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Female Reproductive Tract |
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, female reproductive tract, oviduct, seminal vesicles, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In four species studied to date (hamsters [7], horses [8], cattle [9, 10], and pigs [11, 12]), binding of sperm to oviductal epithelium was found to be carbohydrate mediated in a species-specific manner. For example, binding of hamster sperm to its homologous oviductal epithelium is mediated by sialic acid [7], whereas binding of stallion sperm is mediated by galactose [8]. In cattle, there is a protein on sperm that recognizes and binds a fucosylated ligand on the oviductal epithelium [9, 10]. Recently, Green et al. [11] demonstrated that the binding of boar sperm to oviductal epithelium was competitively inhibited by maltose, lactose, and mannose.
Suarez [13] postulated that substances secreted into the oviduct fluid just prior to ovulation promote sperm release by stimulating capacitation and hyperactivation. Capacitated sperm lose binding affinity for oviductal epithelium [14, 15]; therefore, alterations in the plasma membrane associated with capacitation may result in a loss or modification of the carbohydrate-binding protein responsible for binding sperm to the epithelium. Bull sperm lose affinity for fucose after capacitation in vitro [16, 17]. Hyperactivation may play a role in enabling sperm to detach from oviductal epithelium as they lose binding affinity. DeMott and Suarez [18] observed that only hyperactivated mouse sperm pulled off of the wall of the oviduct.
We isolated a fucose-binding protein from bull sperm using affinity chromatography and identified it as PDC-109 (
rotein with N-terminus aspartic acid, 
, and carboxy terminus 
ystine, having 
amino acids), based on amino acid sequencing [17]. PDC-109 is one of three major heparin-binding acidic proteins in bovine seminal plasma: bovine seminal protein (BSP)-A1/A2 (PDC-109), BSP-A3, and BSP 30 [19]. PDC-109 is a mixture of BSP-A1 and BSP-A2, which differ only in glycosylation [20]. All are secreted by seminal vesicles. At a seminal plasma concentration of 15–20 mg/ml, PDC-109 is the most abundant of the BSPs [21].
Upon ejaculation, PDC-109 associates with epididymal sperm by binding to choline phospholipids [22–25], specifically those in the outer leaflet of the plasma membrane [26]. Anti-PDC-109 antibody labels fixed ejaculated sperm over the head and sometimes the midpiece [27]. Incubation of sperm with bovine follicular fluid and high-density lipoproteins to capacitate them resulted in a loss of PDC-109, as detected by Western blots [28].
Because sperm do not acquire PDC-109 until ejaculation, we undertook this study to determine whether bovine epididymal sperm can bind to oviductal epithelium and if not whether seminal plasma PDC-109 enables them to do so. We also tested seminal plasma PDC-109 to determine whether it could restore the ability of capacitated sperm to bind to oviductal epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Routine laboratory chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.
A modified Tyrode balanced salt solution, Tyrode albumin lactate pyruvate (TALP), was used for semen dilution, sperm incubation, and oviductal explant preparation and incubation. TALP consisted of 99 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.39 mM NaH2PO4, 10 mM Hepes, 2 mM CaCl2, 1.1 mM MgCl2, 25.4 mM sodium lactate, 0.11 mg/ml sodium pyruvate, 6 mg/ml BSA (Fraction V; Calbiochem, La Jolla, CA), and 5 µg/ml gentamycin (pH 7.4, 290 mOsm).
Hepes balanced salts (HBS) solution was prepared by adding 25 mM Hepes to 130 mM NaCl, 5 mM KCl, 0.36 mM NaH2PO4, 0.49 mM MgCl2, and 2.4 mM CaCl2 (pH 7.4, 290 mOsm).
Isolation and Purification of PDC-109 (BSP-A1/A2)
Semen collected from bulls at Genex/CRI (Ithaca, NY) was transported to the laboratory and supplemented with a serine and cysteine protease inhibitor cocktail used at the manufacturer's recommended concentrations (Complete EDTA-free; Roche Molecular Biochemicals, Indianapolis, IN). Seminal plasma was obtained by centrifugation (3000 x g for 15 min) and filtration (0.2-µm cellulose acetate) to remove sperm and particulate debris, assayed for protein content using the DC Protein Assay Kit (BioRad, Hercules, CA), and stored at -20°C.
PDC-109 was isolated from seminal plasma according to the method of Gasset et al. [24]. Aliquots of seminal plasma containing 50–100 mg protein were applied to heparin-Sepharose CL-4B columns (1 x 20 cm) equilibrated with binding buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 7.4). Following binding and extensive washing, heparin-binding proteins were eluted with binding buffer containing 10 mM o-phosphorylcholine. Eluates were concentrated, dialyzed against 20 mM Tris-HCl (pH 6.5) in 1 M NaCl, and applied to diethylaminoethyl-Sephadex equilibrated with the same buffer. Binding and washing were followed by elution of PDC-109 with 10 mM o-phosphorylcholine in column buffer. Eluate fractions were pooled, concentrated, and dialyzed against PBS, water, or HBS, depending upon subsequent applications. Purity of PDC-109 was assessed by SDS-PAGE with silver staining [29].
Purified BSP-A3 was obtained from Dr. Puttaswamy Manjunath (University of Montreal and Guy Bernier Research Center, Montreal, PQ, Canada).
Antibody Production
Two Flemish Giant Chinchilla-cross rabbits were injected subcutaneously with 100 µg PDC-109 in Freund complete adjuvant immediately after blood was collected to obtain preimmune serum. Second and third injections (150 µg and 50 µg, respectively) were administered in incomplete adjuvant, as were final boosts (150 µg) 14 days prior to collection of the last blood sample [30]. Antibody titers were determined by ELISA using PDC-109-coated plates and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG. The IgG fraction of immune serum was purified by protein A-affinity chromatography (Immunopure Plus (A); Pierce, Rockford, IL) following the manufacturer's protocol. Specificity and cross-reactivity of antibodies were determined by Western blotting with purified PDC-109, BSP-A3, and unfractionated seminal plasma. Serum and purified IgG were stored as aliquots at -80°C until needed.
Rabbit polyclonal antiserum against BSP-A3 was kindly provided by Dr. Puttaswamy Manjunath [31].
Gel Electrophoresis and Western Blotting
SDS-PAGE of seminal plasma, sperm extracts, PDC-109, and BSP-A3 was performed according to the method of Laemmli [32]. For extraction, both ejaculated and epididymal sperm were washed three times by 10-fold dilution with protein-free HBS (10 min at 170 x g) and then treated with 1.5% SDS for 30 min at room temperature. Sperm were removed by centrifugation at 13 000 x g for 15 min, and proteins were recovered from supernatants by precipitation with 10% trichloroacetic acid on ice for 30 min. Precipitates were washed with ethanol, air dried, solubilized in 1.5% SDS, and assayed for protein content (DC Protein Assay Kit). Gels were stained with silver nitrate [29] or electrophoretically transferred to polyvinylidene fluoride membranes [33]. Immunodetection was performed on blots blocked for 3 h with 3% BSA in PBS. Blots were probed for 2 h with anti-PDC-109 IgG (1 µg/ml) or with anti-BSP-A3 antiserum (1:5000) in Tris-buffered saline containing 0.05% Tween-20, washed, and incubated with HRP-conjugated goat anti-rabbit IgG (1:200) for 1 h. Reactive proteins were detected by enhanced chemiluminescence (Super Signal; Pierce), and images were recorded on radiographic film.
Preparation of Epididymal Sperm
Testes with attached epididymides and vasa deferentia were obtained from an abattoir (Taylor Packing Co., Wyalusing, PA) and transported to the laboratory on ice. The vas deferens was dissected free of connective tissue and uncoiled. A blunt 25-ga needle was inserted into the lumen, and approximately 5 ml of TALP was infused into the vas deferens and caudal epididymis. A cut was made in the cauda, and epididymal sperm were flushed into a 15-ml polypropylene tube, washed twice by centrifugation in 5 ml TALP for 10 min (170 x g), and resuspended in TALP at 5 x 106 cells/ml. Only samples with motility >85% were used.
Preparation of Ejaculated Sperm
Bull semen was provided by Genex/CRI, was diluted 5-fold in TALP immediately after collection, and was transported to the laboratory in a 37°C warm water jacket. Within 60 min of collection, sperm were washed thrice in 5 ml TALP (170 x g for 10 min), resuspended in TALP at 5 x 106 cells/ml, and incubated at 39°C and 5% CO2 in water-saturated air until assayed. Only samples with motility >85% were used.
Immunofluorescent Labeling of Sperm
Ejaculated or epididymal sperm were prepared as described above. Sperm treated with PDC-109 were washed twice by centrifugation through warmed BSA-free TALP and then fixed with 1% (w/v) paraformaldehyde in PBS for 15 min at room temperature. Sperm were diluted 20-fold in 0.2 M glycine to stop fixation and then pelleted by centrifugation. Sperm were resuspended in PBS, air dried onto slides, and blocked with 3% (w/v) BSA in PBS. Ejaculated sperm were first incubated with an anti-PDC-109 primary antibody or with preimmune serum (control), followed by a fluorescein-conjugated goat anti-rabbit IgG secondary antibody. Epididymal sperm were stained directly with fluorescein-conjugated anti-PDC-109 antibody.
Preparation of Oviductal Explants
Bovine oviducts were collected at an abattoir (Taylor Packing) and transported to the laboratory in PBS containing 6 mM penicillin and 5 mM streptomycin in containers on ice. Oviducts taken from cows in various cycle stages were used, because stage had no effect on sperm binding in previous studies [1]. Oviducts from four cows were pooled for each replicate. Explants of oviductal epithelium were prepared as described previously [9, 16]. The isthmic portion of the oviduct was dissected free of connective tissue and rinsed in PBS. The epithelium was extruded in sheets by squeezing the oviduct with fine tweezers, fragmented by pipeting, centrifuged for 1 min (170 x g), transferred to TALP, and allowed a minimum of 30 min incubation at 39°C and 5% CO2 to form everted vesicles with apical ciliated surfaces oriented outwardly. Explants were used within 6 h of slaughter.
Sperm-Binding Assays
Oviductal explants were centrifuged (170 x g for 1 min in 5 ml TALP), and 10 µl of the pellet was added to 50 µl TALP. Ejaculated or epididymal sperm (5 x 106/ml) were then added in 20-µl aliquots. After a 15-min incubation at 39°C in 5% CO2, loosely bound sperm were removed from explants by pipetting through three 75-µl droplets of TALP. The explants were transferred to a 35- x 10-mm Petri dish and covered with prewarmed silicon oil to prevent dehydration during observation.
Explants were videotaped on a 39°C microscope stage using a Zeiss Axiovert Microscope (Carl Zeiss, Thornbrook, NY). Videotaping was performed using a Dage CCD-72 black-and-white video camera (Dage-MTI, Michigan City, IN) in combination with a Panasonic AG-7300 Super-VHS videocassette recorder (Panasonic Industrial Co., Secaucus, NJ) and a time/date recorder (For-A Corporation of America, Los Angeles, CA). At least eight microscope fields of each treatment group were recorded and assessed for sperm-binding density.
Determination of Binding Density
To determine the density of sperm bound to explants, the video recordings were reviewed to count the number of sperm bound to each explant. A video image of each explant was digitized using a Power Macintosh 7100/Av (Apple Computers, Cupertino, CA), and the surface area was determined by employing National Institutes of Health Image (internet-based freeware at http://rsb.info.nih.gov/nih-image/). The binding density was calculated by determining the number of sperm bound per 0.1 mm2 of explant surface. Approximately 12.3 x 104 ± 2.0 x 104 mm2 (mean ± SD) of explant surface were analyzed per treatment for each experiment performed.
Binding of Epididymal Sperm
Epididymal sperm were treated with either 250 µg/ml PDC-109 or TALP diluent (with 6 mg/ml BSA) for 20 min at 39°C and then washed by centrifugation for 12 min (100 x g) to remove unbound PDC-109. The sperm pellet was resuspended in TALP to a concentration of 5 x 106 cells/ml and added to explants. Ejaculated sperm were used as a positive control for sperm binding. As described above, sperm/explant complexes were transferred through three TALP droplets to remove loosely bound sperm and then placed under silicon oil for video recording. The experiment was repeated four times, each time using sperm from a different bull and explants pooled from four cows.
Competitive Inhibition of Ejaculated Sperm Binding
To determine whether excess unbound PDC-109 could competitively inhibit sperm binding to epithelium, washed ejaculated sperm were added to oviductal explants that had been pre-treated for 20 min with 100 µl of varying concentrations of PDC-109 (250 µg/ml, 125 µg/ml, 62.5 µg/ml, or buffer alone). Density of bound sperm was determined as described above. The experiment was repeated three times, each time using sperm from a different bull and explants pooled from four cows.
Restoring Binding in Capacitated Sperm
To demonstrate that PDC-109 could restore the binding of capacitated sperm to explants, sperm were capacitated in TALP + 10 µg/ml heparin at 39°C for 4 h [34]. Uncapacitated controls were incubated for 4 h in TALP without heparin. Because heparin agglutinates sperm, 20 µg/ml protamine sulfate was added at the end of the incubation to deagglutinate the sperm. Sperm were washed by microcentrifugation (100 x g for 15 min) to remove heparin and protamine sulfate, resuspended in TALP, incubated with 240 µg/ml PDC-109 or TALP diluent for 20 min, washed in TALP to remove unbound PDC-109, and tested for their capacity to bind to explants. BSA was present at 6 mg/ml in all treatments. The experiment was repeated three times, each time using sperm from a different bull and explants pooled from four cows.
Statistical Analysis
All data are expressed as mean ± SEM. The data were analyzed using ANOVA followed by a Tukey honestly significant difference pairwise comparison test. Dose dependence was analyzed using linear regression. Software used was Minitab (State College, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because PDC-109 is secreted by the seminal vesicles, sperm should not be exposed to it until they come in contact with seminal plasma. Accordingly, anti-PDC-109 did not label any proteins extracted from epididymal sperm (Fig. 1B, lane 2) nor did it label fixed epididymal sperm (Fig. 3A). However, following treatment with 250 µg/ml PDC-109, epididymal sperm were labeled brightly over the acrosomal region (Fig. 3C).
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Anti-PDC-109 antibody would not label live sperm. To demonstrate that PDC-109 binds to the external surface of the plasma membrane, we conjugated fluorescein directly to PDC-109. When added to epididymal sperm, the labeled PDC-109 bound to the plasma membrane overlying the acrosome (Fig. 4), thereby demonstrating its surface localization and availability to bind sperm to oviduct.
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We next evaluated whether epididymal sperm would bind to oviductal explants. As anticipated, the binding density of epididymal sperm was significantly lower than that of ejaculated sperm. When epididymal sperm were pretreated with 250 µg/ml PDC-109 and then washed to remove unbound protein, sperm-binding density was increased to the level observed in ejaculated sperm, indicating that PDC-109 conferred on epididymal sperm the ability to bind explants (Fig. 5). Furthermore, ejaculated sperm were prevented from binding by the addition of excess soluble PDC-109 to the wells containing explants, and the inhibition was dose dependent (Fig. 6).
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Because we had already established that capacitation diminishes the ability of bull sperm to bind to fucose and to oviductal epithelium [14], we examined changes in sperm PDC-109 content as a result of capacitation. Sperm incubated for 4 h in TALP containing 10 µg/ml of heparin to promote capacitation [34] showed a decrease in the amount of PDC-109 recovered from extracts (Fig. 7, lane 3) in comparison to sperm incubated for 4 h in TALP alone (Fig. 7, lane 1) or in TALP containing heparin and glucose (Fig. 7, lane 2), which is known to inhibit capacitation of bull sperm [36].
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We next tested the ability of PDC-109 to restore binding of capacitated sperm to epithelium. Sperm incubated 4 h in TALP containing heparin and then washed to remove heparin bound to epithelium in significantly lower numbers than did sperm preincubated 4 h in TALP without heparin. When sperm capacitated by incubation in heparin were washed to remove heparin, treated with 240 µg/ml PDC-109, and then washed to remove unbound protein, binding was restored to the level of uncapacitated sperm (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-L-Fuc[1,4]-ß-D-Gal[1,3]-D-GlcNAc as the trap, to isolate a fucose-binding protein from extracts of ejaculated sperm [17]. The isolated protein blocked binding of ejaculated sperm to oviductal epithelium [17]. The identification of the isolated protein as PDC-109, a protein produced in the seminal vesicles and ductus deferens [35], prompted us to test whether epididymal sperm could bind to epithelium and, if not, whether PDC-109 derived from seminal plasma could enable them to bind. Epididymal sperm lack PDC-109 [27]. Consequently, no labeling was observed for fixed epididymal sperm stained with fluorescein-conjugated anti-PDC-109 antibody. However, when sperm were treated with purified seminal plasma PDC-109 prior to fixation, they were labeled brightly over the acrosomal region. Live sperm were not labeled with anti-PDC-109, either as whole IgG or Fab fragment, possibly because the antigenic epitopes of PDC-109 are buried within the plasma membrane and thus inaccessible to the antibody in live sperm. Structural studies have indicated that the molecule is inserted into the outer leaflet of the lipid bilayer in an orientation parallel to the surface but with segments reaching down to the 14th carbon atom of the acyl chains of the membrane lipids [37]. To test whether our purified seminal plasma PDC-109 was exposed on the sperm surface so that it could bind to oviductal epithelium, we conjugated fluorescein isothiocyanate (FITC) directly to PDC-109 and added this conjugate to live epididymal sperm. The epididymal sperm were labeled intensely over the acrosomal region of the plasma membrane, which is the region where sperm bind to epithelium. Desnoyers and Manjunath [22] reported that anti-PDC-109 antibody labeled the region overlying the acrosome and the midpiece of air-dried sperm.
Very few epididymal bull sperm bound to the oviductal explants. However, addition of seminal plasma PDC-109 increased the number of epididymal sperm binding to the level of ejaculated sperm, which indicates that PDC-109 alone can enable sperm to bind to epithelium. However, a few epididymal sperm did bind to explants. We do not know whether this binding was spurious or represented a weak affinity of epididymal bull sperm for epithelium. Other researchers have reported the existence of an epididymal heparin-binding protein [38], although little is known about the protein at this time. Additional studies are needed to determine whether this epididymal protein plays a part in mediating oviductal sperm binding.
Epididymal hamster sperm can bind to conspecific oviductal epithelium and do so via carbohydrate recognition [7], but the identity of the carbohydrate-binding protein is not known and it may or may not be homologous to PDC-109. Under some circumstances, artificial insemination with bovine epididymal sperm can result in pregnancy [39]; however, it is not known whether the epididymal sperm in this instance formed a reservoir in the oviduct. The careful timing of artificial insemination may reduce the need for a reservoir. If reservoir formation is essential for fertilization, then some redundancy in binding proteins might be expected.
Addition of seminal plasma PDC-109 to explants prior to adding ejaculated sperm in a competitive binding inhibition assay significantly reduced binding of sperm in a dose-dependent manner. The reduction in sperm-binding density was comparable to that observed when using the protein purified from sperm extracts [17], confirming the identity of the oviduct-binding protein on sperm as PDC-109.
We have shown for the first time that PDC-109 can restore the ability of capacitated sperm to bind to epithelium. Capacitation decreases the ability of sperm to bind to oviductal epithelium [14] and to fucose [16]. PDC-109 is lost from sperm during capacitation in vitro by contact with follicular fluid containing high-density lipoprotein [28]. Our Western blot analysis also showed a decrease in the amount of PDC-109 on sperm incubated with heparin to capacitate them. Previously, we demonstrated that addition of PDC-109 purified from sperm extracts could restore the ability of capacitated bull sperm to bind fucose [17]. In this study, we demonstrated that seminal plasma PDC-109 can restore the ability of capacitated bull sperm to bind to oviductal epithelium. In vivo, loss of PDC-109 from sperm during capacitation could therefore account for release of sperm from the oviductal reservoir. The loss of PDC-109 could be initiated in vivo by an increase in heparinlike glycosaminoglycans in the oviduct [40–42] or by follicular fluid entering the oviduct with the cumulus mass.
There is evidence that PDC-109 plays a role in sperm capacitation as well as in reservoir formation. PDC-109 promotes efflux of cholesterol from epididymal sperm [43] and enhances capacitation of epididymal sperm in vitro [28]. Promotion of cholesterol efflux may occur indirectly. PDC-109 appears to reduce the motion of cholesterol in membranes indirectly by forming strong associations with choline phospholipids [44]. Because capacitation is thought to be delayed until sperm are released from the reservoir [4, 13], it may seem contradictory that PDC-109 can participate in both sperm storage and capacitation. However, PDC-109 does not actually induce capacitation per se but rather enhances capacitation in the presence of high-density lipoproteins, such as those found in bovine follicular fluid [28, 45]. Thus, PDC-109 would not initiate capacitation until promoters became available in the oviduct.
We have demonstrated that bovine seminal plasma PDC-109 can bind epididymal sperm to oviductal epithelium and can restore epithelial binding ability to capacitated sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: S.S. Suarez, Department of Biomedical Sciences, T5-006 Veterinary Research Tower, Cornell University, Ithaca, NY 14853. FAX: 607 253 3541; sss7{at}cornell.edu
Received: 4 September 2002.
First decision: 9 October 2002.
Accepted: 30 April 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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