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Biology of Reproduction 63, 1058-1066 (2000)
© 2000 Society for the Study of Reproduction, Inc.

ARTICLES

Radioimmunoassays for Bull Seminal Plasma Proteins (BSP-A1/-A2, BSP-A3, and BSP-30-Kilodaltons), and Their Quantification in Seminal Plasma and Sperm1

Veronica Nauca, and Puttaswamy Manjunath2,a

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three proteins, BSP-A1/-A2, BSP-A3, and BSP-30 kilodaltons (collectively called BSP proteins), represent the major proteins of bovine seminal plasma (BSP). At ejaculation, these proteins bind to the sperm surface and induce molecular changes in the plasma membrane that are deemed to be essential for sperm capacitation. The present study was carried out to develop specific radioimmunoassays (RIAs) for the quantification of each of the BSP proteins in BSP and sperm. RIAs were developed using polyclonal antibodies raised in rabbits against each BSP protein. The purified and iodinated BSP proteins were used as standard and tracer, respectively. The RIAs that were developed were shown to be specific for each protein and the cross-reactivity toward various antigens was negligible (<2%). The average sensitivity limit was 5 ng/ml of sample for BSP-A1/-A2 and BSP-A3, and 40 ng/ml of sample for BSP-30-kDa. The concentration of BSP proteins was determined by analyzing the RIA data with spline function. BSP proteins represented 40% to 57% of seminal plasma total protein (25% to 47% of BSP-A1/-A2, 3% to 5% of BSP-A3, and 3% to 7% of BSP-30 kDa) and 4% to 6% of sperm total protein (2.5% to 4% of BSP-A1/-A2, 0.4% to 0.9% of BSP-A3, and 0.5% to 1% of BSP-30-kDa). We also determined the concentration of BSP proteins that were sperm-bound after semen cryopreservation in Tris-egg yolk-glycerol extender. A significant decrease (70%–80%) in sperm-bound BSP proteins was noted after cryopreservation. The availability of reliable RIA procedures should aid in the further understanding of the role of BSP proteins in sperm function as well as their effect on sperm cryopreservation.

seminal vesicles, sperm capacitation/acrosome reaction, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of genetic markers to identify bulls of high breeding value represents one of the ways of genetic gain achievement in dairy farming. Several studies have been performed in an attempt to uncover the relationship between sperm quality and fertility [1–3]. Thus, sperm morphology and motility, the number of sperm per insemination, percentage of acrosome-reacted sperm, and in vitro fertilization have been extensively evaluated as an indication of sperm ability to fertilize an egg. Evidence suggests that seminal plasma, which is a complex mixture of secretions originating from the testis, epididymis, and accessory glands, contains factors that modulate the fertilizing ability of sperm [4–7]. Seminal plasma factors may also have an influence on sperm storage; however, their role in storage remains controversial [8]. There is evidence revealing that seminal plasma prevents premature capacitation of sperm [9] and protects sperm from peroxidative damage [10–12]. However, seminal plasma has also been shown to have a deleterious effect on bovine sperm during semen storage at ambient temperatures [5, 6] and a damaging effect during semen cooling and freezing [13, 14].

Recent studies have shown that proteins from BSP may modulate sperm properties [15, 16]. Killian et al. [17] have shown that two proteins (26 kDa, pI 6.2; 55 kDa, pI 4.5) predominate in higher-fertility bulls, and two proteins (16 kDa, pI 4.1; 16 kDa, pI 6.7) predominate in lower-fertility bulls. The 26-kDa fertility-associated protein has been identified as lipocalin-type prostaglandin D synthase and the 55-kDa protein has been identified as osteopontin [18, 19]. Another group of seminal plasma proteins associated with sperm fertility has been isolated on the basis of their heparin binding property [20]. This group represents five classes of heparin-binding proteins (HBPs) with molecular weights ranging from 14 to 31 kDa [21]. The average concentration of total HBP represents 19.2 mg and 0.14 mg per ml of ejaculate in bovine seminal plasma and sperm membrane, respectively [16]. Although all HBPs may bind to sperm surfaces [22], the concentration of the 30-kDa HBP, named fertility-associated antigen (FAA), on bovine sperm has been paired with a greater fertility potential [23]. FAA represents 0.8% of total HBP and has been identified as a deoxyribonuclease I-like protein [24].

The major protein fraction of bovine seminal plasma is represented by three acidic proteins, designated as BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins) [25]. These proteins are secretory products of the seminal vesicles and ampullae [26–29], and their biochemical characteristics have been well-described [30]. The BSP-A1/-A2 and BSP-A3 have molecular weights of 15–16.5 kDa, pI 4.7–5.2, whereas the BSP-30-kDa protein has a molecular weight of 28–30 kDa, pI 3.9–4.6 [31]. All three BSP proteins bind to sperm at ejaculation [30, 32] via their interaction with the choline phospholipids of the sperm membrane [33]. These proteins also bind to heparin, a glycosaminoglycan that has been implicated in bovine sperm capacitation, and to apolipoprotein A-I in either the free form or associated with high-density lipoprotein (HDL) [30, 34, 35]. Our studies have shown that these BSP proteins accelerate the capacitation of bovine epididymal sperm induced by heparin and HDL [36, 37]. It is proposed that these proteins promote heparin-induced capacitation by increasing the number of heparin binding sites on the sperm surface [21, 32, 36, 38].

More recent studies have shown that these BSP proteins facilitate capacitation by promoting cholesterol efflux from sperm membranes [39]. Because sperm membrane cholesterol has an important role in modulating membrane bilayer fluidity and stability [40, 41], its efflux may perturb membrane structure and thereby lead to capacitation [42–44]. In the context of sperm cryopreservation, cholesterol efflux may lead to a decrease in sperm resistance to cold shock [45, 46]. Thus, changes induced by the BSP proteins in the sperm membrane may have influence on sperm fertilizing ability and the success of the cryopreservation process.

To better understand the role of the major proteins of BSP in postejaculation events related to sperm membrane stability and sperm fertility and storage, we have developed radioimmunoassays (RIAs) for each BSP protein. The RIAs have allowed the assessment of the concentration of these BSP proteins in seminal plasma, ejaculated sperm, and frozen-thawed sperm.


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

BSA (fraction V), lactoperoxidase, and leupeptin were from Sigma Chemical Company (St. Louis, MO). 125I was purchased from Amersham (Oakville, ON, Canada). Goat anti-rabbit gamma globulin (goat anti-RGG) was from Medicorp Inc. (Montréal, PQ, Canada). Pansorbin cells were from Calbiochem Corporation (La Jolla, CA). Sephadex G-25, protein A-sepharose, and a low molecular weight (LMW) electrophoresis calibration kit were from Pharmacia, Biotech Inc. (Baie d'Urfé, PQ, Canada). Phenylmethylsulfonyl fluoride (PMSF), polyethylene glycol (PEG), acrylamide, bis acrylamide, and other electrophoresis products were obtained from ICN Biomedicals, Inc. (Cleveland, OH). Immobilon-P membranes and an enhanced chemiluminescence (ECL) reagent kit were purchased from Mandel Scientific (Boston, MA). Benzamidine hydrochloride hydrate was from Aldrich Chemical Company (Milwaukee, WI). Tween-20 (enzyme grade) and EDTA were from Fisher Scientific (Nepean, ON, Canada). SDS and Triton X-100 were from Bio-Rad (Mississauga, ON, Canada). L-1-Chlor-3-(4-tosylamido)-7-amino-2-heptanol-hydrochloride (TLCK) was from Boehringer Mannheim (Laval, PQ, Canada). All other chemicals used were of analytical grade and obtained from commercial suppliers.

Semen samples were obtained from Holstein bulls using an artificial vagina at the Centre d'Insémination Artificielle du Québec Inc. (CIAQ, Inc., St. Hyacinthe, PQ, Canada). After collection, each semen sample was divided into two portions. The first fractions were used for the assessment of BSP proteins in seminal plasma and sperm, the second portion of ejaculates was extended in a Tris-egg yolk-glycerol (TYG) medium and cryopreserved using 0.25 ml French straws (20 x 106 sperm per straw).

Preparation of BSP Proteins

BSP-A1/-A2, BSP-A3, and BSP-30-kDa proteins of BSP were isolated using gelatin-agarose affinity chromatography as described earlier [27]. The adsorbed fractions were resolved on Sephadex G-75 and G-200 columns under the conditions described earlier [26]. The BSP proteins isolated by this procedure were lipid-free. The purity of the proteins was assessed by SDS-PAGE [47].

Preparation of Antibodies and Immunoblot Analysis

The polyclonal antibodies against the BSP proteins were raised in male rabbits as described previously [48, 49]. The antibodies were then purified by affinity chromatography on a protein A-sepharose column and their specificity was assessed by immunoblot analysis. For this purpose, the denatured and reduced BSP proteins were subjected to SDS-PAGE and the separated proteins were transferred onto Immobilon-P membranes [50]. Immunodetection was achieved as described previously [51] using an ECL reagent kit.

Preparation of Seminal Plasma and Sperm

Fresh semen was diluted with 50 mM PBS (1:9) and centrifuged in a microcentrifuge at 5000 x g for 10 min. The supernatants were transferred into 1.5-ml tubes, recentrifuged to eliminate the remaining cells, and stored at -20°C for further analysis of seminal plasma proteins. All seminal plasma samples were diluted (1/200 000 for BSP-A1/-A2 and 1/25 000 for BSP-A3 or BSP-30-kDa proteins) in RIA buffer prior to the evaluation of BSP proteins.

The pellets from the first centrifugation were resuspended in 50 mM PBS and washed three times at 5000 x g, and the proteins in the sperm membranes were solubilized as described later.

Cryopreserved semen samples were subjected to a thawing procedure (40°C water bath, 1 min) and centrifuged at 3000 x g for 10 min to remove seminal plasma and cryoprotective extender. The pellets were resuspended in 50 mM PBS and washed three times at 5000 x g, and the sperm membrane proteins were solubilized as described in the next section.

Preparation of Sperm Membrane Protein Extract

The sperm pellets that were obtained from fresh and cryopreserved semen (see earlier) were suspended in 100 µl TETN250 (Tris 25 mM pH 7.5, 5 mM EDTA, 250 mM NaCl, 1% Triton X-100) with the addition of 5 mM benzamidine hydrochloride, 1 mM TLCK, 100 µM PMSF, pepstatin (3 µg/ml), leupeptin (5 µg/ml), sonicated for 10 sec at 50 W, and incubated for 15 min at room temperature. The cells were centrifuged at 10 000 x g for 10 min to remove the cellular debris. The supernatants containing the protein extract were stored at -20°C until use for protein estimation and RIA.

Protein Determination

The total protein concentration in both seminal plasma samples and solubilized sperm protein extracts was determined by the modified Lowry procedure [52], using BSA as a standard.

Iodination Procedure

The iodination of the BSP proteins was performed by the lactoperoxidase method as described previously [53]. Briefly, 50 µl of 1.63 mg/ml EDTA, 0.5 mCi of 125I, 50 µl of 100 µg/ml lactoperoxidase, and 100 µl of 0.0003% H2O2 were added to the reaction tube containing the respective BSP protein (5 µg in 25 µl phosphate buffer). The contents in the reaction tube were mixed and the reaction was stopped at the end of 5 min by adding 70 µl of buffer A (50 mM phosphate buffer pH 7.4 containing 0.1% BSA and 0.2% sodium azide). The reaction mixture was chromatographed on a Sephadex G-25 column (25 x 1 cm) using buffer A to separate the labeled BSP proteins from free 125I. Fractions of 1 ml were collected and the radioactivity in 5-µl aliquots of each fraction was determined in a gamma counter (1272 CliniGamma, Pharmacia Wallac, Finland). The fractions under the first peak, corresponding to the iodinated protein, were pooled, aliquoted (100 µl), and frozen at -80°C until use.

The purity of the iodinated proteins was assessed by SDS-PAGE [47]. After electrophoresis, the gel was dried for 1 h in a Gel-Slab Drier (Model 224; Bio-Rad) and then exposed to a Fuji Medical x-ray film to detect the iodinated protein bands.

Determination of Antibody Concentration

Titration of each of the anti-BSP protein antibodies was performed in preliminary experiments. For each antibody dilution, two zero tubes and two tubes with sufficient standard to cause a decrease in binding were used. The data obtained provided three important measures that could be plotted against antibody dilution. First, the difference in signal with or without standard ({Delta}b); second, the standard deviation (SD) in the measurement of the zero standard at the antibody dilution; and last, the ratio, {Delta}b/SD. The antibody concentration chosen was the one that produced the lowest {Delta}b/SD value.

Choice of the Antigen-Antibody Complex Precipitation Method

Two precipitation methods were compared. The first one consisted of the use of Pansorbin cells (Staphylococcus aureus cells) as precipitating agent in the immunoassay. In the second method, the antigen-antibody complex was precipitated using second antibodies (goat anti-RGG).

Radioimmunoassay Procedure

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 assay tubes containing the labeled (40 000–50 000 cpm) and unlabeled antigens and the primary antibodies in a total volume of 0.4 ml were incubated overnight (20 h, 37°C in humidified chamber). After 20 h of incubation, 50 µl of 10% goat anti-RGG was added to each tube (except to the total count tubes). The tubes were further incubated for 12–16 h and 500 µl of 10% PEG were added to each assay tube (except to the total count tubes). The assay tubes were vortexed and the antibody-antigen complex was separated by centrifugation (2200 x g, 15 min). The supernatant was aspirated and the radioactivity associated with each pellet was determined in a gamma counter.

A spline function was used to generate a standard curve for each BSP protein and to assess the concentration of these proteins in individual samples. Tubes of total counts, nonspecific binding, and total binding also accompanied each assay. The reference BSP proteins used for the standard curve was measured by weighing freeze-dried highly purified proteins on a Cahn microbalance (Model C-31; Fisher Scientific, Fairlawn, NJ).

Data Analysis

Data were analyzed for significant differences by a Student's t-test on paired observations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Native and Iodinated Antigens

Each purified BSP protein was found to be homogeneous as judged by SDS-PAGE under reducing conditions (Fig. 1). Electrophoresis revealed a doublet at 16–16.5 kDa for BSP-A1/-A2 (lane 3), a single band at 15 kDa for BSP-A3 (lane 4), and a single band at 28 kDa for BSP-30 kDa (lane 5). After establishing the purity of the proteins, each BSP protein was subjected to iodination (Fig. 2). The specific activities of the radiolabeled proteins (first peak) were between 85 and 140 µCi/µg protein. Each radiolabeled protein was shown to have a molecular weight similar to the native BSP protein and no interprotein contamination was observed (Fig. 3).



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  		FIG. 1. SDS-PAGE of purified BSP proteins. Purified proteins (5 µg) were reduced, denaturated, and separated on 15% polyacrylamide gel and stained with Coomassie Blue R-250 dye. Lane 1, 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). Lane 2, cBSP. Lane 3, BSP-A1/-A2. Lane 4, BSP-A3. Lane 5, BSP-30 kDa



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  		FIG. 2. Chromatography of the iodinated BSP proteins on a Sephadex G-25 column. The iodinated mix was loaded on the column (25 x 1 cm), which was equilibrated and eluted with 50 mM PBS containing 0.1% BSA and 0.2% sodium azide, pH 7.4. Fractions of 1 ml were collected at a flow rate of 1 ml/min. The eluted iodinated proteins (first peak) were pooled and preserved at -80°C



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  		FIG. 3. SDS-PAGE of iodinated BSP proteins. The radiolabeled proteins (50 000 cpm) were reduced, denaturated, and separated on a 15% polyacrylamide gel. The gel was dried and then exposed to x-ray film for 3 h at room temperature. Lane 1, 125I-BSP-A1/-A2. Lane 2, 125I-BSP-A3. Lane 3, 125I-BSP-30 kDa

Specificity of Antigen-Antibody Interaction

Immunoblotting was performed to verify the specificity of the antigen-antibody interaction. A doublet was detected by anti-BSP-A1/-A2 in the alcohol precipitates of crude (cBSP) and purified BSP-A1/-A2 (Fig. 4A). Anti-BSP-A3 and anti-BSP-30-kDa recognized only the BSP-A3 and the BSP-30-kDa proteins, respectively (Fig. 4, B and C).



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  		FIG. 4. Immunoblotting of BSP proteins. Proteins were separated by SDS-PAGE, transferred to Immobilon-P, and probed with purified antibodies directed against the BSP proteins. A) Anti-BSP-A1/-A2; B) anti-BSP-A3; C) anti-BSP-30 kDa. Lane 1, cBSP. Lane 2, BSP-A1/-A2. Lane 3, BSP-A3. Lane 4, BSP-30 kDa

The specificity of the polyclonal antibodies for the native and iodinated antigens was further tested by RIA (Fig. 5). The antibodies raised against the BSP-A1/-A2 bound to BSP-A1/-A2 protein and a dose-dependent displacement curve was obtained. The percentage of BSP-A3 and BSP-30-kDa cross-reactivity with anti-BSP-A1/-A2 was less than 2% as calculated at 50% displacement (Table 1). The antibodies raised against the BSP-A3 and BSP-30-kDa did not show any cross-reactivity with any protein other than the relevant antigens (Table 1).



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  		FIG. 5. Immunochemical interrelationship of BSP-A1/-A2, BSP-A3, and BSP-30 kDa. A) 125I-BSP-A1/-A2 (approximately 45 000 cpm). Samples were incubated with anti-BSP-A1/-A2 at a final dilution of 1:4000 in a total volume of 0.4 ml at 37°C for 20–24 h. The bound and free proteins were separated by the addition of second antibody. B) Experimental conditions were similar except that labeled protein and antibody were 125I-BSP-A3 and anti-BSP-A3 (1:40 000 dilution), respectively. C) Experimental conditions were as in A except that the labeled protein and antibody were 125I-BSP-30 kDa and anti-BSP-30 kDa (1:4000 dilution), respectively. BSP-A1/-A2 ({diamondsuit}), BSP-A3 (+), and BSP-30-kDa (X)


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  		TABLE 1. Cross-reactivity among BSP proteins (established by RIA)

Radioimmunoassay Validation

The workable range of the standard curve was 0.4 to 500 ng/tube with a sensitivity of 5 ng/ml for BSP-A1/-A2 and BSP-A3, and 40 ng/ml for BSP-30-kDa. The intra-assay variation assessed as the coefficient of variation of duplicate assay samples was equal to 12.3% for BSP-A1/-A2, 8.4% for BSP-A3, and 4.4% for BSP-30-kDa. To assess the interassay variation, a solution of ethanol-precipitated BSP (cBSP) was prepared and added to multiple RIAs. The calculation of the standard deviation as a percentage of the mean value was taken as the interassay variation coefficient and represented 16.9% for BSP-A1/-A2, 14% for BSP-A3, and 10.1% for BSP-30-kDa.

An improved assay specificity was achieved by using the second antibody precipitation method compared with precipitation with Pansorbin cells. Thus, nonspecific binding expressed as a percent of total counts was reduced by 1.4 ± 0.2 (P = 0.09), 1.9 ± 1.2 (P = 0.3), and 2.6 ± 0.6 (P = 0.02) times for BSP-A1/-A2, BSP-A3, and BSP-30-kDa, respectively. The addition of 10% PEG prior to separation of the bound fraction resulted in an increase in pellet strength. Dilutions of BSP showed good parallelism with the standard curve of each purified BSP protein (data not shown). The recovery of BSP proteins from cBSP was 110%.

Quantification of BSP Proteins in Bovine Seminal Plasma

The concentration of each BSP protein was evaluated by respective RIAs in the seminal plasma of 25 individual bull ejaculates. The results (Table 2) indicate that BSP proteins represent 31.4–46.7 mg/ml in seminal plasma where the total protein fraction was 73.5–93 mg/ml. The variation in the concentration of total proteins and BSP proteins was also observed in split ejaculates from the same bull (compare ejaculates 2 and 3 or 4 and 5 of each bull). The major protein of BSP was BSP-A1/-A2. The average ratio of BSP-A1/-A2, BSP-A3, and BSP-30-kDa was 10:1:1, respectively.


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  		TABLE 2. Concentration of total protein and BSP proteins in bovine seminal plasma.a

Quantification of BSP Proteins in Ejaculated Sperm Membranes

The total protein and concentrations of each of the individual BSP proteins were assessed in detergent extracts of washed sperm. The results (Table 3) show that all three BSP proteins were present in the sperm membranes even after several washings and represented 80–135 µg, whereas the total protein fraction was 1.8–2.6 mg in membranes obtained from 1 ml of semen. The average percentage of BSP proteins versus the total protein fraction was 5% and their ratio was 4:1:1 for BSP-A1/-A2, BSP-A3, and BSP-30-kDa, respectively.


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  		TABLE 3. Concentration of total protein and BSP proteins in sperm membranes from 1 ml of bovine ejaculates.a

Quantification of BSP Proteins in Frozen-Thawed Sperm

The total protein and contents of BSP proteins in frozen-thawed washed sperm membrane extracts were also assessed. The results (Table 4) indicated that BSP proteins represented 1–1.43 µg, whereas the total protein fraction was 118–143 µg in sperm membranes obtained from 1 ml of extended semen. Thus, BSP proteins constituted an average of 1% of the total protein fraction after semen cryopreservation. The average ratio of BSP proteins in frozen/thawed sperm was 3:1:1 for BSP-A1/-A2, BSP-A3, and BSP-30-kDa, respectively.


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  		TABLE 4. Concentration of total protein fraction and BSP proteins in sperm membranes from 1 ml of frozen-thawed bovine semen.a

To compare the concentration of sperm-bound BSP proteins before and after semen cryopreservation, we recalculated the concentrations of each of the three proteins per 106 sperm for all samples (Fig. 6). A decrease in sperm-bound BSP proteins was noted in freeze-thawed sperm samples and represented an average of 84%, 79%, and 74% for BSP-A1/-A2, BSP-A3, and BSP-30-kDa, respectively. The concentration of total protein did not show significant changes after semen cryopreservation. An average decrease of 10% was noted in the total protein fraction when estimated for all 25 samples.



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  		FIG. 6. Cryoinduced changes in sperm bound proteins. Each BSP protein and total protein (TP, inset) were assessed prior to and after semen cryopreservation. Values are expressed as mean ± SEM of five different ejaculates per bull. * P < 0.05 compared with the concentration of respective protein in fresh sperm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sperm surface properties are continuously altered following ejaculation by the removal or addition of a variety of protein and lipid constituents, which play important roles in sperm capacitation [54–56]. Because BSP proteins represent the major protein fraction of BSP and interact with sperm membrane phospholipids upon ejaculation [33], we hypothesized that variations in their concentration may influence sperm fertilizing ability and storage success. For this reason, we were interested in developing a sensitive method for the quantitative assessment of each BSP protein. RIA was chosen considering its reliability due to the very high specificity of the immune reaction combined with the high sensitivity of radioisotope detection. The development of the RIA was possible because of the purity of the isolated BSP proteins (Fig. 1). The enzymatic iodination using the lactoperoxidase method in the presence of a trace of hydrogen peroxide did not oxidize BSP proteins, thus allowing preparation of a nondamaged tracer for each BSP protein (Fig. 3). The specificity of polyclonal antibodies against each BSP protein was confirmed by immunoblotting (Fig. 4). The immunoreactivity of both native and iodinated antigens was revealed by RIA. The cross-reactivity of antigens was evaluated by RIA (Fig. 5), which showed negligible (<2%) recognition of BSP-A3 and BSP-30-kDa by the anti-BSP-A1/-A2 (Table 1). The other two antibodies did not cross-react with any protein other than relevant antigens (Table 1). Other parameters such as dilutions of primary and secondary antibodies, incubation time, temperature, and assay buffer were standardized to increase the assay sensitivity.

In the current study, we assessed the concentration as well as the distribution of BSP proteins in bovine seminal plasma and in sperm (fresh and frozen-thawed) membrane protein extracts. The evaluation of BSP proteins in seminal plasma samples showed that these three proteins constituted an average 47% of the total protein fraction (Table 2). The concentration of BSP-A1/-A2 was much higher compared with other seminal plasma proteins, and this protein alone represented an average of 38% of the total protein fraction, whereas BSP-A3 and BSP-30-kDa represented 3% to 4% of the total protein fraction. In the next experiment, we evaluated the concentration of BSP proteins that bind strongly to ejaculated sperm membranes. Several washings were performed in order to remove all traces of seminal plasma proteins that do not bind to sperm membranes. Even after three washings, approximately 5% of the total protein on sperm surfaces corresponded to BSP proteins. These results confirm previous studies [33, 51] in which we have shown that BSP proteins bind to sperm membrane phospholipids. More recent studies by Müller et al. [57] also indicate the incorporation of BSP proteins into sperm membranes.

Our study revealed that all semen samples contain BSP proteins in seminal plasma and also associated with sperm. In addition, availability of the specific RIAs allowed us to detect a variation in the concentration of BSP proteins in different bull ejaculates (Tables 2 and 3). This variation was not limited to different males but was also observed among the ejaculates of the same bull (Tables 2 and 3). Because BSP proteins bind to sperm membranes and potentiate membrane cholesterol efflux and sperm capacitation [32, 36–39, 58], the observed variations in their concentration suggest that these proteins may influence bull fertility. Any correlation of fertility with the concentration of BSP proteins or each BSP protein could not be established in the current study because the number of bulls used in the study was limited to only five animals. To establish any such correlation would require screening several hundred ejaculates, particularly because the differences in the fertility index (expressed as nonreturn rate) among Holstein bulls is 67% ± 5%.

Although several studies explain the role of BSP proteins in bull sperm capacitation [21, 36–39], their role in semen conservation remains elusive. As stated earlier, BSP proteins initiate cholesterol efflux from sperm membranes immediately after ejaculation, and this could trigger a decreased stability of ejaculated sperm to a series of stress factors. On the other hand, BSP proteins were shown to inhibit phospholipase A2 [59, 60], and their presence on the sperm surface is believed to decrease the susceptibility of sperm to capacitate in the absence of capacitation factors [36]. Thus, we became interested in comparing the concentration of sperm-bound BSP proteins on individual bull spermatozoa prior to and after semen cryopreservation. The data illustrated in Figure 6 reveal that there are changes in the sperm surface proteins after semen cryopreservation. Whereas there was an average decrease of 10% in the total protein content after cryopreservation, there was a dramatic decrease (70%–80%) of each of the BSP proteins (Fig. 6). Further work is underway to determine the factors that are involved in the removal of sperm-bound BSP proteins during semen cryopreservation.

Taking into account the properties of BSP proteins, the observed decrease in their concentration on sperm surface could indicate that modifications do indeed occur in sperm membranes during cryopreservation. The induced modifications may further reduce membrane barrier properties and may lead to a premature capacitation of thawed sperm, a phenomenon reported in a series of previous studies [61–67].

In conclusion, the use of highly sensitive and specific RIAs for the measurement of each BSP protein in both seminal plasma and sperm opens new possibilities into the investigation of the role of these proteins in postejaculation maturation of sperm and their effect on cryopreservation. At the same time, the determination of BSP protein content on sperm surface may be an index of individual bull fertilizing ability or post-thaw status of sperm membranes.


   ACKNOWLEDGMENTS
 
The collaboration of Mr. Yves Brindle (CIAQ) and Mr. Tom Kroetsch (Gencor) is highly appreciated. The authors thank Dr. K. Roberts for proofreading the manuscript.


   FOOTNOTES
 
First decision: 7 March 2000.

1 This work was supported by grants from the National Science and Engineering Research Council of Canada and the Cattle Breeding Research Council of Canada. Back

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

Accepted: May 16, 2000.

Received: January 26, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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