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Regular Article |
a Department of Cell Biology and the Center for Recombinant Gamete Contraceptive Vaccinogens, and
b the W.M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia, Charlottesville, Virginia 22908
c Department of Anatomy & Cell Biology, Queens University, Kingston, Ontario, Canada K7L 3N6
ABSTRACT
Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.
reproductive immunology, sperm
INTRODUCTION
Obstruction of the male reproductive tract by disease or by procedures such as vasectomy is often followed by increases in antisperm antibodies [1–3]. Although the induction of an immune response to spermatozoa in this way has been observed repeatedly in most species, including humans, there is relatively little information on the identity of the specific sperm components that are recognized by the postobstruction autoimmune response.
Early work on defining sperm autoantigens in the experimental allergic orchitis model in guinea pigs yielded only semipurified preparations [4]. An autoantigenic sperm surface molecule, sialoglycoprotein RSA-1, was subsequently cloned from the rabbit, and a related family of peptides was identified in several species [5]. More recently, a novel sperm-specific peptide antigen was identified and cloned using a monoclonal antibody obtained from a vasectomized mouse [6]. In the case of human sperm, postvasectomy sera react with nuclear protamines [7], DNA polymerase [8], nuclear autoantigenic sperm protein [9], and the sperm-specific glycoprotein FA-1 [10]. The mass and charge of other human sperm antigens have been determined by one-dimensional (1-D) and two-dimensional (2-D) Western blotting [11–14] or by immunoprecipitation [15] with postvasectomy antisera, and thus the apparent molecular masses of some of these proteins are known [16].
When extracts of rat sperm proteins were probed with sera from vasectomized, isoimmunized, or prepubertally obstructed rats, Western blots displayed a common repertoire of "immunodominant" antigens, including proteins of approximately 82–78, 68 or 63, 57, 42, 36, and 22 kDa [17–19]. A relatively small set of protein autoantigens is recognized by antibodies in postobstruction sera, despite the fact that 1500 or more sperm proteins are discernable in mammalian spermatozoa by high-resolution 2-D gel electrophoresis [20]. Thus, the apparent molecular mass and isoelectric point (pI) of some of the autoantigenic proteins are now known from 1-D and 2-D immunoblots, but to our knowledge the only rat sperm autoantigen identified and sequenced is sperm mitochondria-associated cysteine-rich protein (SMCP) [21], a major structural element of the mitochondria in the midpiece of the sperm tail that was formerly known as mitochondrial capsule selenoprotein (MCS) [22].
The SMCP was cloned and characterized in our laboratory by screening a rat testis expression library with postvasectomy and hyperimmune sera [21], an effective but time-consuming approach. Subsequently, we used 2-D gel electrophoresis with isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension to separate sperm proteins. Western analysis of blots from 2-D gels using postobstruction rat sera [23] provided increased information about the molecular weight (MW) and pI of postobstruction autoantigens and supported the hypothesis that there is a relatively small set of sperm proteins that can be regarded as dominant postobstruction sperm autoantigens. In the present study, we used Western blotting with postobstruction sera, coring of protein spots from preparative gels, and microsequencing by mass spectrometry to identify sperm autoantigens.
MATERIALS AND METHODS
Animals and Surgical Procedures
Lewis rats purchased from Charles River Laboratories (Wilmington, MA) were used throughout this study. Adult males, weighing 225–275 g, received a bilateral vasectomy or a sham operation, and sera were collected 3 mo later [18, 23]. The surgical procedures were performed under aseptic conditions with general anesthesia as previously described [18, 23]. Blood was obtained by cardiac puncture from each rat and allowed to coagulate for 3–4 h at 4°C prior to centrifugation at 2000 x g for 15 min. Sera were then collected and stored at -70°C. Investigations were conducted with the approval of the Animal Research Committee of the University of Virginia School of Medicine and in accordance with the Guide for the Care and Use of Laboratory Animals and other relevant publications. Ten serum samples were studied. The reaction of one postvasectomy serum on Western blot analysis was extremely intense, making it difficult to resolve individual immunoreactive spots; thus, that sample was eliminated from the study. Calculations of the percentages of postobstruction sera that bound individual antigens were based on nine samples.
Sperm Samples
Sperm were prepared from the cauda epididymidis of Lewis rat retired breeders as previously described [23]. The animals were killed by inhalation of halothane, and the epididymides and vasa deferentia were removed. An irrigating cannula was inserted into the distal end of the vas, and a small cut was made in the middle of the cauda epididymidis. M-199 buffer (Gibco BRL, Grand Island, NY) with 3.5 mM sodium pyruvate and 
0.2 mg/ml polyvinyl alcohol was used to flush the sperm from the vas and distal cauda. The sperm were pelleted by centrifugation at 400 x g for 8 min and washed with buffer. Sperm were solubilized in lysis buffer [24] containing 2% NP-40, 100 mM dithiothreitol (DTT), 9.8 M urea, 0.8% (w/v) 3.5–10 pH ampholines, and multiple protease inhibitors [20] at a final concentration of 9 x 107 cells/ml for 30 min at 4°C. The insoluble material was pelleted by centrifugation at 10 000 x g for 10 min, and the supernatant was used as the loading sample for IEF gels.
Two-Dimensional Gel Electrophoresis
The IEF gels were run using the methods previously described [20]. Acrylamide rods (15 x 0.15 cm) for IEF had the gel composition described by Hochstrasser et al. [25] and contained an ampholine composition (v/v) of 30% 3.5–5 pH, 20% 5–7 pH, 10% 7–9 pH, and 40% 3.5–10 pH. Thirty microliters (0.15–0.2 mg of protein determined by the bicinchoninic acid method; Pierce, Rockford, IL) of the sperm extract was applied to each IEF gel. The loaded sample was covered with overlay buffer containing 5% NP-40, 0.4% (w/v) 3.5–10 pH ampholines, 8 M urea, and 100 mM DTT. The gels were focused for 17 700 total volt-hours divided as follows: 200 V for 2 h, 500 V for 5 h, 800 V for 11 h, and 2000 V for 3 h. The IEF gels were then loaded on the second dimension SDS-PAGE gels of 9–16% acrylamide, 1.5 mm thickness, 16 x 16 cm. Six gels were run simultaneously using a Protean II multicell apparatus (Bio-Rad, Hercules, CA) at 245 mA for 4–5 h. Proteins were stained in the gel with silver nitrate or were electroblotted onto nitrocellulose membranes using the BioRad Trans-Blot Cell as previously described [20]. The silver nitrate staining was done in the absence of glutaraldehyde using the method of Shevchenko [26], which is compatible with analysis by mass spectrometry. Proteins of interest were cut out of these gels and were submitted to the W.M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia.
In preliminary experiments, use was made of nonequilibrium pH gradient electrophoresis (NEPHGE [20]) in an effort to study additional sperm proteins of more basic pI than those visualized by IEF [20]. Because the number of immunoreactive proteins and their resolution was greater with IEF than with NEPHGE, we elected to concentrate on IEF in the present study and to leave NEPHGE analysis for future experiments.
Western Blot Analysis
The membranes containing sperm proteins blotted from 2-D gels were washed in PBS twice for 5 min and then blocked with 5% dry nonfat milk in PBS with 0.05% Tween-20 (PBS-tw) for 1 h. Each primary pre- or postobstruction serum sample was incubated with the membrane at a 1:250 dilution in PBS-tw overnight at 4°C while rocking. The blots were then washed three times for 5 min each in PBS-tw prior to incubation for 1 h with the secondary antibody, goat anti-rat IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) diluted 1:5000 in PBS-tw. The membranes were again washed three times for 5 min each in PBS-tw, and proteins were visualized using an enhanced chemiluminescence Western blotting detection reagent kit from Amersham (Buckinghamshire, UK). Other blots of sperm proteins were incubated with a 1:75 000 dilution of a rabbit polyclonal antiserum raised against isolated rat outer dense fibers [27] and subsequently were exposed to the secondary antibody, goat anti-rabbit IgG-horseradish peroxidase conjugate at 1:5000. These blots were washed in PBS-tw three times for 5 min each and stained with the peroxidase substrate, TMB (3,3',5,5'-tetramethylbenzidine and 0.01% H2O2) (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Immunoblots were scanned with a flat bed scanner (Umax, Fremont, CA) at a resolution of 300 dpi. The resulting digitized images were analyzed using Melanie II 2D software (BioRad). The total number of features (spots) in each blot was determined using the following parameters: number of smooths, 4; threshold for Laplacian function, 4; threshold for partial derivatives, 1; saturation, 90; peakedness increase, 100; minimum feature perimeter, 40. These detection parameters were selected conservatively so that too few rather than too many spots would be detected, and automatic detection was followed by manual spot editing to add clearly visible spots, to delete scanning artifacts (e.g., bubbles), and to separate spots. Spot editing and analyses were conducted in unison by two of the authors (J.R. and F.C.L.J.) to increase objectivity. After completion of spot detection and manual identification of a few pairs of matching spots (seed matches), each pair of blots (pre- and postvasectomy) was matched to identify reactive spots nonspecific to vasectomy. These nonspecific spots were deleted from the postvasectomy images, resulting in nine digitized images containing only spots with specific postvasectomy immunoreactivity. Using these nine images, a synthetic image was generated containing all specific spots. The labeling feature of the Melanie II software was utilized to identify which and how many animals had contributed to a specific spot in the synthetic image. The silver-stained gel to be used for microsequencing was scanned, and spots were detected and edited as described for the immunoblots. Spots on the silver gel were then matched with the synthetic image containing all specific immunoreactive spots.
Mass Spectrometry Analysis
Of 28 spots that reacted with more than 50% of the postvasectomy sera and showed no reaction with sera obtained preoperatively, 20 were cut from the gels for mass spectrometric analysis based on visual assessment of the intensity of staining on 2-D Western blots, density and location on the computer-generated composite of postvasectomy blots, and separation from other proteins on Coomassie- or silver-stained gels (the eight spots that were not used were close to other proteins). One additional spot (no. 4) was included because of its intense staining with every postvasectomy serum sample, even though it stained lightly with some preimmune sera. The 21 protein spots were cut from 1.5-mm-thick acrylamide gels. The gel pieces were deposited in siliconized microcentrifuge tubes, which had been rinsed previously with ethanol, water, and ethanol, and were destained overnight at 22°C in 0.5 ml of 50% methanol and 5% acetic acid. The pieces were dehydrated in 200 µl acetonitrile and dried completely in a vacuum centrifuge. Proteins were reduced in 50 µl of 10 mM DTT for 30 min prior to alkylation in 50 µl of 100 mM iodoacetamide for 30 min at 22°C. The samples were dehydrated in 200 µl acetonitrile, hydrated in 200 µl ammonium bicarbonate, and again dehydrated with 200 µl acetonitrile before being dried completely in a vacuum centrifuge. The gel pieces were rehydrated for 5 min in 50 µl of an ice-cold solution containing 20 ng/µl sequencing grade modified porcine trypsin. After removing any excess trypsin solution, enzymatic digestion was allowed to proceed overnight at 37°C. Peptides produced in the digest were collected by serial extraction, first with 50 µl of 50 mM ammonium bicarbonate and then twice with 50 µl of 50% acetonitrile and 5% formic acid. The extracts were combined in a siliconized, rinsed (ethanol, water, ethanol) microcentrifuge tube and concentrated to 20 µl in a vacuum centrifuge.
Peptides were sequenced using a microcapillary column liquid chromatography tandem mass spectrometry system comprised of a Finnigan LCQ ion trap mass spectrometer (ThermoQuest, San Jose, CA) with a nanospray ion source (Protana, Odense, Denmark) interfaced to a self-packed 8-cm x 75-µm inside diameter Jupiter 10-µm C18 reverse-phase capillary column (Phenomenex, Torrance, CA). Peptide extracts (0.5–5.0 µl) were injected, and peptides were subsequently eluted from the column using a gradient of 2–85% acetonitrile/0.1 M acetic acid over 30 min (flow rate of 0.25 µl/min). The microspray ion source was operated at 2.8 kV. Analysis of the digest was accomplished using a full data-dependent acquisition routine. In this procedure, a full scan (MS) spectrum to determine peptide molecular weights was acquired in one scan. Product ion (MS/MS) spectra were acquired in the next four scans, to determine amino acid sequence, before the cycle was repeated. Approximately 500 MS/MS spectra, which range in abundance over several orders of magnitude, were provided by this means of analysis, although not all MS/MS spectra were derived from peptides. Database searches to identify proteins were performed on the MS/MS spectra by automatic batch analysis for each spot using Sequest (Thermoquest) with the nonredundant and expressed sequence tag databases. Amino acid sequence data were analyzed additionally using the Genetics Computer Group (GCG) analysis program package (Madison, WI).
RESULTS
The 2-D gel electrophoresis of rat sperm extracts achieved the separation of hundreds of proteins (Fig. 1). Exposure of 2-D Western blots of sperm proteins to serum from individual vasectomized animals resulted in immunostaining of multiple protein spots, whereas sera from the same animals prior to obstruction showed little reaction (Fig. 2), and sera from animals that received a sham vasectomy bound many fewer spots (Fig. 3). Computer analysis of the staining patterns of individual sera was used to generate a composite image of the sperm antigens recognized postvasectomy (Fig. 4), in accord with the idea that a consensus set of sperm antigens bound antibodies from individual sera with high frequency, as previously reported [23]. A set of 28 proteins was identified that reacted with between 50% and 100% of the postvasectomy sera and showed no significant staining with sera from rats that had received a sham operation. Ten spots (Table 1) were initially selected for study based on the incidence of staining with postvasectomy sera, intensity of reaction, separation from other proteins, and distribution in different parts of the gel (i.e., representing a range of molecular weights and isoelectric points; Fig. 1).
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Analysis of data obtained by mass spectrometry and searching databases using the Sequest search algorithm revealed matches to rat, mouse, and/or human proteins, including proteins of the outer dense fibers of the sperm tail. Subsequent database searches (GCG) permitted alignment (Fig. 5) of the peptide sequences of all the outer dense fiber proteins obtained by mass spectrometry to the rat outer dense fiber protein odf1 (M88759, MW = 27 351) or odf2 (U62821, MW = 68 672). Eight of the initial 10 spots were identified as outer dense fiber proteins, 4 (1–4) matching odf2 and 4 others (6, 8–10) corresponding to odf1. Spot 5 was identified as rat triosephosphate isomerase (P48500, MW = 26.9, pI = 6.45), and spot 7 contained peptides matching both odf1 and triosephosphate isomerase. In most cases, the identifications were made using multiple peptides (Table 1) and thus provided strong matches. Exceptions were spots 8 and 10, each of which was identified using only one peptide.
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The smaller of the two outer dense fiber proteins, odf1 (spots 6–10; MW 27.5), appeared to be phosphorylated in some cases. Spot 9 showed phosphorylation at Ser 153; Ser 180 was mostly unphosphorylated, although a small amount was phosphorylated at this position. In addition, one of the serines 104–109 was phosphorylated, but with this method we could not determine exactly which residue was involved. Spot 10 was also phosphorylated at Ser 153, and Ser 241 showed phosphorylation, but Ser 180 was observed only in the unphosphorylated form. No phosphorylation was detected in spots 6 and 8.
Because the results on the initial set of spots demonstrated a restricted array of proteins, the experiment was expanded. Eleven different protein spots reactive with postvasectomy serum were cut from 2-D gels and analyzed by mass spectrometry (spots 11–21, Table 1). Peptide sequences successfully identified known proteins in all cases. Multiple peptides from one of the spots (no. 12) clearly matched the larger outer dense fiber protein, odf2, and peptides from three spots (14, 16, 19) matched the smaller odf1 protein (Fig. 5). Each of five other spots (11, 15, 18, 20, 21) revealed peptides that matched two different proteins in the database, one of each pair being of outer dense fiber origin (Table 1). Thus, spot 11 contained peptides that resembled T complex protein 1, with two substitutions from the known sequence (135539, MW = 60.4, pI = 5.86) as well as peptides that matched odf2. Peptides from triosephosphate isomerase were detected in two spots (18, 21), and sequences of Ke6 or steroid dehydrogenase (1103844, MW = 26.6, pI = 6.10) were detected in one spot (no. 15), along with those of the odf1. The small outer dense fiber protein (odf1) identified in spots 14 and 16 was phosphorylated at Ser 153. Only 2 of the 11 spots (13, 17) did not contain any outer dense fiber peptides, matching instead to F-actin capping protein (D47755, MW = 31.3, pI = 5.47) and to rat small glutamine-rich tetratricopeptide repeat containing protein (SGT; 3006088, MW = 34.1, pI = 5.05).
Outer dense fiber proteins were clearly identified in the rat sperm proteome by microsequencing. Having identified specific proteins, it became of interest to complement this finding by staining rat sperm protein extract with antisera raised against outer dense fiber proteins. Anti-outer dense fiber serum bound numerous sperm protein spots (Fig. 6). Comparison of spots (Fig. 1) subsequently shown by mass spectrometry to contain odf peptides with spots stained by the anti-odf serum (Fig. 6) showed several areas of comigration. Included were arrays at 76–70, 66, 60, 50, and 43 kDa with pIs of between 5.2 and 5.7, a less acidic group of proteins at 31–28 kDa and pI 5.9–6.5, and a spot (no. 10) at 25 kDa and pI 5.8.
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DISCUSSION
Outer Dense Fiber Proteins as Autoantigens
The main aim of this study was to begin to identify the repertoire of autoantigens recognized by serum antisperm antibodies after obstruction of the male tract. We used mass spectrometry to analyze peptides in protein spots cut from 2-D gels. We anticipated obtaining the sequences of a variety of protein autoantigens both known (in the databases) and novel, but the majority of the spots contained peptides that matched only two proteins, odf1 and odf2, both of which are known components of the outer dense fibers of the sperm tail. Repeated identification by mass spectrometry of peptides unique to odf1 and odf2 clearly establishes the presence of these proteins in the spots. We conclude that the outer dense fiber proteins, odf1 and odf2, are dominant postobstruction autoantigens because they were identified with much greater frequency than all other sperm proteins combined.
Isolation of rat outer dense fibers followed by SDS-PAGE analysis revealed multiple protein components. Although there have been differences between studies (summarized by Kim et al. [28]), major components included polypeptides of approximately 84, 80, 32, 27.5, 20, and 14.4 kDa [28–32]. Subsequent molecular cloning identified genes encoding two major outer dense fiber proteins, now termed odf1 (also known as RT7 and Odf 27) and odf2 (also known as Odf 84 and KTT4 [33–37]). Outer dense fibers of human sperm also contain multiple proteins, the major ones being ODF1 and ODF2, which are very similar to the odf1 and odf2 of rats and mice [38]. Both odf1 and odf2 are believed to be rodlike proteins [35, 39] that interact through leucine zippers and other motifs [36, 39–41]. Newly identified proteins such as Spag 4 and Spag 5 also interact in a complex way with one another and with the odf proteins [39]. The emerging picture of molecular interactions between the various cytoskeletal proteins of the sperm tail offers the hope of more clearly understanding the function of the outer dense fibers, which have been speculated to provide elastic recoil for the sperm tail [42] or to protect sperm against shear forces [43].
Selection of the spots for microsequencing was done on the basis of their reactivity on Western blots with at least half of the postvasectomy sera tested. Despite selecting reactive spots in different areas of the 2-D gels, the outer dense fiber proteins were identified repeatedly. In general, spots corresponding to the smaller of the two outer dense fiber proteins, odf1, were found in the lower portions of the gels, and those corresponding to odf2 were located in the regions of higher molecular mass. Although it is not possible at present to relate specific forms of odf1 and odf2 to each spot, multiple transcripts encoding odf proteins have been observed. For example, there appear to be three or four testis-specific transcripts for odf2 in rat or bull [35, 44] and at least two transcripts for odf1 [34]. Identification of only one odf2 gene by Southern analysis [35] suggests alternative splicing as an explanation of differences in mass and isoelectric point of the various spots. Posttranslational modifications such as proteolytic cleavage or phosphorylation furnish additional opportunities for variation. In our study, several peptides were found by mass spectrometry to be phosphorylated on serine residues (e.g., spots 9, 10, 14, 16). The significance of these phosphorylations is unknown, but they might have a role in regulating interactions between the proteins.
There are several possible explanations for the high frequency with which outer dense fiber proteins were identified as autoantigens. First, outer dense fiber proteins are abundant; isolated fiber polypeptides compose approximately 40% of total sperm proteins [31]. Second, the outer dense fibers are resistant to degradation. They are among the last remnants of spermatozoa when the cells undergo degeneration [45], they are present within spermatic granulomas for prolonged periods following sperm extravasation [46], and they are visible even within lysosomes in phagocytic cells [47]. Moreover, outer dense fibers are known to be resistant to solubilization in vitro [31], perhaps because they have a high content of disulfide bonds, which are believed to be associated with zinc [29, 48, 49]. These observations suggest that outer dense fibers provide a large supply of antigen that is released slowly over a long period of time as a stimulus to the immune system. In addition, outer dense fibers are unique to spermatozoa. They are not produced until after puberty, and then in only the developing spermatids in the testis [50]. Thus, they may escape identification as self proteins during development of the immune system [51] and appear likely to stimulate an immune response when no longer sequestered behind the blood-testis and blood-epididymal barriers. The male duct system frequently ruptures after obstruction [45, 52, 53], forming spermatic granulomas in which sperm come in contact with macrophages, lymphocytes, and other immune cells [52, 54].
Other Possible Autoantigenic Proteins Identified
Although the two outer dense fiber proteins are clearly autoantigens, the status of the other sperm proteins identified by peptide analysis is less certain. In several cases in which peptides from other proteins were detected, these proteins were accompanied by sequences from odf1 or odf2. Thus, the immunostaining on Western blot may have been due to the presence of the odf, whereas the additional peptides were observed as a result of overlap of proteins within the 2-D gels.
Among the other proteins identified by microsequencing of individual immunoreactive spots was F-actin capping protein (beta subunit), which binds to the plus (more rapidly growing) ends of actin filaments. The capping proteins are found in both muscle and nonmuscle cells, where they block the exchange of actin subunits from the ends of filaments [55]. Although its precise localization has been debated in different species, actin is present in spermatozoa [56, 57], so its associated proteins may also be expected to be present and to perform functions similar to those in other cells. Triosephosphate isomerase, which was identified alone in one spot and accompanying odf peptides in three other spots, is presumably an ubiquitous cytosolic component of intermediary metabolism, and it is difficult to surmise why it appears as an autoantigen under these circumstances. Similarly, hydroxyisobutyrate dehydrogenase is found in several tissues, where it normally has a mitochondrial localization [58]. T-complex protein 1, alpha subunit, is a chaperone that assists in the folding of other proteins [59]. Some heat shock proteins are also chaperones that are expressed in male germ cells [60] and may play a role in establishing and maintaining useful configurations of sperm components. The Ke6 gene is interesting because it encodes a protein in the alcohol dehydrogenase family that is downregulated in a model of polycystic kidney disease [61]. Possibly more pertinent to spermatozoa is the observation that the Ke6 protein is structurally homologous to mammalian steroid dehydrogenases. Small glutamine-rich tetratricopeptide repeat (TPR)-containing protein has been observed to bind the NS1 protein of certain parvoviruses in cell lines [62], but its possible role in sperm remains unknown.
Identification of Peptides from More Than One Proteinin a Spot
In most cases, matches to proteins in a database were accomplished through aligning sequences of multiple peptides, up to and as many as 19. Multiple peptide matches from a single spot conferred a high degree of confidence that the spot contained the protein identified. In four cases, microsequence was available for only one peptide (spots 8, 10, 16, 17). However, the observation that the 17 amino acids of spot 10 exactly matched a sequence in the database made it very likely that the spot contained odf1, and even the shorter match of 8 available amino acids strongly linked odf1 to spots 8 and 16. Spot 17 (10 amino acids) was not an odf protein.
In some instances, peptides from two proteins were identified in the same spot. Given the fact that mammalian sperm contain 1500 or more proteins resolvable on silver-stained 2-D gels [20] and that there are probably numerous other proteins present in amounts too small to be visible, it is not surprising that some overlap occurred, even in the large-format 2-D gels used here. One of the strengths of mass spectrometry is its ability to identify more than one protein in such a case. However, when peptides from two proteins were identified, it is uncertain which of the two was immunoreactive. Nevertheless, this limitation of interpretation concerning a few of the protein spots does not alter the general conclusion that outer dense fiber proteins are important autoantigens; microsequencing of the majority of spots revealed matches to a single protein.
ACKNOWLEDGMENTS
The authors are indebted for assistance and advice to Dr. Michael Wolkowicz and other members of the Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia.
FOOTNOTES
First decision: 12 September 2000.
1 Supported by the National Institutes of Health (NIDDK P50 DK45179, NICHD HD U54-29099), the Fogarty International Center (D43 TW/HD 00654), and the NICHD/NIH through cooperative agreement U54 HD28934 as part of the Specialized Cooperative Centers Program in Reproduction Research; the Andrew W. Mellon Foundation; and Schering AG. 
2 Correspondence: Charles J. Flickinger, Department of Cell Biology, School of Medicine, University of Virginia Health System, P.O. Box 800732, Charlottesville, VA 22908-0732. FAX: 804 982 3912;cjf{at}virginia.edu
Accepted: December 18, 2000.
Received: August 10, 2000.
REFERENCES
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