic fragments of BSP-30-kDa. Alternatively, it is also possible that the 12–14-kDa immunoreactive bands detected in the present study (Figs. 3C and 4C) may correspond to a novel BSP-30-kDa-like protein reported recently [47].

BSP proteins are the factors in seminal plasma that stimulate lipid efflux from epidydimal sperm membrane [19, 20]. The present study shows that a continuous contact of ejaculated sperm with seminal plasma that contains BSP proteins stimulates a continuous efflux of sperm cholesterol and choline phospholipids (Fig. 7, undiluted semen). This effect was observed even after dilution of the semen ~10 times with TG extender. Thus, the dilution of semen did not prevent the effect of seminal plasma on the sperm membrane. This result corroborates a study by Parks showing that in the absence of EY in the extender, there is a decrease in sperm cholesterol and phospholipid content during incubation [48]. Interestingly, the presence of EY in the extender stimulated a sperm lipid gain during incubation, and it is EY-LDF that is responsible for this lipid gain (Fig. 8). At each incubation time, no significant differences were observed between lipid gain in semen diluted with EY-TG and LDF-TG extenders. Therefore, cholesterol and choline phospholipids from LDF are added to sperm, or whole molecules of LDF bound to sperm during incubation of semen diluted with EY-TG or LDF-TG extender. Some studies suggest that EY-LDF binding occurs when semen is stored in EY extender, and this binding is important for sperm protection [30, 49].

In the presence of EY in the extender, sperm cholesterol and choline phospholipids gain were two times less in semen incubated at 4°C (Fig. 8) than in semen incubated at 37°C (Fig. 7). This can be explained by the phase transition that occurs in biological membranes during cooling. During cooling, biological membranes become less fluid and are less susceptible for exchange of lipids. It is possible that the change in the lipid phase of the sperm membrane and the phospholipid film of LDF molecule during cooling prevents partially the exchange of lipid from LDF to sperm membrane or the binding of LDF to sperm. In a similar manner, in semen diluted with TG extender, the lipid loss from sperm membrane was two to three times less in semen incubated at 4°C (Fig. 8) than semen incubated at 37°C (Fig. 7).

The presence of EY or LDF in the extender maintained sperm motility during 24 h of incubation at 4°C (Table 2). It has been shown that sperm motility could be maintained more than 8 days when semen is diluted with EY-TG extender and cooled [50] and that EY is the extender constituent that is responsible for maintaining motility [25, 48, 51]. Furthermore, after 24 h of incubation, motility was better in semen diluted with extender containing LDF than whole EY. This can be explained by the fact that EY is known to protect sperm, but it is also known to contain factors that inhibit sperm respiration [52] and decrease motility [29], and it may be possible that those factors are not present in the LDF. Whether semen was diluted with TG or EY-TG extender, sperm viability and motility were better at 4°C (Table 2) than at 37°C (Table 1). When the semen is stored at 4°C, the metabolic rate of sperm is lowered, and this could contribute to the extension of sperm survival. Seminal plasma has been shown to be deleterious to sperm functions by several workers, and the present study supports this notion.

Extensive modification of the lipid content of the sperm membrane, such as removing cholesterol and phospholipids, can compromise the sperm ability to fertilize an oocyte. In the present study, seminal plasma stimulates continuous cholesterol and choline phospholipid efflux in undiluted semen (Fig. 7) or in semen diluted with extender containing neither EY nor LDF (Figs. 7 and 8). However, a continuous exposure of sperm to seminal plasma did not stimulate acrosome reaction (Tables 1 and 2). Furthermore, neither the cooling procedure (second experiment) nor lipid efflux from sperm membrane affected sperm acrosomal integrity during incubation of semen. Seminal plasma contains factors that prevent premature capacitation, such as decapacitation factor and acrosome stabilizing factor [5, 8]. Those factors may contribute to maintaining acrosomal integrity during incubation of undiluted or semen diluted with TG extender. However, some studies indicate that premature capacitation of sperm occurs when extended semen is cooled and incubated at 4°C [53, 54]. In the current study, the capacitation status of sperm was not investigated.

In summary, the current studies revealed that sperm contact with seminal plasma results in gradual loss of choline phospholipids and cholesterol from the sperm plasma membrane. This effect was persistent even when semen was diluted 10 times. Interestingly, when semen was diluted with EY, the opposite effect was observed with sperm (cholesterol and phospholipid gain and decreased association of BSP proteins to the sperm surface). Since the same effect was mimicked by the LDF, it is obvious that it is the LDF that is responsible for this effect. Moreover, the prevention of BSP binding to sperm and lipid efflux from sperm membrane resulted in the increase in semen quality (motility). In view of the present study, we suggest that the EY in extender protects sperm in two ways. First, the association of LDF with BSP proteins protects sperm by preventing BSP proteins in seminal plasma to bind to sperm and intrinsically damage sperm membrane by removing lipid. Second, the lipid from LDF or the whole molecule of LDF could associate with the sperm membrane and preserve the integrity of the plasma membrane during sperm preservation.


   FOOTNOTES
 
1 This work was supported by a grant from the Canadian Institute of Health Research. Back

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

Received: 5 September 2003.

First decision: 17 September 2003.

Accepted: 27 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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