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Laboratories for Reproductive Biology and Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, seminal vesicles, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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36–46 kDa); however, both monomer (18–23 kDa) and multimer forms are present [1]. Immunohistochemical localization of Eppin revealed particularly strong staining in the ciliated cells of the epithelium of the ductuli efferentes as well as strong staining of spermatozoa within the lumen [1]. Eppin is present on the surface of mouse spermatozoa both before and after capacitation [2] and, on the basis of protein expression data from Western blots and from immunohistochemical localization, a significant quantity of Eppin appears to be bound to the surface of human ejaculate spermatozoa [1]. During ejaculation, spermatozoa encounter a change in extracellular environmental conditions, particularly osmotic changes that require cell-volume adjustments when mixing with the secretions from the seminal vesicles and prostate. Bound surface molecules undergo a variety of modifications and/or removal and peripheral membrane proteins (decapacitation factors) are removed as spermatozoa begin capacitation in the female reproductive tract (see, e.g., [3, 4]). Caudal epididymal spermatozoa are in an immotile state and, immediately following ejaculation in humans, sperm continue to appear immotile in the semen coagulum until liquefaction occurs and progressive motility begins [5].
The role of semenogelin (Sg) during ejaculation has been reviewed by Robert and Gagnon [6]. Briefly, it is currently thought that Sg I and Sg II are initially protected from proteolysis by protein C inhibitor (PCI) in the seminal vesicles and that PSA (prostate-specific antigen, a serine protease) in the prostatic secretions is inactive, inhibited by high concentrations of zinc. PCI, a serine protease inhibitor, binds to both PSA and Sg II [7, 8]. During ejaculation, the mixing of Sg with prostatic secretions chelates most of the free zinc, which triggers release of PCI, aggregation of Sg and fibronectin, and activates PSA. PSA cleaves the coagulum proteins, resulting in the release of Sg proteolytic fragments. Extensive work by Gagnon's laboratory [9–14] has demonstrated that Sg is bound to the sperm surface and that the sperm motility inhibitory factor in semen is the N-terminal (amino acids 69–160) of Sg I. Cleavage of Sg I by PSA during liquefaction removes Sg I from the sperm surface. Failure to remove Sg I from the sperm surface results in loss of motility [9] and inhibition of capacitation [15].
The studies reported here, that Eppin and Sg I are bound together on human ejaculate spermatozoa, strengthen the concept that sperm-surface protein complexes are present on ejaculate spermatozoa for at least two functions: 1) protection through antimicrobial activity and 2) survival and preparation of spermatozoa for fertility in the female reproductive tract. A recent report on the N-terminal of Sg [16] and our recent study of Eppin [17] demonstrate the complexes' antimicrobial properties. The importance of Eppin for the preparation of spermatozoa for fertility is evidenced by our report that antibody to Eppin in the male reproductive tract of immunized male monkeys, which disrupts Eppin's function, renders the males infertile [18].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant Protein Production
An Eppin cDNA (nucleotides 70–423) lacking part of the N-terminal secretory sequence was generated by PCR using the eppin-1/Bluescript clone [1] as template. This PCR was performed with Pfx Platinium Polymerase (Invitrogen, Carlsbad, CA) and cloned into pET-100D/TOPO (Invitrogen). In a similar manner, a human semenogelin cDNA (nucleotides 82–849) was generated by PCR using a human seminal vesicle cDNA library as template (a gift from Dr. Frank R. French, University of North Carolina, Chapel Hill, NC) and cloned into pET-100D/TOPO.
To produce N- and C-terminal fragments of Eppin, PCR products were generated using an LA Taq Kit (Takara Bio, Shiga, Japan) with appropriate restriction enzyme sites added to the primers to allow directional cloning into pFLAG-MAC (Sigma). The N-terminal Eppin cDNA construct (nucleotides 73–288) expresses amino acids 18–76 (Eppin18–76) and the C-terminal Eppin construct (nucleotides 283–423) expresses amino acids 75– 133 (Eppin75–133).
Two recombinant fragments of Sg (gi| 2144902) N-terminal, Sg24–163 (amino acids 24–163) and Sg164–283 (amino acids 164–283), were produced from PCR products generated using an LA Taq Kit (Takara Bio) with appropriate restriction enzyme sites added to the primers to allow directional cloning into pFLAG-MAC (Sigma).
All constructs were verified by sequencing and expressed in DH5-
. Bacterial lysates were purified on Ni-NTA agarose (pET-100D/TOPO) or anti-FLAG-M2 affinity gels (pFLAG-MAC).
Antiserum Production
Production of antisera to recombinant human Eppin (anti-rEppin) has been described [1]. Affinity-purified rabbit antisera to N-terminal amino acids 20–39 of mouse Eppin were made by Bethyl Laboratories (Montgomery, TX). Cysteine residue 33 was changed to an alanine. These antisera (anti-Q20E) react with both mouse and human Eppin. Affinity-purified rabbit antisera to a human semenogelin peptide (anti-S20H, aa 104– 123) were made by Bethyl Laboratories (Montgomery, TX). Monoclonal antibodies to human semenogelin ([19]; MHS-5) were kindly provided by Dr. John Herr, University of Virginia.
Mass Spectrometry Identification of Eppin Binding Partners
Biotinylated human spermatozoa were immunoprecipitated with anti-Eppin antibodies and analyzed by SDS-PAGE. The resulting gel was stained overnight with 0.01% Bio-Rad R-250 Coomassie (Bio-Rad, Hercules, CA) in 10% acetic acid. Protein staining bands were excised, digested with trypsin, and analyzed by matrix assisted laser desorption/ionization (MALDI)/time of flight (TOF). MALDI TOF and MALDI TOF/ TOF were performed on an AB 4700 Voyager—Proteomics Discovery System (Applied Biosystems, Foster City, CA). The resulting peptide peaks were searched against the MSDB and NCBI databases using the MASCOT search engine. Mass spectrometry (MS) identification was done in the University of North Carolina-CH Proteomics Core.
Western Blot Analysis
Proteins were separated on reducing 10%–20% gradient gels (Bio-Rad) or, for Figure 1C, on reducing NuPAGE 4%–12% Bis-Tris gels (Invitrogen) and transblotted to Immobilon-P (Millipore) and either stained for protein with amido black or blocked with Tris buffered saline (TBS) (50 mM Tris pH 7.4, 150 mM NaCl) containing 3% BSA for 60 min at room temperature and probed with primary antibodies as described [1]. Two micrograms of recombinant protein were loaded per lane. Primary antibodies were used at a 1:2000 dilution and secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG, 1:2000) were either alkaline phosphatase labeled and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate or peroxidase labeled and developed with chemiluminescence using Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) according to the manufacturer's instructions.
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For far-Western blots, proteins were immobilized on Immobilon-P, blocked as above, incubated 1–2 h or overnight (4°C) in protein probes, washed, and detected with primary and secondary antibodies as described above. The protein concentrations were determined using the Micro BCA Protein Detection Reagents (Pierce).
Labeling and Quantitative Binding Assay
Labeling of 20 µg of rEppin or rSg with 125I was carried out with the Iodo-gen direct method (Pierce) according to the manufacturer's instructions and the free 125I removed with a micro Bio-spin 6 chromatography column (Bio-Rad). Proteins were immobilized on Immobilon-P, blocked as above, incubated for 1–4 h in either 125I rEppin or 125I rSg, and exposed for autoradiography overnight. The in vitro 125I-rSg binding assay was a modification of that described previously [20]. In this assay, 4 µg of rEppin were immobilized on a nitrocellulose membrane (0.45 µm) using a Bio-Dot microfiltration apparatus (Bio-Rad) and the membrane washed with Tris buffered saline-Tween (TBST) (50 mM Tris pH 7.4, 150 mM NaCl with 0.05% Tween 20) and blocked with 5% BSA in TBST. Triplicate bio-dots on a membrane with or without Eppin (control) were incubated in increasing amounts of 125I-rSg overnight at 4°C, washed in TBST, cut into 1-cm squares, each containing a single dot, and counted in a 
counter. To demonstrate competition for binding, increasing amounts of unlabeled rSg were added to the 125I-rSg Eppin bio-dot incubation mixtures.
Immunoaffinity Chromatography
Affinity chromatography was carried out with a Seize-X immunoprecipitation kit (Pierce) using affinity-purified anti-Q20E antibodies or antirecombinant Eppin antibodies. Human spermatozoa were washed several times in Biggers-Whitten-Whittingham (BWW) medium by centrifugation at 500 x g for 5 min and biotinylated with EZ-Link NHS-biotin (Pierce). Labeled sperm were washed three times in PBS, sonicated for 30 sec on ice, centrifuged (12 000 rpm, 10 min), and the supernatant applied to the anti-Eppin antibody Seize-X beads. Human seminal plasma, diluted 1:4 with Seize-X immunoprecipitation kit binding buffer (Pierce), was applied to the affinity-purified anti-Q20E antibody Seize-X beads. Columns were washed and eluted according to the manufacturer's instructions. Preimmune IgG from the immune rabbits served as the control for nonspecific binding to immunoaffinity beads with the same samples.
Immunofluorescence
Human spermatozoa were washed twice in BWW medium and once with PBS by centrifugation at 500 x g for 5 min and resuspended in PBS. Spermatozoa were fixed in 3.7% formaldehyde in PBS, washed, blocked with normal goat serum, and resuspended in PBS. For indirect immunofluorescence, spermatozoa were probed with mouse monoclonal antiserum to recombinant human semenogelin ([19]; MHS-5) and affinity-purified anti-Eppin (Q20E) at 1:100 dilutions. Antibody binding was detected with Alexa Fluor-488- or Alexa Fluor-568-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and Alexa Fluor-488-labeled goat anti-mouse IgG (Molecular Probes) at 1:5000 dilution.
Fluorescent images were obtained from a Zeiss Axiophot microscope equipped with Plan-apochromat 63 x 1.4 NA (numerical aperture) and Plan-neofluar 100 x 1.3 NA objectives, chroma filter sets (Chroma Technology Corp., Brattleboro, VT), and Zeiss filter set 25 (emission at 460, 530, and 610 nm) that allows detection of colocalized Alexa Fluor 488 and Alexa Fluor 568 probes (Molecular Probes). Images were recorded with a Zeiss AxioCam using AxioVision software (Zeiss MicroImaging, Thornwood, NY) and exported to Adobe Photoshop 5.0 to assemble Figure 2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoprecipitation of Eppin from human seminal plasma with affinity-purified antisera to Eppin amino acids 20– 39 (anti-Q20E) results in the coprecipitation of Eppin in its monomer (1x), dimer (2x), 4x, and 8x forms (arrow and asterisks, Fig. 1C, lane 1) and semenogelin (Sg), visualized as a 27-kDa protein (asterisk, Fig. 1C, lane 2) that corresponds to that identified by MALDI/TOF/MS. Smaller peptide fragments of 18–22 kDa from Sg are also immunoprecipitated with Eppin (Fig. 1A, lane 2, and Fig. 1C, lane 2).
Immediately following ejaculation in humans, sperm appear trapped in the semen coagulum as liquefaction and progressive motility begin [5]. As shown in Figure 2C, Eppin (red) and semenogelin (green) are both present within the coagulum and strongly colocalize in large aggregates (yellow, Fig. 2C). At higher magnification (Fig. 2D), semenogelin-coated sperm heads (green, arrow) are clearly bound to a patchwork of Eppin-Sg complexes within the coagulum. On individual spermatozoa within the mass of the coagulum, Eppin and semenogelin are often seen to colocalize (arrow, yellow) on the head and particularly in the postacrosomal region (Fig. 2E).
To study the binding of Eppin and semenogelin in more detail, in vitro experiments with recombinant Eppin (rEppin) and recombinant semenogelin (rSg) were carried out. As shown in Figure 3, when rEppin and rSg are incubated together for 3 h and immunoprecipitated with anti-Eppin (anti-Q20E), rSg (arrow) can be detected in the immunoprecipitate with monoclonal MHS-5. Immunoprecipitation with anti-Sg similarly results in the detection of Eppin in the immunoprecipitate (data not shown).
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Additionally, Eppin-semenogelin binding can be demonstrated by far-Western blotting. Figure 4A (lane 3) demonstrates the monomer (arrow) and multimer forms (*) of rEppin recognized by the anti-Q20E antiserum. When the blot is incubated in rSg, washed, and probed with anti-Sg (anti-S20H), all forms of rEppin bind rSg (lane 1). Antiserum to Sg (anti-S20H) does not recognize rEppin (lane 2). Control blots of an irrelevant recombinant protein (NASP) probed with rSg demonstrate that nonspecific binding to other His-tagged proteins does not occur (Fig. 4B, lane 5) and that anti-S20H does not recognize NASP or the His-tag (lane 6). Similarly, rSg on a blot binds rEppin by far-Western blot analysis (Fig. 4C). The 35-kDa rSg and incomplete translation products (lane 1) are recognized by antisemenogelin (lane 2). When the blot is incubated in rEppin, washed, and probed with anti-Eppin (anti-Q20E), the rSg binds rEppin (lane 3). Anti-Eppin does not recognize Sg (lane 4).
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Quantitative binding data (Fig. 5A) demonstrate that increasing amounts of 125I-rSg bind to 4 µg of rEppin in a saturable manner and that increasing amounts of unlabeled rSg compete for binding sites (Fig. 5B). The binding of 125I-rSg to rEppin can be visualized on blots probed with 125I-rSg. Figure 6A demonstrates that rEppin on a blot (lane 1) binds 125I-rSg (lane 2) and that, when competed with 10x unlabeled rSg, the binding is significantly reduced (lane 3). Conversely, rSg on a blot binds 125I-rEppin (data not shown).
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To address the specificity of Eppin-semenogelin binding, we expressed fragments of both proteins. The Eppin N-terminal fragment (Eppin18–76) appears as a single protein-stained band with only weak multimer formation (Fig. 6B, lane 1) while the Eppin C-terminal fragment (Eppin75–133) appears as several protein-stained bands (Fig. 6B, lane 3), indicating that the multimer bands of intact Eppin (Figs. 4A and 6A) may be a result of C-terminal intermolecular interaction. 125I-rSg binds to the C-terminal, Eppin75–133 fragment (Fig. 6B, lanes 3 and 4) and not to the N-terminal, Eppin18–76 fragment.
Incubation of 125I-rEppin with the two recombinant fragments of the Sg N-terminal, Sg24–163 (Fig. 7, lane 1; amino acids 24–163) and Sg164–283 (Fig. 7, lane 3; amino acids 164–283) demonstrates that Eppin binding is restricted to Sg164–283 (Fig. 7, lanes 3 and 4). The Sg164–283 fragment contains the only cysteine residue (Cys239) present in Sg. Therefore, we tested whether the reduction and carboxymethylation of Cys239 would block the binding of 125I-rEppin. As shown in Figure 7, reduction and carboxymethylation of Cys239 blocks binding of 125I-rEppin to Sg164– 283 (lane 6), while the nonreduced Cys239 does not (lane 5). Consequently we conclude that the C-terminal amino acids of Eppin (75–133) contain a binding site for semenogelin. Semenogelin amino acids 164–283 contain a binding site for Eppin and a nonreduced cysteine residue is necessary for this binding.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The physiological significance of the Eppin-Sg complex bound on the surface of ejaculate spermatozoa lies in its ability to provide antimicrobial activity for spermatozoa, which has been reported for both Eppin- [17] and semenogelin-derived peptides [16] and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract [18]. Additionally Eppin may protect spermatozoa from proteolytic attack by allowing cleavage of Sg bound to Eppin but not of Eppin itself. Semenogelin that is coimmunoprecipitated with Eppin from spermatozoa and seminal plasma has an apparent molecular weight of 
27 kDa, which is smaller than full-length Sg (52 kDa). Therefore, the recovered Sg fragments bound to Eppin (Fig. 1) have probably been cleaved by PSA (a serine protease with restricted chymotrypsin-like specificity) or similar protease in the ejaculate [9]. PSA cleaves Sg almost exclusively at tyrosine and leucine residues [21], and there are two known cleavage sites (Leu-246 and Leu-261), either of which would provide an N-terminal semenogelin peptide fragment of approximately 27 kDa. Our finding that the Eppin binding site on Sg is within amino acids 164– 283 would allow PSA to cleave Sg and the 27-kDa fragment would still remain attached to Eppin on the sperm surface. However, continued PSA activity would mostly likely remove Sg from its Eppin-binding site. Similarly, the sperm motility inhibitory factor in semen described by Gagnon's laboratory [9] (Sg amino acids 85–136 in [21]; gi| 2144902 amino acids 108–160) is located within the Sg fragment bound to Eppin. It too would most likely be removed with continued PSA activity because its presence can inhibit capacitation [15].
As reported previously [1], native Eppin occurs as multimers in both seminal plasma and in the epididymis. These are thought to form by the intermolecular interaction of the 14-cysteine residues. Mass spectroscopy studies on reduced and carboxymethylated recombinant forms of Eppin have determined that the actual mass of the dimer is 33 kDa and that it is unlikely that every cysteine is carboxymethylated (unpublished results). The present study demonstrates that, while multimer recombinant forms of Eppin can bind Sg (Fig. 4), only the native monomers binds Sg in the overlay technique (Fig. 1). Moreover, it is the Eppin75–133 C-terminal fragment (Fig. 6) that binds Sg (Sg164–283, Fig. 7), and this sequence contains the only cysteine in human Sg I (Cys-239), which is necessary for Eppin binding. If a disulfide linkage occurs between Sg and Eppin, it might allow several Sg molecules (or fragments) to bind Eppin, multiplying Eppin's effectiveness as a binding site. Quantitative determination of Sg binding to Eppin (Fig. 5) shows that the binding is saturable; however, a calculation of the number of binding sites is not realistic because the 4 µg Eppin used in the microtiter wells was in a highly multimeric state of unknown molecular weight.
In conclusion, the Eppin-Sg complex found on human ejaculate spermatozoa is thought to be part of a larger network of protein complexes on the sperm surface that provides a protective shield before capacitation in the female reproductive tract. Such sperm-coating proteins function in innate immunity [17], antimicrobial activity, and inhibition of proteases that may directly attack the sperm plasma membrane. Anti-Eppin antibodies may disrupt the Eppin-Sg complex and inhibit the proper removal of Sg by PSA, forming the basis for the inhibition of fertility by anti-Eppin antibodies [18]. Colocalization of Eppin and Sg in the sperm-seminal plasma coagulum (Fig. 2, C and D) allows one to visualize the unquestionable need for spermatozoa to escape this coagulum to become fertile and proceed along the female reproductive tract.
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FOOTNOTES |
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2 Correspondence: Dr. Michael G. O'Rand, Department of Cell and Developmental Biology, CB# 7090, 212 Taylor Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090. FAX: 919 966 1856; morand{at}unc.edu
3 Current address: Department of Urology, Duke University, Durham, NC 27710
Received: 21 September 2004.
First decision: 6 October 2004.
Accepted: 3 December 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) indicates the dimer (33–36 kDa). The position of molecular weight standards (kDa) is indicated on the left. C) Western blot of immunoprecipitation of Eppin and semenogelin from human seminal plasma with affinity-purified anti-Q20E antibody. Lane 1: arrow (
), the monomer (16–18 kDa); asterisks (*), the multimers of Eppin; lane 2: asterisk (*), semenogelin (27 kDa). The position of molecular weight standards (kDa) is indicated on the right. Lane 1: probed with anti-Q20E (1:2000), secondary antiserum peroxidase-labeled goat anti-rabbit IgG (1: 2000) developed with chemiluminescence; lane 2: probed with antisemenogelin, MHS-5 monoclonal antibody (1:2000) secondary antiserum peroxidase-labeled goat anti-mouse IgG (1:2000) developed with chemiluminescence. D) Mass spectrometric identification of semenogelin I. An immunoprecipitate from biotinylated human spermatozoa with anti-Eppin antibodies was analyzed by SDS-PAGE, Coomassie blue stained, and the protein bands trypsin digested for identification. The sequences of the labeled peaks are shown in 
) indicate Eppin staining on sperm tails. Areas of colocalization appear yellow. 63x objective. D) Spermatozoa and coagulum labeled with affinity-purified anti-Eppin (Q20E) and detected with Alexa Fluor-568-labeled goat anti-rabbit antibodies and MHS-5 monoclonal anti-Sg antibodies and detected with goat anti-mouse Alexa Fluor-488. Arrow (
) or without rEppin (
). Data represent one typical experiment of several. B) Displacement of 125I-rSg from rEppin with increasing amounts of unlabeled rSg (
). Data represent one typical experiment of several
