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
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Abstract |
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Key words: antisperm antibodies/IgG/infertility/spermatozoa/sperm protein
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Introduction |
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Materials and methods |
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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, 1999) by masturbation and sperm protein extract (SPE) and was prepared as previously described (Chiu et al., 2002
). 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 416 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, 1976) 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 SpragueDawley 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., 2002). 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 310, 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 310, 7 cm; Bio-Rad) overnight at room temperature using a rehydration tray according to the manufacturers instructions (Bio-Rad).
The sperm proteins were focused using a focusing tray (Bio-Rad) according to the manufacturers 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 2448 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 (-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 6003500 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 4001800 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 502000 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 Freunds adjuvant (Invitrogen New Zealand Limited, Auckland, New Zealand). This procedure was repeated to obtain antigen for three booster immunizations except that incomplete Fruends adjvuant (SigmaAldrich, 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 Gills 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, 1999), and post-swim up sperm was subjected to calcium ionophore-induced acrosome reaction (WHO, 1999
). 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
1 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 Gills 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.
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Results |
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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., 2000). 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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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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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.
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Discussion |
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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., 1998). 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., 1996
; Goldman et al., 1998
). 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., 1999
).
Alpha-lactalbumin is a calcium binding protein the evolution of which precedes the divergence of mammals and birds (Prager et al., 1988; Permyakov et al., 2000
). 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., 2000
). 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., 1995
). The activity of galactosyltransferase can be inhibited by alpha-lactalbumin (Benau et al., 1988
; Ross et al., 1993
). 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., 1989
). 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., 2002
). 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., 2002
). Jones et al. (1982)
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., 2003) (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.
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Acknowledgements |
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References |
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Submitted on April 30, 2003; resubmitted on September 5, 2003; accepted on September 26, 2003.