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Regular Article |
a Centre de Recherche en Biologie de la Reproduction (CRBR),
b Département des Sciences Animales, and
c Unité d'Ontogénie et Reproduction CHUQ-CHUL, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract, sperm, sperm maturation, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The distal cauda epididymides is the site where mature spermatozoa of virtually all mammalian species are stored for several days or weeks in a viable and fertile condition before ejaculation [1, 10, 11]. Little is known about the epididymal secretions and the molecular mechanisms that allow sperm survival and preservation of sperm function during cauda epididymal storage [10, 11]. At least 3 purified proteins (>100 kDa, 67 kDa, and 35 kDa) from hamster cauda epididymal fluid were able to enhance sperm survival when hamster caudal sperm were diluted and motility was activated in vitro [12, 13]. However, the association with the sperm surface and the functional mechanism at a molecular level for these secretory epididymal proteins has not yet been determined.
The characterization of secretory epididymal proteins involved in maturation, motility, and survival of bovine spermatozoa was undertaken using bovine frozen-thawed sperm. Bovine epididymal epithelium fluid (BEEF) from cauda epididymides was able to protect sperm against oxidative damage and to enhance frozen-thawed sperm motility and survival in vitro. In addition, we have characterized a limited number of BEEF proteins that bind specifically to the sperm surface. The 2 major epididymal sperm-binding proteins were identified as the alpha and beta clusterin subunits. The possible roles of clusterin and other potential epididymal proteins acting as sperm motility-maintaining factors or as survival or protection factors were investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RPMI-1640 and retinol were obtained from ICN (Montreal, PQ, Canada), and fetal bovine serum (FBS) was obtained from Medicorp (Montreal, PQ, Canada). The antibiotic/antimycotic saline solution (0.9% NaCl solution, 10 000 IU/L penicillin, 100 mg/L streptomycin, and 250 µg/L amphotericin B), BSA, sodium bicarbonate, Hepes, sodium pyruvate, bovine insulin, bovine apo-transferrin, testosterone, dihydrotestosterone, hydrocortisone, mineral oil, Percoll, hydrogen peroxide, Tween-20, and the electrophoresis grade chemicals acrylamide, bisacrylamide, Tris(hydroxymethyl)aminomethane (TRIS), SDS, 2-mercaptoethanol, and glycerol were purchased from Sigma Chemical Company (St. Louis, MO). Nonidet P-40 was obtained from Calbiochem Corporation (La Jolla, CA), and urea and ampholytes (Biolyte pH ranges 3–10 and 5–7) were from Bio-Rad Laboratories (Hercules, CA). The bicinchoninic acid reagent (BCA), the sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin), the NeutrAvidin-horseradish peroxidase conjugate, and the luminol/enhancer reagent (SuperSignal) were purchased from Pierce Chemical Company (Rockford, IL).
Extraction of BEEF
Whole epididymides were recovered from 16 sexually mature bulls immediately after arrival at slaughterhouse. These bulls were killed because the phenotypic characteristics of F1 females did not meet dairy industry criteria of the Centre d'Insémination Artificielle de Québec (CIAQ, St. Hyacinthe, PQ, Canada). Cauda epididymides were dissected free of fat and connective tissues in an antibiotic-containing saline solution (Sigma). BEEF was recovered from distal cauda epididymides by retrograde flushing of caudal sperm with 10 ml of a prewarmed mineral oil solution. BEEF and caudal sperm were separated by centrifugation at 10 000 x g for 15 min. BEEF samples were thus extracted and processed for protein concentration and indentificatin of pH range and osmolarity. The ability of each collected BEEF sample to maintain sperm motility was assayed after a 6-h incubation period (data not shown). Ten different samples were then pooled, and 2 ml (54 mg) was concentrated by ultrafiltration through a cellulose membrane concentrator with a cutoff of 10 kDa (Amicon, Beverly, MA). The retained fraction (>10 kDa) was washed twice and concentrated 2-fold (1 ml) in PBS (20 mM phosphate, 150 mM NaCl, pH 7.4). The <10-kDa fractions were pooled, lyophilized, and reconstituted in PBS. The protein concentrations of 2 BEEF fractions were determined using the BCA reagent, with BSA as the standard. The protein samples were kept at -80°C.
Incubation Medium for Sperm Motility Assays
Washed postthawed spermatozoa were incubated in a defined culture medium, epididymal cell medium (ECM) [14], at 38.5°C in a humidified atmosphere containing 5% CO2. ECM was prepared with RPMI-1640 medium and buffered with 2.38 mM sodium bicarbonate and 10 mM Hepes. The pH was adjusted to 7.35 before filtration. ECM was supplemented with 1 mM sodium pyruvate, 100 nM bovine insulin, 5 µg/ml bovine apo-transferrin, 1 µg/ml retinol, 200 nM testosterone, 1 µM dihydrotestosterone, and 200 nM hydrocortisone before use, as previously described [14–16].
Motility of Frozen-Thawed Spermatozoa in Total and Fractionated BEEF
Cryopreserved bovine semen samples were prepared from a pool of semen from 5 bulls selected by CIAQ. The same pool was used throughout this study. Straws of cryopreserved spermatozoa were thawed in a 37°C water bath for 1 min and washed twice by centrifugation at 500 x g for 10 min in Tyrode modified medium supplemented with 6 mg/ml BSA and 1 mM pyruvate [17]. Spermatozoa were suspended in a final cell density of 200–400 x 106 spermatozoa/ml. Five BEEF samples were chosen to compare the effects of total BEEF, BSA, and FBS on sperm motility, and 15–20 x 106/ml of freshly thawed spermatozoa were first incubated for 3 and 6 h using the following concentrations: 0 (ECM control), 0.5, 1, 2, and 5 mg/ml. Fractionated BEEF samples were tested at protein concentrations of 0.25 mg/ml for the <10-kDa fraction plus 4.75 mg/ml BSA and at 2.5 mg/ml for the >10-kDa fraction plus 2.5 mg/ml BSA. Postthawed sperm were also incubated in ECM with no protein at all and in ECM containing 2.5 mg/ml and 5 mg/ml BSA as positive controls. Incubation assays were performed at 38.5°C, pH 7.4, in a humidified atmosphere containing 5% CO2. Two mixed aliquots of sperm suspension were placed on a prewarmed MicroCell counting chamber (Conception Technologies, San Diego, CA). Motion parameters were recorded using a computer-assisted sperm analyzer (CASA; Hamilton Thorn Research, Beverly, MA) after a 6-h incubation period.
Sperm Motility Analysis by CASA
Sperm motion parameters were assessed using the following parameters as previously described [14]: frames acquired, 20; frame rate, 30 Hz; minimum contrast, 7; minimum size, 5; LO/HI size gates, 0.4–1.5; LO/HI intensity gates, 0.4–1.5; nonmotile head size, 16; nonmotile brigthness, 14; threshold straighness, 60%; medium average path velocity (VAP) value, 80 µm/sec; low VAP value, 5 µm/sec; slow cells motile, no. Cells with a path velocity of <5 µm/sec were counted as nonmotile and were not included in the mean of motion parameters. All cells moving at a path velocity of 80 µm/sec and having a threshold straightness of 60% define a progressive motile sperm. These parameters differ slightly from those proposed by the manufacturer (Hamilton Thorn Research).
Nature and Stability of BEEF Active Factors
To partly characterize the active factors in fractionated BEEF, postthawed sperm were incubated in untreated and heat-treated ECM (10 min at 100°C) containing 1) no protein, 2) 5 mg/ml BSA, or 3) 2.5 mg/ml BSA plus 2.5 mg/ml BEEF >10 kDa. Sperm motility was evaluated by CASA after a 6-h incubation period. In a second set of experiments, frozen-thawed sperm were first incubated in ECM containing 1) 5 mg/ml BSA or 2) 2.5 mg/ml BSA plus 2.5 mg/ml BEEF >10 kDa for 2 h. In each experiment, the 2 samples were separated in 2 equal fractions after the 2-h incubation period. One sample from each fraction was replaced in the incubator (BSA and BEEF samples), and spermatozoa in the other sperm sample fractions were washed twice (2 min at 500 x g) in Tyrode modified medium and placed in ECM + 5 mg/ml BSA (BSA-washing-BSA and BEEF-washing-BSA samples). Motility was recorded hourly by CASA before and after washing. Another similar experiment was designed to test the ability of BEEF to protect thawed sperm against oxidative injury induced by H2O2. In this set of experiments, H2O2 was added to the medium at a final concentration of 150 µM after a 2-h preincubation period. Sperm motion parameters were recorded by CASA hourly before H2O2 addition and after 1 h, 2 h, 3 h, and 4 h incubation.
BEEF Protein Biotinylation
BEEF proteins from >10-kDa fractions were processed for biotin labeling (biot-BEEF) to identify the proteins interacting with the sperm surface. Protein biotinylation and biotinylated protein detection was performed according to the manufacturer's instructions (Pierce Chemical Co.). A >10-kDa BEEF extract (8–10 mg/ml) was previously dialyzed twice against 0.1x PBS (pH 8.0) during 48 h, lyophilized, and reconstituted with water (1x PBS). A 10-mg/ml protein sample was incubated with 2.5–3.0 µM NHS-LC-biotin at room temperature for 2 h. The biot-BEEF proteins were extracted by elution in a desalting column with PBS (pH 7.8). The protein-containing fractions were pooled and stored at 4°C until ready for use.
Electrophoretic Characterization and Specific Binding of Sperm Surface-Interacting BEEF Factors
Postthawed bovine sperm (25 x 106) were incubated for 3–5 h in ECM containing 5 mg/ml BSA and different concentrations of biot-BEEF (7, 15, and 32 µg/ml). Binding specificity of biot-BEEF proteins to the sperm surface was evaluated by incubation of thawed sperm in ECM with biot-BEEF at 30 µg/ml, followed by the addition of an excess of 0, 50-, and 150-fold unbiotinylated BEEF or FBS. Preincubated spermatozoa were washed 3 times through a two-layer Percoll gradient (45% and 60%), followed by several washes in Tyrode modified medium to eliminate unbound biotinylated proteins. Recovered spermatozoa were counted, and sperm protein samples were lysed in a one-dimensional SDS-PAGE reducing buffer (Tris buffer containing 2% SDS, 5% 2-mercaptoethanol, and 10% glycerol). Approximately 2–4 x 106 lysed spermatozoa were separated onto a denaturing 12.5% acrylamide gel according to a standard protocol of Laemmli [18]. Separated proteins were transferred onto a nitrocellulose membrane (Amersham, Piscataway, NJ) using a semidry blotting system (Millipore, Bedford, MA). Unspecific sites were blocked for a minimal period of 1 h in a blocking solution (PBS, pH 7.4, containing 5% nonfat milk) and washed twice for 5 min in PBS plus 0.1% Tween-20. Blotted membranes were incubated with NeutrAvidin-horseradish peroxidase conjugate (a modified avidin derivative) for 1 h using a 1:40 000 or a 1:10 000 dilution in PBS (pH 7.4). Chemiluminescent detection of the biot-BEEF/avidin complexes was performed using a luminol/enhancer reagent (Pierce Chemical Co.).
Isoelectrophoretic Focusing Analysis
Isoelectric focusing (IEF) was conducted under equilibrated conditions as described by Bérubé et al. [19]. Slab gels were prepared on glass tubes 14 cm in length and 1.5 mm thick (Bio-Rad Laboratories) that were filled with 4% polyacrylamide, 8 M urea, 2.5% ampholytes (0.5% pH 3–10 and 2% pH 5–7), and 2% Nonidet P-40 (Calbiochem). The gels were allowed to polymerize for at least 2 h with a 20-µl 8 M urea layer on the slab top. The anode reservoir was replenished with 25 mM H3PO4, and the cathode reservoir was replenished with 0.1 M NaOH. The 20-µl 8 M urea layer was replaced with 20 µl of denaturing IEF lysis buffer (8 M urea, 0.5% ampholytes pH 3–10, 2% ampholytes pH 5–7, 2% Nonidet P-40, and 1% 2-mercaptoethanol), and a pre-equilibration step was run gradually at 200 V for 15 min, 300 V for 30 min, and 400 V for 30 min. Protein samples (30–50 µl) were loaded on the slab's top gel, and gels were run at 400 V for 16 h. Isoelectric points were deduced directly from the pH pattern by measuring the pH of the 0.5-cm dissolved pieces of gel control in deionized water for 3 h. For two-dimensional SDS-PAGE, the slab gels were placed on the top of a 12.5% acrylamide gel (1 mm thick and 15 cm in length; Bio-Rad Laboratories) and run at 15 mA/gel for 1 h and 20–22 mA/gel for 4 h.
Identification of Sperm Surface-Interacting BEEF Factors by the Matrix-Assisted Laser Desorption Ionization Time-of-Flight Technique
Approximately 10–15 x 106 Percoll-enriched spermatozoa preincubated with biot-BEEF >10-kDa proteins and aliquots containing 150–200 µg of biot-BEEF >10-kDa proteins were firstly separated by IEF in parallel. Proteins separated by two-dimensional SDS-PAGE (12.5% acrylamide; reducing conditions: 5% v/v 2-mercaptoethanol) were transferred onto a nitrocellulose membrane using a semidry blotting system (Millipore) to identify epididymal sperm-binding proteins. The selected spots of interest were cut out from 2 gels containing 150–200 µg of biot-BEEF >10-kDa proteins and stained by the Coomasie brilliant blue technique. Knowing that selected proteins were blocked at the N-terminal side by biotin, proteins were digested with trypsin for peptide mass spectrometric analysis using the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) technique. Identification was performed by mass fingerprinting in a peptide mass computer databank (www.expasy.ch/tools) at the Service Protéomique de l'Est du Québec (St. Foy, Canada).
Statistical Analysis
Data were analyzed with the statistical analysis program Statview 4.5 (1992–1995; Abacus Concepts, Berkeley, CA). All values from sperm motion parameters were expressed as mean ± SEM. Significant difference between the treatments was determined by a one-way ANOVA using both the Fisher protected least significant difference (PLSD) test and the Scheffé test. Differences were considered significant at P < 0.05.
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RESULTS |
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The average protein content of BEEF was evaluated at 22.4 ± 1.6 mg/ml (n = 10). The pH and the osmolarity were recorded at 6.2 ± 0.2 pH units and 300 ± 15 mOsm/kg, respectively. These results were in accordance with data reported by Zaneveld [20] for bovine cauda epididymal fluid. Sperm motility from 5 different experiments, using 5 different BEEF samples, was computerized by CASA to express the results as a percentage of sperm motility. The initial motility average of freshly thawed sperm was 79% ± 5%. FBS was less efficient than was BSA at maintaining sperm motility, but both were less efficient than BEEF (Fig. 1). The positive effects of BEEF on sperm motility obtained at 0.5 and 1 mg/ml were comparable to those obtained with BSA at 1 and 2 mg/ml, respectively. The maximum beneficial effect was obtained at 2 mg/ml BEEF, greater than the maximum effect of BSA at 5 mg/ml. When thawed sperm were incubated with BEEF at 5 mg/ml, there was a dramatic decrease in the positive effects on sperm motility. Sperm motility was not restored after sperm washing or BEEF dilution in the same sample (data not shown). When frozen-thawed bovine spermatozoa were incubated with lower concentrations of total BEEF (0.5–2 mg/ml) in ECM, the average pH value and osmolarity of the medium were 7.2 ± 0.2 pH units and 275 ± 10 mOsm/kg, respectively. By the addition of total BEEF at 5 mg/ml, the pH of culture medium decreased to a value near 6.2.
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Optimal conditions to conjugate Sulfo-NHS-LC-biotin to a protein or a peptide must be achieved using a buffer without primary amine molecules. In addition, TRIS or glycine can compete with the biotinylation reaction. Subsequently, total BEEF was fractionated through a cellulose membrane with a 10-kDa cutoff and dialyzed against a PBS solution. The ability of 2 fractions to maintain sperm motility was tested. Total BEEF is approximately 92.5% >10-kDa fraction. Consequently, the following concentrations were also assayed for sperm motility: 1) 0.25 mg/ml for the <10-kDa fraction supplemented with 4.75 mg/ml BSA and 2) 2.5 mg/ml for the >10-kDa fraction supplemented with 2.5 mg/ml BSA. The percentage of motile and progressive motile spermatozoa after a 6-h incubation period was higher in postthawed bovine sperm incubated in ECM + 2.5 mg/ml BEEF >10 kDa than it was for sperm incubated in ECM without proteins or in ECM containing 2.5 or 5 mg/ml BSA (Fig. 2). The number of motile and progressive spermatozoa in the <10-kDa (containing 4.75 mg/ml BSA) fraction was similar to that for sperm incubated in 5 mg/ml BSA. Immediately after sperm washing, total and progressive motility were evaluated at 77% and 37%, respectively. After a 6-h incubation period, these parameters decreased respectively to 33% and 13% in ECM + 2.5 mg/ml BSA. However, in ECM + 2.5 mg/ml BEEF >10 kDa, total and progressive motility were 65% and 32%, respectively (Fig. 2).
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BEEF Active Factors Are Heat Sensitive, Sustain Sperm Motility, and Protect Sperm Against Oxidative Injury
High temperatures cause protein denaturation and affect enzyme activity. Thus, 2 samples from the active >10-kDa BEEF fraction were heat treated at 100°C for 10 min before incubation with thawed sperm for 6 h (Fig. 3). A negative control without protein and a positive control containing 2.5 mg/ml BSA were also included. Each experiment was performed 3 times in duplicate. Initial sperm motility was 67% ± 3% (Fig. 3). Compared with untreated samples, sperm motility decreased to 19% ± 5%, 33% ± 2%, and 48% ± 5% in ECM without proteins, ECM + 2.5 mg/ml BSA, and ECM + 2.5 mg/ml BEEF >10-kDa fraction, respectively. Sperm motility in heat-treated samples was not modified in ECM without proteins or for in ECM + BSA, respectively. However, sperm motility in heat-treated BEEF samples decreased to 33% ± 4%.
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In a second set of experiments, thawed sperm were washed twice after a 2-h preincubation period in ECM containing 2.5 mg/ml BEEF >10 kDa + 2.5 mg/ml BSA and in ECM + 5 mg/ml BSA (Fig. 4). Sperm samples were washed twice and placed in ECM + 5 mg/ml BSA (BSA-washing-BSA and BEEF-washing-BSA samples) or incubated continuously in the same medium (BSA and BEEF samples). By measuring the motion parameters hourly, it appeared that frozen-thawed sperm motility was better maintained in medium supplemented with BEEF than in medium supplemented with BSA over a 7-h incubation period. The positive BEEF effect on sperm motility was not affected even though BEEF-preincubated sperm were washed twice in Percoll and incubated in medium supplemented with BSA (BEEF-washing-BSA). Sperm motility was thus significantly higher in BEEF-washing-BSA samples than in BSA samples (BSA and BSA-washing-BSA; Fig. 4).
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Our preliminary studies showed that bovine sperm motility was negatively affected when incubated in ECM containing 50 µM H2O2. This effect was immediate (<15 min) and was not prevented by the presence of BEEF in the medium. We tested the capacity of BEEF >10 kDa (2.5 mg/ml) to protect sperm cells against the deleterious effects of 150 µM H2O2 added after a 2-h preincubation period with BEEF (Fig. 5). Sperm motility in ECM + BSA and ECM + BEEF were 48%–50% and 58%–60%, respectively, after a 1-h incubation period and 50%–52% and 59%–62%, respectively, after a 2-h incubation period (Fig. 5). Sperm motility recorded immediately after H2O2 decreased in ECM + BSA from 35% to 37% and declined progressively to 21%–24% over a 4-h incubation period. In contrast, sperm motility in BEEF + H2O2 was similar to that in ECM + BSA after H2O2 addition, was slightly decreased at 1 h after H2O2 addition, but was increased significantly and was similar to that in BEEF 2 h after H2O2 addition. Sperm motility in BEEF + H2O2 was similar to that in BEEF alone but higher than that in BSA and much higher than that in BSA + H2O2 until the end of incubation. In all sperm motility assays where BEEF was tested, sperm remained homogeneously dispersed in the medium or were easily resuspended by gentle agitation. In contrast, sperm agglutination was observed in medium containing only BSA, and vigorous agitation was needed to help sperm swim.
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Limited Number of Proteins from Sperm Preincubated with BEEF Bind Specifically to Sperm Surface
Western blot of proteins extracted from preincubated and Percoll-enriched spermatozoa were probed with NeutrAvidin-horeseradish peroxidase (Fig. 6). Under our sperm incubation conditions, reduced SDS-PAGE revealed 3 major and some minor epididymal proteins associated with the sperm plasma surface. These proteins have an apparent molecular mass of 36, 38, and 48 kDa (Fig. 6A). The density of biotinylated protein bands decreased when the same experiment was performed in the presence of an excess of 50- and 150-fold nonbiotinylated BEEF (Fig. 6B). The specific binding of biot-BEEF proteins was also addressed by incubation of sperm with the same protein excess of FBS. In this case, biot-BEEF proteins were not displaced, as noted by the constant intensity of protein bands (data not shown).
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Two-dimensional SDS-PAGE was performed in parallel with proteins extracted from spermatozoa preincubated with BEEF and total biot-BEEF protein samples. Major sperm-associated proteins were cut out of the gel, and the mass of trypsin-generated peptide fragments was measured by MALDI-TOF and screened for identification in the Swiss-Prot databank. Three single bands having an apparent mass of 36, 38, and 48 kDa and a doublet of about 50–52 kDa were screened (Fig. 7). Under reducing conditions, the 36-kDa and 38-kDa proteins were respectively identified as the beta chain and the alpha chain of bovine clusterin. The other sperm-associated protein of about 48 kDa was identified as the ß-adrenergic receptor kinase 2 (or G-protein-coupled receptor kinase 3 [GRK3]). The doublet of about 50–52 kDa was identified as 2 different proteins: bovine antithrombin-III (AT-III; 49.1 kDa, isolelectric point of 6.02) and bovine fibrinogen gamma-B chain (47.7 kDa, isolelectric point of 5.5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine spermatozoa are also protected against rapid loss of sperm motility in vitro by large proteins purified from bovine oviductal fluid [22, 23], by conditioned medium from cultured bovine oviductal cells [24, 25], and by coincubation with cultured cells from different tissues [26–28], including bovine epididymal epithelial cells [14, 16]. Because neither the nature nor the identities of the majority of the potential sperm motility-maintaining factors have been determined, it is presently impossible to estimate the extent to which we understand sperm survival at the molecular level. Within the female reproductive tract, a bovine oviductal fluid 60-kDa catalase has been suggested as playing an important role in bovine sperm survival [23, 24]. This enzyme activates the decomposition of hydrogen peroxide into water and oxygen and inhibits further sperm-membrane lipid peroxidation. In addition, this active catalase protein binds to mammalian spermatozoa and enhances sperm survival and motility in vitro [23, 24]. The identification of epididymal proteins binding to the sperm surface during sperm storage in vitro in the present study suggest a similar sperm protection mechanism.
Bovine clusterin is present along the entire length of the epididymis and in the epididymal fluid [29, 30]. Various subunit isoforms (
or ß 40 kDa, 38 kDa, and ß 36 kDa) are associated with the bovine caudal sperm membranes [29, 30]. Testicular and epididymal isoforms of bovine clusterin mediate cell aggregation and actively prevent complement-mediated sperm destruction [30]. Another study provided direct evidence that promotion of cell interactions by serum human clusterin protects porcine proximal tubular cells against oxidative damage induced by the action of exogenous and endogenous peroxide [31]. A complete abrogation of oxidative damage was found at a concentration of clusterin that only minimally increased aggregation (2.5 µg/ml). Higher concentrations (20–50 µg/ml) were needed to obtain a partial cytoprotective effect in the complete absence of cell aggregation. In our system, the protection provide by BEEF against the deleterious effects of peroxidative attack was not due to restricted access of hydrogen peroxide to critical cellular targets, because hydrogen peroxide is freely diffusible across cellular membranes. The protective effect also was not due to sperm cell aggregation per se, because in the experiments where BEEF >10 kDa (2.5 mg/ml) was challenged to protect sperm against the peroxidative membrane attack, sperm remained homogeneously dispersed in medium or were easily resuspended by gentle agitation. In contrast, sperm agglutination in medium containing only BSA was observed, and vigorous agitation was needed to allow sperm to swim. This observation suggests that some agglutination and peroxidative sperm damage were abrogated in our system. From these results, it is reasonable to propose that bovine epididymal clusterin may bind to the sperm surface and could act as an anti-peroxidation-like factor. Functionally, it will be crucial to purify the bovine epididymal clusterin monomers and develop anti-bovine clusterin antibodies to determine the effect of clusterin in a sperm protection mechanism.
Other sperm surface-interacting proteins, such as AT-III and ß-adrenergic receptor kinase 2 (or GRK3) and those that bind slightly or not at all to the sperm surface, need to be considered as beneficial factors in our system. AT-III was purified from porcine ovarian follicular fluid [32] and has been identified as a motility-stimulating protein and a chemoattractant for boar sperm [33]. In addition, AT-III was also detected on the zona pellucida of ovulated eggs flushed from swine oviducts and was proposed to play an important role in the sperm-egg interaction [34]. Other studies indicated that human sperm contains small amounts of fibrinogen-like and thrombin-like proteins [35], the tissue factor, and the factor VII (fVIIa), all known to be important in initiating blood coagulation [36]. In vitro, fibrinogen compounds reacted with thrombin to form a fibrin clot, and thrombin inhibition by AT-III was necessary to allow complete conversion of fibrinogen to fibrin [37]. A part of this process was also observed in human seminal plasma in vitro [35]. The concomitant presence of AT-III-like protein and fibrinogen-like substances in BEEF and the binding at the bovine sperm surface suggest the existence of another function for these epididymal proteins: the AT-III activity could protect membrane-sperm from inappropriate proteolytic degradation, and a system similar to that in seminal plasma could regulate sperm coagulation-like processes. Future studies will address these possibilities and are needed to identify other secretory epididymal proteins implicated in these processes.
The presence of GRK3 in BEEF, its binding to the sperm surface, and its role in the sperm protection mechanism is intriguing and remains unclear. GRK3 is a G-protein-coupled receptor, is a member of the olfactory receptor family [38], and is implicated in olfactory desensitization. The beta and alpha subunits of G-protein were detected by a Western blot analysis in the tail membranes of bovine spermatozoa [39]. GRK3 was also detected by the Western blot technique in the midpiece of mature rat sperm and was colocalized with beta arrestin 2, another olfactory desensitization receptor [40]. As proposed by Vanderghaegen et al [38], the expression pattern of these receptors in the testis and on the sperm surface is consistent with a role as a sensor for unidentified chemicals, possibly involved in the control of mammalian sperm motility and/or fertilization.
Bovine cauda epididymal fluid can be considered a complex mixture of survival or sperm-maintaining factors that can be retained in vitro and that are independent of or overlap other sperm survival mechanisms. To date, the proposed mechanisms regulating sperm protection during epididymal storage could implicate potent inhibitors of complement-mediated cell lysis [30], the enzymes involved in glutathione conjugation and metabolism that provide protection against oxidative damage [4], and potential proteolysis activity regulators such as serine- and cysteine-protease inhibitors that may protect membrane-sperm from inappropriate proteolytic degradation [3, 6–8]. Several critical questions regarding the molecular sperm protection mechanism remain to be resolved. We do not known whether any sperm-associated epididymal proteins act to maximize membrane integrity and prevent premature acrosome reaction or act as antiagglutination factors to regulate the eventual induction of capacitation and to extend sperm longevity. Determination of what other molecules are associated with the epididymal proteins identified here and of respective binding sites at the sperm membrane should provide new insights to better explain the molecular mechanism of sperm protection. We have developed a bovine epididymal cell culture system in which each potential survival factor and its molecular mechanism can be explored in vitro [14, 16].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The Natural Sciences and Engineering Research Council (NSERC)-Canada supported this work. C.R.-M. holds a postdoctoral fellowship award from the NSERC. This study was partially presented at the 33rd Annual Meeting of the Society for the Study of Reproduction (USA), at Madison, WI, July 2000. 
2 Correspondence: Marc-André Sirard, CRBR, Département des Sciences Animales, Université Laval, Sainte-Foy, PQ, Canada G1K 7P4. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca
Accepted: August 28, 2001.
Received: May 9, 2001.
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