Bovine Seminal Plasma Proteins PDC-109, BSP-A3, and ... - BioOne

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Jun 21, 2006 - TanYa M. Gwathmey,3 George G. Ignotz,3 Jacob L. Mueller,4 Puttaswamy Manjunath,5 and Susan S. Suarez2,3. Department of Biomedical ...
BIOLOGY OF REPRODUCTION 75, 501–507 (2006) Published online before print 21 June 2006. DOI 10.1095/biolreprod.106.053306

Bovine Seminal Plasma Proteins PDC-109, BSP-A3, and BSP-30-kDa Share Functional Roles in Storing Sperm in the Oviduct1 TanYa M. Gwathmey,3 George G. Ignotz,3 Jacob L. Mueller,4 Puttaswamy Manjunath,5 and Susan S. Suarez2,3 Department of Biomedical Sciences,3 College of Veterinary Medicine, and Department of Molecular Biology and Genetics,4 Cornell University, Ithaca, New York 14853 Department of Medicine,5 University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada H1T 2M4 the seminal vesicles and represent approximately 70% of the total protein content of bovine seminal plasma [1, 2]. At ejaculation, sperm become coated with these proteins. The three major BSPs are PDC-109 (also referred to as BSP-A1/-A2), BSP-A3, and BSP-30-kDa. They are small acidic proteins with apparent molecular weights of 15–16 kDa (PDC-109 and BSP-A3) and 28–30 kDa (BSP-30-kDa) and isoelectric point ranging from 3.6 to 5.2. PDC-109 constitutes the most abundant protein of bovine seminal plasma (15–25 mg/ml) [1–3] and exists in two forms as BSP-A1 and BSP-A2, which differ only in degree of glycosylation [1]. BSPs coat epididymal sperm by binding to plasma membrane phospholipids [4–8]. Each BSP is composed of a unique N-terminal domain followed by two fibronectin type II (FN2) domains in tandem that are separated by a short linker polypeptide chain [9–11]. Each FN2 domain of PDC-109 has been shown to contain a choline phospholipid-binding site [12]. In placental mammals, when ejaculated sperm deposited in the female tract reach the oviduct, they bind to the oviductal epithelium and form a storage reservoir. Binding to oviductal epithelium prolongs the motile life span of sperm in vitro, even when only apical membrane vesicles of oviductal epithelium are substituted for whole cells [13–15]. Previously, a protein was isolated from bovine sperm membrane extracts that mediates binding of sperm to oviductal epithelium and was identified as PDC-109 [16, 17]. Herein, we report for the first time (to our knowledge) that PDC-109 and the other two BSPs can each maintain bull sperm motility in the presence of vesicles of oviductal epithelium. Redundancy in function indicates the importance of sperm binding to epithelium in promoting fertilization and suggests a broadening of adaptability to variations in female tracts. Therefore, we examined BSPs for similarities and differences in three-dimensional structure and distribution of surface electrical potential to explain similarities in function and to discover differences that could enable sperm to interact with a broader range of molecules in female tracts.

ABSTRACT On ejaculation, sperm become coated with proteins secreted by the male accessory sex glands. In the bull, these proteins consist predominantly of the bovine seminal plasma family of proteins (BSPs): PDC-109 (BSP-A1/-A2), BSP-A3, and BSP-30kDa. PDC-109 plays a role in forming an oviductal sperm reservoir by enabling sperm to bind to oviductal epithelium. Because PDC-109 has high sequence identity with the other BSPs, we tested BSP-A3 and BSP-30-kDa for the capacity to bind sperm to oviductal epithelium. BSP-A3 and BSP-30-kDa each increased binding of epididymal sperm to epithelium and were as effective as PDC-109 in competitively inhibiting binding of ejaculated sperm. Because binding extends the motile life of sperm, BSPs were tested for the ability to maintain sperm motility. BSP-treated epididymal sperm incubated with plasma membrane vesicles from bovine oviductal epithelium maintained progressive motility longer than untreated sperm. To our knowledge, this is the first report of this protective effect of BSPs. Similarities in function among the BSPs were reflected in their three-dimensional structure, whereas surface maps of electrostatic potential indicated differences in binding affinities and kinetics. Such differences may provide sperm with greater adaptability to variations among females. Altogether, these results indicate that BSPs play a crucial role in fertilization by maintaining sperm motility during storage. fallopian tubes, male reproductive tract, oviduct, sperm, sperm motility and transport

INTRODUCTION In the male genital tract, secretions from the testes, epididymis, seminal vesicles, and other accessory glands contribute to the complex mixture of fluid and proteins that comprise seminal plasma. A family of heparin-binding proteins called bovine seminal plasma proteins (BSPs) are secreted by 1

Supported by a Canadian Institutes of Health Research grant to P.M. and by National Research Initiative Competitive Grant no. 200435203-14952 from the USDA Cooperative State Research, Education, and Extension Service to S.S.S. The contributions of J.L.M. were supported by grant HD38921 from the National Institutes of Health to M.F. Wolfner. 2 Correspondence: Susan S. Suarez, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, T5–006 Veterinary Research Tower, Ithaca, NY 14853. FAX: 607 253 3541; e-mail: [email protected]

MATERIALS AND METHODS Media and Reagents Routine laboratory chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. TALP (Tyrode albumin lactate pyruvate) medium, a modified Tyrode balanced salt solution, was used for semen dilution, oviductal explant preparation, and sperm incubations. It consists 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, sodium pyruvate (0.11 mg/ml), BSA (6 mg/ml) (fraction V; Calbiochem, La Jolla, CA), and gentamycin (5 lg/ml) (pH 7.4, 290 mOsmolal [mOsm]). Hepes balanced salts (HBS) solution was used

Received: 20 April 2006. First decision: 4 June 2006. Accepted: 14 June 2006. Ó 2006 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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GWATHMEY ET AL. water jacket. Within 60 min of collection, sperm were washed three times in 5 ml of TALP (170 3 g, 10 min), resuspended in TALP at 5 3 106 cells/ml, and incubated at 398C (bovine core body temperature) under 5% CO2 in watersaturated air until used in binding assays. Only samples with motility exceeding 85% were used.

Epididymal Sperm

FIG. 1. A) BSPs enhance binding of epididymal sperm to oviductal epithelium. Data are presented as mean 6 SEM for samples from five bulls. *Different from control (P , 0.01). B) MLC did not enhance binding of epididymal sperm. Data are presented as mean 6 SEM for samples from four bulls. *Different from control (P , 0.001).

To obtain fresh epididymal sperm, testes with attached epididymides and vasa deferentia were acquired from an abattoir (Cargill Taylor Beef, Wyalusing, PA; or Cudlins Meat Market, Newfield, NY) or from Genex/CRI and transported on ice to the laboratory. Epididymal sperm were flushed from the caudal epididymis by retrograde perfusion with TALP medium. The sperm obtained were washed twice by centrifugation in 5 ml of TALP for 10 min (170 3 g) and resuspended in TALP at 5 3 106 cells/ml. Only samples with progressive motility exceeding 85% were used. Most samples were highly motile. Frozen epididymal sperm cryopreserved in Tris-egg yolk extender and stored in liquid nitrogen were generously provided by ABS Global, Inc. (Deforest, WI). Straws were thawed in a 378C water bath for 35 sec. The sperm suspension was then layered over a 45%–90% discontinuous Percoll gradient and centrifuged for 15 min at 600 3 g according to the method by Parrish et al. [20]. After discarding each layer of the gradient, the pellet of sperm was resuspended in 5 ml of fresh TALP medium and washed for 10 min at 170 3 g. Centrifugation through Percoll selected for highly motile sperm, and only samples with a final progressive motility exceeding 85% were used.

Preparation of Oviductal Explants for protein storage and dilution and 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).

Purification of BSPs PDC-109 was isolated from seminal plasma as previously described [7, 17], with some modifications. Briefly, semen collected from bulls at Genex/CRI (Ithaca, NY) was transported to the laboratory within 1 h of collection. Following supplementation with a serine and cysteine protease inhibitor cocktail (Complete EDTA-free; Roche Molecular Biochemicals, Indianapolis, IN), semen was centrifuged (3000 3 g, 15 min) and filtered through 0.2-lm cellulose acetate filters to remove sperm and particulate debris and then assayed for protein content using the BioRad DC Protein Assay Kit (Hercules, CA). The seminal plasma obtained was stored at 208C until use. Aliquots of seminal plasma containing 50–100 mg of protein were applied to heparin-sepharose CL-6B columns (1 3 20 cm) that were equilibrated with binding buffer (50 mM Tris-HCl, 150 mM NaCl, and 5 mM EDTA [pH 7.4]). After extensive washing, heparin-binding proteins were eluted with 10 mM o-phosphorylcholine in binding buffer. The eluates were concentrated and dialyzed against 20 mM Tris-HCl (pH 6.4) in 1 M NaCl and then applied to DEAE-Sephadex equilibrated with the same buffer. Washings were repeated, 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 on subsequent applications. Purity of PDC-109 was assessed by SDS-PAGE with silver staining [18]. BSP-A3 and BSP-30-kDa were isolated from bovine seminal plasma as previously described [19], with some modifications. Semen was centrifuged (3000 3 g, 15 min) to remove sperm and particulate debris. Proteins were precipitated from the resultant seminal plasma by the addition of nine volumes of cold ethanol with stirring for 1 h at 48C. The precipitate was recovered by centrifugation, washed with ethanol, dried, and dissolved in PBS. Seminal plasma proteins were then applied to a gelatin-agarose affinity column (1.5 3 30 cm), and unbound proteins were removed by washing with PBS. BSPs were eluted with a linear urea gradient (0–4 M) in PBS. Peak fractions corresponding to BSP-A3 and BSP-30-kDa were pooled, dialyzed against 50 mM ammonium bicarbonate, and lyophilized. BSP-A3 and BSP-30-kDa were purified to homogeneity by HPLC using a reverse-phase lBondapak phenyl column [1]. The solvent system used consisted of solvent A (0.13% [v/v] heptafluorobutyric acid in water) and solvent B (0.13% heptafluorobutyric acid in acetonitrile). Proteins were solubilized in solvent A before column application, and purity of resolved proteins was judged by SDS-PAGE and amino acid composition.

Ejaculated Sperm Bull semen was provided by Genex/CRI, diluted fivefold in TALP immediately after collection, and transported to the laboratory in a 378C warm

Bovine oviducts were collected from a slaughterhouse (Cargill Taylor Beef or Cudlins Meat Market) and transported partially submerged in PBS in covered containers placed over ice. Oviducts taken from cows in various cycle stages were used, as previous findings indicated that cycle stage has no effect on sperm-epithelial binding [21]. Oviductal isthmi from a single cow were used in each experiment. Explants of oviductal epithelium were prepared as described previously [22]. The isthmic portion of the oviduct was dissected free of connected tissue and rinsed in PBS. The epithelium was extruded in sheets by squeezing the oviduct with fine tweezers, fragmented by pipetting, centrifuged for 1 min (170 3 g), transferred to TALP in a 35-mm Petri dish, and allowed a minimum of 30-min incubation at 398C under 5% CO2 to form everted vesicles with apical ciliated surfaces oriented outwardly. Explants were used within 6 h of slaughter and showed ciliary motility.

Preparation of Oviductal Apical Plasma Membrane Vesicles (OAPMVs) Epithelial sheets were extruded from isthmuses of oviducts (8–10 pairs) by squeezing with tweezers into ice-cold PBS supplemented with protease inhibitors (Complete; Roche Molecular Biochemicals) and washed twice by allowing them to settle. Cell pellets were collected by centrifugation (100 3 g for 5 min), yielding 0.9–1.2 g of wet tissue weight. Apical plasma membranes were prepared according to the method by Murray and Smith [23]. Briefly, this involved homogenizing the cells, collecting membranes by differential centrifugation, and using Mg2 þ precipitation to remove all but the apical plasma membranes from the preparation. Purified membranes were suspended in HBS, and an aliquot was removed for estimation of protein content. Typically, 600-1000 lg of apical plasma membrane protein was recovered in membrane vesicles from the isthmic portions of 8–10 pairs of oviducts.

Binding of Epididymal Sperm to Oviductal Explants Oviductal explants were centrifuged (170 3 g for 1 min in 5 ml of TALP), and 10 ll of the pellet was added to 50 ll of TALP in 0.5-ml Eppendorf tubes. Fresh epididymal sperm were treated with approximately 15 lM of PDC-109 (250 lg/ml), BSP-A3 (250 lg/ml), or BSP-30-kDa (500 lg/ml) for 20 min at 398C and then washed by centrifugation for 12 min (100 3 g) to remove unbound protein. The sperm pellet was resuspended in TALP to a concentration of 5 3 106 cells/ml, and 20-ll aliquots were added to explants. After 15-min incubation at 398C under 5% CO2, loosely bound sperm were removed from explants by pipetting though three 75-ll droplets of warm TALP. The explants were transferred to a four-well culture plate (each treatment group in an individual well), covered with warm silicone oil, and topped with the accompanying lids. The experiment was repeated four times, each time using sperm from a different bull and explants from a different cow. Explants were videotaped on a 398C microscope stage of a Zeiss Axiovert 35 Microscope (Carl Zeiss Inc., Thornbrook, NY) using a 303 Hoffman modulation objective and a 1.63 magnifier. Videotaping was performed using a

BOVINE SEMINAL PLASMA PROTEINS AND SPERM STORAGE

FIG. 2. Excess BSPs in solution competitively inhibit ejaculated sperm binding to oviductal epithelium. Data are presented as mean 6 SEM for samples from four bulls, with control normalized to 100%. *Different from control (P , 0.01). Dage CCD-72 black-and-white video camera (Dage-MTI, Inc. 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 later assessed for spermbinding density.

Competitive Inhibition of Ejaculated Sperm Binding to Oviductal Explants To determine whether excess unbound BSP could competitively inhibit sperm binding to epithelium, washed ejaculated sperm were added to oviductal explants in the presence of 100 ll of PDC-109 (250 lg/ml), BSP-A3 (62.5 lg/ ml), and BSP-30-kDa (62.5 lg/ml), to reflect the relative concentrations of protein found on the sperm surface [2], or buffer alone. Loosely bound sperm were removed, and explants were videotaped for determination of spermbinding density as already described. The experiment was repeated four times, each time using sperm from a different bull and explants from a different cow.

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FIG. 3. BSPs prolong the progressive motility of fresh epididymal sperm coincubated with OAPMVs. Data are presented as mean 6 SEM for samples from six bulls. *Different from control (P , 0.01). TALP medium and incubated in microtiter wells at 398C under 5% CO2 for 5 h. The wells were videorecorded at 5 h using a 403 objective, and the recordings were reviewed to determine the percentage of progressively motile sperm (of 200 sperm).

Statistical Analysis All data are expressed as mean 6 SEM. The data were analyzed using ANOVA followed by Tukey honestly significant difference pairwise comparison. Linear regression was applied to analyze dose dependence. STATISTIX (Tallahassee, FL) statistical software was used for analyses.

Generating Comparative Structural Models of BSP-A3 and BSP-30-kDa

The numbers of sperm bound to explants were counted by reviewing the videotapes. Next, videotape images of the explants were digitized using a Scion CG7 frame grabber and Scion Imaging 1.62c software (Scion Corp., Frederick, MD). The surface area of each explant was determined using National Institutes of Health Image (Internet-based freeware at http://rsb.info.nih.gov/nih-image/). Sperm-binding density was calculated by determining the number of sperm bound per (0.1 mm2) of explant surface. Approximately 1.23 3 105 lm2 of explant surface was analyzed per treatment for each experiment performed.

The three BSP sequences were obtained from GenBank: PDC-109 (BSPA1/-A2) (P02784), BSP-A3 (P04557), and BSP-30-kDa (P81019). The known structure of PDC-109 (1H8P) was used as a template on which to thread BSPA3 and BSP-30-kDa using the MODELLER6v2 program [24]. Only the FN2 domains of BSP-A3 and BSP-30-kDa were comparatively structurally modeled to PDC-109, because a three-dimensional structure of the N-terminal domain of PDC-109 had not been solved due to lack of electron density [25]. On generation of the two structural models, each model was then inspected visually to ensure that the four conserved disulfide bridges were in the correct spatial orientation. Each model was also quality-checked using the ‘‘WHAT_CHECK’’ [26] program (http://www.cmbi.kun.nl/gv/servers/WIWWWI/), which found no major structural errors for the BSP-A3 or BSP-30-kDa models that were not already present in PDC-109 [25]. Electrostatic potentials and root-mean-square distance (RMSD) calculations were performed using Swiss PDBviewer [27]. The three-dimensional coordinates of the theoretical models are included as PDB (Protein Data Bank) files in the supplemental material (available online at http://www.biolreprod.org).

Assays of Maintenance of Sperm Motility

RESULTS

Fresh epididymal sperm were incubated with approximately 15 lM PDC109, BSP-A3, BSP-30-kDa, or HBS diluent in 100 ll of TALP for 20 min at 398C under 5% CO2, centrifuged for 12 min at 100 g, and resuspended in 100 ll of TALP. The sperm (10 ll) were then added to microtiter wells containing 90 ll of protein equivalent (40 lg/ml) of bovine oviductal apical plasma membrane vesicles (OAPMVs) in TALP and coincubated at 398C under 5% CO2. Videotaped recordings of sperm motility were obtained at 0, 1, 3, 5, and 8 h of coincubation using a 403 objective. The percentages of progressively motile sperm were determined for each treatment and time point by reviewing recordings. At least 200 sperm were evaluated per treatment and time point. Frozen-thawed epididymal sperm, prepared as already described, were used to evaluate dose-dependent effects of BSPs on sperm motility. Sperm were treated with 15 lg/ml, 7.5 lg/ml, 1500 ng/ml, 300 ng/ml, 60 ng/ml, 12 ng/ml, or 2.4 ng/ml of PDC-109, BSP-A3, BSP-30-kDa, or HBS buffer. Then, 10-ll aliquots of washed sperm suspension were added to 90 ll of OAPMVs in

BSP-A3 and BSP-30-kDa Enhance Sperm Binding to Oviductal Epithelium

Determining Sperm-Binding Density

To determine if BSP-A3 and BSP-30-kDa enhance binding of sperm to oviductal epithelium, fresh epididymal sperm were coated with PDC-109, BSP-A3, or BSP-30-kDa. BSP-A3 and BSP-30-kDa significantly increased binding above control values (Fig. 1A), as previously observed with PDC-109 [17]. To verify that the effects observed with the BSPs were specific, we tested a small acidic protein of similar charge and size to the BSPs, myosin light chain (MLC). As shown in Figure 1B, addition of 15 lM MLC to epididymal sperm did not significantly affect the binding of sperm to the epithelium.

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to bind epithelium, we sought to determine if the prolonged maintenance of sperm motility could be attributed to the action of the BSPs. Fresh epididymal sperm that were treated with PDC-109, BSP-A3, or BSP-30-kDa and coincubated with OAPMVs maintained a forward progressive motility significantly longer than untreated sperm coincubated with OAPMVs or than untreated sperm alone (Fig. 3). Furthermore, at 5 h of incubation, there was a dose-dependent effect on maintenance of motility with BSPs in a range of 2.4 ng/ml-15 lg/ml (Fig. 4). Comparative Structural Modeling of BSPs

FIG. 4. BSPs increase the percentage of frozen-thawed epididymal sperm remaining motile after coincubation with OAPMVs for 5 h. The regression of dose on percentage motility was significant for PDC-109, BSP-A3, and BSP-30-kDa (P , 0.01). Symbols encompass the mean 6 SEM for six replicates.

Previously, it was demonstrated that excess PDC-109 in solution inhibits the binding of ejaculated sperm to oviductal epithelium in a competitive and dose-dependent manner [17]. Likewise, preincubation of oviductal explants with BSP-A3 or with BSP-30-kDa was equally effective as PDC-109 at reducing binding density of ejaculated sperm (Fig. 2). The protein concentrations used in these experiments reflect the relative amount of proteins found on the surface of sperm (i.e., a 4:1:1 ratio of PDC-109:BSP-A3:BSP-30-kDa) [2]. These same experiments were conducted using equal protein concentrations, yielding similar results (data not shown). BSPs Prolong the Motile Life Span of Sperm Because binding of sperm to oviductal epithelium prolongs the motile life span of sperm and because BSPs enable sperm FIG. 5. Amino acid sequence alignments of PDC-109, BSP-A3, and BSP-30-kDa. Residues conserved among all three proteins are indicated by asterisks. The FN2 domains are indicated above the sequences. P indicates aromatic residues that bind phosphorylcholine; H, heparin-binding residues.

The amino acid sequence identity between the FN2 domains of the three BSPs are PDC-109 and BSP-A3 (71.4%), PDC109 and BSP-30k-Da (53.8%), and BSP-A3 and BSP-30-kDa (57.5%). The tyrosine 30, 54, 75, 100, and 108 residues and the tryptophan 58 and 106 residues of the FN2 domains, which are important for binding PDC-109 to phosphorylcholine in sperm plasma membranes [23], are conserved among the three BSPs (Fig. 5). Residue 60, also involved in phosphorylcholine binding, has a conservative change of a tyrosine to a phenylalanine in BSP-30-kDa. In contrast, conservation is not the case for the residues with side chains exposed on the opposite face, which are predicted to bind to heparin and which would face the oviductal epithelium. The heparin-binding residues of PDC-109 (Arg57, Lys59, Arg64, Lys68, Lys85, and Lys107), which had been identified using heparinsepharose binding to protect bound Arg and Lys residues from chemical modification [28], are more divergent in BSP-A3 and BSP-30-kDa than the phosphorylcholine-binding residues and show conservative and radical amino acid substitutions (Fig. 5). To obtain a better understanding of the interactions of the key residues involved in sperm and oviduct membrane binding, comparative structural models were generated for the FN2 domains of BSP-A3 and BSP-30-kDa, based on the established structure of PDC-109 FN2 domains [29]. The structural models generated for BSP-A3 and BSP-30-kDa thread tightly to the

BOVINE SEMINAL PLASMA PROTEINS AND SPERM STORAGE

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FIG. 6. Three-dimensional structures of the FN2 domains of BSP-30-kDa and BSP-A3 threaded over the established structure of PDC-109. Superposition of PDC-109 (red), BSP-30-kDa (blue), and BSP-A3 (green), illustrating the secondary structural elements of the N-terminal FN2 domain (left) and C-terminal FN2 domain (right). (A video clip of this image may viewed in Supplement 3, available online at http://www. biolreprod.org.)

˚ and 0.43 A ˚ RMSD PDC-109 structure (Fig. 6). Only 0.42 A separate the modeled structures from PDC-109 for BSP-A3 and BSP-30-kDa (352 atoms involved in each comparison), respectively. The binding of all three BSPs to sperm appears to occur via a similar mechanism, namely, utilization of a hydrophobic pocket formed by aromatic residues within each FN2 domain. Because the heparin-binding residues are not as well conserved among the BSPs as the phosphorylcholine-binding sites residues, differences in electrostatic or surface charge potential along the face of the BSPs involved in binding to heparin and oviductal epithelium were investigated. This face on PDC-109 and BSP-A3 exhibits positive electrostatic surface potential (Fig. 7), while this face on BSP-30-kDa primarily exhibits a negative electrostatic surface potential. Unexpectedly, on examination of the sperm-binding face of the three BSPs, we found a positive electrostatic potential for PDC-109 and BSP-30-kDa and a negative electrostatic field for BSP-A3. This suggests that there are differences in the binding affinities and kinetics of the BSPs for the sperm surface, as well as for heparin and the oviductal surface. DISCUSSION Like PDC-109, we demonstrate herein that BSP-A3 and BSP-30-kDa promote binding of sperm to oviductal epithelium. We also demonstrate, for the first time to our knowledge, that each of these three BSPs prolongs the maintenance of sperm motility in the presence of oviductal epithelium, presumably by enabling sperm to interact with the epithelium. These results confirm that PDC-109, as well as the other two BSPs, plays an important role in sperm storage in the female. The first functional role identified for PDC-109 was that it promotes capacitation of bull sperm, particularly by stimulating lipid efflux from the plasma membrane [30–32]. Second, Yu et al. [33] demonstrated that (in vitro) protein kinase C activity is inhibited by PDC-109 and proposed that this serves to prevent premature acrosome reaction of sperm in the female tract. Herein, we demonstrate a third role for PDC-109, which is prolonging sperm survival during storage in the oviduct. BSPA3 and BSP-30-kDa share this third role with PDC-109, as well as the first role [30, 31]. The three roles are related in that all are involved in maintaining sperm in an appropriate state in the female tract until the oocyte reaches the site of fertilization. The role of the BSPs in storing sperm may seem at odds with

FIG. 7. Electrostatic potential maps of the FN2 domains of the three BSPs: PDC-109, BSP-A3, and BSP-30-kDa. Blue indicates positive charge; red, negative charge. The N-terminal FN2 domain is on the left. The sperm-binding face is indicated by arrows.

their role in stimulating lipid efflux during capacitation; however, the BSPs do not stimulate lipid efflux during capacitation unless certain capacitating agents are present, such as heparin or high-density lipoprotein [30–32]. Therefore, sperm could remain stabilized by binding to the oviductal epithelium via the BSPs until capacitating agents enter the storage area to initiate capacitation. During capacitation, PDC-

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109 (and probably the other two BSPs) are lost from the sperm [17]. Once capacitated, sperm lose binding affinity for the oviductal epithelium [17]; therefore, the loss of BSPs during capacitation plays a major role in release of sperm from the reservoir. It is well documented that sperm motility, viability, and fertilizing capacity are maintained when sperm are bound to epithelium of the female reproductive tract (reviewed by Suarez [34, 35]) and that cultured oviductal epithelial cells and their secretions have a beneficial effect on sperm motility [36– 38]. Boilard et al. [39] showed a concentration-dependent effect of OAPMVs on maintenance of bull sperm motility. Because binding of sperm to epithelium prolongs motility, and BSPs enable sperm to bind epithelium, we evaluated the effects of the BSPs on sperm motility in the presence of OAPMVs. It had already been reported that BSPs alone (in the absence of epithelium) do not improve survival of epididymal sperm after 5 h of incubation [40]. We determined that PDC-109, BSP-A3, and BSP-30-kDa each prolongs the motile life span of epididymal sperm in the presence of OAPMVs. This is a direct result of epithelial binding, as epididymal sperm coincubated with OAPMVs in the absence of BSPs lose motility faster over time than epididymal sperm coincubated with OAPMVs in the presence of BSPs. Redundancy in function of the BSPs may exist to ensure that a vital function is carried out under a variety of circumstances or conditions, such as variation among females and in the timing of insemination with respect to the onset of ovulation. Also, the primary role of each BSP could differ or the BSPs could function synergistically. For example, when epididymal sperm are coincubated with purified BSPs, the proteins work synergistically (not additively) to induce capacitation, as measured by lysophosphatidylcholine-induced acrosome reaction [40]. The amino acid residues that interact with phosphorylcholine are conserved among PDC-109, BSP-A3, and BSP-30kDa, indicating similarities in the sites in which the BSPs interact with the sperm plasma membrane. However, the map of electrostatic potential of BSP-A3 showed a strong negative surface potential on the sperm-binding face, whereas the maps of PDC-109 and BSP-30-kDa showed positive surface potentials. These differences could explain why The´rien et al. [31, 41] observed that BSP-A3 is less effective at stimulating phospholipid and cholesterol efflux from the sperm plasma membrane than PDC-109 and BSP-30-kDa. PDC-109 interaction with heparin is predicted to occur via two arginines (57 and 64) and four lysines (59, 68, 85, and 107), which form a patch of basic residues [28]. The heparinbinding region is present on the face of the protein opposite to the phosphorylcholine-binding face. The heparin-binding face would also be the surface by which sperm interact with the oviductal epithelium, although it is not known whether the heparin-binding residues are identical to those binding oviductal epithelium. Amino acid residues involved in heparin binding do not demonstrate the same evolutionary conservation observed for phosphorylcholine-binding residues, suggesting that there are differences in the interactions of the individual BSPs with heparin. These differences are borne out by the differing elution profiles of the three BSPs on heparin affinity columns. Therefore, differences are also likely to exist in the interactions of the BSPs with oviductal ligands. Furthermore, differences in the FN2 surface electrostatic potentials among the BSPs indicate that the proteins must differ from each other with regard to binding characteristics for heparin and oviductal ligands. The N-terminal domains of the BSPs also differ considerably from each other, and these domains could affect

kinetics of binding to the sperm plasma membrane and the oviductal epithelium. Seminal plasma proteins from several other species share heparin- and phosphorylcholine-binding properties similar to the BSPs of bovine, including the following: bison (BiSV-16kDa, BiSV-17-kDa, BiSV-18-kDa, and BiSV-28-kDa) [42]; stallion (HSP-1, HSP-2, and HSP-12-kDa) [43–45], boar (pb1) [44, 46], goat (GSP-14-kDa, GSP-15-kDa, GSP-20-kDa, and GSP-22-kDa) [47], and ram (RSP-15-kDa, RSP-16-kDa, RSP22-kDa, and RSP-24-kDa) [48]). Phosphorylcholine-binding proteins from seminal plasma of several species (humans, hamsters, mice, rats, and pigs) share antigenic determinants with the BSPs [49], suggesting that comparative modeling can be utilized to generate predictive structures for seminal plasma proteins in other species. Because many seminal plasma proteins are rapidly evolving [50], an examination of the sequence and structural differences of BSP homologs in closely related organisms may provide greater insight into the dynamics of interactions of sperm with oviductal membranes. ACKNOWLEDGMENTS We thank Dr. Michael Kaproth and Genex/CRI for donating fresh semen samples; Dr. Robert Hillman for surgical retrieval of epididymides; ABS Global, Inc., and Dr. Marjorie Faust for providing frozen ejaculated and epididymal sperm samples; and Dr. Jan Scarlett for her assistance with the statistical analyses.

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