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a Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
b Department of Animal Science & Food Technology, Texas Tech University, Lubbock, Texas 73409
ABSTRACT
To establish a systematic strategy for characterizing fertilization proteins of sperm cells, we prepared alloantisera by immunizing gilts with salt-washed membranes from boar spermatozoa. The antisera recognized a unique subset of sperm membrane proteins that migrated with Mr 7500–66 000 in SDS-PAGE under nonreducing conditions. The antisera did not recognize proteins of erythrocyte membranes, and tissue absorption experiments further confirmed that the alloantigens were sperm-specific proteins. Each of these sperm-specific membrane proteins (SSMPs) possessed one or more disulfide bonds that were essential for its interaction with alloantibody. Enzymatic deglycosylation revealed that most of the SSMPs were glycoproteins, and their alloantigenicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs identified each comprise at least one extracellular domain. Two-dimensional electrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed acidic pIs (range 4.2–4.8). By indirect immunofluorescence, the SSMPs localized to the cell surface overlying all major regions of the sperm cell. We conclude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions in fertilization.
acrosome reaction, fertilization, gamete biology, sperm, sperm capacitation
INTRODUCTION
Animal fertilization comprises a series of several overlapping and interdependent cellular events [1, 2]. In mammals, fertilization begins upon sperm transfer to the female and is complete when the male and female genetic complements merge to form a diploid zygote. The complexity of this process is amplified by interspecies differences that are readily apparent in the diversity of sperm morphology and the inefficiency of heterospecific fertilization [2–6]. The relative species-specificity of fertilization probably derives from variation in fertilization proteins and the cellular events they mediate [2, 4, 7, 8]. Considering the complexity of fertilization, a correspondingly complex repertoire of fertilization proteins would seem to be required. Because neither the size of this repertoire nor the identities of its protein constituents have been determined, it is presently impossible to estimate the extent to which we understand fertilization at the molecular level.
Spermatozoa are among the most highly specialized of all animal cells. The activities of sperm-specific proteins are important for this cell's only function-fertilization. Studies focusing on individual fertilization events have led to the discovery of several unique sperm-specific proteins. Biochemical [7, 9–11], immunological [12–14], and molecular genetic [15–17] methods have been applied in various species. Different proteins that appear to mediate the same event have been discovered using these diverse experimental strategies [7, 11, 18–22], and this has sometimes led to conflicting conclusions about molecular mechanisms. It is unclear whether these conflicts are a function of incomplete understanding, interspecies variation, or both. Thus, systematically characterizing the complete repertoire of sperm-specific fertilization proteins in a single species would improve our understanding of fertilization.
Sperm-specific proteins induce alloantibodies when they are injected into females of the same species [23–25]. They are major molecular targets of the alloimmune response to whole spermatozoa or sperm fractions. Proteins that are not sperm-specific are perceived as self, so the immune response to them is generally small compared to nonself antigens. In humans and other mammals, antisperm alloantibodies cause infertility [14, 25, 26], apparently by binding to and inhibiting the activities of fertilization proteins. Experimentally induced auto- or alloantibodies to spermatozoa, sperm fractions, or to sperm-specific proteins inhibit fertilization in vitro [27–34] and in vivo [24, 30, 35]. Thus, sperm alloantigens are sperm-specific proteins that are likely to have important functions in fertilization.
Studies in mice, guinea pigs, rabbits, and humans documented the feasibility of identifying sperm-specific proteins with auto- or alloantisera [12, 30, 34, 36–38]. Such proteins in any subcellular compartment are of interest because of their potential functions in fertilization. However, membrane proteins are of particular interest because they are likely to be receptors or adhesion molecules with important functions in recognition and signaling events of fertilization. In this paper we report use of alloantisera to identify and characterize sperm-specific membrane proteins (SSMPs) in the pig, a large animal species from which these proteins can be isolated and further studied. Our results represent a step toward the goal of determining how the concerted actions of multiple proteins mediate fertilization in a single mammalian species.
MATERIALS AND METHODS
Isolation of Sperm Membranes
Particulate fractions containing plasma membranes were isolated from pig-ejaculated spermatozoa. The sperm-rich fractions of fresh ejaculates (200–400 ml each) were immediately diluted fourfold in warmed extender (AndroHep; Minitube of America, Verona, WI), transported to the laboratory in insulated containers, checked microscopically to assess semen parameters (cell count, motility), and then filtered through a 70-µm screen to remove acellular debris. Cells (>90% motile) were washed with 2 x 1 L HNE (10 mM Na-Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA) by centrifugation for 5 min at 400 x g, suspended in 1 L HNE containing 1 mM diisopropylfluorophosphate (DFP; Sigma Chemical Co., St. Louis, MO), and then disrupted by N2 cavitation at 650 psi, which preferentially removes the sperm plasma membrane [39]. Particulate fractions containing the released plasma membranes were then isolated by differential high-speed and ultracentrifugation as for isolation of membranes from epididymal spermatozoa [7, 40]. To remove peripherally bound proteins that might be acrosomal or not of germ cell origin (e.g., from seminal fluid), isolated particulate fractions were washed twice with 1 M NaCl by ultracentrifugation (235 000 x g, 45 min, 2°C), and stored at -70°C.
Isolation of Erythrocyte Membranes
Packed erythrocytes (1.5 L) from 3 L of pig blood were suspended in 7.5 L of hypotonic buffer (HE-DFP: 5 mM Na-HEPES pH 7.5, 1 mM EDTA, 0.1 mM DFP) and centrifuged (17 700 x g, 15 min, 2°C, Beckman JA-17 rotor). The pellet was resuspended in HE-DFP, and the swollen cells were disrupted in portions with a 200-ml Potter-Elvejem homogenizer (300 rpm, 10 strokes, 0°C). The homogenate was filtered through a 25-mesh screen and centrifuged at low speed (1000 x g, 15 min, 2°C) to remove debris and unlysed cells. The supernatant suspension was recentrifuged (17 700 x g, 15 min, 2°C) to recover a crude particulate fraction that was then washed sequentially with 3 x 800 ml of HE-DFP (17 700 x g, 30 min, 2°C). The final pellet was resuspended in 50 ml HE-DFP and stored at -70°C. The SDS-PAGE profile of this preparation corresponded to that previously described for pig erythrocyte membranes [41].
Protein Assays
Protein concentrations were measured with bicinchoninic acid (BCA Assay; Pierce Chemical Co., Rockford, IL), using BSA as a standard [42].
Antisera Production
Gilts were immunized with pig sperm membranes prepared as described above. Primary injections (i.m.) consisted of either 80 mg membrane protein/animal (alloimmune group, n = 2) in Freund complete adjuvant [43] or adjuvant only (nonimmune control group, n = 2). Similarly, booster injections (i.m.) at 6 and 10 wk postprimary each consisted of incomplete adjuvant ± 80 mg membrane protein. Sera were harvested 3 wk after the final boost and stored at -70°C.
Electrophoresis
Polyacrylamide gel electrohoresis (8–15% linear gradient gels) in the presence of SDS was done according to Laemmli [44]. When desired, protein disulfides were reduced with 10 mM dithiothreitol (5 min, 100°C, in 1x SDS-PAGE sample buffer), and the sulfhydryls formed were alkylated with 24 mM iodoacetamide (5 min, 22°C). Two-dimensional electrophoresis consisted of isoelectric focusing (IEF) in the first dimension, and SDS-PAGE (10–15% linear gradient) in the second dimension [45, 46]. The IEF was done in 1.5-mm tube gels containing 9 M urea, 2% Triton X-100, and 2% (w/v) ampholines (Amersham/Pharmacia, Piscataway, NJ); gels for IEF over pH 3.5–10 contained only ampholine 3.5–10, and gels for increased resolution in the acidic range contained a 4:1 ratio of ampholines 4–6 and 3.5–10. Molecular weights were estimated by comparing migration of SSMPs with those of protein standards (Broad Range; BioRad Laboratories, Richmond, CA), and pIs were estimated by measuring the pH of 5-mm IEF gel sections. Gels were routinely stained first with Coomassie blue, then silver-stained [47, 48].
Western Blotting
For Western blotting, proteins separated by SDS-PAGE were transferred electrophoretically (10–12 V·h/cm, 22°C) to nitrocellulose membranes (Micron Separations Inc., Westborough, MA) [40, 49]. For all Western blotting experiments, incubations were at 22°C with constant orbital shaking, unbound antibody and/or peroxidase conjugates were removed by washing (2 x 10 sec, 2 x 15 min) with TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20), and bands were visualized by chemiluminescence (SuperSignal HRP; Pierce Chemical Co.). Proacrosin was detected as described [40] with a 40 000-fold dilution of rabbit antiserum to the zymogen purified from guinea pig testes [48], and heterotrimeric G-proteins were similarly detected with a 20 000-fold dilution of rabbit antiserum to the Gß subunit (antiserum U-49, kindly provided by A. Gilman, UT Southwestern Medical Center, Dallas, TX). Blots were incubated at least 1 h with diluted antisera, washed to remove unbound antibody, incubated 1 h with horseradish peroxidase (HRP)-conjugated antibody to rabbit immunoglobulin (BioSource International, Camarillo, CA) diluted 40 000-fold in TBST, and then washed again prior to immersion in chemiluminescent substrate. SSMPs were detected with alloantisera to sperm membranes. After blocking for 
1 h with 5% nonfat dry milk in TBST, blots were incubated at least 1 h in antisera diluted 10 000-fold in milk-TBST, washed, incubated 1 h with HRP-conjugated protein A (Pierce Chemical Co.) diluted 10 000-fold in TBST, washed again, and developed. Artifactual signal from nonspecific binding of HRP-protein A to band 1 of pig erythrocyte membranes [41] was removed digitally from some images in Figure 2. Biotinylated proteins from immunoprecipitations were detected with HRP-conjugated streptavidin (BioSource International) as described [40], except that blots were blocked for 30 min with 2% BSA in TBST prior to incubation with the HRP conjugate.
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Absorption of Antisera
Acetone powders of pig testis (no epididymis) and spleen were prepared as described by Tabor [50]. Pooled alloantisera were diluted 50-fold in TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 % Tween 20) containing a cocktail of group-directed protease inhibitors: 1 mM EDTA for inhibition of Ca2+-dependent proteases and metalloproteases, 1 mM DFP for irreversible inhibition of serine proteases, and 10 µM N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-agmatine] (E-64; Boehringer Mannheim, Indianapolis, IN) for inhibition of thiol proteases. Diluted sera (300 µl) were absorbed three times sequentially with acetone powder (5 mg per absorption) by vortexing 30 min at 22°C and microfuging (22 000 x g, 15 min, 2°C). The supernatant fractions from the final absorptions were then diluted another 100-fold with TBST and used immediately to probe Western blots.
N-Deglycosylation
Glycoproteins were N-deglycosylated with peptide-N-glycosidase (PNGase) F from F. menigosepticum (Oxford GlycoSystems, Bedford, MA). Briefly, 100 µg of sperm membrane protein was solubilized at 5 mg/ml in the vendor-supplied reaction buffer containing 0.5% SDS. This solution was then diluted twofold by addition of Triton X-100, aprotinin, E-64, and DFP to final concentrations of 1.25% (v/v), 100 µM, 20 µM, and 1 mM, respectively. Deglycosylation was carried out by adding 4 U of enzyme (0.2 U/µl) and incubating at 37°C for up to 3 h. A parallel reaction incubated without added enzyme was run as a control for possible endogenous hydrolase activities. Samples (10 µl each) were removed from the reactions at various times and mixed with 2x SDS-PAGE sample buffer in preparation for analysis by Western blotting.
Immunoprecipitations
Sperm proteins in isolated particulate fractions were biotinylated on amino groups [40] with sulfosuccinimidyl-6-(biotinamido) hexanoate (Pierce Chemical Co.), then solubilized at 10 mg/ml in 1% (w/v) SDS. The solubilized, biotinylated proteins were then diluted to 1 mg/ml with HNE containing 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 20 µM E-64, and 100 µM aprotinin (Boehringer Mannheim). To preclear, 1 µl of pooled nonimmune control sera were added to 400 µl of diluted extract, and the mixture was incubated for 1 h at 0°C. Protein A-Sepharose (10 µl of a 50% slurry; Amersham/Pharmacia) was then added, the suspension was rocked for 1 h at 4°C, and the beads were collected by centrifugation (10 000 x g, 15 sec, 4°C). The precleared supernatant solution was then used for immunoprecipitation with pooled alloimmune sera (1 µl/100 µl) and Protein A-Sepharose as for the preclearing. The preclear and immunoprecipitated beads were each washed by centrifugation with 3 x 1 ml of HNE containing 1% Triton X-100, and bead-bound immune complexes were eluted with 50 µl of 1x SDS-PAGE sample buffer.
Immunofluorescence
Pig spermatozoa obtained as for membrane isolations were washed once by centrifugation (5 min, 400 x g) with PBS (10 mM NaPO4, 150 mM NaCl, pH 7.4). The washed cell pellet was gently resuspended in buffered 4% paraformaldehyde (freshly prepared), and the cells were fixed for 15 min [51]. Fixed cells were diluted 20-fold with PBS, dropped on polylysine-coated coverslips, and allowed to settle and attach for 60 min [51]. Coverslips were blocked for 30 min with PBS containing 3% BSA, then incubated 60 min with 400-fold dilutions of antisera in PBS-BSA. After washing 3 x 5 min with PBS, the coverslips were incubated in a Texas red conjugate of protein A (Pierce Chemical Co.) diluted 500-fold in PBS-BSA. The coverslips were then washed again with PBS, mounted with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL), and viewed with a Zeiss Axiovert fluorescence microscope. All manipulations for immunofluorescence were done at 22°C.
RESULTS
We first tested whether immunizing gilts with sperm membranes would elicit an alloimmune response to sperm proteins similar to that observed in smaller mammals. To maximize chances that the dominant immunogens would be membrane proteins of germ cell origin, peripheral proteins were stripped from the membranes with salt. This was necessary because acrosomal matrix proteins and proteins from epididymal or seminal fluid are sometimes present in preparations of boar sperm membranes [40, 52]. Western blots confirmed that this procedure removed most contaminating proacrosin, a marker for acrosomal matrix proteins (Fig. 1). The remaining salt-wash-resistant proacrosin may represent a persistent contaminant or truly membrane-associated protein as postulated by others [53]. The salt-washed fraction contained Gß subunits of heterotrimeric G-proteins, confirming the presence of plasma membrane-containing receptors and signaling components with potential functions in fertilization.
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Alloantisera to the salt-washed membranes recognized at least 10 antigens ranging from Mr 7500 to Mr 66 000 on Western blots of sperm membrane proteins (disulfides not reduced, Fig. 2). Sera from two different alloimmune gilts identified similar profiles of sperm protein antigens, but did not recognize antigens from pig erythrocyte membranes. Nonimmune control sera or pooled preimmune sera did not react with any sperm proteins. Alloantisera absorbed with an acetone powder of boar testis recognized only one (Mr 26 000) sperm protein (Fig. 3). In contrast, absorption with spleen tissue powder did not alter the pattern of bands recognized.
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The alloantibodies did not bind to sperm membrane proteins that had undergone disulfide bond reduction (Fig. 4). N-deglycosylation of sperm membrane proteins altered the electrophoretic mobilities of all but one of the SSMPs (Fig. 5). The deglycosylation reaction was complete within 30 min, as no further changes in mobilities were observed even after 3 h of incubation. The mobilities of SSMPs from a parallel reaction without added enzyme did not change.
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The alloantisera also recognized solubilized SSMPs in an immunoprecipitation assay (Fig. 6). The pattern of immunoprecipitated SSMPs resembled the pattern of SSMPs detected on Western blots probed with alloantisera (compare to Fig. 2). Nonimmune control sera did not precipitate significant amounts of sperm proteins.
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The SSMPs were characterized further by two-dimensional electrophoresis and Western blotting (Fig. 7). When first dimension gels were focused over a broad pH range, fewer than 20 immunoreactive proteins were detected (Fig. 7a) even though silver-staining indicated that more than 100 proteins were resolved (Fig. 7b). Several acidic SSMPs and a train of four Mr 26–30 000 immunoreactive proteins with basic pI were evident. The four basic proteins were classified as a single SSMP (Table 1) because this pattern is reminiscent of a single polypeptide modified post-translationally with variable numbers of an acidic moiety (i.e., phosphate or sialic acid). When resolution in the acidic range of the first-dimension gels was enhanced, 13 SSMPs with acidic pIs were detected (Fig. 7, c and d).
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In indirect immunofluorescence, the alloantisera detected SSMP antigens associated with all major structures of paraformaldehyde-fixed spermatozoa (Fig. 8, a–d). On cells immunostained with alloantisera, fluorescence was notably punctate on the apical head and the principal piece of the tail (Fig. 8a). Staining was stronger and more uniform on the postacrosomal head and tail midpiece of these cells. No fluorescence was evident on cells stained with nonimmune control sera (Fig. 8c).
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DISCUSSION
Sperm-specific proteins mediate key molecular events of the fertilization process, but these proteins have not been characterized systematically in any one species. Here we have tested the hypothesis that, as for small mammals [12, 30, 34, 36, 37], alloantisera can be used to identify and characterize sperm-specific membrane proteins in swine. Because pig sperm membranes can be obtained in large quantities, future purification and characterization of these proteins will not be hampered by the limitations in starting material inherent in small animal studies.
Our absorption results demonstrate that all but one of the alloantigens identified are present in the testis where, as proteins destined for insertion in the sperm membrane, they are presumably synthesized by germ cells. The failure of testis to absorb immunoreactivity to the Mr 26 000 antigen indicates that this protein becomes associated with spermatozoa after they exit the testis. The absence of the alloantigens in both spleen and erythrocytes confirms that they are not proteins commonly present in somatic tissues. Although these results do not unequivocally rule out the possibility that one or more of these proteins is expressed in other somatic tissues, this is unlikely given the nature of the immunization regimen and the results obtained from previous studies in smaller mammals [12, 24, 25, 30]. Thus, we conclude that the sperm membrane antigens detected are indeed sperm-specific proteins.
The striking inability of the alloantisera to recognize any disulfide-reduced sperm membrane proteins demonstrates that the SSMPs must contain at least one disulfide bond each, and the alloantibodies bind to structures that are dependent on these bonds. Furthermore, deglycosylation revealed that most SSMPs are N-linked glycoproteins, and the reactivity of the immune sera with multiple SSMPs is not dependent on the presence of N-linked oligosaccharides. Collectively, these results indicate that the epitopes recognized by the antisera are protein, but they are not linear strings of amino acids that could easily be mimicked by synthetic peptides. Rather, they appear to be conformational epitopes that require at least some protein tertiary structure.
Although the pattern of proteins observed in our silver-stained two-dimensional gels resembled those reported by Russell et al. [54, 55] for boar sperm membranes, protein disulfides were reduced in these and other [56, 57] previous studies, so direct comparisons with our results were not possible. Consequently, we are unable to determine which of the proteins we detected are already known, and which are novel. The sequencing of cDNAs encoding these proteins will enable straightforward comparisons to sequence data generated by other studies. Such comparisons, even though limited by the relative paucity of data from swine, should be especially useful for assigning potential functions to genes identified by large-scale, random sequencing projects.
By design, the SSMPs identified in this work are the immunodominant proteins of the pig sperm membrane. Some of these proteins may be immunodominant because they are abundant components of the membrane. For example, SSMPs 4, 5, and 13 were readily visible in silver-stained two-dimensional gels of membranes (Fig. 7). In contrast, SSMPs 10–12 were not visible; such proteins may instead be strongly immunogenic because they possess unusual structures. Because some SSMPs may be minor sperm membrane components that lack unusual structures, the 14 proteins identified in this study represent a minimal estimate of the SSMP repertoire. More SSMPs may be identified by immunizing gilts with membranes from which the immunodominant proteins have been removed.
Three types of SSMPs have previously been identified: 1) products of genes transcribed only by germ cells; 2) germ cell-specific variants of genes that are also transcribed in somatic cells; and 3) proteins that are produced by male somatic tissues (e.g., the epididymis or accessory glands) and become associated with the cell during epididymal maturation or ejaculation. Each of these SSMP types includes proteins with functions in fertilization [14, 52, 58]. Of the 14 SSMPs we identified, only SSMP1 falls in the third category. The fact that SSMP1 was not removed during our isolation procedure suggests that it binds with high affinity to the sperm membrane. This protein is therefore unlikely to be removed during sperm transit through the female reproductive tract and could function in gamete interactions despite its not being of germ cell origin. Indeed, the size, pI, and post-testicular origin of this protein suggest it could be the swine homolog of P26h, which is an epididymal protein that appears to function in sperm-zona pellucida adhesion [59]. Our use of salt-washed membranes favored identification of integral membrane proteins, so it is not surprising that the other 13 SSMPs identified are synthesized in the testis. Determining whether these 13 proteins are type 1 or type 2 SSMPs is not possible without further study.
SSMPs are likely to be receptors, cell-signaling components, or adhesion molecules that mediate a variety of fertilization events [1, 60]. Several sperm surface receptors have not been discovered, including a novel progesterone receptor that triggers the acrosome reaction [61], a receptor for a chemoattractant in follicular fluid [62], and receptors for motility-promoting [63] and decapacitation factors [64]. Spermatozoa may also possess unique signal transduction molecules that interact with these receptors. Finally, although various investigators have discovered adhesion molecules that function during sperm interaction with the zona pellucida [7, 11, 14, 18, 19, 40] or the oocyte membrane [14, 65], controversies still remain, and other yet unknown sperm components may also be important in these processes. Our immunofluorescence on aldehyde-fixed cells demonstrated that pig SSMPs are localized primarily (but perhaps not exclusively) on the surface of all regions of the sperm cell (head and tail), suggesting that these proteins might function in several fertilization events. Systematic characterization of pig SSMPs, regardless of whether they are abundant or rare, should clarify the fundamental molecular bases of these events and lead to a comprehensive understanding of fertilization in a single species.
SSMPs are potentially good targets for contraceptives that function by disrupting gamete interactions. The presence of disulfides and glycosylation in the SSMPs we identified, as well as localization of these proteins to the surface of aldehyde-fixed, unpermeabilized cells, indicates that they are present in an extracellular compartment that is accessible to pharmacological agents. By immunizing gilts we have defined a group of proteins that may be especially suitable for development of female immunocontraceptives. Our results do suggest strongly that immunization with properly folded and disulfide-bonded forms of these proteins would be required to elicit antibodies capable of neutralizing their activities in fertilization.
ACKNOWLEDGMENTS
We are grateful to Debbie Alberts in the laboratory of Doug Stocco for assistance with IEF, John McGlone for providing boar semen, and Sam Prien for helpful comments.
FOOTNOTES
First decision: 31 March 2000.
1 Supported by grants from the TTUHSC Office of Research and the National Institutes of Health (HD-35166) to D.M.H. N.P.H. was supported by a State of Texas line item for efficient pork production. 
2 Correspondence: Daniel M. Hardy, Dept. of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 Fourth St., Lubbock, TX 79430. FAX: 806 743 2990; daniel.hardy{at}ttmc.ttuhsc.edu
3 Current address: Department of Animal Sciences, North Carolina State University, Raleigh, NC 27695.
Accepted: August 4, 2000.
Received: March 9, 2000.
REFERENCES
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