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Biology of Reproduction 61, 428-435 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Sperm Mitochondria-Associated Cysteine-Rich Protein (SMCP) Is an Autoantigen in Lewis Rats1

John C. Herr2,a,c, David Thomasa, Leigh Ann Busha,c, Scott Coonroda,c, Vrinda Khole3,a,c, Stuart S. Howardsb,c, and Charles J. Flickingera,c

a Department of Cell Biology, b Department of Urology, and c The Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A common repertoire of rat sperm antigens have previously been identified by Western blotting of sperm proteins with sera obtained after vasectomy or isoimmunization with sperm. Aside from a determination of their apparent masses, however, the biochemical characteristics of these antigens have remained unknown. In this study, a rat testis cDNA expression library was screened with polyclonal antibodies obtained from rats immunized with isologous spermatozoa to identify and sequence a full-length clone encoding rat sperm mitochondria-associated cysteine-rich protein (SMCP). The open reading frame of SMCP was expressed in the pET22b vector, and recombinant SMCP (rec-SMCP) was purified. Sera from rats that had been vasectomized or hyperimmunized with isologous sperm specifically recognized rec-SMCP whereas preimmune sera from these experimental groups did not react. Rabbit antiserum produced to rec-SMCP recognized rec-SMCP on Western blots and precisely immunolocalized SMCP to the mid-piece of rat sperm. On Western blots against sperm extracts, the rabbit antibody recognized a major protein band of approximately 22–25 kDa that co-migrated with bands of identical mass that were recognized by sera from hyperimmune or vasectomized rats. These findings demonstrate that SMCP is a sperm autoantigen, recognized following vasectomy, and an isoantigen, recognized by antibodies generated through isologous immunization with sperm.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autoimmunity to sperm can be induced by immunization with isologous or autologous spermatozoa, resulting in autoimmune orchitis and aspermatogenesis of the testis [1–3]. Obstruction of the vas deferens following vasectomy also results in development of autoantibodies to sperm in most species, including the majority of men [1, 4–6], as well as testicular alterations [7–12] and lesions in various parts of the male ducts including the vas and epididymis [13]. Persistence of serum antisperm antibodies has been associated with infertility in men following vasectomy reversal (vasovasostomy) [14, 15] and in otherwise unexplained cases of both male and female infertility [14, 16–18].

Sperm antigens are considered to induce autoimmune responses because many are "differentiation or neo-antigens" [19], tissue-specific gene products that do not appear until puberty when meiosis is initiated [2]. Before puberty during neonatal induction of self-tolerance, these differentiation antigens may not be recognized as self antigens by the immune system [2]. The later stages of male germ cells are normally sequestered from contact with the immune system by the blood-testis barrier in the seminiferous epithelium [20] and by junctions between epididymal epithelial cells, the so-called blood-epididymal barrier [21]. However, obstruction of the vas deferens leads to release of both spermatozoa and/or soluble sperm antigens and induction of antibodies, which correlate with testicular alterations [10, 22]. Interestingly, unilateral vasectomy in rats shows histological changes in both the obstructed and the contralateral testis along with elevations in antisperm antibodies [23]. Obstruction of the male reproductive tract during prepubertal development is also followed by formation of antisperm antibodies, but only after sexual maturation when sperm appear in the epididymis [24, 25]. Complete occlusion is not even necessary since injury to the vas at a prepubertal state also leads at a later time to the production of antisperm antibodies [26].

Although antisperm antibodies arise after hyperimmunization with isologous sperm, after vasectomy, or after the onset of puberty in rats in which the vas deferens has been obstructed prepubertally, very few sperm autoantigens have been biochemically characterized. Western blots of sperm antigens reacted with sera from vasectomized, isoimmunized, or prepubertally obstructed rats show a common repertoire of immunodominant antigens, including proteins of 82–78, 68 or 63, 57, 42, 36, and 22 kDa [27–29]. High-resolution two-dimensional gel electrophoresis over a broad isoelectric range demonstrates more than 1400 silver-stained sperm proteins [30]. Remarkably, from this large field of potential sperm immunogens, relatively few consistently appear in the patterns of immunoreactive rat sperm proteins observed on Western blots reacted with sera from different animals. Although the apparent molecular masses and isoelectric points of some of the few autoantigenic proteins are now known from one- and two-dimensional immunoblots, to our knowledge no rat sperm autoantigenic protein sequences have been published.

Sperm mitochondria-associated cysteine-rich protein (SMCP), previously known as mitochondrial capsule selenoprotein (MCS) and mitochondrial capsule protein (MCP), is a major structural element of the mitochondria in the midpiece of the sperm tail [31]. (Since there are recent questions about the selenium content of this protein [see Discussion], we use the designation SMCP.) This report presents evidence that SMCP is an antigen in both auto- and iso-immune responses to sperm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Lewis Rat Testis cDNA Expression Library

Poly(A)+ mRNA was isolated and purified from freshly harvested Lewis rat testis using guanidine isothiocyanate extraction and oligo(dT)-cellulose chromatography as described by Freemerman et al. [32, 33]. A testis expression library was constructed using Stratagene's Zap cDNA Synthesis Kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The system used an oligo(dT) primer with an XhoI site and EcoRI adapters to generate double-stranded cDNA from 5 µg of the poly(A)+ RNA. Samples were size-fractionated before ligation into Uni-Zap XR Vector Arms (Stratagene) and were then packaged into lambda coat proteins with Gigapack II Gold extracts (Stratagene). The library was amplified once through Stratagene's XL-Blue MRF' strain of Escherichia coli.

Screening of Library

Following procedures described by Maniatis [34], the expression library was screened with a 1:500 dilution of Lewis rat hyperimmune sera produced by immunizing rats with isologous cauda epididymal sperm [28]. Approximately 5 x 106 phage were plated in the first round of screening. Initial clones were isolated to purity, converted into Bluescript SK+ plasmid through phagemid rescue as described in Stratagene's protocol, and sequenced using the deoxy chain termination method and Sequenase (United States Biochemical Corp., Cleveland, OH) [32]. A cDNA probe from the longest of these clones, 1-1-1-3-2, was used to screen approximately 1 x 107 phage for additional clones [34]. Sixteen micrograms of clone 1-1-1-3-2 plasmid was digested simultaneously with EcoRI/XhoI (Promega, Madison, WI), and the insert was isolated by electrophoresis on a 1% agarose gel followed by electro-elution. The resulting fragment was labeled with [{alpha}-32P]dCTP using the Gibco-BRL Nick-Translation Kit protocols (Gibco, Grand Island, NY) and was used to isolate the full-length clone.

Expression of Recombinant SMCP (rec-SMCP) Protein

Full-length expression constructs of SMCP were engineered in the pET-22b+ expression vector (Novagen, Madison, WI). The constructs included 24 bases of 5 prime sequence because when this work was initiated the literature indicated that the start codon for SMCP was further upstream [35] than the currently accepted start codon. The vector confers an N-terminal pelB signal peptide to the recombinant protein (rec-protein) to enhance cytosolic expression and adds 6 histidine residues to the C terminus for purification by Ni-affinity chromatography. The following oligonucleotide primers were synthesized by the Biomolecular Research Facility at the University of Virginia: 137 (5'-GGGGAATTCTCAGAAACTCCAACTCTAAAG) and 595 (5'-CCCCTCGAGCTTGGTCTTCTTCTGGTTCCA). For directional cloning into the vector, the 5' primer 137 contains an EcoRI site (shown in bold italics), and the reverse complement 3' primer 595 contains an XhoI site (bold italics). In addition, the 5' primer contains a spacer nucleotide (underlined G) to preserve the vector reading frame in the recombinant construct. Polymerase chain reaction fragments corresponding to the sequence (nucleotides 137–595) were amplified using the primer set 137/595. All of the recombinant DNA procedures were carried out as described by Maniatis [34]. Both the engineered inserts and the vector DNA were digested with EcoRI and XhoI sequentially. The digested vector DNA was treated with calf intestine phosphatase before ligation. Ligation was carried out at 16°C overnight [34]. Five milliliters of each ligation reaction was transfected into competent BL-21 E. coli (Novagen) and selected for ampicillin resistance on NZY-ampicillin agar plates. Colonies were selected and the isolated plasmids were analyzed by EcoRI/XhoI digestion for the corresponding recombinant inserts. The constructs were sequenced, as above, from both sides of the inserts to ensure that the correct reading frame for the vector was maintained. To increase the overall yield of expressed protein, the construct was transfected into BL-21 pLysS E. coli (Novagen), which controls the transcription rate of the gene, preventing cell death due to overproduction of the protein.

Production and purification of the rec-SMCP protein was performed as described by Reddi et al. [36]. Briefly, 10 L of 3-strength Terrific Broth [37] with 50 µg/ml carbenicillin was inoculated with 200 ml of an overnight culture of the pET-22b construct in BL-21 pLysS host bacteria. The cells were induced with 1 mM isopropyl thiogalactoside (IPTG) when they had reached a density of A600 = 0.97, and the induction of the rec-protein continued for 5 h. The cells were harvested, pelleted under slow-speed centrifugation, and stored at -20°C. Purification of the soluble rec-protein was accomplished by Ni-affinity chromatography [36].

Generation of Polyclonal Ascites and Sera

Mouse anti-rat 137/595 rec-SMCP polyclonal ascites was generated in the Lymphocyte Culture Center (LCC) at the University of Virginia. Preimmune blood samples were obtained from the tail veins of three 6- to 8-wk-old female Balb/c mice. The immunogen for each injection was 20 µg of the Ni-affinity-purified 137/595 rec-SMCP, which was gel-purified by SDS-PAGE. The ~22.5 kDa acrylamide slice was emulsified in complete Freund's adjuvant for the first two injections and incomplete Freund's adjuvant for the third. Each animal received 3 injections at Weeks 0, 5, and 11, half of the immunogen being injected s.c. and half intraperitoneally. The abdomen was pristane-primed at Week 11, and the production of an ascites tumor was established by injection of nonsecreting SP2/0-Ag14 [38] mouse myeloma cells 10 days after priming. Sham ascites fluids were generated in an identical manner, after immunization with acrylamide slices lacking any protein. Ascites fluids from both sets of mice were collected over several weeks and were delipidated by centrifuging the fluid and removing the ascites from beneath the lipid layer.

Rabbit anti-rat 137/595 rec-SMCP polyclonal sera were generated in 3 female New Zealand white rabbits (7–8 lbs). After the preimmune sera were collected, each animal received an injection of 300 µg of rec-protein, gel-purified as described above. Half of the emulsion was injected i.m. and half into the popliteal lymph node. After the first immunization with Freund's complete adjuvant, three booster immunizations were administered, every fifth week, using Freund's incomplete adjuvant, and serum samples were collected 1 wk after the final boost.

Analysis of Rec-Protein and Polyclonal Antibodies

SDS-PAGE and Western blotting Proteins were solubilized in Laemmli buffer [39]. A 7.5–15% gradient or 10% linear separating gel was run at 30 mA for 4 h, and proteins were either silver-stained or electrotransferred onto 0.45-µm NitroPure nitrocellulose (Micron Separations Inc., Westborough, MA) at 100 mAmp for 14 h. The blots were stained with 0.1% amido black and were destained in 10% methanol and 10% acetic acid. The membrane was cut into strips that were blocked in 5% milk in PBS with 0.05% Tween-20 (PBS-tw) for 2 h. Rat serum samples were preabsorbed with an E. coli lysate, and the primary sera were incubated overnight at 4°C at varying dilutions. After the primary antibody incubation, the strips were washed 3 x 5 min in PBS-tw. The peroxidase-conjugated secondary antibodies were diluted 1:10 000 in the case of the goat anti-rabbit IgG or 1:5000 for the rabbit anti-rat IgG. The dilutions were made in PBS-tw, and incubation was for 1.5 h at 22°C. The strips were washed thrice for 5 min in PBS-tw, and each blot was developed using 0.2% diaminobenzidine substrate enhanced with 0.1% nickel chloride and 0.1% H2O2 in 0.1 M Trizma base.

Olmstead elution of antibody To purify anti-SMCP antibodies from the rabbit polyclonal sera, the immunoaffinity method of Olmstead was used [27, 40]. One hundred micrograms of purified rec-SMCP was loaded on an SDS-PAGE curtain gel, which was run and transferred as described previously. The 22- to 25-kDa rec-SMCP region was cut horizontally from the blot, blocked for 2 h in 5% milk, and incubated overnight at 4°C with a 1:1000 dilution of rabbit anti rec-SMCP polyclonal antibody (preimmune or postimmune). After washing for 3 x 30 min in PBS-tw, the purified antibody was eluted from the strip by incubating twice for 6 min each with 1 ml of 0.2 M glycine/0.5 M NaCl (pH 3.5). After incubation, each 1 ml of antibody solution was immediately neutralized with NaOH, and 1% w:v of BSA was added to stabilize the antibody. The strip was placed in 100 mM Tris/0.5 M NaCl (pH 8.0) and was incubated for 15 min. The strip was then incubated overnight with the original antibody solution, and the process was repeated a second time on the next day.

Immunofluorescence Adult Lewis rats were killed, and the cauda epididymidis and vas deferens were removed. Sperm were obtained by inserting an irrigating cannula into the proximal vas and flushing the contents from the vas and distal cauda of the epididymis with TE culture medium [41]. The sperm were washed twice with TE culture medium and collected by centrifugation. Five milliliters of medium was layered on top of the pellet, and the sperm were allowed to swim up for 1 h at 37°C in a 5% CO2 incubator. The swim-up sperm were counted using a hemocytometer and were diluted to a concentration of 1 x 106 sperm/ml. Twenty microliters of the sperm suspension was added per well (2 x 105 sperm) onto poly-L-lysine coated slides, and the sperm were allowed to bind to the slide for 7 min before the excess sperm were removed. The slides were dried at 40°C and then methanol-fixed for 10 min. After three 5-min washes in PBS, all subsequent incubations were carried out in a humid chamber. The preparations were blocked in 10% normal goat serum (NGS) in PBS-tw for 30 min. The rabbit anti-rec-SMCP primary antibody was diluted 200-fold with 10% NGS in PBS-tw and was incubated overnight at 4°C. The slides were then washed 3 x 5 min in PBS-tw, and the secondary antibody, goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, PA), was applied at a 1:100 dilution in 10% NGS in PBS-tw for 2 h at 37°C. The slides were washed 3 x 5 min in PBS-tw, and the Slow Fade-Light Antifade Kit (Molecular Probes, Inc., Eugene, OR) was used to reduce the fading rate of the fluorescein.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
By screening a Lewis rat testis cDNA library with antisera produced by injection of isologous spermatozoa, a partial-length clone of 293 base pairs (bp) was obtained. After labeling, this was used to re-screen the same library to obtain a full-length cDNA clone, which encoded a protein with the sequence presented in Figure 1. This sequence is identical to that reported for rat mitochondrial capsule selenoprotein by Adham et al. [42, 43], a protein more recently renamed SMCP [44].



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  		FIG. 1. Nucleotide sequence of rat SMCP. The deduced amino acid sequence for the open reading frame is shown on the second line. The arrows indicate the sequence of cDNA inserted into the pET 22b expression vector. Sequences recognized by the primers used for cloning are underlined.

The cDNA encoding the complete open reading frame for SMCP, including 24 bases of 5 prime sequence, was inserted into the pET 22b expression vector with inclusion of a PelB leader to increase protein export (Fig. 2). The terminal codon (bp 594–596) was deleted (Fig. 2), and the expressed protein was purified by immobilized metal immunoaffinity chromatography using the six terminal histidines provided by the vector.



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  		FIG. 2. Diagram of the SMCP sequence showing the region included in rec-SMCP. A cDNA of 842 bases was sequenced, with an open reading frame (ORF) beginning at base 158 and ending at base 596. The rec-protein begins 24 bp upstream (5') of the start site because of previous uncertainty concerning the start site of the rec-SMCP (see text).

Migration in SDS-PAGE of the expressed rat SMCP, inclusive of the 26 amino acids of the PelB leader, is shown in Figure 3, lanes I and P. The bulk of this material ran at approximately 22–25 kDa, and in addition a band at 36–40 kDa was observed in these preparations, possibly indicating the presence of dimers of the expressed rec-SMCP. When recombinant rat SMCP was gel-purified and injected into rabbits, antisera were generated that showed reactivity with the 22- to 25-kDa band corresponding to rec-SMCP (Fig. 3).



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  		FIG. 3. Silver-stained gel and Western blot demonstrating stages of expression and purification of rat rec-SMCP. Lanes were loaded with an equivalent number of BL-21 bacterial cells or with 1 µg of purified rec-SMCP, and immunostaining was carried out using a 1:30 000 dilution of rabbit anti-rec-SMCP polyclonal serum. Lane B contains blank BL-21 cells not induced, while lane L contains the same blank cells after induction with IPTG. Lane N contains BL-21 cells that possess the SMCP insert without induction, whereas lane I contains the same cells after induction with IPTG. Lane P contains rec-SMCP protein purified by Ni-affinity chromatography. M, Molecular weight markers.

In order to understand the pattern of immunoreactivity with SMCP in sperm, antiserum generated to the rec-SMCP was incubated with sperm protein extracts and was found to react with a broad band ranging from 16 to 24 kDa (Fig. 4), with some high molecular immunoreactive forms also being noted, e.g., at ~40 kDa. To study further the nature of the high molecular mass material, an immunoreagent specific to the 22- to 25-kDa form of the rec-SMCP was generated using the Olmstead elution method from a nitrocellulose strip containing only the 22- to 25-kDa form of mitochondrial capsule protein. This mono-specific antiserum to the recombinant 22- to 25-kDa form stained the higher molecular mass forms of the rec-SMCP at 36–40, 50–53, and 70 kDa, as well as the 22- to 25-kDa form from which the antibody was eluted, supporting the belief that the higher molecular mass forms were generated by complexing of SMCP, either with itself or with another protein (Fig. 5). Both the original polyclonal antisera to rec-SMCP as well as the Olmstead-enriched monospecific serum to the 22- to 25-kDa band recognized these polymorphic forms (Figs. 4 and 5). It may also be noted that when two-dimensional gels of sperm extracts were run, blotted, and immunostained with anti-rec-SMCP ascites, the higher molecular mass forms of SMCP showed considerable charge heterogeneity (data not shown).



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  		FIG. 4. Western blot of purified rec-SMCP (0.6 µg per lane) and sperm protein extract (18.2 µg per lane) probed with the rabbit anti-rec-SMCP polyclonal serum. A, Amido black stain; P, preimmune rabbit serum; M, postimmune rabbit anti-rec-SMCP antiserum at a 1:50 000 dilution. The R lanes are negative controls probed with goat anti-rabbit IgG secondary antibody alone. Positions of molecular weight markers are on the left.



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  		FIG. 5. Western blot of rec-SMCP reacted with an antibody purified by the Olmstead elution technique by elution from the 22- to 25-kDa rec-SMCP band. Lane R was stained with the rabbit polyclonal anti-rec-SMCP (1:50 000 dilution), while lane O was reacted with the Olmstead-purified antibody. Note that the Olmstead-purified antibody, which was eluted from the 22- to 25-kDa band, reacted with the same higher molecular weight proteins at 36–40 kDa, 50–53 kDa, and 70 kDa in addition to the 22- to 25-kDa band in a pattern identical to the polyclonal serum. M, Molecular weight standards, identified on the left; A, amido black stain; N, secondary antibody alone control; and P, preimmune rabbit serum.

Immunocytochemical staining using rabbit antisera to rec-SMCP showed intense staining of a portion of the tails of virtually all the sperm (Fig. 6A). Careful comparison of immunofluorescence (IF) and differential interference contrast (DIC) images (Fig. 6; A and B, E and F) showed localization of the staining to the anterior portion of the sperm tail beginning immediately behind the sperm head; i.e., in the region corresponding to the midpiece of the tail. Immunofluorescence controls exposed to preimmune serum under the same conditions as the immune test serum produced no staining (Fig. 6; C and D, G and H). Thus, the immunoreactivity of the antiserum produced to rec-SMCP corresponded to the location of the helical array of mitochondria within the sperm midpiece.



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  		FIG. 6. IF of rat sperm probed with the pre- and postimmune rabbit polyclonal serum raised against rec-SMCP. For each IF field, the corresponding DIC image is shown to its right. All primary sera were used at a 1:200 dilution. Exposure of sperm to the postimmune serum in A and E shows staining of an antigen localized on the anterior portion of the sperm tail. To our knowledge, this is the first immunoreagent generated to rec-SMCP. C and G show an absence of staining with the preimmune serum under identical conditions. A–D) x320; E–H) x365; published at 78%.

In order to determine whether the SMCP is an iso- and an auto-antigen, rec-SMCP and sperm protein extracts were reacted with sera 1) from animals that had been hyperimmunized with isologous sperm, or 2) from vasectomized rats containing autoantibodies to rat sperm (Fig. 7). Both hyperimmune serum (Fig. 7A, lane 3) and postvasectomy serum (Fig. 7A, lane 5) recognized rec-SMCP. Staining of sperm extracts was much more complex because both of these antisera were raised to whole sperm and thus bound multiple proteins (Fig. 7B). Reactivity of antibodies in hyperimmune and postvasectomy sera with rec-SMCP demonstrated that this sperm molecule was both an isoantigen and an autoantigen.



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  		FIG. 7. Western blot demonstrating that SMCP is both an auto- and an iso-antigen in the rat using recombinant and native SMCP. Rec-SMCP and rat sperm protein extracts were separated by SDS-PAGE, blotted, and immunostained with polyclonal rabbit anti-rec-SMCP serum (lane 7), postvasectomy serum (lane 5), and hyperimmune serum from rats immunized with isologous sperm (lane 3). The blot demonstrates that all three types of immune sera immunoreacted with rec-SMCP as well as with proteins in the sperm extract at the anticipated mass for SMCP. Since postvasectomy and hyperimmune sera were raised against autologous or isologous sperm, they also reacted as expected with several other sperm protein bands. Negative controls are as follows: rabbit anti-rat IgG secondary antibody alone (lane 2), prevasectomy serum (lane 4), preimmune rabbit polyclonal serum (lane 6), and goat anti-rabbit IgG secondary antibody alone (lane 8). Lane 1, amido black stain. Molecular weight markers are on the left.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
On the basis of the observations that postvasectomy sera and sera from animals hyperimmunized with isologous spermatozoa recognize a common repertoire of sperm antigens [28, 29], hyperimmune serum was used to screen a cDNA library. Complementary DNA clones encoding the sperm mitochondrial capsule protein were identified with this hyperimmune serum, and a complete open reading frame was obtained for rat SMCP that corresponded identically to the rat mitochondrial capsule protein sequence recently published [42]. A full-length recombinant form of SMCP was expressed with a short PelB leader sequence, and antibodies generated to the recombinant protein in rabbits recognized both recombinant and "native" SMCP. Moreover, postvasectomy sera recognized recombinant and native SMCP, confirming that this molecule is an autoantigen. In addition, sera from animals hyperimmunized with isologous spermatozoa recognize SMCP, indicating that it is an isoantigen.

SMCP, identified here both as an sperm autoantigen and isoantigen for the first time, is an unusual protein believed to comprise part of the sperm mitochondria. The sperm tail contains many unique, highly differentiated structures. The mitochondria are very unusual in being arranged head to tail in a helix around the outer dense fibers in the mid-piece of the mammalian spermatozoon [45]. In this position, the individual mitochondria assume a crescentic shape, and there is evidence that they are held in place or stabilized in this array by a thickening of the outer mitochondrial membranes known as the mitochondrial capsule [46, 47]. SMCP is rich in cysteine and proline, found in repeat domains [48], and it is believed that SMCP has a structural role in the mitochondrial capsule [31]. This paper presents the first report of the expression and purification of a rec-SMCP and the generation of a monospecific antibody to the protein.

The precise immunofluorescent localization of SMCP to the midpiece in our study reinforces the generally accepted view that SMCP is a component of the mitochondrial capsule. Although there appears to be agreement about the composition and general location of the cysteine- and proline-rich SMCP in the sperm tail, the relation of SMCP to selenium-containing proteins of the spermatozoa has been more problematic. The selenium content of sperm proteins is of interest because reduced sperm motility and disorganization of sperm mitochondria occur in rats with a selenium deficiency [49, 50]. Pallini et al. [46] described three main proteins as comprising the mitochondrial capsules of bull sperm, including a 20-kDa protein of unusual composition, rich in cystine (17.9%) and proline (26.5%), and containing selenium. Calvin et al. [51, 52] subsequently studied a 17-kDa protein in rat sperm mitochondrial capsules with a molecular mass of 17 kDa that labeled with radioactive selenium, which they believed was identical to the smallest of the proteins identified by Pallini et al. [46] in the bull. Thus, the cysteine- and proline-rich mitochondrial protein became known as mitochondrial capsule selenoprotein, or MCS [48]. However, molecular cloning of cDNAs and sequencing of the MCS gene revealed several possible transcriptional start sites (AUG codons) [44]. In the mouse, use of one of the more 5 prime AUG codons provided three in-frame UGA codons [35], opal stop codons that are read by a tRNA for selenocysteine in the context of secondary structure [53]. However, in the rat and human, the comparable UGA codons are in a different reading frame [42, 43]. More recent evidence suggests that it is the third of the three AUG codons that constitutes the start site for SMCP in the mouse [44] and that the reading frame contains no UGA (selenocysteine) codons [44]. If SMCP indeed contains no selenium, the selenium-containing protein of sperm mitochondria may instead be the enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) [44, 54].

The gene for both mouse and human SMCP is composed of two exons. The human SMCP gene maps to Q21 of chromosome 1 [43]. SMCP is a single-copy gene in both mice and humans, and expression of SMCP appears to be testis-specific [48]. A dramatic reduction in the level of the RNA for SMCP is seen in mice in which gene targeting was used to selectively eliminate the cAMP response element modulator CREM, a transcription factor active in regulating a number of postmeiotic genes [55].

The identification of the cysteine- and proline-rich mitochondrial protein SMCP as an auto- and iso-antigen of rat sperm is not surprising in view of its expression only in the testis and in sperm [48]. SMCP mRNA appears to be transcribed postmeiotically in round spermatids in mice [56] and rats [42]. Furthermore, SMCP is under translational regulation, and the protein is synthesized mainly in elongating spermatids [44, 57]. Since it is found only in the highly differentiated structures of the sperm tail and is expressed only after puberty, and since it is normally sequestered behind the blood-testis and blood-epididymal barriers, SMCP might be expected to be viewed as foreign by the immune system. After vasectomy or obstruction of the epididymis, contact of cells of the immune system with large amounts of SMCP can take place when rupture of the tract in the epididymis and/or vas deferens leads to formation of spermatic granulomas [58–61], which are cyst-like foci of chronic inflammation containing sperm, lymphocytes, plasma cells, etc. Additional ways in which immune cells can contact spermatozoa include migration of macrophages into the lumen of the efferent ductules [60].

Other sperm and testicular autoantigens have been identified previously by various means. In experimental allergic orchitis in guinea pigs, semipurified preparations have been defined that are capable of inducing aspermatogenesis [62, 63]. The surface autoantigenic rabbit sialo glycoprotein, RSA1, has been cloned, and a related family of peptides has been identified in the mouse and human [64, 65]. Recently, a novel sperm-specific peptide antigen has been identified and cloned using a monoclonal antibody obtained from a vasectomized mouse [66]. Postvasectomy sera from men bind several human sperm proteins such as nuclear protamines [67], DNA polymerase [68], the sperm-specific glycoprotein FA1 [69], and other antigens identified by immunoprecipitation [70] or blotting with postvasectomy sera [71–74].

Although immunoblotting has identified numerous immunoreactive protein spots, the molecular identity of only a few are known. Now that SMCP has been cloned and expressed, and identified as both an auto- and an iso-antigen in rats, use of rec-SMCP may prove of interest in experimental models of autoimmune orchitis. In the future, recombinant human SMCP might be useful as a target antigen for assessing the incidence of anti-SMCP antibodies in both men and women with antisperm antibody-mediated infertility.


   FOOTNOTES
 
1 Supported by the NIH-HD U54–29099, P30–28934, P50-DK45179; the Andrew W. Mellon Foundation; and Schering AG. Back

2 Correspondence: John C. Herr, Department of Cell Biology, University of Virginia, Box 439, Health Sciences Center, Charlottesville, VA 22908. FAX: 804 982 3912; jch7k{at}virginia.edu Back

3 Current address: Vrinda Khole, Institute for Research in Reproduction (ICMR), JM Street, Mumbai, India. Back

Accepted: March 17, 1999.

Received: December 29, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tung KSK. Autoimmunity of the testis. In: Dhindsa D, Schumacker G (eds.), Immunologic Aspects of Infertility and Fertility Regulation. New York: Elsevier-North Holland; 1980: 33–91.
  2. Tung KSK, Menge AC. Sperm and testicular autoimmunity. In: Rose NR, Mackay IR (eds.), The Autoimmune Diseases. New York: Academic Press; 1985: 537–590.
  3. Tung KSK, Teuscher C. Mechanisms of autoimmune disease in the testis and ovary. Hum Reprod Update 1995; 1:35–50.[Abstract/Free Full Text]
  4. Alexander NJ. Immunologic and morphologic effects of vasectomy in the rhesus monkey. Fed Proc 1975; 34:1692–1697.[Medline]
  5. Alexander NJ, Anderson DJ. Vasectomy—consequences of autoimmunity to sperm antigens. Fertil Steril 1979; 32:253–260.[Medline]
  6. Rose NR, Lucas PL. Immunologic consequences of vasectomy: II. Two-year summary of a prospective study. In: Lepow IH, Crozier R (eds.), Vasectomy: Immunologic and Pathophysiologic Effects in Animals and Man. New York: Academic Press; 1979: 533–573.
  7. Flickinger CJ. The effects of vasectomy on the testis. N Engl J Med 1985; 313:1283–1285.[Medline]
  8. Bedford JM. Adaptations of the male reproductive tract and the fate of spermatozoa following vasectomy in the rabbit, rhesus monkey, hamster and rat. Biol Reprod 1976; 14:118–142.[Abstract]
  9. Tung KS. Allergic orchitis lesions are adoptively transferred from vasoligated guinea pigs to syngeneic recipients. Science 1978; 201:833–835.[Abstract/Free Full Text]
  10. Flickinger CJ, Herr JC, Howards SS, Caloras D, Yarbro ES, Spell DR, Gallien TN. The influence of vasovasostomy on testicular alterations after vasectomy in Lewis rats. Anat Rec 1987; 217:137–145.[CrossRef][Medline]
  11. Tung KS, Alexander NJ. Monocytic orchitis and aspermatogenesis in normal and vasectomized rhesus macaques (Macaca mulatta). Am J Pathol 1980; 101:17–30.[Abstract]
  12. Jarow JP, Budin RE, Dym M, Zirkin BR, Noren S, Marshall FF. Quantitative pathologic changes in the human testis after vasectomy. A controlled study. N Engl J Med 1985; 313:1252–1256.[Abstract]
  13. Flickinger CJ, Howards SS, Herr JC. Effects of vasectomy on the epididymis. Microsc Res Tech 1995; 30:82–100.[CrossRef][Medline]
  14. Linnet L, Hjort T, Fogh-Anderson P. Association between failure to impregnate after vasovasostomy and sperm agglutinins in semen. Lancet 1981; 1:117–119.[CrossRef][Medline]
  15. Parslow JM, Royle MG, Kingscott MMB, Wallace DMA, Hendry WF. The effects of sperm antibodies on fertility after vasectomy reversal. Am J Reprod Immunol 1983; 3:28–31.
  16. Haas GG, Cines DB, Schreiber AD. Immunologic infertility: identification of patients with antisperm antibody. N Engl J Med 1981; 303:722–727.[Abstract]
  17. Mathur S, Baker ER, Williamson HO, Derrick FC, Teague KJ, Fudenberg HH. Clinical significance of sperm antibodies in infertility. Fertil Steril 1981; 36:486–495.[Medline]
  18. Marshburn PB, Kutteh WH. The role of antisperm antibodies in infertility. Fertil Steril 1994; 61:799–811.[Medline]
  19. Boyse EA, Old LJ. Some aspects of normal and abnormal cell surface genetics. Annu Rev Genet 1969; 3:269–290.[CrossRef]
  20. Setchell BP, Uksila J, Maddocks S, Pollanen P. Testis physiology relevant to immunoregulation. J Reprod Immunol 1990; 18:19–32.[CrossRef][Medline]
  21. Hoffer AP, Hinton BT. Morphological evidence for a blood-epididymis barrier and the effects of gossypol on its integrity. Biol Reprod 1984; 30:991–1004.[Abstract]
  22. Flickinger CJ, Howards SS, Carey PO, Spell DR, Kendrick SJ, Caloras D, Gallien TN, Herr JC. Testicular alterations are linked to the presence of elevated antisperm antibodies in Sprague-Dawley rats after vasectomy and vasovasostomy. J Urol 1988; 140:627–631.[Medline]
  23. Chehval MJ, Martin SA, Alexander NJ, Winkelmann T. The effect of unilateral injury to the vas deferens on the contralateral testis in immature and adult rats. J Urol 1995; 153:1313–1315.[CrossRef][Medline]
  24. Pedersen J, Rubenson A, Nilsson LA. Formation of antisperm autoantibodies in rats vasectomized at prefertile age. Scand J Urol Nephrol 1983; 17:277–281.[Medline]
  25. Flickinger CJ, Herr JC, Baran ML, Howards SS. Temporal appearance of antisperm antibodies during sexual maturation of rats after obstruction of the vas deferens. J Androl 1995; 16:75–79.[Abstract/Free Full Text]
  26. Pedersen J, Rubenson A, Nilsson LA. Occurrence of sperm antibodies in adult rats after prefertile traumatic vas lesions. Scand J Urol Nephrol 1987; 21:1–4.[Medline]
  27. Handley HH, Flickinger CJ, Herr JC. Post-vasectomy sperm autoimmunogens in the Lewis rat. Biol Reprod 1988; 39:1239–1250.[Abstract]
  28. Flickinger CJ, Howards SS, Bush LA, Baker LA, Herr JC. Antisperm autoantibody responses to vasectomy and vasovasostomy in Fischer and Lewis rats. J Reprod Immunol 1995; 28:137–157.[CrossRef][Medline]
  29. Flickinger CJ, Baran ML, Howards SS, Herr JC. Sperm autoantigens recognized by autoantibodies in developing rats following prepubertal obstruction of the vas deferens. J Androl 1996; 17:433–442.[Abstract/Free Full Text]
  30. Naaby-Hansen S, Flickinger CJ, Herr JC. Two-dimensional gel electrophoretic analysis of vectorially labeled surface proteins of human spermatozoa. Biol Reprod 1997; 56:771–787.[Abstract]
  31. Kleene KC. The mitochondrial capsule selenoprotein—a structural protein in the mitochondrial capsule of mammalian sperm. In: Burk RF (ed.), Selenium in Biology and Human Health. New York: Springer-Verlag; 1994: 135–148.
  32. Freemerman AJ, Wright RM, Flickinger CJ, Herr JC. Cloning and sequencing of baboon and cynomolgus monkey intra-acrosomal protein SP-10: homology with human SP-10 and a mouse sperm antigen (MSA-63). Mol Reprod Dev 1993; 34:140–148.[CrossRef][Medline]
  33. Freemerman AJ, Flickinger CJ, Herr JC. Characterization of alternatively spliced human SP-10 mRNAs. Mol Reprod Dev 1995; 41:100–108.[CrossRef][Medline]
  34. Sambrook J, Fritsch EF, Maniatis TE. Molecular Cloning: A Laboratory Manual. Plainview, NY: Cold Spring Harbor Laboratory Press; 1989.
  35. Karimpour I, Cutler M, Shih D, Smith J, Kleene KC. Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: identification of three in-phase TGA selenocysteine codons. DNA Cell Biol 1992; 11:693–699.[Medline]
  36. Reddi PP, Castillo JR, Klotz K, Flickinger CJ, Herr JC. Production in Escherichia coli, purification and immunogenicity of acrosomal protein SP-10, a candidate contraceptive vaccine. Gene 1994; 147:189–195.[CrossRef][Medline]
  37. Tartof KD, Hobbs CA. Improved media for growing plasmid and cosmid clones. Bethesda Res Lab Focus 1987; 9:12.
  38. Shulman M, Wilde CO, Kohler G. A better cell line for making hybridomas secreting specific antibodies. Nature 1978; 276:269–270.[CrossRef][Medline]
  39. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
  40. Olmstead JB. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem 1981; 256:11955–11959.[Abstract/Free Full Text]
  41. Almeida EAC, Huovila A-PJ, Sutherland AE, Stephens LE, Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A, Primakoff P, Myles D, White JM. Mouse egg integrin {alpha}6ß1 functions as a sperm receptor. Cell 1995; 81:1095–1104.[CrossRef][Medline]
  42. Adham IM, Tessmann D, Soliman KA, Murphy D, Kremling H, Szpirer C, Engel W. Cloning, expression, and chromosomal localization of the rat mitochondrial capsule selenoprotein gene (MCS): the reading frame does not contain potential UGA selenocysteine codons. DNA Cell Biol 1996; 15:159–166.[Medline]
  43. Aho H, Schwemmer M, Tessmann D, Murphy D, Mattei G, Engel W, Adham IM. Isolation, expression, and chromosomal localization of the human mitochondrial capsule selenoprotein gene (MCSP). Genomics 1996; 32:184–190.[CrossRef][Medline]
  44. Cataldo L, Baig K, Oko R, Mastrangelo MA, Kleene KC. Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm. Mol Reprod Dev 1996; 45:320–331.[CrossRef][Medline]
  45. Otani H, Tanaka O, Kasai K-I, Yoshioka T. Development of mitochondrial helical sheath in the middle piece of the mouse spermatid tail: regular dispositions and synchronized changes. Anat Rec 1988; 222:26–33.[CrossRef][Medline]
  46. Pallini V, Baccetti B, Burrini AG. A peculiar cysteine-rich polypeptide related to some unusual properties of mammalian sperm mitochondria. In: Fawcett DW, Bedford JM (eds.), The Spermatozoon: Maturation, Motility, Surface Properties and Comparative Aspects. Baltimore: Urban & Schwartzenberg; 1979: 141–151.
  47. Pallini V, Bacci E. Bull sperm selenium is bound to a structural protein of mitochondria. J Submicrosc Cytol 1979; 11:165–170.
  48. Kleene KC, Smith J, Bozorgzadeh A, Harris M, Hahn L, Karimpour I, Gerstel J. Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse. Dev Biol 1990; 137:395–402.[CrossRef][Medline]
  49. Wu SH, Oldfield JE, Whanger PD. Effects of selenium, vitamin E, and antioxidants on testicular function in rats. Biol Reprod 1973; 8:625–629.[Abstract]
  50. Wu SH, Oldfield JE, Shull LR, Cheeke PR. Specific effect of selenium deficiency on rat sperm. Biol Reprod 1979; 20:793–798.[Abstract]
  51. Calvin HI, Cooper GW, Wallace E. Evidence that selenium in rat sperm is associated with a cysteine-rich structural protein of the mitochondrial capsules. Gamete Res 1981; 4:139–149.[CrossRef]
  52. Calvin HI, Grosshans K, Musicant-Shikora SR, Turner SI. A developmental study of rat sperm and testis selenoproteins. J Reprod Fertil 1987; 81:1–11.[Abstract]
  53. Berry MJ, Banu L, Chen Y, Mandel SJ, Kieffer JD, Harney JW, Larsen PR. Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region. Nature 1991; 353:273–276.[CrossRef][Medline]
  54. Roveri A, Casasco A, Maiorini M, Dalan P, Calligaro A, Ursini F. Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification. J Biol Chem 1992; 267:6142–6146.[Abstract/Free Full Text]
  55. Nantel F, Monaco L, Foulkes NS, Masquilier D, LeMeur M, Henriksen K, Dierich A, Parvinen M, Sassone-Corsi P. Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 1996; 380:159–162.[CrossRef][Medline]
  56. Shih DM, Kleene KC. A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse. Mol Reprod Dev 1992; 33:222–227.[CrossRef][Medline]
  57. Kleene KC. Multiple controls over the efficiency of translation of the mRNAs encoding transition proteins, protamines, and the mitochondrial capsule selenoprotein in late spermatids in mice. Dev Biol 1993; 159:720–731.[CrossRef][Medline]
  58. Flickinger CJ, Yarbro ES, Howards SS, Herr JC, Caloras D, Gallien TN, Spell DR. The incidence of spermatic granulomas and their relation to testis weight after vasectomy and vasovasostomy in Lewis rats. J Androl 1986; 7:285–291.[Abstract/Free Full Text]
  59. Herr JC, Howards SS, Spell DR, Carey PO, Kendrick SJ, Gallien TN, Handley HH, Flickinger CJ. The influence of vasovasostomy on antisperm antibodies in rats. Biol Reprod 1989; 40:353–360.[Abstract]
  60. Flickinger CJ. The fate of sperm after vasectomy in the hamster. Anat Rec 1982; 202:231–239.[CrossRef][Medline]
  61. Flickinger CJ, Herr JC, Baran ML, Howards SS. Testicular development and the formation of spermatic granulomas of the epididymis after obstruction of the vas deferens in immature rats. J Urol 1995; 154:1539–1544.[CrossRef][Medline]
  62. Hagopian A, Limjuco GA, Jackson JJ, Carlo DJ, Eylar EH. Experimental allergic aspermatogenic orchitis. IV. Chemical properties of sperm glycoproteins isolated from guinea pig testes. Biochim Biophys Acta 1976; 434:354–364.[Medline]
  63. O'Rand MG. Antigens of spermatozoa and their environment. In: Dhindsa D, Schumacher G (eds.), Immunological Aspects of Infertility and Fertility Regulation. New York: Elsevier-North Holland; 1980: 155–171.
  64. Kong M, Richardson RT, Widgren EE, O'Rand MG. Sequence and localization of the mouse sperm autoantigenic protein, Sp17. Biol Reprod 1995; 53:579–590.[Abstract]
  65. Lea IA, Richardson RT, Widgren EE, O'Rand MG. Cloning and sequencing of cDNAs encoding the human sperm protein, Sp17. Biochim Biophys Acta 1996; 1307:263–266.[Medline]
  66. Nakamura S, Tsuji Y, Komori S, Koyama K. Identification and characterization of a sperm peptide antigen recognized by a monoclonal antisperm antibody derived from a vasectomized mouse. Biochem Biophys Res Commun 1994; 205:1503–1509.[CrossRef][Medline]
  67. Samuel T, Linnet L, Rumke P. Post-vasectomy autoimmunity to protamines in relation to the formation of granulomas and sperm agglutinating antibodies. Clin Exp Immunol 1978; 33:261–269.[Medline]
  68. Witkin SS, Higgins PJ, Bendich A. Inhibition of viral reverse transcriptase and human sperm DNA polymerase by anti-sperm antibodies. Clin Exp Immunol 1978; 33:244–251.[Medline]
  69. Naz RK, Deutsch J, Phillips TM, Menge AC, Fisch H. Sperm antibodies in vasectomized men and their effects on fertilization. Biol Reprod 1989; 41:163–173.[Abstract]
  70. D'Cruz OJ, Haas GG, Lambert H. Heterogeneity of human sperm surface antigens identified by indirect immunoprecipitation of antisperm antibody bound to biotinylated sperm. J Immunol 1993; 151:1062–1074.[Abstract]
  71. Lee CY, Lum V, Wong E, Menge AC, Huang YS. Identification of human sperm antigens to antisperm antibodies. Am J Reprod Immunol 1983; 3:183–187.
  72. Hald J, Naaby-Hansen S, Egense J, Hjort T, Bjerrum OJ. Autoantibodies against spermatozoal antigens detected by immunoblotting and agglutination. A longitudinal study of vasectomized males. J Reprod Immunol 1987; 10:15–26.[CrossRef][Medline]
  73. Naaby-Hansen S. The humoral autoimmune response to vasectomy described by immunoblotting from two-dimensional gels and demonstration of a human spermatozoal antigen immunochemically crossreactive with the D2 adhesion molecule. J Reprod Immunol 1990; 17:187–205.[CrossRef][Medline]
  74. Naaby-Hansen S, Bjerrum OJ. Auto- and iso-antigens of human spermatozoa detected by immunoblotting with human sera after SDS-PAGE. J Reprod Immunol 1985; 7:41–57.[CrossRef][Medline]



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