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Laboratories for Reproductive Biology and Department of Cell & Developmental Biology,3 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Laboratory of Reproductive Medicine and Department of Urology,4 The First Affiliated Hospital of Nanjing Medical University, Jiangsu Nanjing 210029, China
ABSTRACT
Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.
clusterin, contraception, eppin, gamete biology, lactotransferrin, male reproductive tract, semenogelin, seminal plasma, sertoli cells, sperm, spermatozoa, testis
Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa [1, 2] and originates from genes expressed in Sertoli cells and epididymal epithelial cells [3]. As spermatozoa move into the human ductuli efferentes they encounter epididymal eppin, which continues to be present in the caput and cauda regions of the epididymis [1]. As spermatozoa continue their journey they enter the ejaculatory duct, encountering seminal vesicle secretions and prostatic secretions as they pass through the prostatic urethra. In this viscous mix of fluids, semenogelin (SEMG1), a major protein component of seminal fluid, coats spermatozoa and binds eppin on the sperm surface [4, 5, 6]. Immediately following ejaculation when spermatozoa are trapped in the ejaculate coagulum [4], eppin becomes an integral part of that coagulum [6], and active prostate-specific antigen (PSA, a serine protease) cleaves SEMG1, resulting in the release of several proteolytic fragments [7]. Our earlier studies demonstrated that the C-terminal of eppin (amino acids 75–133) binds an SEMG1 fragment (amino acids 164–283) that contains SEMG1's only cysteine (amino acid 239) [2]. Furthermore, our earlier studies found that eppin has strong antibacterial activity and could permeabilize the inner and outer membranes of E. coli [8], indicating a potential protective function on spermatozoa. Fertility studies of eppin-immunized male monkeys [9] demonstrated that effective and reversible male immunocontraception in primates is possible, and suggested that antibodies directed at eppin have the potential of disrupting an essential step in the prefertilization preparation of spermatozoa in the male reproductive tract and in the ejaculate that is initially deposited in the vagina.
Two human isoforms of the eppin protein are expressed; one is secreted, and one lacks a signal sequence [1]. The secreted form of eppin (CAB37635) has a theoretical pI of 8.52 and a calculated molecular weight of 15 283.72. Analysis of native eppin by SDS-PAGE indicates that the monomer form has an apparent molecular weight of 16–18 kDa and the dimer an apparent molecular mass of 33–36 kDa [2]. Higher multimer forms of eppin are often seen in seminal plasma, and analysis of recombinant human eppin indicates that multimer forms (2x, 3x, 4x), which are stable to boiling in SDS in the presence of reducing agents, easily form in vitro [2].
Eppin has a saturable binding site (receptor) on human swim-up spermatozoa, as demonstrated by the saturation kinetics of 125I-recombinant human eppin binding to fertile, live, human swim-up spermatozoa from patients attending the University of North Carolina at Chapel Hill In Vitro Fertilization Clinic [6]. To facilitate the identification of possible eppin receptors we have isolated eppin from ejaculate supernatant (seminal plasma) and washed ejaculate spermatozoa and identified the protein components that copurify with eppin. Lactotransferrin (LTF), clusterin (CLU), and eppin copurify as an eppin protein complex (EPC) that is subsequently associated with semenogelin (SEMG1) in the ejaculate. The identification of the EPC suggests that LTF receptors, CLU receptors, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC. Additionally, we have localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, suggesting that the EPC is present on spermatozoa from the time they leave the seminiferous tubule.
All chemicals and reagents used in this study were molecular biology grade purchased from Sigma-Aldrich (St. Louis, MO). Immobilon-P and N transfer membranes were purchased from Millipore (Bedford, MA). Human semen samples were obtained from Dr. S. Beyler (Department of Obstetrics and Gynecology, University of North Carolina Hospital, Chapel Hill, NC) and this study was approved by The Committee on the Protection of the Rights of Human Subjects at the University of North Carolina, School of Medicine, Chapel Hill, NC. Human testicular sections were obtained through the University of North Carolina Laboratories for Reproductive Biology Molecular Histology Core Tissue Collection.
Isolation of the Native Eppin Complex
Ammonium sulfate precipitation. Native eppin throughout this study refers to eppin derived from biological sources, not synthetic or recombinant in origin. Human seminal plasma from frozen ejaculates (6–8 ml) was pooled for each purification preparation and fractionated with 25% ammonium sulfate. The precipitate was removed by centrifugation (10 000 x g, 20 min) and the supernatant brought to 70% saturation. The 70% saturated supernatant was centrifuged (1500 x g, 30 min) and the resulting pellet dissolved in PBS (pH 7.4) and dialyzed against PBS in a 10 000 MWCO membrane (Millipore).
Size exclusion chromatography. Aliquots (1.8 ml) of the dialyzed ammonium sulfate fraction were applied to a BioSep SEC S3000 column (600 x 21.2 mm) with a precolumn (75 x 21.2 mm; Phenomenex, Torrance, CA) equilibrated in 10 mM Tris-HCl (pH 8.0), 25 mM NaCl, 3 mM sodium azide, and 2 mM benzamidine. The isocratic column run of 28 min (7.5 ml/min) was monitored at 280 nm and 214 nm, and fractions were collected every 15 sec. Fractions from multiple runs were pooled.
Ion exchange chromatography. Eppin-positive fractions from the BioSep SEC S3000 column were pooled, dialyzed, and concentrated in Amicon Ultra Centrifugal 10 000 MWCO filters (Millipore) against 20 mM Na2PO4 (buffer A; pH 6.0) and applied to a Shodex IEC CM-825 column (8 mm x 75 mm; Phenomenex). Multiple injections in buffer A were made to load the entire sample onto the column. After the final injection was made and allowed to wash through the column for 5 min in 100% buffer A, a linear gradient was developed as follows: 0%–20% buffer B (20 mM Na2PO4 [pH 6.0], 0.5 M NaCl) over 20 min followed by 20%–100% buffer B over 10 min. Fractions were collected every 0.5 min for 40 min, and the elution was monitored at 280 nm.
Reverse phase chromatography. Eppin-positive fractions were pooled and loaded onto a 300 Å 15 µ Delta Pak C-18 column (3.9 x 300 mm; Waters, Milford, MA). The column was run 5 min at initial conditions (15% acetonitrile [ACN], 0.1% trifluoroacetic acid [TFA]) followed by a 60-min gradient with 85% ACN and 0.1% TFA at 1.0 ml/min. Fractions were collected every minute, and the elution was monitored at 280 nm and 214 nm.
Isolation of the native eppin complex from spermatozoa. Human spermatozoa were prepared from frozen ejaculates by thorough washing (1200 rpm, 5 min, 3 times) in PBS. The pellet was resuspended in PBS, sonicated (1 min) on ice, and extracted with chloroform and methanol (3:1). The organic-aqueous (phospholipid) interface was removed and placed in 4M urea in SDS-PAGE sample buffer without reducing agents and analyzed by SDS-PAGE. The location of eppin-positive bands was determined by Western blotting with anti-eppin antibody. A gel lane containing an identical sample, which had not been blotted, was incubated in 50 mM dithiothreitol (Sigma-Aldrich) for 1 h and separated in the second dimension with reducing agent. The resulting gel was stained with 0.01% Bio-Rad R-250 Coomassie (Bio-Rad Laboratories, Hercules, CA) and the protein staining bands located below the positions of the eppin bands were excised for mass spectrometry analysis.
Mass Spectrometry Identification of Eppin Protein Complex
SDS gels (one-dimensional or two-dimensional) were stained overnight with 0.01% Bio-Rad R-250 Coomassie in 10% acetic acid. Protein staining bands were excised, digested with trypsin, and analyzed by MALDI/TOF (matrix-assisted laser desorption ionization/time of flight). 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 identification was done in the UNC-CH Proteomics Core.
Enzymatically active PSA was obtained from EMD Biosciences (San Diego, CA). The chromogenic substrate 3-carbomethyoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroaniline hydrochloride (diaPharma Group, West Chester, OH) was prepared by dissolving 12.5 mg in 30 ml deionized H2O, and aliquots were frozen until use. Assay buffer consisted of 0.1 M Tris-Cl (pH 8.3) and 1.0 M NaCl with or without 10 mM CaCl2. PSA, sample, and assay buffer were pipetted into duplicate wells in 96-well plates for a total volume of 200 µl. Substrate (100 µl) was pipetted into each well to initiate the reaction. The OD at 405 nm was read immediately and every 10 min in a plate reader for a period of 2 h.
SDS-PAGE and Western Blot Analysis
HPLC fractions were analyzed on Criterion 10–20% gradient gels (Bio-Rad), transferred to Immobilon P (Millipore) and stained for protein with amido black or blocked with Tris-buffered saline (50 mM Tris [pH 7.4], 150 mM NaCl) containing 3% BSA (60 min, room temperature) and probed with primary antibodies as described [2]. Primary antibodies were rabbit anti-eppin and rabbit anti-SEMG1 [2], mouse anti-CLU (Upstate, Lake Placid, NY), goat anti-LTF (Bethyl Laboratories, Montgomery, TX) and mouse anti-LTF (QED Bioscience Inc., San Diego, CA). Rabbit anti-LTF receptor antiserum was a gift from Dr. Bo Lönnerdal (University of California, Davis, CA). Secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG, 1:2000) were alkaline-phosphatase labeled and developed with NBT-BCIP (nitro blue tetrazolium/5-bromo-4chloro-3-indolyl phosphate) as substrate. Far-Western blotting was carried out as described [2].
Immunohistochemistry and Immunofluorescence
Sections of testis were deparaffinized, endogenous peroxidase activity was blocked with methanol-H202, the sections blocked with 2% normal goat serum, and incubated overnight at 4°C with one of the primary antisera used for Western blotting. The color was developed with an ABC kit (Vector Laboratories Inc., Burlingame, CA) and diaminobenzidine substrate. Stained slides were overlaid with osmium vapors and counterstained with 0.05% toluidine blue. Images were recorded with a Zeiss AxioCam using AxioVision software (Zeiss MicroImaging, Thornwood, NJ); images in Figure 7, E–G, were taken with Nomarski optics.
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For immunofluorescence studies, the following primary antibodies were used at 1:100 dilutions: rabbit anti-recombinant human eppin [2], monoclonal mouse anti-LTF as used for Western blotting, and anti-CLU 
(N-18), an affinity purified goat polyclonal antibody made against a peptide mapping near the N-terminus of human CLU alpha (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was detected with Alexa Fluor-488 labeled goat anti-rabbit IgG, Alexa Fluor-568 labeled donkey anti-goat IgG, or Alexa Fluor-568 labeled goat anti-mouse IgG (Invitrogen/Molecular Probes, Eugene, OR) at a 1:5000 dilution. Fluorescent images were obtained from a Zeiss Axiophot microscope equipped with a Plan-neofluar 100x 1.3 NA objective, 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) and exported to Adobe Photoshop 5.0 to assemble Figures 7 and 8.
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Purification of the Native Eppin Protein Complex
Figure 1 illustrates typical chromatographs from the BioSep SEC S3000 size exclusion column (Fig. 1A) and the Shodex IEC CM-825 cation exchange column (Fig. 1B). Eppin eluted from the CM-825 column in four fractions (#72–75, shaded area), which were pooled for analysis. Figure 2, A and B, illustrates an SDS-PAGE analysis of the CM-825 pool. Amido black staining of the Western blot for protein (Fig. 2A, lane 1) indicated two strongly staining bands at approximately 98 and 72 kDa (arrows), two lesser staining bands of 48 and 38 kDa, and several lower-molecular-mass bands below 26 kDa. Antibody staining of the blots of nonreduced samples (Fig. 2A, lanes 2–6) demonstrated the presence of eppin, CLU, and LTF in the 98- and 72-kDa bands and SEMG1 in bands below 26 kDa. Antibody staining of the blots of reduced samples (Fig. 2B, lanes 2–6) demonstrated the presence of two major eppin bands at 70–72 kDa and 38–48 kDa (lane 4), CLU monomers in the 48- and 38-kDa bands, and nonreduced CLU subunits in the upper 82- to 96-kDa band (lane 2). LTF remained as a doublet band. Additional hydrolytic fragments of SEMG1 are present at 48 kDa as well as below 26 kDa under reducing conditions. Mass spectrometry analysis confirmed the presence of LTF and CLU copurifying with eppin in the 98- and 72-kDa bands (data not shown; however, identical mass spectrometry results are shown in Figure 5).
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Further analysis of EPC was carried out on a reverse phase Delta Pak C-18 column, as illustrated in Figure 1C. All the peaks were analyzed by SDS-PAGE and Western blotting for the presence of eppin, SEMG1, CLU, and LTF. SEMG1, although bound to eppin in the ejaculate complex, is separated from the complex under the reverse phase buffer conditions and eluted in peaks 1 and 2 (Fig. 1C, peaks 1 and 2; Fig. 2C, lane 1 bracket; Western blot data not shown). LTF eluted as a doublet predominantly in peak 3 (Fig. 2C, lane 2; Fig. 2D, lanes 1 and 2), whereas CLU and eppin were predominantly in peak 6 (Fig. 2C, lane 4; Fig. 2D, lanes 4–7). Smaller amounts of LTF, CLU, and eppin were found in peaks 4 and 5. Antibody staining of a nonpeak fraction (control) is shown in Figure 2D, lane 3. Coelution of eppin with CLU and LTF in ACN and TFA buffers indicates its strong interaction with these proteins in the EPC.
Eppin modulates the hydrolysis of SEMG1 by inhibiting PSA enzyme activity [10]. PSA assays of the CM-825 pool indicated that the EPC contained PSA inhibitory activity (Fig. 3). Increasing amounts of the EPC from 2–8 µg inhibited PSA activity, whereas addition of 2–64 µg of BSA did not. In a typical preparative run, 0.6 mg of EPC could be prepared from 290 mg of seminal plasma protein. PSA assays of the reverse phase Delta Pak C-18 column fractions indicated that the PSA inhibitory activity had been lost as a result of treatment with the reverse phase column buffers (data not shown).
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Purification of the Native Eppin Protein Complex from Spermatozoa
A second approach was undertaken to analyze proteins present with eppin on the sperm surface. Washed human spermatozoa were sonicated and extracted with chloroform-methanol, and the organic-aqueous (phospholipid) interface was collected and diluted into 4M urea/SDS sample buffer. The sample was analyzed by SDS-PAGE in two dimensions. After the nonreducing first dimension, the gel lane was turned 90° and analyzed by reducing SDS-PAGE in the second dimension. Western blotting after the first dimension determined that the EPC contained two strongly staining protein bands at approximately 98 and 72 kDa, similar to those seen in Figure 2A, lane 1. Figure 4 demonstrates the Coomassie Blue-stained second-dimension SDS gel and indicates the spots that were excised for MS/MS analysis. Spots A6 and A7 (determined by Western blotting) correspond to the 98- and 72-kDa protein bands, respectively, and spot A10_36 was derived from spot A7 under reducing conditions. Spots below 37 kDa were not analyzed. The results of the MS/MS analysis of spot A6 are shown in Figure 5. Spot A6 contained LTF (78 kDa) and CLU (HUMAPOJ NID; sulfated glygoprotein-2, a disulfide linked heterodimer). Spots A7 and A10_36 contained LTF and the reduced alpha subunit of CLU, respectively (data not shown). Under these isolation conditions very little SEMG1 was present, and what is present migrates to 
27 kDa. Eppin cannot be detected as a MS fragment because it is not hydrolyzed by trypsin under the conditions used to prepare samples for MS analysis.
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The Native Eppin Protein Complex: Eppin, LTF, and CLU
To confirm the association of eppin with LTF and CLU, antibodies to clusterin were used to immunoprecipitate CLU from human sperm lysates. As shown by reducing SDS-PAGE (Fig. 6A), anti-CLU antibodies will immunoprecipitate the EPC (protein stain, left panel) and this complex stains positively with antibodies (Western blot, right panel) to eppin (lane 1, asterisks), SEMG1 (lane 2), and LTF (lane 3). Control antibodies did not stain the EPC bands (lane 4). Identical results were obtained using anti-LTF antibodies to immunoprecipitate the complex (Fig. 6B). Anti-LTF antibodies will immunoprecipitate the EPC (protein stain, left panel) and this complex stains positively with antibodies (Western blot, right panel) to eppin (lane 1, asterisks), CLU (lane 2, asterisks), SEMG1 (lane 3), and LTF (lane 4). The eppin bands, two asterisks in Figure 6A, lane 1 (right panel) and three asterisks in Figure 6B, lane 1 (right panel), indicate bands of eppin similar to the major bands seen in Figure 2B, lane 4. The CLU bands, two asterisks in Figure 6B, lane 2 (right panel), indicate bands of CLU similar to those seen in Figure 2B (lane 2). Control antibodies did not stain the EPC bands (lane 5).
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Further experiments using Far-Western blotting indicate that recombinant eppin on a blot will bind both native CLU and native LTF, whereas recombinant SEMG1 on a blot does not. An example of a Far-Western blot of recombinant eppin and recombinant SEMG1 is shown in Figure 6C. Blots containing recombinant SEMG1 (Fig. 6C, lanes 1 and 2) and recombinant eppin (Fig. 6C, lanes 5 and 6) were incubated with or without native LTF (+Lf/-Lf), washed, and probed with antibodies to LTF. Lanes 3 and 4 demonstrate that SEMG1 does not bind LTF, and lanes 7 and 8 demonstrate that eppin (monomer and multimer forms) binds LTF. Similar data were obtained with CLU (data not shown).
Localization of Eppin, LTF, and CLU in the Testis
Expression of the genes for the components of the eppin protein complex has been reported to occur in Sertoli cells of the testis [3, 11] (see mammalian reproductive genetics database [12]). To confirm that Sertoli cells contain eppin, LTF, and CLU in the human testis, immunohistochemistry was performed on sections of human testis. As shown in Figure 7, eppin was localized primarily in the Sertoli cells (white arrows, Figure 7A), though myoid cells and spermatogenic cells were occasionally positive. Both LTF (Fig. 7C) and CLU (Fig. 7D) were found primarily in Sertoli cells of the testis (arrows), while spermatogenic cells were weakly positive for LTF and accumulations of CLU were often seen along the luminal surface (Fig. 7D). Sections of human testis probed with control antibodies, IgG, or second antibody only (not shown) or anti-eppin antibodies absorbed with recombinant eppin (Fig. 7B) were negative. Testicular spermatozoa were found to be labeled with eppin, LTF, and CLU (arrows, Fig. 7, E, F, G, respectively). From these observations it appears that in human testis the EPC components are present in Sertoli cells, and that they most likely coat testicular spermatozoa.
Localization of Eppin, LTF, and CLU on Ejaculate Spermatozoa
To determine whether the EPC found on testicular spermatozoa was present on ejaculate spermatozoa, washed ejaculate spermatozoa were double labeled with either anti-eppin and anti-CLU or anti-eppin and anti-LTF. As shown in Figure 8 (eppin-CLU series A–D), eppin and CLU colocalize on the sperm tail. The arrows (Fig. 8, B–D) indicate a punctate spot that is eppin positive (B) and CLU positive (C), and in the merged image (D) appears orange-yellow as both eppin and CLU colocalize. Similarly, in the eppin-LTF series (Fig. 8, E–H), eppin and LTF colocalize on the sperm tail. The arrows (Fig. 8, F–H) indicate two punctate spots at which eppin (F) and LTF (G) are positive. In the merged image both spots appear strongly orange-yellow, indicating the colocalization of eppin and LTF. The colocalization of eppin with CLU and LTF demonstrates that the EPC is present on ejaculate spermatozoa. Control images probed with second antibodies only (no primary antibody) or anti-eppin antibodies absorbed with recombinant eppin as shown in Figure 7, were negative (data not shown).
The isolation and identification of a protein complex containing four interacting proteins—eppin, LTF, CLU, and SEMG1—from seminal plasma and spermatozoa demonstrate the complicated nature of protein-protein interactions on the surface of human ejaculate spermatozoa. Although numerous proteins have been identified as being on the surface of human spermatozoa, relatively little is understood about their functional interactions. Based on data presented here and previously published work [1, 13, 14, 15], eppin, LTF, and CLU are synthesized in the testis and are present in Sertoli cells and on testicular spermatozoa, indicating that the EPC is most likely assembled from secreted components on the surface of spermatozoa during the last steps of spermiogenesis. Although the EPC of eppin, LTF, and CLU may be formed on testicular sperm, it is likely that the EPC present on ejaculate sperm is also of epididymal origin because eppin [1], CLU [13, 16], and LTF [15] are all also secreted by epididymal epithelial cells. In fact, it has been reported that testicular CLU is removed from epididymal fluid [16]. Consequently, the final EPC present on ejaculate spermatozoa may be a mixture of testicular and epididymal contributions with the addition of SEMG1 from seminal vesicle fluid during ejaculation.
Analysis of the data presented in Figure 1C, Figure 2, and Figure 4 indicates that the most likely association within the EPC under nonreducing conditions would be an eppin monomer (18 kDa) bound to a CLU dimer (82 kDa; and ß subunits) and an eppin monomer bound to LTF (72 kDa) in the higher molecular mass band (
98 kDa) in the EPC observed after SDS-PAGE. In the lower molecular mass band (72 kDa), LTF is present along with an eppin dimer (36 kDa) associated with a CLU monomer (38–41 kDa). Since immunoprecipitation of either CLU or LTF results in the precipitation of all four components of the complex (Figure 6), eppin dimers in vivo may be able to form associations with LTF and CLU dimers simultaneously, suggesting that the formation of eppin multimers on the sperm surface could assemble a very large network or lattice of protein-protein interactions. In vivo, monomeric eppin binds SEMG1 through cysteine 239 [2], implying that the EPC network would have multiple binding sites for SEMG1. Moreover, the eppin-SEMG1 interaction through disulfide exchange suggests that eppin might bind CLU through a similar exchange at cysteine residues, explaining the inseparable nature of eppin and CLU in reverse phase HPLC.
Recent studies [17, 18] on three-dimensional protein networks have suggested that "multi-interface hubs" are central proteins that have more than one interaction within a complex and are often conserved and essential for function. Eppin genes are highly conserved within the eppin gene cluster on mouse chromosome 2 and human chromosome 20 [1, 6], and eppin covers the sperm surface [1, 5] such that the EPC may provide a surface network, particularly over the tail, as seen in Figure 8. SEMG1 does not bind LTF (Figure 6C) or CLU, and in preliminary experiments in vitro we have been unable to detect LTF binding CLU in the absence of eppin. LTF, CLU, and SEMG1 all have eppin in common, implicating eppin as a central player in a network of protein-protein interactions on the human sperm surface. LTF, SEMG1 [19], and eppin [8] have strong microbicidal properties, and CLU can inhibit MT6-MMP metalloproteinase activity [20]; consequently one important function of the EPC may be the protection of spermatozoa. Additionally, SEMG1 inhibits sperm motility [7] and capacitation [5] while on the sperm surface bound to eppin, and SEMG1's hydrolysis by PSA is required for forward motility and fertility [5, 7]. Eppin plays an essential role in fertility [9], and the EPC may regulate the sperm's transition from the protected environment of the coagulum to the capacitated sperm in the oviduct.
One of the objectives of our study was to ascertain the possible associations of eppin with the sperm surface. Eppin has been demonstrated to be a sperm surface receptor for SEMG1 [2, 6], and the identification of the components of the EPC implies that surface receptors for CLU, LTF, and eppin itself could bind the EPC to the surface. Although apoER2 has been described as a CLU receptor in the epididymis [16], and MT6-MMP a CLU binding protein on neutrophils [20], spermatozoa do not appear to have CLU receptors. However, CLU is known to bind a variety of hydrophobic molecules [21, 22] and may function in several biological pathways, including as an extracellular chaperone [23].
LTF, on the other hand, has a well-defined receptor characterized from human small intestine [24], which has been reported to be anchored to the plasma membrane by glycophosphatidylinositol. The presence of LTF in the EPC raises the possibility that a LTF receptor might be present on human spermatozoa, because LTF receptor mRNA has been identified in human testis [24]. In preliminary experiments we confirmed that human testis expresses the LTF receptor mRNA and found that Percoll gradient human spermatozoa treated with PI-PLC release LfR into the supernatant. Therefore the human LfR appears to be glycophosphatidylinositol linked to human spermatozoa; however, whether the EPC specifically binds the receptor in a receptor-ligand relationship has been difficult to determine and is currently under investigation. Additional studies are also currently underway to ascertain whether the EPC might associate with other receptors on spermatozoa.
ACKNOWLEDGMENTS
The authors thank Gail Grossman of the UNC Laboratories for Reproductive Biology Molecular Histology core facility for preparing the stained testis sections.
FOOTNOTES
1Supported by grant CIG-96-06 from the CICCR Program of CONRAD, D43TW-HD00627 from the Program for International Training and Research in Population and Health from the Fogarty International Center, NICHHD, and by grant HD048843 from NICHHD (M.O.). 
Correspondence: 2Michael 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; e-mail: morand{at}unc.edu
Received: 19 January 2007.
First decision: 5 March 2007.
Accepted: 5 June 2007.
REFERENCES
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  M. G. O'Rand, E. E. Widgren, S. Beyler, and R. T. Richardson Inhibition of Human Sperm Motility by Contraceptive Anti-Eppin Antibodies from Infertile Male Monkeys: Effect on Cyclic Adenosine Monophosphate Biol Reprod, February 1, 2009; 80(2): 279 - 285. [Abstract] [Full Text] [PDF]
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