SPRASA, a novel sperm protein involved in immune-mediated infertility

W.W.C. Chiu, E.K.L. Erikson, C.A. Sole, A.N. Shelling and L.W. Chamley1

Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand

1 To whom correspondence should be addressed. e-mail: l.chamley{at}auckland.ac.nz


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Antisperm antibodies (ASA) may be an important cause of infertility, but current tests for the detection of ASA have poor prognostic value. Identification of the sperm proteins that ASA bind to may aid the development of more useful diagnostic tests. METHODS: One- and two-dimensional PAGE and western blotting analyses, as well as amino acid sequencing, were used to identify a novel sperm protein reactive with ASA (SPRASA) from infertile men. An antiserum reactive with SPRASA was produced by immunizing a rabbit with SPRASA excised from two-dimensional gels. This antiserum was used to demonstrate the localization of SPRASA on the sperm. RESULTS: Amino acid sequences derived from SPRASA matched those of a theoretical protein, XP-085564. This protein is derived from the C-type lysozyme/alpha-lactalbumin gene family. Immunohisto chemistry indicates that SPRASA is localized to the acrosome. Western blot analysis revealed that 50 unselected individuals did not have antibodies that reacted with SPRASA. CONCLUSION: Only ASA from infertile men react with SPRASA, suggesting that this novel protein may be important in the processes of fertility. The identification of SPRASA as the antigen for infertility-associated ASA raises the possibility of developing first, antigen-specific tests for ASA, and secondly, more targeted treatment for immune-mediated infertility.

Key words: antisperm antibodies/IgG/infertility/spermatozoa/sperm protein


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infertility is estimated to affect up to 20% of couples of reproductive age and poses a significant health problem (Templeton et al., 1990Go; Mosher et al., 1991Go). Antisperm antibodies (ASA) are found in ~10% of infertile couples (Ayvaliotis et al., 1985Go; Clarke et al., 1985Go; Collins et al., 1993Go). However, ASA are also present in up to 5% of fertile men (Sinisi et al., 1993Go; Heidenreich et al., 1994Go). The presence of ASA in the fertile population indicates that not all ASA cause infertility. The controversy over the role of ASA in infertility to some extent reflects the inadequacies of current diagnostic techniques for ASA, such as the mixed agglutination assay (MAR) (Jager et al., 1978Go; Hendry et al., 1982Go) and immunobead test (Clarke et al., 1985Go). These tests measure the gross binding of antibodies to sperm (the percentage of sperm bound) but do not examine the antigenic specificities of the ASA. It is reasonably obvious that some sperm antigens are not essential to the processes of fertilization. Therefore, antisperm antibodies that bind to these antigens, as long as they do not cause aggregation of sperm or cause interactions with the cervical mucus, are unlikely to impede fertility. Whereas sperm proteins that have essential roles in fertilization, such as receptors for oocytes, could be neutralized by the binding of antisperm antibodies leading to infertility. Therefore, the development of tests that can distinguish the antigenic specificities of ASA that cause infertility would improve the diagnostic value of ASA testing. In order to develop such a test, we previously examined the specificities of ASA from men with vasectomy reversal by western blotting, and found that only ASA from infertile men were reactive with sperm proteins of ~16 kDa (Chiu et al., 2002Go). In this study we have used the ASA from these infertile men to isolate a low molecular weight protein and identify it as a novel sperm protein that we have called SPRASA (sperm protein reactive with ASA).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Participants
This investigation was approved by the regional ethics committee and all samples were obtained after the participants provided informed consent. Two men, undergoing investigations for infertility after 1 year of inability to impregnate their partners, have previously been shown to demonstrate strong reactivity against sperm proteins of ~16 kDa (Chiu et al., 2002Go). These men have a positive (>80%) sperm/MAR sperm surface antibody test (Ferti-PRO, Intermed Scientific Ltd, Auckland, New Zealand). As previously published, these men had sperm counts of >10 x 106/ml and >75% motility (Chiu and Chamley, 2002Go). Sera from these two men were used to identify SPRASA in this work.

Blood was also obtained from 50 unselected individuals and the serum separated by centrifugation and then stored at –80°C until use.

Preparation of sperm proteins
Normal semen was collected from donors (WHO, 1999Go) by masturbation and sperm protein extract (SPE) and was prepared as previously described (Chiu et al., 2002Go). Briefly, the semen was allowed to liquefy at room temperature for 30 min and divided into aliquots containing 50 x 106 sperm in 1.5 ml microfuge tubes. Sperm were then separated from the seminal plasma by centrifugation (1500 g) for 15 min at 4°C. Each aliquot of sperm was washed by suspension in 1 ml of phosphate-buffered saline (PBS) (pH 7.4) and centrifuged at 1500 g at 4°C. The supernatant was discarded and the washing procedure repeated three times.

The sperm aliquots were subsequently suspended in 250 µl of protease inhibitor (PI) buffer (benzamidine hydrochloride hydrate 1 mmol/l, pepstatin A 1 µmol/l; N Tosyl L phenylalanine chloromethyl ketone 100 µmol/l in Tris 25 mmol/l/EDTA 10 mmol/l pH 8.0 buffer) and stored at –80°C until use. All materials, unless otherwise stated, were obtained from Sigma (Sydney, Australia).

SPE were prepared freshly before use as follows. Stored sperm aliquots were thawed and the sperm were solubilized by mixing with Triton X-114 (1% v/v) (Boehringer Mannheim, Auckland, New Zealand) by repeated pipetting with a micropipette. The samples were then incubated for 4–16 h at 4°C on a rotating platform. Debris was removed by centrifugation at 13 000 g for 15 min at 4°C. The concentration of the extracted protein in the supernatant was determined by Bradford microprotein assay (Bradford, 1976Go) using bovine serum albumin dissolved in PI buffer as standard protein.

Rat testicular sperm proteins were prepared from sperm extracted from the testes of an adult male rat. Briefly, a Sprague–Dawley rat was sacrificed and its testes were excised. The capsules of the testes were incised and the seminiferous tubules were removed and minced in a Petri dish containing PBS (pH 7.4). The sperm were allowed to disperse from the seminiferous tubules and aspirated using a Pasteur pipette. The aspirate was transferred to a centrifuge tube and the mixture was allowed to settle for 10 min. The suspended sperm were aspirated and centrifuged at 1500 g for 10 min. The sperm were subsequently resuspended with PBS (pH 7.4) and adjusted to aliquots of 50 x 106 sperm in microfuge tubes. The sperm pellet was washed by resuspending it in 1 ml of PBS (pH 7.4) followed by centrifugation 1500 g at 4°C. The washing procedure was repeated twice. The procedure for extracting proteins from the rat sperm then followed that used for human sperm described above.

PAGE and western blotting
One-dimensional PAGE and western blot analysis were performed as described previously (Chiu et al., 2002Go). In addition, western blots of SPE were probed with SPRASA antiserum diluted 1/500.

Isoelectric focusing and two-dimensional electrophoresis of sperm proteins
For Coomassie Blue identification of sperm proteins, 160 µg (80 µl) of sperm protein extracts mixed with Ready Prep reagent three [5 mol/l urea, 2 mol/l thiourea, 2% (w/v) CHAPS, 2% (w/v) SB 3–10, 40 mmol/l Tris, 0.2% (w/v) Bio-Lyte 3/10 (Ready Prep; Bio-Rad, Auckland, New Zealand)] and 0.00001% bromophenol blue to a total volume of 180 µl. The protein sample was mixed, vortexed briefly and centrifuged at 13 000 g for 1 min. The sample was then passively rehydrated into IPG strips (pH 3–10, 7 cm; Bio-Rad) overnight at room temperature using a rehydration tray according to the manufacturer’s instructions (Bio-Rad).

The sperm proteins were focused using a focusing tray (Bio-Rad) according to the manufacturer’s instructions. The focusing conditions were as followed: prefocusing at 500 V for 15 min, followed by linear voltage ramping for 4 h to 4000 V and final focusing to 80 000 Volt hours at 20°C. Current was limited to 50 µA per gel and the focusing run was terminated when the current dropped to approximately one-fifth of the current that was originally set. The focusing process would usually take 24–48 h. The focused strips were either immediately resolved in their second dimension or stored at –80°C until later analyses.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis
The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses were carried out by the Australian Protein Analyzing Facilities, Australia.

Briefly, protein spots that were the antigens for ASA were identified by western blot of two-dimensional electrophoresis gels of SPE. Spots corresponding to these were excised from Commassie Blue stained gels and the spots were washed three times with 50% acetonitrile, 25 mmol/l NH4HCO3, pH 7.8, and dried down. One hundred and twenty nanograms of trypsin (Promega, Madison, WI, USA) in 25 mmol/l NH4HCO3, pH 7.8, was added to the dried gel spot and incubated at 37°C overnight. The resulting peptides were extracted from the gel with a 50% (v/v) acetonitrile, 1% (v/v) TFA solution and mixed with an equal volume of matrix ({alpha}-cyano-4-hydroxycinnamic acid, 8 mg/ml in 70% v/v AcN, 1% v/v TFA) and allowed to air dry. MALDI-MS was performed with a Micromass TofSpec 2E Time of Flight Mass Spectrometer. A nitrogen laser (337 nm) was used to irradiate the sample. The spectra were acquired in reflectron mode in the mass range 600–3500 Da. A near-point calibration was applied and gives a typical mass accuracy of ~100 p.p.m. or less.

Electro-spray ionization mass spectrophotometry/mass spectrophotometry
Electro-spray ionization mass spectrophotometry/mass spectrophotometry (ESI-TOF MS/MS) was performed by the Australian Protein Analyzing Facilities. Briefly, protein spots were obtained from two-dimensional gels as described above and the proteins contained in them sequenced by ESI-TOF MS/MS using a Micromass Q-TOF MS equipped with a nanospray source and data manually acquired using borosilicate capillaries. Data were acquired over the m/z range 400–1800 to select peptides for MS/MS analysis. After peptides were selected, the MS was switched to MS/MS mode and data collected over the m/z range 50–2000 with variable collision energy settings.

Immunization of animals with SPRASA
Two-dimensional gels of SPE were stained with Coomassie Blue then placed on damp glass plates that had previously been cleaned with high-pressure liquid chromatography grade methanol (BDH Laboratory Supplies, Poole, UK). Glass plates and gels were then visualized on a light box and the protein spots containing SPRASA were excised with a number 20 disposable scalpel (Swaun-Moston, Sheffield, UK). The excised protein spots were then placed in a 1.7 ml Eppendorf tube and stored at –80°C until the spots from at least seven gels had been collected. The stored protein spots were allowed to thaw for 10 min at room temperature and placed onto a clean glass plate then macerated using a number 20 disposable scalpel. The macerated gel spots were placed in a 1.7 ml Eppendorf tube and suspended in 1 ml PBS (pH 7.4), and then emulsified with 1 ml of complete Freund’s adjuvant (Invitrogen New Zealand Limited, Auckland, New Zealand). This procedure was repeated to obtain antigen for three booster immunizations except that incomplete Fruend’s adjvuant (Sigma–Aldrich, Sydney, Australia) was used for the boosters. A New Zealand white rabbit was immunized by subcutaneous injection of the emulsified proteins at multiple sites. Following immunization, blood was obtained from the central ear artery of the rabbit and the serum was separated by centrifugation and stored at –20°C.

Immuno-localization of SPRASA on washed sperm
Half a million freshly washed normal donor spermatozoa (in 10 µl of PBS, pH 7.4) were obtained from three individuals, pipetted onto microscope slides (BioLab Scientific, Auckland, New Zealand) and allowed to dry overnight. The slides were fixed in cold acetone for 10 min in a fume hood and then allowed to air dry for 1 h. Slides were then wrapped in foil and stored at –20°C.

Stored slides were allowed to thaw for 10 min at room temperature. Normal goat serum 10% in PBS (pH 7.4) containing 0.05% Tween 20 (PBST), used as a blocking solution, was incubated on the slides at room temperature for 10 min, and the slides were then washed three times with PBST. The primary antisera (SPRASA antiserum 1/50) or rabbit anti-CD46 (1/200; Santa Cruz, Global Sciences, Auckland, New Zealand), or an irrelevant control rabbit antiserum (1/50) were diluted in blocking solution, applied to the slides and incubated for 2 h at 20°C. To quench endogenous peroxidases, 3% hydrogen peroxide in methanol was then incubated on the slides for 5 min at room temperature, and the slides were then washed three times with PBST. Biotin-conjugated goat anti-rabbit IgG (Jackson Laboratories, Austrailian Laboratory Services, Auckland, New Zealand) diluted 1:1000 in blocking solution was then incubated on the slides at 20°C for 1 h. The slides were washed three times with PBST and streptavidin-conjugated horseradish peroxidase (HRP) (Serotec, Oxford, UK) diluted 1:1000 in blocking solution was incubated on slides at 20°C for 1 h. The slides were washed three times with PBST, and 50 µl of 3-amino-9-ethylcabazole (AEC; DAKO Corporation, Christchurch, New Zealand) was incubated on slides at 20°C for 10 min. The slides were then washed in de-ionized water for 2 min. Following the washing, the slides were counterstained with Gill’s haematoxylin (Amber Scientific, Belmont, Australia) for 30 s. Slides were immediately washed in tap water, changing the water frequently until the water remained clear. Coverslips (BioLab Scientific) were then mounted on the slides with aquamount (BDH Laboratory Supplies). The slides were allowed to air dry for 1 h and then were viewed using a light microscope (Ernst Leitz GmbH, Wetzlar, Germany). The images were photographed using a Nikon Coolpix990 digital camera.

Immuno-localization of SPRASA on unfixed sperm
Swim up sperm was prepared from three donors according to the WHO laboratory manual (WHO, 1999Go), and post-swim up sperm was subjected to calcium ionophore-induced acrosome reaction (WHO, 1999Go). The sperm was then incubated for 60 min with the SPRASA-reactive antiserum (diluted 1/100), anti-CD46 (diluted 1/100; Santa Cruz) or an irrelevant control antiserum (diluted 1/100) at 37°C in a 5% CO2 humidified atmosphere. The sperm were then washed with PBS (pH 7.4) and aliquots of ~x 106 sperm were smeared onto glass microscope slides, air dried and fixed with cold acetone. Normal goat serum 10% in PBST used as a blocking solution, was incubated on the slides at room temperature for 10 min, and the slides were then washed three times with PBST. To quench endogenous peroxidases, 3% hydrogen peroxide in methanol was then incubated on the slides for 5 min at room temperature, and the slides were then washed three times with PBST. The biotin-conjugated goat anti-rabbit IgG diluted 1:1000 in blocking solution was then incubated on the slides at 20°C for 1 h. The slides were washed three times with PBST and streptavidin-conjugated HRP diluted 1:1000 in blocking solution was incubated on slides at 20°C for 1 h. The slides were washed three times with PBST, and 50 µl of 3-amino-9-ethylcabazole was incubated on slides at 20°C for 10 min. The slides were washed in de-ionized water for 2 min. Following the washing the slides were then counterstained with Gill’s haematoxylin for 30 s. Slides were immediately washed in tap water, changing the water frequently until the water remained clear. Coverslips were then mounted on the slides with aquamount. The slides were allowed to air dry for 1 h and then viewed using a light microscope (Ernst Leitz GmbH, Wetzlar, Germany). The images were photographed using a Nikon Coolpix990 digital camera.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification of SPRASA by two-dimensional electrophoresis and western blot analysis
Proteins were extracted from normal donor sperm and separated by two-dimensional electrophoresis. Separate two-dimensional western blots were probed with serum from two men who remained infertile 1 year after vasectomy reversal to identify the ~16 kDa sperm protein reactive with IgG ASA (SPRASA). Both men had been previously shown to have intense reactivities to ~16 kDa sperm proteins (Chiu et al., 2002Go). In this two-dimensional western blot system, reactivity with proteins of 16 and 19 kDa was observed for both infertile men (Figure 1). Serum from two control fertile men did not react with either the 16 or 19 kDa protein spot (Figure 1). The 16 and 19 kDa proteins had isoelectric points of 5.7 and 5.4, respectively. These two protein spots were identified on Coomassie Blue-stained SDS–PAGE (Figure 1). The two proteins were subjected to characterization by peptide mass fingerprinting (PMF) and peptide microsequencing.



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Figure 1. Two dimensional SDS–PAGE and western blot identification of the ~16 kDa sperm protein(s) reactive with IgG. (A) Human sperm protein extract (160 µg) was resolved by two-dimensional SDS–PAGE and stained with Coomassie Blue. Western blots of SPE (50 µg) were similarly resolved then electrotransferred to nitrocellulose membranes and probed with serum IgG ASA from two infertile men (B and C), and serum from two fertile men (D and E). Serum IgG ASA from the two infertile patients showed strong reactivities to the two sperm proteins marked by arrows (<) on the Coomassie Blue-stained gel (A).

 
Identification of SPRASA by peptide mass fingerprinting and tandem MS/MS
In order to characterize the 16 and 19 kDa sperm proteins reactive with IgG ASA from infertile men, these proteins were subjected to MALDI-TOF PMF analysis. The protein spots from four Coomassie Blue-stained gels were pooled for the purpose of obtaining enough protein for peptide sequencing. The proteins were subjected to trypsin digestion and MALDI-TOF analysis. There was no match of the PMF results with the PMF database (not shown), but the spectra for both proteins contained many identical peaks, suggesting that they are isoforms of the same protein.

Given the lack of a match with the MALDI-TOF PMF databases, ESI-TOF MS/MS analysis was carried out to determine the amino acid sequences of the tryptic peptides obtained from the 16 and 19 kDa proteins. The amino acid sequences of three tryptic peptides were obtained from both the 16 and 19 kDa proteins in this manner (Table I). The sequences of the peptides from both proteins were the same. Searches of the National Center for Biotechnology Information (NCBI) and OWL databases demonstrated that the three peptides matched the amino acid sequence of a theoretical protein with the accession code XP-058864 in the NCBI protein database (Figure 2). This is a putative protein encoded by a gene from the C-type lysozyme/alpha-lactalbumin family. This gene family is phylogenetically ancient and encodes alpha-lactalbumin and lysozyme. The two proteins have similar structures but different functions (Permyakov et al., 2000Go). However, one of the matching peptides (peptide three) was mismatched by one residue against XP-058864. There was an extra glycine in the sequenced peptide that is not present in XP-058864; however, this may be an artefact of the PAGE, which employs glycine containing buffers. The protein encoded by XP-058864 has not previously been identified, and we have named it sperm protein reactive with ASA (SPRASA).


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Table I. Amino acid sequences of tryptic-digested peptides separated by ESI-TOF MS/MS
 


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Figure 2. Homology of peptides sequenced from the 16 and 19 kDa sperm protein, relevant to infertility, to a theoretical peptide. Three of the deduced peptide sequences of the two sperm proteins reactive with IgG ASA are identical. They shared identity with the deduced amino acid sequence of a gene (NCBI database accession code XP-058864) encoding a theoretical protein. The theoretical protein belongs to the C-type lysozyme/alpha-lactalbumin family.

 
Using the same peptide matching/searching strategies as above, the fourth peptide (Table I) from the 19 kDa isoform did not match either XP-058864 or any other entries in the databases. This peptide was not present in tryptic digests of the 16 kDa protein and we believe this peptide is derived from an unrelated contaminant.

Immunoreactivity of SPRASA to alpha-lactalbumin
In order to investigate whether an antibody raised against alpha-lactalbumin showed any reactivity with SPRASA, western blots of SPE and breast milk were probed with a rabbit alpha-lactalbumin antiserum. This antiserum reacted with an ~16 kDa protein in breast milk but did not react with SPE (data not shown).

Western analysis for ASA reactive with SPRASA
In order to examine the prevalence of SPRASA-reactive ASA, we probed western blots of SPE with serum from 50 unselected patients (Figure 3). Serum from one of the infertile man with ASA to SPRASA was used as positive control (Figure 3). None of the 50 unselected persons had antibodies reactive with SPRASA.



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Figure 3. Western blot analysis of ASA in the serum of unselected subjects. Western blots of sperm proteins were probed with serum from 25 (representative examples of 50 screened) unselected patients (lanes 1–25). Western blot of sperm proteins probed with serum from an infertile man with known ASA reactivity to SPRASA (lane 26, arrow <).

 
Localization of SPRASA on sperm
An antiserum reactive with SPRASA was prepared by immunizing a New Zealand white rabbit with SPRASA excised from two-dimensional gels. This antiserum was used to probe sperm that had been extensively washed and smeared onto microscopic slides. The analysis indicated that SPRASA was localized to the acrosome (Figure 4). Not all sperm stained with the SPRASA antiserum. In order to investigate whether the staining of sperm with the SPRASA antiserum was related to the acrosomal status of the sperm, the staining patterns of SPRASA and CD46, an inner acrosomal membrane protein, were investigated in sperm obtained from four donors. Counting 10 random high-power fields of sperm from the four donors showed that 57% of sperm were immunoreactive with SPRASA. Similarly, 68% of sperm were immunoreactive with CD46 (Figure 4). The percentages of sperm stained by these two antisera were not statistically significantly different (P = 0.22). Non-viable sperm are known to non-specifically bind to antibodies, and in order to ensure that the binding of both the CD46 and SPRASA antisera to sperm was specific we also examined the binding of a control irrelevant antiserum to the sperm. Only 6.4% of sperm stained with a control irrelevant antiserum (P = 0.01). In contrast, the SPRASA-reactive antiserum bound to only 3.5% of unfixed sperm from three donors that had been prepared by swim up, but when this swim up sperm was exposed to calcium ionophore the binding increased to 19.1%. The irrelevant control antiserum bound to <1% of both swim up and calcium ionophore-treated (post-swim up) sperm from these three donors. These results suggest that SPRASA, like CD46, is an inner acrosomal protein.





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Figure 4. The localization of SPRASA on sperm. Photomicrographs demonstrating the staining pattern on normal human sperm of antisera reactive with (A) SPRASA, (B) CD46 and (C) an irrelevant control antiserum.

 
Some sperm proteins are known to be conserved between species (Ward et al., 2000Go). A western blot was carried out to determine whether a protein(s) similar to SPRASA is present in rat testicular sperm. Normal human sperm proteins and rat testicular sperm proteins were resolved using 4–15% gradient SDS–PAGE. The proteins were electrotransferred and the western blots were probed with human sera from two fertile men without ASA detected by MAR and from two infertile patients with sperm ASA (Figure 5). A wide range of rat testicular sperm proteins were bound by human ASA. Notably, human ASA from infertile, but not fertile, men reacted with proteins of molecular weights similar to SPRASA. This suggests that these proteins may be conserved between humans and rat (Figure 5).



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Figure 5. Sperm proteins reactive with IgG ASA have similar immunoreactivity to rat testicular proteins. Western blots of rat testicular (lanes 2 and 4) and human sperm proteins (lanes 1 and 3) were probed with serum from two infertile men with SPRASA-reactive ASA (lanes 1, 2 and 3, 4). SPRASA is present in both human sperm and the rat testicular sperm (arrow <).

 
Sequence analysis of SPRASA (protein XP-085564)
The NCBI database entry for XP-085564 did not provide any information regarding the tissue expression of this protein. The amino acid sequence of XP-085564 was matched to other proteins using the BLAST-link feature of NCBI protein database. The sequence of XP-085564 showed homology to several proteins, but most significantly to: (i) an unnamed protein from mouse (Mus musculis) (BAB24544) (67% protein identity); and (ii) a human protein similar to lysozyme C-1 (AAH21730) (41% protein identity). The high degree of identity between the proteins suggests that these proteins may have a conserved function through evolution.

Analysis of the NCBI human expressed sequence tag (EST) database indicates that SPRASA has 15 matching EST clones. The origin of these sequences was primarily from the testes; however, it is of interest that ESTs were also isolated from germ cell tumour samples. Sixteen ESTs were found for the homologous mouse protein, BAB24544 (NCBI locus accession code AK006357; gene identifier 12839451). All of these ESTs were derived from mouse testicular cDNA libraries. These data suggest that SPRASA and its mouse homologue, BAB24544, are expressed mainly in the testis or testis-derived tissue.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have used a combination of immunoassays and protein sequencing techniques to identify SPRASA, a previously undescribed sperm protein, as the antigen for ASA from two men who remained infertile following vascetomy reversal despite having reasonable sperm counts. Antibodies reactive with SPRASA were not found in any of the 50 unselected persons screened in this study or in any of the six fertile men screened in a previous study (Chiu et al., 2002Go). This raises the possibility that the SPRASA-reactive ASA could contribute significantly to the pathogenesis of infertility in the two men who have these antibodies.

SPRASA belongs to the C-type lysozyme/alpha-lactalbumin family of proteins. Lysozyme is a phylogenetically ancient antimicrobial factor produced by mammalian apocrine glands (Goldman et al., 1998Go). It has the ability to lyse certain bacteria by degrading peptidoglycans from cell walls and protects elastin from degradation by preventing leukocytic elastase from interacting with elastin, thereby accounting for the involvement of lysozyme in the immunity of mucosal surfaces (Park et al., 1996Go; Goldman et al., 1998Go). The role, if any, of lysozyme in reproduction is unknown, but one study has shown that lysozyme is elevated in seminal plasma of 14% of infertile men, suggesting a potential link of this protein with fertility (Uleova-Gallova et al., 1999Go).

Alpha-lactalbumin is a calcium binding protein the evolution of which precedes the divergence of mammals and birds (Prager et al., 1988Go; Permyakov et al., 2000Go). Alpha-lactalbumin is one of the components of lactose synthase, the enyme complex that catalyses the final step in lactose synthesis from glucose in the lactating mammary gland (Permyakov et al., 2000Go). The function of alpha-lactalbumin in reproduction is poorly understood. Alpha-lactalbumin binds to and modulates the specificity of galactosyltransferase, a sperm ligand for oocyte ZP3 that is essential for fertilization (Cardullo et al., 1995Go). The activity of galactosyltransferase can be inhibited by alpha-lactalbumin (Benau et al., 1988Go; Ross et al., 1993Go). Bovine mammary alpha-lactalbumin can suppress capacitation and hyperactivation of mouse sperm and the ability of sperm to bind to zona pellucida of oocytes (De Geyter et al., 1989Go). Sperm from transgenic rats expressing a heterozygous human alpha-lactalbumin gene (LAC3) are defective and immotile, and are not capable of fertilizing oocytes using ICSI (Hirabayash et al., 2002Go). However, ICSI using round spermatids from these rats appeared to produce live birth rates comparable to control experiments using spermatids from Wistar rats (Hirabayashi et al., 2002Go). Jones et al. (1982)Go suggested that sperm proteins with alpha-lactalbumin-like activities act in concert with epididymal glycosidase to regulate the modification of sugars on membrane-bound glycoproteins. It is interesting to speculate that if SPRASA has alpha-lactalbumin-like properties, it potentially could interact with galactosyltransferase on sperm in the process of fertilization. Hence, ASA directed against this antigen could interfere with fertilization.

By screening EST databases we have identified that genes for proteins homologous to SPRASA are expressed in the testis of rats and mice. Expression of these cDNAs appears to be limited largely to the testis, with the only other site of expression being the brain. Our own western blotting results also show that ASA from the two infertile men whose antibodies led us to identify SPRASA react with a protein of similar molecular weight to SPRASA in rat sperm. This suggests that a highly conserved homologue of SPRASA exists in the rat, and that this homologue is expressed in sperm.

Using a polyclonal rabbit antiserum we have shown that SPRASA is located on the fore part of the head of washed human sperm. The antisera was reactive with ~60% of washed sperm, but only 3.5% of sperm prepared by swim up. This is a similar percentage of sperm that reacted with a CD46 antiserum. CD46 is an inner acrosomal membrane protein that is externalized following the acrosome reaction. We believe that these antibodies were reacting with spontaneously acrosome-reacted washed sperm, and that the swim up procedure removed most of these spontaneously acrosome-reacted sperm. Induction of the acrosome reaction in the swim up sperm increased the percentage of sperm bound by the SPRASA-reactive antiserum, suggesting that the acrosome reaction must occur for SPRASA to be exteriorized on sperm. Thus, we believe that SPRASA, like CD46, is expressed on the inner acrosomal membrane. We have also shown by immunohistochemistry that a SPRASA homologue is present in other species such as sheep, bull and deer (data not shown), confirming that SPRASA is a highly conserved protein and further suggesting that it has an important role in reproduction.

We have shown that SPRASA has two isoforms, one of 16 kDa and the other of 19 kDa. The origin of these two isoforms is not yet clear. The gene for SPRASA is predicted to produce three alternative transcripts, the largest of which has a predicted molecular weight of 23.4 kDa with a pI of 4.6. The other predicted products of the SPRASA gene have molecular weights of 17.4 kDa (pI 4.6) and 12.6 kDa (pI 5.5). None of these predicted proteins corresponds to the isoforms of SPRASA we have identified. It is possible that the isoforms of SPRASA we have identified are proteolytic products of the 23.4 kDa variant. These proteolytic variants may be naturally occurring products due to post-translational modification or artefacts of the procedure used to isolate the sperm proteins prior to two-dimensional electrophoresis.

While preparing this manuscript we found that another group has also recently identified SPRASA (Mandal et al., 2003Go) (called SLLP-1 by them). These workers identified SPRASA as part of a data-mining program. Their results confirm that SPRASA is an acrosomal protein, and have shown that an antiserum reactive with recombinant SPRASA inhibits the binding of sperm to hamster zona-free oocytes. These results, taken together with our finding that SPRASA-reactive ASA are present only in the serum of infertile men, indicate that SLLP-1/SPRASA is important in the process of fertilization and suggest that the ASA reactive with SPRASA could contribute significantly to the infertility of the men who have these antibodies. Accordingly, tests such as antibody capture enzyme-linked immunosorbent assays may allow the rapid detection of SPRASA-specific ASA in couples seeking infertility treatment. Furthermore, homologues of SPRASA could be used as a target for new contraceptive modalities in various species, including domestic and feral animal populations.


    Acknowledgements
 
We would like to thank the staff of Fertility Plus, Ms M.Merrilees, T.Naysmith, J.Kenny, H.Smith and Dr G.Gudex, as well as Dr G.Clarke, for their assistance in obtaining and preparing samples. We also wish to thank Mr L.McGowan and Mr I.Scott, Agresearch Ltd, for supplying animal sperm. This work was supported by grants from the Maurice and Phyllis Paykel Trust, The University of Auckland Research Committee, the Centre for Reproductive Medicine, University of Auckland, and Uniservices Auckland.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ayvaliotis B, Bronson R, Rosenfeld D and Cooper G (1985) Conception rates in couples where autoimmunity to sperm is detected. Fertil Steril 43,739–742.[ISI][Medline]

Benau DA and Storey BT (1988) Relationship between two types of mouse sperm surface sites that mediate binding of sperm to the zona pellucida. Biol Reprod 39,235–244.[Abstract]

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72,248–254.[CrossRef][ISI][Medline]

Cardullo RA and Wolf DE (1995) Distribution and dynamics of mouse sperm surface galactosyltransferase: implications for mammalian fertilization. Biochemistry 34,10027–10035.[ISI][Medline]

Chiu WWC and Chamley LW (2002) Use of antisperm antibodies in differential display Western blotting to identify sperm proteins important in fertility. Hum Reprod 17,984–989.[Abstract/Free Full Text]

Clarke GN, Elliott PJ and Smaila C (1985) Detection of sperm antibodies in semen using the immunobead test: a survey of 813 consecutive patients. Am J Reprod Immunol Microbiol 7,118–123.[Medline]

Collins JA, Burrows EA, Yeo J and YoungLai EV (1993) Frequency and predictive value of antisperm antibodies among infertile couples. Hum Reprod 8,592–598.[Abstract]

De Geyter C, Cooper TG, De Geyter M and Nieschlag E (1989) Effects of bovine mammary alpha-lactalbumin on hyperactivation and sperm–zona pellucida binding of mouse spermatozoa. Gamete Res 24,415–426.[ISI][Medline]

Goldman AS, Chheda S and Garofalo R (1998) Evolution of immunologic functions of the mammary gland and the postnatal development of immunity. Pediatr Res 43,155–162.[Abstract]

Heidenreich A, Bonfig R, Wilbert DM, Strohmaier WL and Engelmann UH (1994) Risk factors for antisperm antibodies in infertile men. Am J Reprod Immunol 31,69–76.[ISI][Medline]

Hendry WF, Stedronska J and Lake RA (1982) Mixed erythrocyte–spermatozoa antiglobulin reaction (MAR test) for IgA antisperm antibodies in subfertile males. Fertil Steril 37,108–112.[ISI][Medline]

Hirabayash M, Kato M, Aoto T, Sekimoto A, Ueda M, Miyoshi I, Kasai N and Hochi S (2002) Offspring derived from intracytoplasmic injection of transgenic rat sperm. Transgenic Res 11,221–228.[CrossRef][ISI][Medline]

Hirabayashi M, Kato M, Aoto T, Ueda M and Hochi S (2002) Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids. Mol Reprod Dev 62,295–299.[CrossRef][ISI][Medline]

Jager S, Kremer J and van Slochteren-Draaisma T (1978) A simple method of screening for antisperm antibodies in the human male. Detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. Int J Fertil 23,12–21.[ISI][Medline]

Jones R and Brown CR (1982) Association of epididymal secretory proteins showing alpha-lactalbumin-like activity with the plasma membrane of rat spermatozoa. Biochem J 206,161–164.[ISI][Medline]

Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling LC, Coonrod SA, Black MB, Dickman AB, Haystead TA, Flickinger CJ, Her JC. (2003) SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c Lysozyme-like protein of human spermatozoa. Biol Reprod 68,1525–1537.[Abstract/Free Full Text]

Mosher WD and Pratt WF (1991) Fecundity and infertility in the United States: incidence and trends. Fertil Steril 56,192–193.[ISI][Medline]

Park PW, Biedermann K, Mecham L, Bissett DL and Mecham RP (1996) Lysozyme binds to elastin and protects elastin from elastase-mediated degradation. J Invest Dermatol 106,1075–1080.[Abstract]

Permyakov EA and Berliner LJ (2000) alpha-Lactalbumin: structure and function. FEBS Lett 473,269–274.[CrossRef][ISI][Medline]

Prager EM and Wilson AC (1988) Ancient origin of lactalbumin from lysozyme: analysis of DNA and amino acid sequences. J Mol Evol 27,326–335.[ISI][Medline]

Ross P, Vigneault N, Provencher S, Potier M and Roberts KD (1993) Partial characterization of galactosyltransferase in human seminal plasma and its distribution in the human epididymis. J Reprod Fertil 98,129–137.[Abstract]

Sinisi AA, Di Finizio B, Pasquali D, Scurini C, D’Apuzzo A and Bellastella A (1993) Prevalence of antisperm antibodies by SpermMARtest in subjects undergoing a routine sperm analysis for infertility. Int J Androl 16,311–314.[ISI][Medline]

Templeton A, Fraser C and Thompson B (1990) The epidemiology of infertility in Aberdeen. BMJ 301,148–152.[ISI][Medline]

Uleova-Gallova Z, Krauz V, Mohamed AM and Rokyta Z (1999) Immunity to spermatozoa and male fertility. Andrologia 31,318–319.[ISI][Medline]

Ward E and Berger T (2000) Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane. Zygote 8,181–187.[CrossRef][ISI][Medline]

WHO (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. 4th edn, Press Syndicate of the University of Cambridge, Cambridge, UK.

Submitted on April 30, 2003; resubmitted on September 5, 2003; accepted on September 26, 2003.





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