Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print August 22, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.062505
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
77/6/906    most recent
biolreprod.107.062505v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ignotz, G. G
Right arrow Articles by Suarez, S. S
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ignotz, G. G
Right arrow Articles by Suarez, S. S
Agricola
Right arrow Articles by Ignotz, G. G
Right arrow Articles by Suarez, S. S
BIOLOGY OF REPRODUCTION 77, 906–913 (2007)
DOI: 10.1095/biolreprod.107.062505
© 2007 by the Society for the Study of Reproduction, Inc.

Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal Reservoir1

George G Ignotz , Margaret Y Cho , and Susan S Suarez  2

Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853

ABSTRACT

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.

fallopian tubes, oviduct, seminal vesicles, sperm, sperm motility and transport, sperm reservoir, spermatozoa, uterine tube

INTRODUCTION

Oviductal sperm storage reservoirs are widespread in mammals, including cattle [1], mice [2], hamsters [3], pigs [4], sheep [5], and horses [6]. In these eutherian species, the reservoirs are formed as sperm are trapped in the caudal portions of the oviducts by binding to the apical surfaces of the epithelium. Binding to oviductal epithelium prolongs the life span of sperm in vitro [7–9], and thus would preserve sperm during storage. The mechanism of preserving sperm involves inhibiting capacitation, because sperm bound to oviductal epithelium in vitro undergo capacitation-associated events at a slower rate than free sperm [9, 10]. In vivo, as the time of ovulation approaches, sperm are gradually released from the reservoir [2]. This gradual release evidently serves to reduce incidence of polyspermy, because in pigs it has been shown that polyspermy increased when the caudal oviduct was resected [11] or when sperm were inseminated cranial to the entrance of the reservoir in the uterotubal junction [12]. Together, these observations indicate that the reservoir serves to preserve sperm in a fertile state at a site near to the site of fertilization so that sperm can be quickly and reliably provided to the descending oocytes in numbers that would not overwhelm the oocytes' intrinsic blocks to polyspermy.

In several species, sperm binding to oviductal epithelium has been demonstrated to be inhibited by specific carbohydrates [13–18]. These findings indicate that a carbohydrate moiety on a larger molecule, such as a glycoprotein, is a crucial part of the sperm binding site. In Bos taurus, fucose in an {alpha}1–4 linkage to N-acetyl glucosamine, as in the trisaccharide Lewis-a (Lea), has been shown to inhibit sperm binding [15, 16]. Sperm binding was severely reduced when fucose was removed from bovine oviductal epithelium by fucosidase, indicating that fucose is a key part of the sperm-binding site on the epithelium [15]. Using Lea in an affinity purification scheme, a protein was isolated from bull sperm membrane extracts [19] and identified as PDC109 (also known as BSPA1/A2), which is secreted by the seminal vesicles and the male ampulla and is the most abundant protein component of bovine seminal plasma [20]. When sperm released from the caudal epididymis come into contact with seminal vesicle secretions, they adsorb PDC109, because it associates with choline phospholipids in the sperm plasma membrane [21, 22]. Epididymal bull sperm that have not come into contact with seminal vesicle secretions bind poorly to oviductal epithelium, but binding is greatly enhanced if the sperm are exposed to purified PDC109 [23].

PDC109 is a member of the BSP protein family and is one of three major acidic heparin-binding proteins in the seminal plasma, two others being BSPA3 and BSP30K [20]. The BSP proteins consist of unique N-terminal domains followed by two fibronectin type II domains [20, 24, 25]. PDC109 is 109 amino acids long and occurs in two forms: BSPA1 and BSPA2. BSPA1 possesses a single trisaccharide, NeuNAc-Gal-GalNAc, that is O-linked via GalNAc to threonine residue number 11 [26], whereas BSPA2 lacks the full trisaccharide [27]. BSPA3 is composed of 115 amino acids and is not glycosylated [24]. BSP30K is the largest of the three (158 amino acids), due to a long and unique N-terminal domain and six glycosylation sites [25].

Each of these three BSPs alone is able to promote sperm binding to oviductal epithelium [28]. Furthermore, when epididymal bull sperm are coated with any one of these proteins and incubated in the presence of apical plasma membranes derived from oviductal epithelial cells, their motile life span is prolonged [28].

Having established a role for the BSPs in binding sperm to oviductal epithelium and forming the sperm reservoir, we next sought to identify the receptors for the BSPs on the surface of the epithelium.

MATERIALS AND METHODS

Chemicals and Reagents

Unless stated otherwise, all chemicals were from Sigma (St. Louis, MO), including biotinylated Lotus tetragonolobus lectin (LTL), streptavidin-conjugated horseradish peroxidase (HRP), normal rabbit serum, and gelatin (bovine skin Type B, 225 Bloom). Disuccinimidyl suberate (DSS) and SDS-PAGE protein markers were from Pierce (Rockford, IL). EDTA-free Complete protease inhibitors (Roche Applied Science, Indianapolis, IN) were used during protein and membrane isolations. Rabbit polyclonal anti-PDC109 was prepared as previously described [23], and anti-BSPA3 and anti-BSP30K antibodies were provided by P. Manjunath [29]. Mouse monoclonal anti-bovine ANXA1, ANXA2, and ANXA4 were provided by Dr. Ian Varndell, BIOMOL International (Exeter, UK). Mouse monoclonal and rabbit polyclonal anti-ANXA5 antiserum were gifts of J. Mollenhauer [30].

A modified Tyrode balanced salt solution (TALP [31]) was used for semen dilution and oviductal explant preparation and incubation. TALP consisted of 99 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.35 mM NaH2PO4, 10 mM HEPES, 2 mM CaCl2, 1.1 mM MgCl2, 21.6 mM sodium lactate, 1.1 mg/ml sodium pyruvate, 6 mg/ml BSA (Fraction V; Calbiochem, La Jolla, CA), and 1 µg/ml gentamycin (pH 7.4, 290 mOsm/kg).

BSP Protein Purification

Fresh bovine ejaculates were transported to the laboratory at ambient temperature and processed within 30 min of collection. The ejaculates were diluted with an equal volume of sterile PBS containing protease inhibitors. Sperm were removed by centrifugation at 10 000 x g for 10 min, and the supernatant was further clarified by passage through 0.2-µm sterile filters. A crude BSP fraction was obtained by precipitation with 7 volumes of cold ethanol overnight at –20°C. Precipitates were washed twice with ethanol, vacuum dried, and dissolved in PBS (0.9% NaCl, 50 mM KH2PO4, pH 7.5) at 50 mg/ml and stored at –80°C.

Individual BSPs were isolated by gelatin affinity chromatography [32], DEAE partitioning [23, 33], and gel filtration as follows. Bovine gelatin (20 mg/ml in 0.1 M morpholinoethanesulfonic acid [MES], pH 6.5) was coupled to Affigel-15 beaded agarose (Bio-Rad, Hercules, CA) following the manufacturer's suggested protocol for aqueous coupling. The reaction was terminated after 2 h of mixing at room temperature by the addition of 1 ml of 1 M ethanolamine and further incubation for 1 h. The beads were washed on a glass sinter with water, followed by PBS to remove unbound gelatin, packed into a 1.5 x 20 cm glass column, equilibrated with additional PBS containing 0.02% sodium azide, and transferred to a 4°C cold room. Coupling efficiency was approximately 50%, resulting in 12 mg gelatin per milliliter of packed matrix. Two milliliters containing 100 mg crude BSPs were applied to the column and allowed to bind for 1 h, followed by extensive washing with PBS to remove unbound proteins. BSPs were eluted from the column with a linear gradient of 0–7 M urea in PBS developed over 15 column volumes. Fractions (3 ml) were collected across two major peaks and evaluated for BSP content by SDS-PAGE [34] and/or Western blotting. The first peak contained BSP30K and BSPA3, whereas the second contained BSP30K and PDC109.

Fractions within each peak were pooled, concentrated by dialyzing against polyethylene glycol, and further dialyzed against starting buffer for subsequent chromatographic separations. BSP30K and BSPA3 in the first peak were separated by gel filtration on Bio-Gel P10 (Bio-Rad) in 0.5 M NaCl, 50 mM Tris-HCL, pH 8.5. PDC109 and BSP30K in the second peak were separated by absorption to and elution from DEAE Sephadex [33]. Purity of BSPs was assessed by SDS-PAGE and staining of gels with silver nitrate [35]. The BSP30K purified from the two peaks was pooled, and then each purified BSP was dialyzed against PBS and stored in aliquots at –80°C.

Oviduct Apical Plasma Membrane (OAPM) Protein Preparation

Freshly excised bovine oviducts with ovaries and tips of the uterine horns attached were obtained from Cargill Meat Solutions (Wyalusing, PA) and Cudlin's Meat Market (Newfield, NY) and transported to the laboratory semisubmerged in ice-cold PBS containing 100 IU/ml penicillin and 100 µg/ml streptomycin. Oviducts were dissected free of mesentery and blood vessels and were cut at the uterotubal junction and at the ampullary-isthmic junction. Epithelial sheets were extruded from the isthmuses of 8–10 pairs of oviducts by squeezing with tweezers into ice-cold PBS supplemented with protease inhibitors, washed twice by settling, and collected by centrifugation at 600 x g to obtain 0.9–1.2 g wet tissue. Apical plasma membranes were isolated according to Murray and Smith [36] as modified by Boilard et al. [37]. Briefly, cells were homogenized with 20 strokes of a Dounce tissue grinder (Kimble-Kontes, Vineland, NJ) on ice in 60 mM mannitol, 5 mM EGTA, pH 7.4, adjusted with Tris base and supplemented with protease inhibitors. Then, a 1:100 volume of 1 M MgCl2 was added to the homogenate to agglutinate nonapical membranes during a 30-min period on ice. Homogenates were centrifuged at 3000 x g to remove nonapical membranes, and the supernatant was centrifuged at 27 000 x g to recover apical membranes. The pellet was resuspended in homogenization buffer and subjected to a second round of purification to yield a final pellet of oviductal apical plasma membranes. Membranes were suspended in Hepes-buffered balanced salts (HBBS: 25 mM Hepes, 130 mM NaCl, 5 mM KCl, 0.36 mM NaH2PO4, 0.49 mM MgCl2, and 2.4 mM CaCl2, pH 7.4), and 100-µl aliquots containing approximately 100 µg were stored at –80°C. Representative aliquots from each preparation were solubilized by boiling in the presence of 0.5% (w/v) SDS, and protein content was determined [38] to be 600-1000 µg/ml.

BSP Affinity Chromatography

For the identification of oviductal receptors for BSP proteins, affinity columns were made using each BSP bound to its specific antibody covalently bound to protein A-agarose beads (Pierce). Protease inhibitors were included in all column buffers. To bind each anti-BSP antibody to the protein A on the beads, the beads were transferred to small polypropylene columns and washed and equilibrated with PBS, resulting in a final bed volume of 1 ml. A total of 5–10 mg of the appropriate anti-BSP antibody in 3 ml PBS was applied to each column, and then the columns were capped and placed on a rotating shaker for 1 h at room temperature to allow antibody binding. Unbound antibody was removed by washing with 30 column volumes of PBS. Bound antibody then was covalently coupled to protein A by mixing the suspension for 1 h at room temperature in the presence of 3 mM DSS. The column was drained and washed with 5 column volumes of 50 mM glycine, pH 2.5, to quench the reaction and remove noncovalently attached IgG.

To bind each BSP to its antibody on each column, 200 µg of PDC109, BSPA3, or BSP30K was added to the corresponding anti-BSP antibody column along with sufficient PBS to facilitate mixing, and BSPs were allowed to bind by rocking for 4 h at room temperature. Columns were washed with PBS to remove unbound BSPs, then equilibrated with BSA-free TALP.

To capture BSP receptors on the columns, OAPM aliquots were thawed and membrane proteins were extracted with an equal volume of 2% (w/v) octyl β-D-glucopyranoside (OBG) in BSA-free TALP by gentle mixing for 1 h at 4°C in the presence of protease inhibitors. Before addition to the BSP affinity columns, the extracts were precleared by adsorption with protein A-agarose beads for 30 min to remove nonspecifically bound materials. Supernatants were recovered by centrifugation at 1000 x g and applied to the BSP-anti-BSP columns and allowed to bind overnight with constant rocking at 4°C. Unbound material was washed from the columns with BSA-free TALP, and bound materials were eluted with 5 mM EGTA. Fractions were processed for analysis using PAGEprep resin (Pierce), resolved on 12% SDS-PAGE gels, and stained with silver nitrate [35] or colloidal Coomassie blue G (Invitrogen, Carlsbad, CA).

Mass Spectrometry (MS)

Coomassie-stained bands of proteins eluted by EGTA from the BSP-anti-BSP affinity columns were excised and submitted to the Cornell Bioresource Center for identification by nanoLC-MS/MS analysis. Each protein band was placed into a 0.5-ml microtube for in-gel tryptic digestion and manual extraction following a protocol modified from Shevchenko et al. [39]. Extracts were combined, evaporated to dryness by a Speedvac SC110 (Thermo Savant, Milford, MA), and then reconstituted in 0.1% formic acid and 2% acetonitrile. Nanoscale liquid chromatography was performed using an LC Packings Ultimate integrated capillary high-performance liquid chromatography system by injecting peptides into a C18 µ-precolumn cartridge for on-line desalting followed by separation on a PepMap C-18 RP nano column (Dionex, Sunnyvale, CA). The nanoLC was connected in-line to a hybrid triple quadrupole linear ion trap mass spectrometer, 4000 Q Trap from ABI/MDS Sciex (Framingham, MA) equipped with Micro Ion Spray Head ion source.

The data acquisition on the MS was performed using Analyst 1.4.1 software (Applied Biosystems, Foster City, CA) in the positive-ion mode for information-dependant acquisition (IDA) analysis. In IDA analysis, after each survey scan for 400 to 1550 m/z and an enhanced resolution scan, the three highest-intensity ions with multiple charge states were selected for tandem MS (MS/MS) with rolling collision energy applied for detected ions based on different charge states and m/z values. The MS/MS data generated were submitted to Applied Biosystems ProID 1.4 search engine for searching against a bovine database that was created within ProID 1.4. One trypsin-missed cleavage, the carbobamidomethyl modification of cysteine, and a methionine oxidation were used for searching. Protein identification was limited to peptide hits with >95% statistical significance.

Histochemistry

Oviducts were surgically recovered from heifers as previously described [15]. Isthmic portions were separated by dissection into 1-cm segments and immersed in Bouin fixative (1:10 volume ratio) for 22 h. Tissue was dehydrated, embedded in paraffin, cut into 4-µm sections, and mounted on poly-L-lysine-coated slides. Tissue sections were deparaffinized in xylene, then rehydrated through graded dilutions of ethanol (100%, 95%, 70%, and 35%), followed by two washes in PBS. Antigen retrieval was performed by microwaving slides for 20 min in 10 mM trisodium citrate (pH 6). Endogenous peroxidases were inactivated by incubating slides for 15 min in 3% H2O2 in methanol, followed by three 5-min washes in PBS. Sections were blocked with nonimmune goat serum for 30 min, then incubated for 1 h each with a mouse monoclonal anti-ANXA IgG (20 µg/ml), followed by HRP-conjugated goat anti-mouse IgG (diluted 1:100). For detection of fucose, sections were incubated with biotinylated LTL lectin (10 µg/ml) and streptavidin-HRP (200 ng/ml) in place of the antibodies. Antibody and lectin binding were visualized by development with 3,3'-diaminobenzidine tetrahychloride (DAB; Kirkegaard & Perry Laboratories, Gaithersburg, MD) and stopped by washing in water. Slides were counterstained with 2x Gill hematoxylin (Fisher Scientific, Pittsburgh, PA), dehydrated through graded dilutions of ethanol, washed with xylene, and mounted in Permount (Fisher Scientific). Negative controls for antibody-probed sections consisted of omitting the anti-ANXA IgGs and replacing them with normal mouse IgG. Lectin controls consisted of blocking LTL binding with 100 mM fucose.

Annexin Immunoprecipitation

OAPM (100 µg) suspended in HBBS and protease inhibitors was solubilized using 1% OBG. Solubilized proteins were precleared by incubation with protein A-agarose beads, then incubated with anti-ANXA antibodies for 90 min on ice. Monoclonal antibodies were used to immunoprecipitate ANXAs 1, 2, and 4, whereas the polyclonal antibody was used for ANXA5. Immune complexes were captured on protein A-agarose beads for 1 h at room temperature. Unbound materials were removed by washing the beads with HBBS supplemented with OBG and protease inhibitors. Beads were recovered by centrifugation and eluted by boiling in SDS sample buffer.

Western and Lectin Blotting

Following SDS-PAGE, SDS-solubilized OAPM proteins and immunoprecipitated ANXAs were electroblotted [40] to polyvinylidene fluoride (PVDF; Millipore, Billerica, MA), and the blots were blocked with Protein-Free Blocking Buffer (Pierce) supplemented with 0.05% (v/v) Tween-20. Blots were probed with mouse monoclonal anti-ANXA antibodies (1 µg/ml anti-ANXA1 IgG, 500 ng/ml anti-ANXA2 IgG, 1 µg/ml anti-ANXA4 IgG, or 2 µg/ml polyclonal anti-ANXA5 IgG) followed by HRP-conjugated secondary antibodies (1:20 000 dilution), or with 10 µg/ml biotinylated LTL lectin followed by 200 ng/ml streptavidin-HRP. Reactive proteins were visualized by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Pierce) and film detection. Omission of primary antibodies was used to check for anti-ANXA specificity and LTL was preabsorbed with 100 mM fucose to check for fucose specificity of the lectin labeling.

Preparation of Oviductal Explants

Isthmic segments of oviducts obtained as described above were squeezed with fine tweezers to extract sheets of epithelium, which were broken into smaller pieces by gentle pipetting. The pieces were centrifuged for 1 min at 170 x g, transferred to TALP, and allowed 30–60 min of incubation at 38.5°C, 5% CO2 to form vesicles, termed explants, with apical cell surfaces facing outward.

Sperm Preparation

Semen samples from fertile Holstein bulls were diluted in egg yolk extender, individually loaded into 0.5-ml insemination straws at a concentration of 60 x 106 sperm/ml, frozen in liquid nitrogen (LN2), and donated by Genex/CRI (Ithaca, NY). Frozen semen was thawed by immersion in a 37°C water bath for 30 sec and expelled into culture tubes. Semen was overlaid with 1 ml TALP and incubated for 1 h at 38.5°C, 5% CO2, and 95% relative humidity to select for motile sperm via swim-up. After incubation, 750 µl was removed from the top of each tube, and sperm were washed through 5 ml TALP by centrifugation (170 x g, 10 min). Sperm were suspended in warm TALP and adjusted to 50 x 106/ml. The motility of sperm used for binding assays exceeded 85%.

Sperm-Oviduct Binding Assays

Oviductal explants in 5 ml TALP were centrifuged (170 x g, 1 min), and 5 µl of the pellet was added to 10 µl TALP under mineral oil. Next, 5 µl anti-ANXA antibody or mouse control IgG or normal rabbit serum was added to the explants to a concentration of 500 µg/ml and incubated for 30 min at 38.5°C, 5% CO2. Explants were transferred through three droplets of TALP to remove unbound antibody and were placed in fresh 20-µl TALP droplets. Sperm (5 x 106/ml) were added in 10-µl aliquots. After 15 min, loosely bound sperm were removed from explants by pipetting through three droplets of TALP. The explants were transferred to prewarmed slides and covered with coverslips supported by two layers of parafilm, forming a chamber approximately 200 µm deep.

Explants were viewed on a 38.5°C microscope stage using a Zeiss Axiovert Microscope (Carl Zeiss Inc., Thornbrook, NY) with a 30x Hoffman modulation contrast objective (Modulation Optics, Greenvale, NY). Recording was performed using a black-and-white video camera (model CCD72; Dage-MTI Inc., Michigan City, IN) in combination with a LITEON HDD/DVD recorder (Freemont, CA) and a time/date recorder (For-A Corporation of America, Los Angeles, CA). Several microscope fields of each treatment group were recorded and assessed for sperm binding density.

To determine the density of sperm bound to explants, video recordings were analyzed using playback mode, and the number of motile sperm bound to ciliated surfaces of explants was counted. Acetate tracings of video screen images were cut out and weighed to determine explant surface area. Weights were converted to area using the weight of a reference tracing of known area determined by videotaping a micrometer slide with 0.1-mm divisions. The binding density was calculated as the number of sperm bound per (0.1 mm)2 of explant surface. Explant surface areas of 12.3 x 104 ± 2.0 x 104 µm2 were analyzed per treatment for each replicate experiment.

Statistical Analysis

Sperm binding experiments were repeated at least three times, and results are expressed as means ± SEM. All data were analyzed using one-way analysis of variance (ANOVA) to test for statistically significant differences among treatments affecting sperm binding, followed by a Tukey Honestly Significant Difference pairwise comparison test using VassarStats interactive web-based freeware, available at http://faculty.vassar.edu/lowry/VassarStats.html.

RESULTS

Purified BSPs Were Obtained from Seminal Plasma

Gelatin affinity chromatography of BSP proteins produced two peak fractions. The first peak contained BSP30K and BSPA3, and the second contained BSP30K and the two forms of PDC109, namely, BSPA1 and BSPA2 (Fig. 1). The BSP proteins within each peak were separated by gel filtration and DEAE affinity chromatography, respectively, and purity was assessed by SDS-PAGE and Western blotting. Purified BSP30K migrated as a single band of approximately 27 kDa; PDC109 appeared as a doublet of 16 kDa corresponding to BSPA1 and BSPA2, which differ only in glycosylation [27]; and BSPA3 migrated at approximately 14.5 kDa (Fig. 1).


Figure 01
View larger version (53K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Silver-stained SDS-PAGE gels of fractionation of the BSP proteins from seminal plasma. Lane 1: the first peak eluted from the gelatin affinity column contained BSP30K and BSPA3; Lane 2: the second peak contained BSP30K and PDC109. Lanes 3–5: purified BSPA1/A2 (PDC109), BSPA3, and BSP30K, respectively.

Oviductal Receptors for BSPs are Annexins

OAPM proteins captured by BSP-anti-BSP protein A-agarose beads were eluted with EGTA, because sperm binding to oviductal epithelium had been shown to be Ca2+ dependent [16]. EGTA rapidly eluted two relatively abundant proteins of approximately 36 and 34 kDa (Fig. 2). PDC109, BSPA3, and BSP30K affinity columns each yielded protein bands of similar mass. When the bands shown in Figure 2 were excised and analyzed by mass spectrometry, the 36-kDa band was found to contain the annexins ANXA1, ANXA2, and ANXA5, whereas the 34-kDa band was found to contain ANXA4 and ANXA5 (Table 1). ANXA5 has been reported to occur in two forms of 36 and 34 kDa in human endothelial cells, probably due to posttranslational modification [41]. PDC109 captured all four annexins from OAPM, whereas BSPA3 captured ANXA1, ANXA2, and ANXA5, and BSP30K captured ANXA1, ANXA2, and ANXA4. Each BSP protein also was eluted from the column by EGTA. Protein bands of approximately 66 kDa eluted continuously from the PDC109 and BSP30K columns before and after EGTA application.


Figure 02
View larger version (53K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 BSP-binding proteins isolated from OAPM by BSP affinity chromatography, resolved by SDS-PAGE, and stained with Coomassie blue. Arrows indicate bands of approximately 36 and 34 kDa that were eluted by EGTA and subsequently excised for mass spectrometry, which identified them as containing ANXA proteins. A) Fractions from PDC109 affinity column. Lanes 1–2, wash; lanes 3–5, EGTA elution. Arrowhead indicates elution of PDC109. B) Fractions from BSP30K affinity column. Lane 1, wash; lanes 2–5, elution. Arrowhead indicates elution of BSP30K. C) EGTA elution fraction from BSPA3 affinity column. Arrowhead indicates elution of BSPA3.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1 LC-MS/MS Identification of BSP-binding proteins isolated from apical plasma membrane fraction of bovine oviduct epithelium.

Annexins Localize to the Apical Surfaces of Oviductal Epithelium

Immunohistochemistry localized ANXA1, ANXA2, ANXA4, and ANXA5 to the apical surface of the oviductal epithelium, including the cilia (Fig. 3, A–D), where bull sperm are most often seen to bind [1]. The distribution of the annexins on the surface was patchy, which is also how sperm distribute on the epithelial surface of explants in vitro [1]. When isthmic oviduct sections were probed with LTL for the presence of fucosylated molecules, a patchy surface distribution again was observed (Fig. 3G).


Figure 03
View larger version (118K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 Localization of ANXAs and fucose in sections of bovine isthmic oviductal epithelium. Mouse monoclonal antibodies were used to localize ANXAs, and fucose lectin LTL was used to localize fucose. Arrows indicate positive staining associated with the ciliated cell surface. A) ANXA1, B) ANXA2, C) ANXA4, D) ANXA5, E) mouse IgG control, F) second antibody alone control, G) fucose labeling with LTL, and H) LTL labeling blocked by fucose. The scale bars equal 10 µm.

Bovine Oviductal Annexins Contain Fucose

Proteins extracted from OAPM were resolved by SDS-PAGE and transferred to PVDF. The blots were probed with anti-ANXA antibodies to demonstrate the presence of ANXAs in apical membranes, as shown in Figure 4A. Because bull sperm bind to fucose-containing molecules on the oviductal epithelium [15], the oviductal ANXAs were assayed for the presence of fucose. OAPM protein extracts were immunoprecipitated with individual anti-ANXA antibodies, and the captured fractions were resolved by SDS-PAGE, electroblotted, and probed with LTL fucose lectin. As seen in Figure 4B, ANXAs 1, 2, 4, and 5 contain fucose. In the case of ANXA4, a doublet was resolved by the antibody, whereas the lectin produced a broad band at the site. In the case of ANXA5, an additional faint band of approximately 31 kDa was detected.


Figure 04
View larger version (124K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 Anti-ANXA and fucose lectin probing of ANXAs in membranes of oviductal epithelium. A) Western blots of solubilized OAPMs probed with antibodies to ANXA1, ANXA2, ANXA4, and ANXA5. B) ANXAs immunoprecipitated from detergent extracts of OAPMs probed with biotinylated fucose lectin LTL. When stripped and reprobed with LTL in the presence of 100 mM fucose, labeling was abolished (not shown).

Anti-ANXA Antibodies Inhibit Sperm Binding to Oviductal Epithelium

Fresh explants derived from bovine isthmic oviductal epithelium were treated with anti-ANXA IgG or control nonspecific IgG and then rinsed to remove unbound antibodies before sperm were added. When anti-ANXAs 1, 2, and 4 were bound to the surface of explants, sperm binding was reduced by 78%, 67%, and 70%, respectively, compared with explants incubated with nonspecific IgG (Fig. 5A). Similarly, an anti-ANXA5 monoclonal antibody reduced binding by 62%, and a polyclonal anti-ANXA5 antibody reduced binding by 79% (Fig. 5B).


Figure 05
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 Anti-ANXA antibodies inhibit sperm binding to explants of oviductal epithelium. Values represent mean number of sperm bound per (0.1 mm)2 ± SEM. Different letters indicate significant treatment effects, P < 0.01. A) Inhibition of sperm binding by mouse monoclonal antibodies to ANXAs 1, 2, and 4 compared with nonspecific mouse IgG as a control (sperm from four bulls). B) Inhibition of sperm binding by antibodies to ANXA5 (sperm from three bulls). Mouse IgG was used as a control for the mouse monoclonal anti-ANXA5 antibody, and rabbit serum was used as a control for rabbit polyclonal anti-ANXA5 antibody.

DISCUSSION

We have identified the annexins ANXA1, ANXA2, ANXA4, and ANXA5 as candidates for the oviductal epithelium sperm receptors that hold bull sperm in the oviductal reservoir. To our knowledge, this is the first identification of oviductal receptors for mammalian sperm. Our identification of these annexins as candidates was verified by 1) demonstrating that all four ANXAs are present on the apical surface of oviductal mucosal epithelium, particularly on the cilia, where most sperm are seen to bind [1]; 2) showing that antibody to each ANXA blocks sperm binding to oviductal epithelium; and 3) showing that these ANXAs contain fucose, which was previously shown to be a key component of the oviductal sperm receptor [15].

Epididymal bovine sperm bind poorly to oviductal epithelium, but addition of any one of the three BSPs purified from seminal plasma significantly raises the level of binding [28]. Furthermore, addition of any one of the BSPs to epididymal sperm prolongs their motile lives when they are incubated in vitro with plasma membranes derived from the apical surfaces of oviductal epithelium [28]. Thus, each BSP alone can promote sperm binding and preservation in the oviductal reservoir. In our study, each of the BSP proteins captured three or four ANXAs on the affinity columns; however, it is not known whether each ANXA can also act alone to bind and protect sperm or whether they must form complexes with each other or with additional proteins. We did notice that the distributions of the ANXAs seen by immunohistochemistry did not overlap completely, indicating that ANXAs may act somewhat independently of each other.

ANXAs comprise a large family of proteins of diverse but mostly poorly understood functions. Some have been localized within cells or on cellular surfaces, and some are secreted [42]. In our study, the immunolabeling of the epithelial surfaces, along with the ability of anti-ANXA antibodies to block sperm binding, provides strong evidence that the oviductal ANXAs are expressed on the surface of the epithelium. Although annexins lack signal peptides to direct them to the cell surface, other mechanisms have been identified that transport them from the cytoplasm to the plasma membrane [43]. ANXA4 and ANXA5 have been localized to the apical regions of rat oviductal epithelium [44], whereas ANXA1 was detected in extracts of rabbit oviduct epithelium [45]. ANXA1 and ANXA2 are associated with the cilia of quail oviduct epithelial cells [46]. In nonreproductive tissues, ANXA1 and ANXA2 are components of the cilia of the nasal epithelium of rabbits [47], human bronchial epithelium [48], and upper respiratory tract epithelium [49]. Cell surface ANXA2 acts as a plasminogen receptor in endothelial cells [50], and it also mediates cell adhesion between lymphocytes and endothelial cells [51], which might occur via a similar mechanism as sperm binding to oviductal epithelium. In summary, all four of the ANXAs identified in our study have been localized by others to cell surfaces.

ANXA5 conjugated with fluorescein has been shown to bind to the plasma membrane in the acrosomal region of bull [52] and boar [53] sperm. Although ANXA5 is commonly used as a probe to detect cells undergoing apoptosis, other markers of apoptosis showed that ANXA5-labeled boar sperm were not apoptotic [53].

Interestingly, ANXA1 is secreted in fairly high amounts (140 µg/ml) by the human prostate gland into the seminal plasma [54]. A protein with strong homology to human ANXA5 was isolated from rabbit seminal plasma and found to inhibit in vitro fertilization when added to sperm [55]. It is not known whether sperm carry the seminal plasma ANXAs into the oviduct, where they might be replaced by the ANXAs on the oviductal epithelium.

ANXAs have been shown to bind with high affinity heparin, heparan sulfate, and related glycosaminoglycans [56, 57]. This is also true for the BSPs [58, 59]. In cattle, heparin-like glycosaminoglycans are present in oviductal fluid and act as capacitating factors in vitro [31, 60]. Incubation with heparin in vitro for 4 h has been shown to reduce the amount of PDC109 on sperm [23]. Sperm binding to oviductal epithelium is dependent upon the capacitation status of sperm, and only noncapacitated sperm exhibit binding [3, 61, 62]. The release of sperm adhering to oviductal epithelial cells in vitro is induced by glycosaminoglycans [63], and released sperm exhibit characteristics of capacitation, including increased intracellular Ca2+ and protein tyrosine phosphorylation [64]. Because both BSPs and ANXAs bind heparinlike glycosaminoglycans, the glycosaminoglycans in oviduct fluid might competitively disrupt the interaction between BSPs and ANXAs. Thus, theoretically, heparinlike glycosaminoglycans in oviduct fluid could play a role in bull sperm release from the epithelium by capacitating sperm and/or by disrupting the binding of BSPs to ANXAs.

The involvement of multiple species of BSPs and ANXAs in sperm binding underscores the importance of holding sperm in the oviductal reservoir. During evolution, when gene duplication occurs and multiple closely related versions of genes are maintained in the genome, all of which actively produce protein products, it is likely that these gene products serve important functions and provide reproductive advantages to the individuals that produce them. The differences among the duplicated gene products can ensure that the system is functional under a variety of conditions. An example of this is an isoenzyme, which provides catalytic activity under a broad range of conditions [65]. The BSP proteins differ from each other in distribution of surface electrostatic charge [28]. This bestows the BSPs with different binding affinities for the surface of sperm on one face of the molecule and for the oviductal epithelium on the opposite face. Similarly, the various ANXAs on the oviductal epithelium must have different binding affinities and kinetics for the BSPs on sperm. Thus, the duplication of BSP and ANXA proteins on the sperm side and the oviduct side of the interaction, respectively, can provide a finely tuned regulatory system to ensure that sperm are held and kept fertile in the reservoir and then released gradually at the appropriate time to ensure that fertilization (but not polyspermy) takes place.

In conclusion, ANXAs are good candidates for the oviductal receptors for sperm.

FOOTNOTES

1Supported by U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service National Research Initiative CGP grant 2004-35203-14952. Back

Correspondence: 2Susan S. Suarez, Department of Biomedical Sciences, T5-006 Veterinary Research Tower, Cornell University, Ithaca, NY 14853. FAX: 607 253 3541; e-mail: sss7{at}cornell.edu

Received: 30 April 2007.

First decision: 24 June 2007.

Accepted: 21 August 2007.

REFERENCES

  1. Lefebvre R, Chenoweth PJ, Drost M, LeClear CT, MacCubbin M, Dutton JT, Suarez SS. Characterization of the oviductal sperm reservoir in cattle Biol Reprod 1995 531066–1074[Abstract]
  2. Suarez SS. Sperm transport and motility in the mouse oviduct: observations in situ Biol Reprod 1987 36203–210[Abstract]
  3. Smith TT, Yanagimachi R. Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct J Reprod Fertil 1991 91567–573[Abstract/Free Full Text]
  4. Suarez SS, Redfern K, Raynor P, Martin F, Phillips DM. Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir Biol Reprod 1991 44998–1004[Abstract]
  5. Hunter RHF, Nichol R. Transport of spermatozoa in the sheep oviduct: preovulatory sequestering of cells in the caudal isthmus J Exp Zool 1983 228121–128[CrossRef][Medline]
  6. Thomas PGA, Ball BA, Brinsko SP. Interactions of equine spermatozoa with oviduct epithelial cell explants is affected by estrous cycle and anatomic origin of explant Biol Reprod 1994 51222–228[Abstract]
  7. Pollard JW, Plante C, King WA, Hansen PJ, Betteridge KJ, Suarez SS. Fertilizing capacity of bovine sperm may be maintained by binding to oviductal epithelial cells Biol Reprod 1991 44102–107[Abstract]
  8. Chang RC, Sirard MA. Fertilizing ability of bovine spermaotzoa cocultured with oviductal epithelial cells Biol Reprod 1994 52156–162[CrossRef]
  9. Dobrinski I, Smith TT, Suarez SS, Ball BA. Membrane contact with oviductal epithelium modulates the intracellular calcium concentration in equine spermatozoa in vitro Biol Reprod 1997 56861–869[Abstract]
  10. Chian RIC, LaPointe S, Sirard MA. Capacitation in vitro of bovine sperrmatozoa by oviductal cell monolayer conditioned medium Mol Reprod Dev 1995 42318–324[CrossRef][Medline]
  11. Hunter RHF, Leglise PC. Polyspermic fertilization following tubal surgery in pigs, with particular reference to the role of the isthmus J Reprod Fertil 1971 24233–246[Abstract/Free Full Text]
  12. Polge C, Salamon S, Wilmut I. Fertilizing capacity of frozen boar semen following surgical insemination Vet Rec 1970 87424–428[Medline]
  13. DeMott RP, Lefebvre R, Suarez SS. Carbohydrates mediate the adherence of hamster sperm to oviductal epithelium Biol Reprod 1995 521395–1403[Abstract]
  14. Lefebvre R, DeMott RP, Suarez SS, Samper JC. Specific inhibition of equine sperm binding to oviductal epithelium In: Sharp DC, Bazer FW (eds.), Equine Reproduction VI Madison, WI Society for the Study of Reproduction 1995689–696
  15. Lefebvre R, Lo M, Suarez SS. Bovine sperm binding to oviductal epithelium involves fucose recognition Biol Reprod 1997 561198–1204[Abstract]
  16. Suarez SS, Revah I, Lo M, Kolle S. Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognizes Lewis-a trisaccharide Biol Reprod 1998 5939–44[Abstract/Free Full Text]
  17. Green CE, Bredl J, Holt WV, Watson PF, Fazeli A. Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro Reproduction 2001 122305–315[Abstract]
  18. Wagner A, Ekhlasi-Hundrieser M, Hettel C, Petrunkina A, Waberski D, Nimtz M, Töpfer-Petersen E. Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig Mol Reprod Dev 2002 61249–257[CrossRef][Medline]
  19. Ignotz GG, Lo MC, Perez CL, Gwathmey TM, Suarez SS. Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir Biol Reprod 2001 641806–1811[Abstract/Free Full Text]
  20. Salois D, Menard M, Paquete Y, Manjunath P. Complemenary deoxyribonucleic acid cloning and tissue expression of BSPA-3 and BSP-30-kDa: phosphatidylcholine and heparin-binding proteins of bovine seminal plasma Biol Reprod 1999 61288–297[Abstract/Free Full Text]
  21. Desnoyers L, Manjunath P. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipids J Biol Chem 1992 26710149–10155[Abstract/Free Full Text]
  22. Muller P, Erlemann KR, Muller K, Calvete JJ, Topfer-Petersen E, Marienfeld K, Hermann A. Biochemical characterization of the bovine seminal plasma protein PDC-109 with phospholipid vesicles Eur Biophys J 1998 2733–41[CrossRef][Medline]
  23. Gwathmey TM, Ignotz GG, Suárez SS. PDC-109 (BSP-A1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir Biol Reprod 2003 69809–815[Abstract/Free Full Text]
  24. Seidah NG, Manjunath P, Rochemont J, Sairam MR Chrétien. Complete amino acid sequence of BSP-A3 from bovine seminal plasma Biochem J 1987 243195–203[Medline]
  25. Calvete JJ, Mann K, Sanz L, Raida M, Töpfer-Petersen E. The primary structure of BSP-30K, a major lipid-, gelatin-, and heparin-binding glycoprotein of bovine seminal plasma FEBS Lett 1996 399147–52[CrossRef][Medline]
  26. Calvete JJ, Raida M, Sanz L, Wempe F, Scheit KH, Romero A, Töpfer-Petersen E. Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109. Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa FEBS Lett 1994 350203–206[CrossRef][Medline]
  27. Manjunath P, Sairam MR. Purification and biochemical characterization of thee major acidic proteins (BSP-A1, BSP-A2 and A-3) from bovine seminal plasma Biochem J 1987 241685–692[Medline]
  28. Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez SS. Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct Biol Reprod 2006 75501–507[Abstract/Free Full Text]
  29. Nauc V, Manjunath P. Radioimmunoassays for bull seminal plasma proteins (BSP-A1/-A2, BSP-A3, and BSP-30-Kilodaltons), and their quantification in seminal plasma and sperm Biol Reprod 2000 631058–1066[Abstract/Free Full Text]
  30. King KB, Chubinskaya S, Reid DL, Madsen LH, Mollenhauer J. Absence of cell-surface annexin V is accompanied by defective collagen matrix binding in the Swarm rat chondrosarcoma J Cell Biochem 1997 65131–144[CrossRef][Medline]
  31. Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin Biol Reprod 1988 381171–1180[Abstract]
  32. Manjunath P, Sairam MR, Uma J. Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography Biosci Rep 1987 7231–238[CrossRef][Medline]
  33. Gasset M, Saiz JL, Laynez J, Sanz L, Gentzel M, Topfer-Petersen E, Calvete JJ. Conformational features and thermal stability of bovine seminal plasma protein PDC-109 oligomers and phosphorylcholine-bound complexes Eur J Biochem 1997 250735–744[Medline]
  34. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature (Lond) 1970 227680–685[CrossRef][Medline]
  35. Blum H, Beier H, Gross HJ. Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels Electrophoresis 1987 893–99[CrossRef]
  36. Murray SC, Smith TT. Sperm interaction with fallopian tube apical membrane enhances sperm motility and delays capacitation Fertil Steril 1997 68351–357[CrossRef][Medline]
  37. Boilard M, Bailey J, Collin S, Dufour M, Sirard MA. Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach Biol Reprod 2002 671125–1132[Abstract/Free Full Text]
  38. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent J Biol Chem 1951 193265–275[Free Full Text]
  39. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins in silver-stained polyacrylamide gels Anal Chem 1996 68850–858[Medline]
  40. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc Natl Acad Sci U S A 1979 764350–4354[Abstract/Free Full Text]
  41. Bruneel A, Labas V, Mailloux A, Sharma S, Royer N, Vinh J, Pernet P, Vaubourdolle M, Baudi B. Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis Proteomics 2005 53876–3884[CrossRef][Medline]
  42. Rescher U, Gerke V. Annexins—unique membrane binding proteins with diverse functions J Cell Sci 2004 1172631–2639[Abstract/Free Full Text]
  43. Deora AB, Kreitzer G, Jacovina AT, Hajjar KA. An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface J Biol Chem 2004 27943411–43418[Abstract/Free Full Text]
  44. Kaetzel MA, Hazarika P, Dedman JR. Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins J Biol Chem 1989 26414463–14470[Abstract/Free Full Text]
  45. Tsao FH, Chen X, Chen X, Ts'ao CH. Annexin I in female rabbit reproductive organs: varying levels in relation to maturity and pregnancy Lipids 1995 30507–511[CrossRef][Medline]
  46. Chailley B, Pradel LA. Immunodetection of annexins 1 and 2 in ciliated cells from quail oviduct Biol Cell 1992 7545–54[CrossRef][Medline]
  47. Rodrigo JP, Garcia-Pedrero JM, Gonzalez MV, Fernandez MP, Suarez C, Herrero A. Expression of annexin A1 in normal and chronically inflamed nasal mucosa Arch Otolaryngol Head Neck Surg 2004 130211–215[Abstract/Free Full Text]
  48. Ostrowski LE, Blackburn K, Radde KM, Moyer MB, Schlatzer DM, Moseley A, Boucher RC. A proteomic analysis of human cilia: identification of novel components Mol Cell Proteomics 2002 1451–465[Abstract/Free Full Text]
  49. Rodrigo JP, Garcia-Pedrero JM, Pena AE, Fernandez MP, Morgan RO, Suarez C, Herrero A. Expression of annexins Al and A2 in the mucosa of the upper airdigestive tract Acta Otorrinolaringol Esp 2004 55310–314[Medline]
  50. Kang HM, Choi KS, Kassam G, Fitzpatrick SL, Kwon M, Waisman DM. Role of annexin II tetramer in plasminogen activation Trends Cardiovasc Med 1999 992–102[CrossRef][Medline]
  51. Tressler RJ, Updyke TV, Yeatmen T, Nicolson GL. Extracellular annexin II is associated with divalent cation-dependent tumor cell-endothelial cell adhesion of metastatic RAW117 large-cell lymphoma cells J Cell Biochem 1993 53265–276[CrossRef][Medline]
  52. Chaveiro A, Santos P, da Silva FM. Assessment of sperm apoptosis in cryopreserved bull semen after swim-up treatment: a flow cytometric study Reprod Domest Anim 2007 4217–21[Medline]
  53. Gadella BM, Harrison RA. Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells Biol Reprod 2002 67340–350[Abstract/Free Full Text]
  54. Christmas P, Callaway J, Fallon J, Jones J, Haigler HT. Selective secretion of annexin 1, a protein without a signal sequence, by the human prostate gland J Biol Chem 1991 2662499–2507[Abstract/Free Full Text]
  55. Okabe M, Kishi Y, Ying X, Kohama Y, Mimura T, Li SS. Characterization of capacitation inhibitory protein from rabbit seminal plasma: homology with human annexins Biol Pharm Bull 1993 16453–456[Medline]
  56. Ishitsuka R, Kojima K, Utsumi H, Ogawa H, Matsumoto I. Glycosaminoglycan binding properties of annexin IV, V, and VI J Biol Chem 1998 2739935–9941[Abstract/Free Full Text]
  57. Shao C, Zhang F, Kemp MM, Linhardt RJ, Waisman DM, Head JF, Seaton BA. Crystallographic analysis of calcium-dependent heparin binding to annexin A2 J Biol Chem 2006 28131689–31695[Abstract/Free Full Text]
  58. Chandonnet L, Roberts KD, Chapdelaine A, Manjunath P. Identification of heparin-binding proteins in bovine seminal plasma Mol Reprod Dev 1990 26313–318[CrossRef][Medline]
  59. Calvete JJ, Campanero-Rhodes MA, Raida M, Sanz L. Characterization of the conformational and quaternary structure-dependet heparin-binding region of bovine seminal plasma protein PDC-109 FEBS Lett 1999 444260–264[CrossRef][Medline]
  60. Parrish JJ, Susko-Parrish JL, Uguz C, First NL. Differences in the role of cyclic adenosine 3',5'-monophosphate during capacitation of bovine sperm by heparin or oviduct fluid Biol Reprod 1994 511099–1108[Abstract]
  61. Thomas PGA, Ball BA, Brinsko SP. Changes associated with induced capacitation influence the interaction between equine spermatozoa and oviduct epithelial cell monolayers Biol Reprod Monographs 1995 1697–705
  62. Lefebvre R, Suarez SS. Effect of capacitation on bull sperm binding to homologous oviductal epithelium Biol Reprod 1996 54575–582[Abstract]
  63. Talevi R, Gualtieri R. Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro Biol Reprod 2001 64491–498[Abstract/Free Full Text]
  64. Gualtieri R, Boni R, Tosti E, Zagami M, Talevi R. Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro Reproduction 2005 12951–60[Abstract/Free Full Text]
  65. Campbell AM, Heyer LJ. Discovering Genomics, Proteomics, and Bioinformatics San Francisco Benjamin Cummings 2003



This article has been cited by other articles:


Home page
 ReproductionHome page
R. Gualtieri, V. Mollo, G. Duma, and R. Talevi
Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation
Reproduction, July 1, 2009; 138(1): 33 - 43.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
M. G. Gervasi, M. Rapanelli, M. L. Ribeiro, M. Farina, S. Billi, A. M. Franchi, and S. P. Martinez
The endocannabinoid system in bull sperm and bovine oviductal epithelium: role of anandamide in sperm-oviduct interaction
Reproduction, March 1, 2009; 137(3): 403 - 414.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
L. C. Carrasco, R. Romar, M. Aviles, J. Gadea, and P. Coy
Determination of glycosidase activity in porcine oviductal fluid at the different phases of the estrous cycle
Reproduction, December 1, 2008; 136(6): 833 - 842.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
S. S. Suarez
Control of hyperactivation in sperm
Hum. Reprod. Update, November 1, 2008; 14(6): 647 - 657.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
A. Wong, S. N. Albright, J. D. Giebel, K. R. Ram, S. Ji, A. C. Fiumera, and M. F. Wolfner
A Role for Acp29AB, a Predicted Seminal Fluid Lectin, in Female Sperm Storage in Drosophila melanogaster
Genetics, October 1, 2008; 180(2): 921 - 931.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
77/6/906    most recent
biolreprod.107.062505v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ignotz, G. G
Right arrow Articles by Suarez, S. S
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ignotz, G. G
Right arrow Articles by Suarez, S. S
Agricola
Right arrow Articles by Ignotz, G. G
Right arrow Articles by Suarez, S. S

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS