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Gamete Biology |
Department of Obstetrics and Gynecology, Division of Reproductive Biology,3
Departments of Environmental Toxicology and Nutrition,4 Bodega Marine Laboratory, University of California, Davis, Davis, California 94923
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cyclic adenosine monophosphate, epididymis, gamete biology, sperm, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature sperm released from the male tract at ejaculation still must spend additional time in the female tract, or another appropriate environment, before they are competent to fertilize [3]. This final maturation process, termed capacitation, has been recognized for more than 50 years as an essential prerequisite for fertilization, but the changes that take place in sperm during capacitation are still not completely understood [3]. The sperm maturation processes in the male and female tracts appear to be linked. The sperm surface modifications, including addition of surface coats that occurs in the male reproductive tract, appear to establish a stable membrane composition for protection of sperm during transport in the female tract. Subsequently, the modification or removal of these stabilizing factors in the female may allow the sperm-receptor presentation and membrane destabilization that are required for the acrosome reaction and fertilization [4].
The secretory activities of the epididymal epithelium are of major importance in the process of sperm surface remodeling in the male reproductive tract [5]. A number of epididymal proteins are known to be added to the sperm surface, and most of these proteins are synthesized and secreted under strict androgen control [6, 7]. Two separate and unique populations of (glyco)proteins are added to sperm, and these proteins are classified according to their relative affinities for the sperm surface [8, 9]. Some glycoproteins are glycosylphosphatidyl inositol (GPI)-anchored and firmly incorporated into specific membrane domains along the sperm surface [10–12], whereas other epididymal proteins are adsorbed on the sperm surface and not integrated into the lipid bilayer [13, 14]. Most, but not all, of the GPI-linked glycoproteins are utilized for sperm protection within the female reproductive tract [15, 16]. The so-called "maturation antigens" laid down in the epididymis may function for protection, cumulus penetration, zona recognition and binding, as well as sperm-oocyte fusion [14–17].
Cynomolgus macaque sperm have a constituent population of GPI-anchored (glyco)proteins that are released after exposure to phosphatidylinositol-specific phospholipase C (PI-PLC) [18]. Because this group of membrane proteins is completely exposed to the external environment, they have been used to elicit an isoimmune response in female macaques [18]. Following immunization with these PI-PLC-released proteins, together with five different adjuvants, only two glycoproteins (24 and 53 kDa) were consistently recognized as potent isoantigens [18]. The 24-kDa protein was found to be a GPI-anchored CRISP glycoprotein called MAK248, and it has been described in detail previously [18]. The 53-kDa protein was determined to be a cell surface-coating protein that was linked to GPI-anchored protein(s), because it could be partially displaced from the sperm surface with elevation of the ionic strength of the medium [18].
Recently, the defensins, which are members of the innate immune system, have been shown to exist within the male reproductive tract [19]. One such defensin is DEFB126 (ESP13.2), which is epididymis-specific and expressed in large quantities [20]. However, the association of this protein with sperm was not previously demonstrated, and no function for the molecule was envisioned outside the male reproductive tract. In the present study, we show that the previously unidentified 53-kDa glycoprotein is ESP13.2, the ß-defensin DEFB 126, and that it is adsorbed to the entire surface of macaque sperm. We show that the protein is highly immunogenic, and we provide evidence of its function during the sperm capacitation process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) unless stated otherwise.
Semen and Tissue Collection
Animals were housed at the California National Primate Research Center in compliance with the Federal Health Guidelines for Care and Use of Laboratory Animals. Semen samples were collected by electroejaculation from 10 individually caged cynomolgus macaques [21]. Each ejaculate was collected into a 15-ml centrifuge tube containing 5 ml of Hepes-buffered Biggers, Whitten, and Whittingham (BWW) medium (Irvine Scientific, Santa Ana, CA). After a 1-h dispersion of the ejaculate in BWW, the samples were checked for motility, and only those with greater than 70% motile sperm were used in the experiments. The upper 4 ml of the sperm suspension were transferred to another tube for further processing.
Cynomolgus macaque tissues were obtained from three males at the time of elective necropsy. The different regions of the reproductive tract and selected other tissues were dissected, and a small extract of each tissue sample was prepared for PAGE by boiling for 3 min in a reducing SDS solubilizing buffer (Pierce, Rockford, IL). The samples were centrifuged at 2000 x g for 10 min, and the supernatants were stored at -20°C.
Antibody Production and Preparation
The methods for developing isoantibodies to PI-PLC-released macaque sperm surface components were originally reported by Yudin et al. [18]. Briefly, the upper 4 ml from ejaculated sperm suspensions were pelleted (300 x g) for 10 min, resuspended in 1 ml of Dulbecco PBS (DPBS; Life Technologies, Rockville, MD), and layered over a 3.5-ml column of 80% Percoll in DPBS [22]. After centrifugation for 30 min at 300 x g, the resulting pellet was washed twice in DPBS (10 ml), resuspended in PI-PLC (3 U/ml), and gently rolled during the 2-h incubation at 37°C. After 2 h, the sample was centrifuged for 10 min at 1000 x g, and the supernatant was cleared by passage through a 0.22-µm syringe filter. The samples were concentrated fourfold with a Centricon-YM-10 (Millipore, Bedford, MA). Five separate adjuvants were used for immunization with the PI-PLC-released sperm surface glycoproteins [18], but the antibodies developed from the PI-PLC/Montanide ISA 51 (Seppic, Paris, France) injections were used in the present study to demonstrate the production of isoantibodies. The serum was collected 2 wk following the last injection from an immunized female monkey [23].
Intact, fixed cynomolgus macaque sperm were used to immunize rabbits. Thoroughly washed sperm were fixed for 1 h in 2% paraformaldehyde and 0.2% glutaraldehyde/DPBS. After 1 h, the sperm were washed thoroughly over a 2-h period and then mixed with Freund adjuvant for injection. Complete Freund adjuvant was used for the first immunization; incomplete Freund adjuvant was used for subsequent immunizations. The immunizations were performed every 2 wk with 106 sperm/injection, and the rabbits were killed by exsanguination 2 wk following the third injection. Three rabbits were immunized with this protocol.
Antibodies were developed to the purified ESP13.2 antigen. Sperm were washed through 80% Percoll, resuspended twice in 10 ml of DPBS, and pelleted by centrifugation (300 x g) for 10 min. The resulting pellets were resuspended in 2x DPBS (2:8 10x PBS:H2O; 300 mM) for 30 min and then pelleted for 10 min at 300 x g. The supernatant was passed through a 0.22-µm syringe filter. The elevated NaCl extractions from 10 ejaculates were concentrated and electrophoretically separated on a 8–16% gel. The gels were stained with Gel Code Blue (Pierce), and the 53-kDa band was cut out and electroeluted. After complete electroelution, the sample was chemically reduced with 0.1 M dithiothreitol (DTT) and again run on an 8–16% gel. The entire 31- to 35-kDa band was cut from the gel and electroeluted for immunization. After electroelution, the sample was concentrated, and 100-µg aliquots were injected into each rabbit in a series of four immunizations as described above.
All serum samples (macaque and rabbit) were initially heat-inactivated (56°C for 30 min) and then precipitated with ammonium sulfate (0.24 g/ml). The ammonium sulfate was added slowly over a 4-h period at 4°C. The precipitated immunoglobulin (Ig) was pelleted and resuspended in DPBS and dialyzed overnight. The Ig was aliquoted and stored at -20°C.
Amino Acid Sequence Analysis
Upper and lower regions of the Gel Code Blue-stained, 31- to 35-kDa band that resulted from the purification and reduction of the 53-kDa band from NaCl-extracted sperm preparations were cut from the gel and submitted to the Molecular Structure Lab, University of California, Davis, for amino acid sequencing. The upper and lower portions of the 31- to 35-kDa band were proteolytically cleaved and extracted. Sequencing was done on an ABI Q-Star XL Hybrid LC/MS/MS system (Foster City, CA). All chemistry programs and data collection/analysis were performed using standard Procise protocols. The individual amino acids were cleaved from the protein using standard Edman chemistry and resolved on the standard ABI RP columns using standard solvents. Absorbance was monitored at 269 nm and results displayed and reports generated using the ABI 610a V.2,1 software running on a Mac G3 (Apple Computer, Inc., Cupertino, CA). The sequence data were compared to the existing DNA databanks for any sequence similarity.
Sperm Preparation
As described above, sperm remained in Hepes-BWW for 1 h after ejaculation and then were pelleted at 300 x g for further processing. Next, sperm were capacitated overnight as described previously [24]. Briefly, sperm were washed through a 3.5-ml column of 80% Percoll and suspended in BWW medium containing 30 mg/ml of BSA buffered with 35.7 mM sodium bicarbonate ("capacitation medium"). Following overnight incubation at 28°C and 5% CO2, sperm were placed into a 37°C incubator and 5% CO2 for an additional 2 h. This process promotes capacitation in at least a subset of macaque sperm, as determined by the ability of sperm to tightly bind to the zona pellucida and undergo the zona pellucida-induced acrosome reaction. Capacitation of the majority of sperm was synchronized with the addition of 1 mM dbcAMP and 1 mM caffeine ("activators") to sperm suspensions for an additional 1-h incubation at 37°C and 5% CO2 [24]. Sperm were studied at different stages of preparation: before Percoll washing, after Percoll washing, after overnight incubation, and following addition of activators. In some experiments, ejaculates were split into two groups before Percoll washing, one being treated with 1 mg/ml of lignosulfonic acid (LSA). After 1 h of incubation, the LSA-treated and non-LSA-treated sperm were prepared in the same way, as described above.
Fluorescent Immunolocalization of ESP13.2
Only the rabbit anti-ESP13.2-specific Ig was used for the localization experiments. Multiple sperm samples from five different males were fixed (2% paraformaldehyde/DPBS) for 20–30 min at the four different stages of sperm preparation. After fixation, the sperm were thoroughly washed (two to three times) in blocking solution (1% BSA, 0.1% NaN3, and 1% gelatin/DPBS). Sperm samples were suspended in preimmune or primary antibody (10 µg/ml of Ig) and gently rolled for 1 h, then washed thoroughly (three times) in blocking solution and resuspended in a solution of 20 µg/ml of goat anti-rabbit Alexa 488 (Molecular Probes, Eugene, OR) in blocking solution. The samples were again rolled for 1 h and then thoroughly washed and resuspended in a fluorescent stabilization medium (50% glycerol, 0.2% NaN3, and 1% paraformaldehyde/DPBS). Photomicrographs were taken of representative cells using a cooled CCD digital camera (Optomics, Santa Barbara, CA) mounted on a Leitz Laborlux S microscope (Carl Zeiss Vision GmbH, Oberkochen, Germany) equipped with a 200-W mercury fluorescence vertical illuminator and a 1-Lambda Ploemopac incident light fluorescence illuminator employing an I3 filter cube with a BP 450–490 excitation filter, an RKP 0510 dichromatic mirror, and an LP 515 suppression filter. Optics included a 3.3x intraocular magnifier (Scientific Instruments, Sunnyvale, CA) and a Zeiss 63x oil emersion fluorescence objective (JH Technologies, San Jose, CA). Initial images were captured using Magnafire 2.0 software (Optomics) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA) for production of figures.
Fine Structural Localization of ESP13.2
Sperm samples from three different males were washed through 80% Percoll and fixed in 2% paraformaldehyde and 0.2% glutaraldehyde/0.2 M cacodylate buffer. After fixation for 30 min, the sample was washed thoroughly in blocking solution and incubated in preimmune or primary anti-ESP13.2 Ig (10 µg/ml) for 1 h, thoroughly washed in blocking solution, and resuspended in goat anti-rabbit Ig 10-nm gold (E-Y Laboratories, San Mateo, CA). Sperm were gently rolled for 2 h, washed in DPBS, resuspended for 2 h in 2.5% glutaraldehyde, and buffered with 0.2 M cacodylate (pH 7.4). Finally, sperm were washed in 0.2 M cacodylate and postfixed for 1 h with 1% osmium tetroxide in 0.2 M cacodylate. Sperm were dehydrated through a graded alcohol series, infiltrated with Spurrs epoxy (Ted Pella, Inc., Redding, CA), and embedded in an epon-araldite mixture. Sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before viewing with a Philips 401 transmission electron microscope (Eindhoven, The Netherlands).
Electrophoresis and Western Blot Analysis
All gels used were 8–16% Tris-glycine (Invitrogen, Carlsbad, CA). The PI-PLC-released sperm surface (glyco)proteins were solubilized in SDS-nonreducing buffer (Pierce, Rockford, IL) and then split equally and reduced with 100 mM DTT. Concentrated surface extract removed with elevated osmolarity (300 mM) was solubilized with SDS-nonreducing buffer (Pierce), split equally, and reduced with 100 mM DTT. Whole-sperm and PI-PLC-released sperm surface components were treated similarly. Whole-sperm samples from eight different males were individually solubilized in SDS-nonreducing buffer at 50 x 106sperm/ml. These samples were also split into nonreduced and reduced aliquots.
After electrophoresis, the gel was electroblotted to nitrocellulose membranes and blocked for at least 2 h in TBS (50 mM Tris-HCL [pH 7.4] and 0.3 M NaCl) containing 5% nonfat dry milk and 0.1% NaN3. After blocking, the blots were incubated with Ig suspended at 50 µg per 10 ml of TBS with 3% BSA and 0.1% NaN3. After labeling the nitrocellulose blots with the primary antibody, the blots were washed three times for 10 min each in TTBS (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween 20). Blots were subsequently incubated with the appropriate secondary antibody (1:2000): goat anti-rabbit IgG-alkaline phosphatase (Bio-Rad, Hercules, CA) or goat anti-monkey IgG-alkaline phosphatase (Accurate Chemical and Scientific Corp., Westbury, NY). After washing in TTBS, immune complexes were detected using precipitating alkaline phosphatase substrate (1-Step NBT-BCIP; Pierce).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of sperm with PI-PLC released a number of proteins, as shown in the concentrated sample run on nonreducing 8–16% gels (Fig. 1A). When the same gels were blotted onto nitrocellulose and probed with isoantibodies developed in female macaques immunized with PI-PLC-released sperm components (anti-PI-PLC isoantibodies), a potent immunologic response was demonstrated to a broad, diffuse, 53-kDa region of the blot (Fig. 1B). After chemical reduction of the sample with DTT, a different electrophoretic profile was apparent, and the bands recognized by the same antibodies were the 28-, 18-, and 10-kDa components of the MAK248 glycoprotein [18] and a diffuse, broad band that extended from 31 to 35 kDa (Fig. 1, C and D).
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Sperm exposed to 2x DPBS (300 mM) released a population of (glyco)proteins (Fig. 2A). After blotting onto nitrocellulose and subsequent exposure to anti-PI-PLC isoantibodies, a number of bands were recognized, with the 53-kDa band being the most prominent (Fig. 2B, which is representative of three gels). Chemical reduction of the NaCl extract before electrophoresis resulted in a limited number of bands being recognized by the same antibodies, with the 31- to 35-kDa region being the most evident (Fig. 2, C and D).
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Purification and Amino Acid Sequence of the 31- to 35-kDa Sperm Protein
Because of the broad nature of the band, portions of the upper (35-kDa) and lower (31-kDa) regions of the band were sliced for protein extraction and sequencing. Analysis of peptide fragments from the upper segment gave one good sequence; the lower segment had three peptides that were easily sequenced. The sequence of the peptide from the upper region was the same as that of one of the lower peptides, minus one of the initial amino acids. All of the sequences had 100% amino acid identity with a previously described epididymal secretory protein, ESP13.2, which was reported to have a molecular mass of approximately 30 kDa [20]. The amino acid sequence of ESP13.2 (Q9BEE3/AJ236909) showed a structural relationship very similar to that of the ß-defensins and, later, was given the genomic name DEFB126 [25].
The amino acid sequence of ESP13.2 is shown in Figure 3, and the enzymatically cleaved peptides that were sequenced in the present study are shown in brackets. The whole sequence includes the initial 20-amino acid signal, followed by the cysteine core and hydrophobic tail (Fig. 3). The projected molecular weight of the entire protein based on the amino acid sequence is approximately 13 kDa, but based on electrophoretic mobility, the apparent molecular weight is approximately 31–35 kDa, a difference that probably results from glycosylation. In the sequence, no possible regions for N-glycosylation were found, suggesting O-linkage as the sole source of glycosylation. Within the hydrophobic carboxyl region is an abundance of threonine and serine residues; in fact, their contribution within this region is nearly 40%. Eight threonine and serine residues are found that are most likely utilized for O-linked glycosylation, as determined using previously reported criteria [26]. Within the core region of the defensins, the cysteines often are invariably spaced, but ESP13.2 has an additional five amino acids between the C3 and C4 regions. All of the defensins have a strong cationic nature, and ESP13.2 is no exception, having 9 more basic amino acids over the acidic amino acids. The last 45 amino acids give ESP13.2 a strong hydrophobic region with an abundance of apolar amino acid residues.
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Immune Response of Rabbits to Intact Fixed Macaque Sperm
Antibodies raised in rabbits to intact, fixed macaque sperm recognized a prominent band in the 31- to 35-kDa range on Western blots under reducing conditions (Fig. 4) as well as a doublet at 212 kDa that is not seen in Figure 4 (that portion of the blot has been cut off). All three immunized rabbits produced antibodies with the same recognition profile, and the 31- to 35-kDa band was the primary immunoreactive band for the first 4 wk after the initial injection in all three rabbits (data not shown). Whether the reduced sample was obtained from whole sperm, PI-PLC-released sperm surface components, or sperm surface components obtained by NaCl extraction, the same large region of immunoreaction between 31 and 35 kDa was apparent (Fig. 4).
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Immune Response of Rabbits to Purified ESP13.2
The same procedures for gel purification used to obtain peptides for amino acid sequencing were used to obtain ESP13.2 for antibody production in rabbits. These antibodies were used for Western blot analysis of nonreduced and reduced preparations of whole sperm and PI-PLC-released sperm surface components (Fig. 5). In the nonreduced samples, a number of bands were apparent, particularly in the 53- and 66-kDa regions (Fig. 5, B and F), but when the samples were subjected to chemical reduction, one primary band was recognized that ranged from approximately 31 to 35 kDa (Fig. 5, D and H).
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ESP13.2 in the Male Reproductive Tract
Male reproductive tract tissues were solubilized, chemically reduced, and blotted to nitrocellulose before probing with rabbit anti-ESP13.2 Ig. Extracts from the testes and the caput epididymides showed no observable bands on Western blots, even when high concentrations of tissue samples were used for blotting (Fig. 6, A and B). Tissues from three different cynomolgus macaques were used to confirm the absence of expressed ESP13.2 in the testes and caput region of the epididymis (not shown). The first evidence of antibody recognition was observed with corpus epididymal tissue, in which a single, thin band appeared at approximately 35 kDa (Fig. 6C). Extracts of tissue from the caudal region of the epididymis had a broader band of recognition by anti-ESP13.2 Ig than that observed with corpus tissue (Fig. 6, C and D). The preparation of whole sperm that was used for comparison (Fig. 6E) was significantly less concentrated than the tissue samples and allowed clearer delineation of the multiple bands that comprise the entire 31- to 35-kDa region of immunoreactivity to the anti-ESP13.2 Ig (Fig. 6E). Comparison of the different segments of the epididymis and whole sperm shows the addition of anti-ESP13.2 reactive bands in progressive regions along the reproductive tract (compare Fig. 6, C–E).
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ESP13.2 in Other Macaque Tissues
A number of nonreproductive tract tissue samples from macaques were solubilized, reduced, and blotted to nitrocellulose for probing with anti-ESP13.2 Ig (Fig. 7, A–G). Of the tissues tested, only skeletal muscle had a minor band of reactivity with the antibody, which may be a result of components with shared epitopes or nonspecific labeling (Fig. 7E).
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Fine Structural Localization of ESP13.2
Gold particles were found to be evenly dispersed over the entire sperm surface, and no evidence was found of gold concentration in any particular sperm domain (i.e., the anterior head, equatorial segment, posterior head, midpiece, or flagellum) (Fig. 8).
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Fluorescence Localization of ESP13.2
In agreement with the fine structural observations, indirect immunofluorescence showed that sperm were coated with ESP13.2 over the entire surface (Fig. 9A), even after sperm were washed through a 3-ml column of 80% Percoll (Fig. 9B). When sperm were incubated overnight at 28°C in 30 mg/ml of BSA followed by a 2-h incubation at 37°C, a reduction of fluorescence was observed over the head and midpiece, but significant fluorescence was still observed on the sperm tail (Fig. 9C). After 2 h at 37°C, sperm were incubated an additional 1 h with activators before fixation, which resulted in almost complete loss of fluorescent labeling (Fig. 9D). Sperm treated with preimmune Ig under the same conditions described in Figure 9 displayed no immunofluorescence (data not shown). The rapid loss of ESP13.2 following sperm treatment with activators was confirmed by Western blot analysis. Samples that were treated identically to those shown in Figure 9, C and D, were solubilized, reduced, and blotted to nitrocellulose. After sperm were treated with activators, the 31- to 35-kDa band was barely visible (Fig. 10D), as compared to sperm that were incubated overnight but were not exposed to activators (Fig. 10B).
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Effect of LSA on ESP13.2
As shown by indirect immunofluorescence using anti-ESP13.2 Ig, sperm that were incubated with LSA continued to be coated with ESP13.2 even after treatment with activators (Fig. 11). After Percoll washing and incubation in capacitation medium overnight, control sperm lost a portion of the ESP13.2 coating (Fig. 11A). That loss was dramatically increased after treatment with activators (Figs. 9D and 11B). When the sperm were incubated in LSA before Percoll washing and incubation, no loss of the fluorescence labeling was obvious, even after incubation with activators (Fig. 11, C and D). Sperm treated with preimmune Ig under the same conditions described in Figure 11 displayed no immunofluorescence (data not shown).
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The retention of ESP13.2 after sperm treatment with LSA was confirmed by experiments using Western blots. Sperm were exposed to LSA and then washed, incubated, and treated with activators before solubilization and probing for ESP13.2. As indicated by the relative densities of the immunoreactive bands, ESP13.2 was retained on sperm after treatment with activators in comparison to control sperm that were not treated with LSA (Fig. 12, A and B). When the supernatants were evaluated for the presence of ESP13.2 after sperm were treated with activators, the supernatant from control sperm had a significant immunoreactive band at 31–35 kDa, but the supernatant from LSA-treated sperm had only a faint band in this region (Fig. 12, C and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of ESP13.2 mRNA in the macaque epididymis was originally reported by Perry et al. [20]. Subsequently, it became clear that this epididymal secretory protein has a structure that is characteristic of ß-defensins, a class of proteins that are components of the innate immune system [25]. Defensins are small, cationic peptides containing a well-conserved six cysteines that are uniquely paired, distinguishing the 
- from the ß-defensins [29].
The present study extends the work of Perry et al. [20] by demonstrating the presence of ESP13.2 on the surface of ejaculated sperm. Perry et al. were unable to demonstrate labeling of ESP13.2 on sperm using an antipeptide antibody. It is possible that the peptide sequence single epitope to which the antibody was built may not be exposed on sperm but was still recognizable within the corpus tissue because it is not yet a fully mature protein. A coating protein, ESP13.2 can be partially removed from the sperm surface by salt extraction and is lost during the capacitation process. It is also a potent antigen, which stimulates production of an isoantibody when injected into female monkeys [18 and present study]. This is consistent with the research on E-3, a ß-defensin found on the surface of rat sperm, which elicited a potent immune response [30]. This observation also appears to be consistent with the biology of the ß-defensins, which have been reported to elicit a call to the adaptive immune system such that monocytes, polymorphonuclear leukocytes, B cells, and T cells are actively recruited to sites where ß-defensins are elevated [31, 32]. In fact, Fearon [33] reported that ß-defensins could act as potent adjuvants of the adaptive immune system.
The findings of the present study confirm the previous report [20] that ESP13.2 is present within the corpus and cauda epididymides but not in other tissue extracts from macaques. As sperm pass through the male reproductive tract, numerous surface modifications can occur [28]. In the present study, ESP13.2 appeared as a single, 35-kDa band on Western blots of the corpus epididymides under reducing conditions, but blots of ejaculated sperm showed at least three bands (31, 33, and 35 kDa), which most likely result from changes in glycosylation of the protein as sperm move through the epididymis.
The epididymis has been shown to alter the sperm plasma membrane by addition of numerous proteins, some of which are integrated into the plasma membrane and some of which are coating proteins that are adsorbed on the sperm surface [5]. Within the family of sperm-coating proteins, some are tightly attached, but others are easily dissociated from the sperm surface [8, 17]. The synthesis and secretion of sperm-coating proteins by the epididymis, whether GPI-anchored or adsorbed, are thought to be under strict control of androgens [6, 7, 12, 34], although it is not known at present whether the production of ESP13.2 is hormonally regulated. Surface coats acquired in the epididymis often are localized regionally to specific domains of the sperm plasma membrane, but a few are globally adsorbed on the entire sperm surface [1].
In the present study, ESP13.2 was found to be uniformly distributed on the heads and tails of macaque sperm at the time of ejaculation. Western blot analysis with nonreducing gel electrophoresis suggests that either ESP13.2 is bound to a number of different proteins via disulfide bonds or undergoes some self-assembly following solubilization in SDS buffer, utilizing its free cysteine. It can be dissociated from sperm surface proteins under conditions of increased osmolarity, suggesting that ionic interactions with the sperm surface are responsible for at least some of the sperm surface binding. The ESP13.2 released by increased osmolarity could readily be resolved as a single band under reducing conditions. A significant amount of ESP13.2 appears to be attached to a number of GPI-anchored proteins, because it is released along with GPI-anchored proteins following PI-PLC treatment of live cells. It is also possible that ESP13.2 interacts with membrane-spanning proteins, such as ion channels. Neurotoxins have a very similar secondary structure to ß-defensins due to their six-cysteine motif [35]. These toxins are known to bind to and inhibit Na+, K+, and Ca2+ channels [36]. It has been recently shown that scorpion toxins are potent inhibitors of t-type calcium channels in sperm [37]. It seems possible that ESP13.2 could also be associated with certain ion channels on the sperm surface.
The mechanism by which ESP13.2 attaches to macaque sperm proteins is not known, but one possibility centers around the highly charged cationic region, which allows for promiscuous binding to multiple partners because of charge attraction [38]. Another possibility lies in the fact that ESP13.2 has a very hydrophobic motif, which could insert into the anionic phospholipids of the plasma membrane [39]. Defensins can readily undergo oligomerization, which is thought to be critical for their antimicrobial activity [40]. In addition, epididymal secretory proteins can bind to the sperm surface via a DTT-sensitive mechanism [41]. Thus, ESP13.2 may interact with the sperm surface, utilizing its free cysteine. Finally, E-3, a defensin, has a lectin-like motif [30], so molecules such as ESP13.2 could attach to sperm via carbohydrate domains.
After sperm are ejaculated into the female reproductive tract, they must reside there for a period of time before they are capable of fertilization, a process termed capacitation [2, 3]. Capacitation involves reversal of the plasma membrane stabilization that took place in the epididymis, allowing the acrosome reaction to occur in response to a physiologically appropriate stimulus [2]. A variety of physiological changes take place in sperm during capacitation, including membrane lipid bilayer modulation, increased protein phosphorylation, intracellular ion fluctuations, and loss of surface coats [4].
Removal or loss of sperm surface components is thought to be an important part of the capacitation process and appears to be reversible. In some circumstances, surface components, once removed from sperm, can be added back to "decapacitate" the sperm and render them incapable of fertilization [4, 42]. These decapacitation factors are reported to reside on the receptors that regulate sperm motility and zona pellucida recognition [13, 43, 44]. A sperm-coating protein, described as a decapacitation factor, was reported to be linked to GPI-anchored proteins on the posterior head of mouse sperm [44]. The complex of GPI-anchored proteins regulates Ca2+-ATPase, and when the coating protein was removed, Ca2+ transport was stimulated [44]. The linkage of the mouse sperm decapacitation factor to the GPI-anchored proteins involves fucose residues [44]. Similarly, ESP13.2 may reside on one or more functionally unique membrane proteins that are involved in fertilization-related functions.
At the time of ejaculation, ESP13.2 was found to be uniformly distributed over the entire sperm surface, and this global distribution did not change during the capacitation process. In addition, ESP13.2 appears to be tightly attached to all regions of the sperm surface. Centrifugation through 80% Percoll did not remove it from the surface, nor did prolonged incubation in medium containing high concentrations of BSA. The latter treatment has been used successfully to remove surface coats from sperm of other species [45].
Although prolonged incubation in capacitation medium resulted in a reduction of ESP13.2 from the sperm surface, it was only when sperm were exposed to the pharmacologic activators dbcAMP and caffeine that ESP13.2 was almost completely removed from the plasma membrane. It is noteworthy that the incubation-related reduction of ESP13.2 was more apparent on the sperm head and midpiece than on the tail. It has been recognized for some time that macaque sperm require exogenous dbcAMP and caffeine for the final synchronization of capacitation and to ensure maximal fertilization in vitro [46]. Sperm capacitation is the culmination of physiological events that allow sperm to recognize and bind to the zona pellucida, to undergo an acrosome reaction, to maintain hyperactivated motility for penetration of the zona pellucida, and to fuse with the oolemma. All of these events have been reported to be regulated by the second messenger, cAMP [47]. Under the conditions reported here, a percentage of macaque sperm are capacitated without the addition of activators, as demonstrated by hyperactivated motility, tight binding to the zona pellucida, and the zona-induced acrosome reaction (data not shown). These events appear to coincide with the limited release of ESP13.2 that follows prolonged sperm incubation in capacitation medium. Sperm treatment with activators results in the synchronized loss of ESP13.2 from most sperm in the population and their enhanced ability to undergo the same capacitation-related events [24].
Although both activators (dbcAMP and caffeine) appear to be required for the synchronization of capacitation of macaque sperm, evidence suggests that the two compounds have different actions on the sperm cell [48]. Macaque sperm exposed to the phosphodiesterase inhibitor, caffeine, have been shown to initiate zona recognition and binding in four- to fivefold greater numbers than sperm exposed to only dbcAMP or controls, whereas dbcAMP has been shown to be most effective in promoting the acrosome reaction of sperm that have completed zona binding [48]. The addition of caffeine alone, but not of dbcAMP, induced a significant loss of ESP13.2 from the sperm surface (data not shown), suggesting that ESP13.2 may reside on the sperm receptor for zona pellucida and that it must be detached from the receptor before zona recognition and binding.
Clearly, a number of phosphodiesterases (PDEs) exist, and cAMP is anchored in various membrane locations so that it can stimulate protein kinase A, either globally or within discrete domains [47, 49]. The A kinase-anchoring proteins are an expanding group of structural proteins, which allow for the subcellular targeting of messages to specific cellular compartments [50]. Recently, it was shown that PDE1A, a variant of PDE1, is linked to calmodulin and is permanently in a state of activation (cAMP breakdown). The inhibition of one or more PDEs could lead directly to a global cellular response, such as loss of ESP13.2 from the entire sperm surface, or the enzyme could be stimulated locally and a more global response could be attained by activation of a cyclic nucleotide-gated channel, leading to membrane hyperpolarization [47, 51]. At least four separate plasma membrane domains exist in mammalian sperm, and ESP13.2 appears to uniformly coat each region, suggesting that hyperpolarization of the plasma membrane may be responsible for the uniform loss of ESP13.2 even though the exact mechanism is not known [47, 52].
Recently, we reported that LSA completely blocked the fertilization of macaque oocytes in vitro [24]. Macaque sperm treated with LSA before or after washing and capacitation had a dramatically decreased capability to recognize and bind to zona pellucida [24], and zona pellucida-induced acrosome reactions were almost completely blocked [53]. In the present study, when sperm were pretreated with LSA before washing and capacitation, including treatment with activators, no apparent loss of ESP13.2 from the sperm surface was observed, yet sperm exhibited normal hyperactivated motility characteristic of capacitated sperm (data not shown), as has been reported previously for LSA-treated sperm [53]. As such, intracellular physiological changes that result from capacitation still appear to occur in the presence of LSA; however, the release of surface-bound ESP13.2 is inhibited. This suggests that one portion of the capacitation process is perturbed by LSA. The retention of ESP13.2 as a result of LSA treatment likely is the sole basis for its antifertility effect on macaque sperm. The mechanism by which LSA impedes the loss of ESP13.2 is currently being investigated.
Originally, ESP13.2 was described as a major secretory protein of the macaque epididymis, with structural similarities to the defensin class of proteins [20]. Since then, HE2 and ESC42 have been characterized as members of the ß-defensin family of the innate immune system found within the primate epididymal tract [34, 54–56]. Some of these proteins are epididymal in origin and secreted into the lumina of different regions of the epididymis; others come from the testes and are associated initially with the sperm [19].
The innate immune system is the first line of defense against the invasion of pathogens, such as bacteria, fungi, and viruses [57]. The defensins are key to this system and have the capability to kill a broad spectrum of pathogens, combating infections until other lines of immune defense can be mobilized [33]. A role for defensins in protecting the male reproductive tract from infection has only recently been recognized [19, 54]. It now appears that the ß-defensins, which are produced by epithelial cells, can be strongly induced by various stimuli, such as bacterial lipopolysaccharides. It was recently noted that secretion of ß-defensins in the epididymis of mice and rats is androgen-dependent but is also stimulated by lipopolysaccharides [58, 59]. If the production of ESP13.2 is under androgen control, it would be secreted continuously, as required for its function as a sperm-coating protein involved in fertilization.
An additional function for ESP13.2 can be envisioned in sperm protection, because the protein coats the entire sperm surface and is abundant within the epididymis. In mice, Bin1b is a hormonally regulated epididymal defensin, but its production is also stimulated by microbial assault [59]. It is not known whether ESP13.2 can function as an antimicrobial agent. Our amino acid sequence data suggest that sperm surface ESP13.2 is not cleaved, as would be required for antimicrobial activity. Metalloproteases, which are numerous on the sperm surface, have the capability to enzymatically cleave defensins and, thus, to release the hydrophobic tail from the antimicrobial peptide, but to our knowledge, no evidence suggests that this action takes place on sperm. The ESP13.2 may not have an antimicrobial function, which is possible, because unlike other defensins, it has a unique, five additional amino acid sequence between the C3 and C4 regions. Because this inner core of the defensin molecule is very well conserved, the structure of ESP13.2 suggests either that its primary function is involved with capacitation and fertilization, rather than with immunity, or that this variation may be related to other, yet undiscovered antimicrobial activities.
In primates, sperm are ejaculated into the vagina and must traverse a barrier of mucus to cross the cervix and gain access to the upper female reproductive tract [60]. Cervical mucus is a well-studied biological fluid that presents immunological and physical barriers to pathogen invasion [61]. The function of sperm surface defensins in the female tract is not readily apparent. The functions of sperm surface-coating proteins in the female reproductive tract before fertilization also are poorly understood. Their possible functions include alteration of surface charge to enhance sperm migration through cervical mucus [61] and prevention of premature or inappropriate acrosome reactions in the female tract [1]. Within the female tract, ESP13.2 may or may not act as a component of the immune system, but it clearly is involved in the sperm capacitation/fertilization process. Further investigation of this function will be important for understanding causes of infertility as well as for contraceptive development.
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FOOTNOTES |
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2 Correspondence: Gary N. Cherr, Bodega Marine Laboratory, University of California, Davis, PO Box 247, Bodega Bay, CA 94923. FAX: 707 875 2089; gncherr{at}ucdavis.edu
Received: 6 February 2003.
First decision: 2 March 2003.
Accepted: 12 May 2003.
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