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Dominant Prostasome Immunogens for Sperm-Agglutinating Autoantibodies of Infertile Men

B. OVE NILSSON

From the ^* Department of Medical Sciences, University Hospital, Uppsala, Sweden; and Department of Medical Cell Biology, Biomedical Center, University of Uppsala, Uppsala, Sweden.

Correspondence to: Prof Gunnar Ronquist, Department of Medical Sciences, University Hospital, S-751 85 Uppsala, Sweden (e-mail: gunnar.ronquist{at}akademiska.se).

Received for publication January 20, 2004; accepted for publication April 21, 2004.

	Abstract

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The presence of naturally occurring anti-sperm antibodies (ASA) is a well-known cause of infertility in men and women, but the antigens for these antibodies are poorly characterized. We have previously shown that prostasomes adhere to sperm cells and that prostasomes are major targets for ASA associated with infertility. These autoantigens have not been characterized. We used 2-dimensional electrophoresis, immunoblotting, and mass-spectrometry to identify the prostasome antigens for these autoantibodies. By these techniques, we revealed that prolactin-inducible protein (PIP) and clusterin were dominant prostasome immunogens for spermagglutinating autoantibodies of 20 patients with immunological infertility. PIP was identified by 19 of 20 (95%) patient sera and clusterin by 17 of 20 (85%). In addition, 10 sporadically occurring prostasomal antigens were identified in this context, viz alcohol dehydrogenase [NADP⁺], annexin I, annexin III, BRCA1-associated ring domain protein 1, heat shock 27-kd protein, isocitrate dehydrogenase, lactoylglutathione lyase, NG,NG-dimethylarginine dimethylaminohydrolase 1, peroxiredoxin 2, and syntenin 1.

     Key words: Antisperm antibodies (ASA), autoantibody, MALDI-TOF, prostasomes, human

A proteomic analysis of human prostasomes applying microcapillary liquid chromatography–tandem mass spectrometry revealed that they contained at least 139 proteins (Utleg et al, 2003). They were enzymes (35%), transport/structural proteins (19%), GTP proteins (14%), chaperone proteins (6%), signal transduction proteins (17%), and novel proteins (9%). Some of these components were already detected by other techniques in prostasomes (Ronquist et al, 1997; Ronquist and Nilsson, 2001).

We recently reported that circulating human anti-sperm antibodies (ASA) from immunoinfertile men and women recognized prostasomes at a high frequency (Carlsson et al, 2004). This shows that prostasomes are major auto-antigens for sperm-agglutinating ASA.

The aim of this study was to identify the proteins corresponding to the prostasome autoantigens. We used 2-dimensional electrophoresis, immunoblotting, and matrix-assisted laser desorption/ionization-time-of-flight (MAL-DI-TOF) to identify the antigens for these autoantibodies. The mass spectrometry results can then be used to identify the proteins by comparing the peptides with MS-Fit (http://prospector.ucsf.edu) and ProFound (http://prowl.rockefeller.edu) or the licensed ProteinLynx Software (Micromass, Manchester, United Kingdom).

	Material and Methods

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Patient Samples

To prove the presence of ASA, sera were derived from immuneinfertile patients who presented a positive Tray agglutination test (Friberg, 1980). The patient sera were tested with a prostasome enzyme-linked immunosorbent assay (ELISA) resulting in the finding that 113 of 116 patients (97%) had autoantibodies against prostasomes (Carlsson et al, 2004). Twenty of these sera with highest ELISA titers of anti-prostasome antibodies were selected for this study. Sera from 10 ASA-negative fertile males were run concomitantly as controls. The study was approved by the Ethical Committee of Uppsala University.

Sample Preparation

Prostasomes were isolated and purified as reported earlier (Wang et al, 2001). In short, semen samples were centrifuged for 20 minutes at 1 000 x g and 20°C to separate spermatozoa and other possible cells from the seminal plasma, which was then pooled (12–15 samples) and ultracentrifuged at 10 000 x g at 4°C for 15 minutes to remove cell debris. The supernatant was subsequently subjected to another ultracentrifugation for 2 hours at 100 000 x g at 4°C to pellet the prostasomes. The prostasomes were resuspended in 30 mmol/L Tris-HCl containing 130 mmol/L NaCl, pH 7.6 (isotonic Tris-HCl buffer). This suspension was further purified on a Sephadex G 200 column (Amersham Biosciences, Uppsala, Sweden), equilibrated with the isotonic Tris-HCl buffer, at 4°C to separate the prostasomes from an amorphous substance (Stegmayr and Ronquist, 1982). The eluant was the isotonic Tris-HCl buffer, and the eluate was monitored at 260 and 280 nm. Those fractions (5–12) with initial elevated ultraviolet absorbances were collected and analyzed for aminopeptidase N activity, a marker enzyme for prostasomes (Ronquist et al, 1988). Ultraviolet-absorbing fractions with high aminopeptidase N activity were pooled and ultracentrifuged at 100 000 x g at 4°C for 2 hours. The pellet representing the prostasomes was resuspended in the isotonic Tris-HCl buffer and adjusted to a protein concentration of 2 mg/mL by the Protein Assay ESL method (Roche Diagnostics, Mannheim, Germany).

Production of Antibodies

Chicken anti-prostasome antibodies were produced by immunizing chickens according to Polson and von Wechmar (1980). Briefly, seminal prostasomes (2 mg/mL) serving as antigen were emulsified with an equal volume of Freunds adjuvant. White Leghorn hens were immunized intramuscularly in the breast muscle with 0.5 mL of the emulsified antigen. After the initial immunization, the animals received 3 booster injections at 2-week intervals. The first immunization was performed with Freunds complete adjuvant and the booster immunizations with Freunds incomplete adjuvant. Eggs were then collected, labeled and stored at 8°C until antibody preparation (Larsson et al, 1993). The antibodies were isolated by polyethylene glycol precipitation.

Two-Dimensional Polyacrylamide Gel Electrophoresis

The purified prostasomes were precipitated with a solution containing 8 mol/L urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 40 mmol/L Tris, and 0.2% (wt/vol) immobilized pH gradient (IPG) buffer 3–10 for 1 hour at 4°C and ultrasonicated on ice for 1.5 minutes at 40 kHz. The extraction was then ultracentrifuged at 100 000 x g and at 4°C for 2 hours. Extracted proteins in the supernatant were collected and their protein concentration determined.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunoblotting were performed with Bio-Rad systems (Hercules, Calif), the Protean isoelectric focusing (IEF) cell and Protean II XI cell, according to the instructions of the manufacturer.

For the first-dimension electrophoresis (IEF), purified prostasomes (40 µg of soluble proteins) were rehydrated with a buffer consisting of 8 mol/L urea, 2% (wt/vol) CHAPS, 50 mmol/L dithiotreitol (DTT), and 0.2% IPG buffer 3–10. The IEF, using IPGs, was carried out by loading 300 µL of sample onto the 17-cm strips (Bio-Rad). Focusing was performed to a total of 65 kV·h using an IPGphor unit (20°C). The focused strips were equilibrated for 30 minutes in a solution containing 6 mol/L urea, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 2% DTT, 40 mmol/L Tris HCl (pH 8.8), and traces of bromphenol blue. Alkylation of free cysteine residues was carried out for 30 minutes in the same solution containing 2.5% iodoacetamide instead of DTT.

The second dimension was carried out in a 10% SDS gel (Tris HCl, 1 mm thick, 17 x 17 cm IPG well), and protein visualization was by silver staining. Stained gels were analyzed with Image Master 2D Elite software (Amersham Biosciences, Uppsala, Sweden). Polypeptide quantities were calculated (ppm of the total integrated optical density).

2D-PAGE Western Blotting

For immunoblotting, the proteins were electro-transferred to nitrocellulose membranes (30 V, 15 hours). The membranes were blocked for 1 hour with 1% bovine serum albumin in phosphate-buffered saline (PBS). For visualization of the antigens, the membranes were incubated overnight with patient and control sera (diluted in PBS, 1:200 for patient and control sera, 1:10 000 for the chicken polyclonal antibody). The following day, the membranes were washed 3 times with PBS-Tween and incubated first with biotinylated anti-human immunoglobulin G (IgG, Zymed Laboratories Inc, San Francisco, Calif) for 1 hour and thereafter with streptavidin alkaline phosphatase (Zymed) for 1 hour. The visualization was made by NBT/BCIP (Roche Diagnostics, Germany). The membranes were also probed with a polyclonal chicken anti-prostasome antibody (Immunsystem AB, Uppsala, Sweden) diluted 1:10 000 in PBS. The detection of bound antibody was similar to the detection of patient samples except that a biotinylated anti-chicken IgG antibody (Zymed Laboratories) was used instead of the biotinylated anti-human IgG antibody.

Mass Spectrometry

Proteins recognized by Western blotting were selected for identification via mass spectrometry. Preparative gels loaded with 200 µg protein from extracted seminal prostasomes were analyzed with Coomassie colloidal stain. In-gel digestion was carried out with trypsin (Jensen et al, 1999) and digests were desalted with ZipTip (Millipore, Sundyberg, Sweden). Peptides were eluted in 70% acetonitrile/5% formic acid. The eluate was mixed 1:1 (vol/vol) with a saturated matrix solution of -cyano-4-hydroxycinnamic acid in 30% acetonitrile/0.1% trifluoroacetic acid. Mass mapping of tryptic peptides was performed with a Voyager De Pro MALDI-TOF (Applied Biosystems, Stockholm, Sweden) or with a TofSpec-2E MALDI-TOF (Micromass) mass spectrometer, both equipped with a nitrogen laser (337 nm) and operated in the positive ion mode. Trypsin fragments of masses 842.50 and 2211.10 daltons were used as internal standards for spectra calibration. Data generated were screened in databases with a mass tolerance of 20 ppm or less. Program used for mass mapping were MS-Fit (http://prospector.ucsf.edu) and ProFound (http://prowl.rockefeller.edu) or the licensed ProteinLynx Software (Micromass).

	Results

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2D-PAGE

Silver-stained SDS-PAGE gel of separated prostasomal proteins revealed over 200 protein spots in the molecular mass range of 8–180 kd having isoelectric points (IPs) in the range of 3.6–8.3 (Figure 1). Approximately 70% of the spots displayed IPs below 7 (ie, reflecting anionic proteins).

View larger version (45K): [in this window] [in a new window]   Figure 1. Two-dimensional gel electrophoresis map (immobilized pH gradients 5–8, silverstained) of prostasome proteins. The numbered spots have been identified by mass spectrometry and database search and are listed in the Table.

View larger version (22K): [in this window] [in a new window]   Figure 2. Immunoblottings of prostasome proteins from a 2-D electrophoresis gel after incubation with (A) serum from an ASA-positive patient (1:200) and (B) chicken polyclonal anti-prostasome antibody (1:10 000).

Mass Spectrometry

Proteins in 12 spots were successfully identified and are summarized with their theoretical isoelectric points (pIs), molecular masses, accession numbers, and gene names in the Table. The two most frequent antigens were prolactin-inducible protein (PIP), recognized by most patient sera (19/20; 95%) and clusterin (17/20; 85%). In addition, 10 sporadically occurring prostasomal antigens were identified viz alcohol dehydrogenase (NADP⁺), annexin I, annexin III, BRCA 1-associated ring domain protein 1, heat shock protein (27 kd), isocitrate dehydrogenase, lactoylglutathione lyase, NG,NG-dimethylarginine dimethylaminohydrolase 1, peroxiredoxin 2, and syntenin 1.

	Discussion

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Several laboratory tests for ASA are available, and the correlation of the results both with different tests and the fertility prognosis has been evaluated in various ways (Meinertz and Hjort, 1986; Dondero et al, 1991, 1997; Collins et al, 1993; Andreou et al, 1995; Peters et al, 1995). The differences noticed are assumed to depend on the cellular location of the sperm antigens, the type of Ig class present, and the pretreatment of the patient's sperm sample (Peters et al, 1995). It seems that the mixed antiglobulin reaction (MAR; Meinertz, 1987) and the tray agglutination test (TAT: Friberg, 1980) are the most preferred methods. MAR requires patient semen and added anti-Ig antiserum to detect a binding of Ig-coated latex particles to the patient's Ig-coated sperm cells and measures the gross binding of antibodies to sperm (sperm bound, %) but does not examine the antigenic specifications of ASA. TAT, on the contrary, requires patient serum and normal semen to detect sperm agglutination that appears when the patient's serum antibodies recognize the antigens on the normal sperm cells. There is a rather good correlation between those two test results. We preferred serum in favor of semen samples because of the high prostasomal antigen concentration in seminal plasma, thus avoiding the problem of antigen excess.

This study demonstrated several prostasome antigens, which served as targets for ASA. Most frequently occurring antigens were PIP and clusterin.

The PIP gene, localized at 7q34 (Lander et al, 2001), is expressed in exocrine glands, such as the salivary and lacrimal glands, as well as in the seminal vesicles (Murphy et al, 1987a; Akiyama and Kimura, 1990). In pathologic conditions, it is overexpressed in breast cysts, breast cancers, and metastases of mammary origin (Murphy et al, 1987a; Losi et al, 1995). The protein binds to CD4 (Autiero et al, 1991) and inhibits CD4+ T-lymphocyte apoptosis (Gaubin et al, 1999). PIP, which is a 16.6 kd was secretory protein, is identical to gp17/secretory actin binding protein (Murphy et al, 1987b) and was previously described to be a prostasomal protein (Utleg et al, 2003). The protein from human seminal plasma and from breast cyst fluid shows different posttranslational modification patterns (Caputo et al, 1998). The functions of PIP are unclear (Autiero et al, 1997; Bergamo et al, 1997), although a role in passive mucosal immune defense has been proposed (Schenkels et al, 1997). The ability of PIP to bind to CD4 (Autiero et al, 1991) and to block CD4-mediated T-cell programmed death (Gaubin et al, 1999) suggests that this factor might modulate the immune response at insemination. Our finding of the occurrence of PIP on prostasomes, which are capable of interacting with spermatozoa, supports the contention that prostasomes, perhaps via PIP action on spermatozoa, can play a role in reproduction (cf Bergamo et al, 1997). With this in mind, it is not unexpected that PIP on prostasomes was the predominant antigen for ASA from our immunoinfertile patients.

Clusterin is a 75–80-kd heterodimeric glycoprotein that was first identified as a component of the fluid from ram rete testis with the ability to enhance cell aggregation in vitro (hence, the name) (Blaschuk et al, 1983). It has a wide array of functions, including roles in reproduction, lipid transport, tissue remodeling, and apoptosis (Tenniswood et al, 1998; Bailey and Griswold, 1999; Hochgrebe et al, 1999). The concentration of clusterin in normal human seminal plasma is considerably higher than that found in serum (O'Bryan et al, 1990). Concentrations up to 15 mg/mL have been found, suggesting that, like the rat and ram homologs, clusterin is a major glycoprotein of the human reproduction tract (O'Bryan et al, 1990). Studies showed that postvasectomy samples had levels of clusterin in the normal range, meaning that this glycoprotein was also to a large extent produced in accessory sex glands (O'Bryan et al, 1990). This is consistent with our finding that clusterin is associated with prostasomes. The general belief is that clusterin in seminal plasma is mainly a seminal vesicle contribution (Spring-Mills, 1980). However, seminal clusterin is not only produced in the testes, epididymides, and seminal vesicles, but also, as shown here, in the prostate gland. This means that the function of the glycoprotein in the reproductive system is more complex than the previously suggested role in spermatogenesis (Sylvester et al, 1984). A significant, positive relationship has been demonstrated between clusterin concentration in the semen sample used for preparation of the sperm cells for in vitro fertilization and the fertilization rate (Liu et al, 1988). This is in line with our present finding of a high proportion of serum antibodies directed against prostasomal clusterin in our immunoinfertile patients.

Sperm studies have been performed with the use of membrane proteins (plasmalemma, inner and outer acrosomal membrane) prepared from spermatozoa, enriched and purified by means of a swim-up procedure, and seminal plasmas containing ASA from 20 infertile patients (Bohring et al, 2001). A total of 18 sperm antigens were identified, and 6 of the recognized proteins were isolated and analyzed by mass spectrometry and peptide matching. They were identified as heat shock proteins (HSPs) 70 and 70-2, the disulfide isomerase ER60, the inactive form of caspase-3, and 2 subunits of the proteasome (Bohring et al, 2001). Hence, there was an essential difference regarding recognized immunogens between the spermatozoa and the human prostasomes.

NG-dimethylarginine dimethylaminohydrolase 1 and the annexins could be mentioned among the other prostasomal antigens, appearing sporadically as targets for the serum antibodies of our ASA patients. Monomethyl-L-arginine has been regarded as the standard nitric oxide synthase inhibitor. This and other methylated arginine compounds are synthesized in vivo by a family of enzymes known as protein arginine methyltransferases. Of the 3 known methylarginine residues formed in mammals, only asymmetrically methylated forms inhibit nitric oxide (NO) synthase. Therefore, endogenously produced, asymmetrically methylated arginines might modulate NO production (Donnelly et al, 1997; Arienti et al, 2002). The activity of the enzyme dimethylarginine dimethylaminohydrolase that hydrolyzes asymmetric methylarginines might exert a regulatory function on the synthesis of NO in the reproductive tract, again emphasizing the influential role of prostasomes in the reproductive process.

Annexins hold a central hydrophilic pore functioning as a Ca²⁺ channel. Because prostasomes are rich in Ca²⁺ and Zn²⁺ (Tvedt et al, 1987; Ronquist, 1998), annexins could be involved in the recruitment of Ca²⁺ to the prostasomes. An increase of sperm Ca²⁺ was proportional to the fusion with prostasomes (Palmerini et al, 1999), and by this annexin action on prostasomes, a subsequent effect on spermatozoa could be registered.

The identification of prostasomal antigens as targets for ASA in blood samples of patients with immunological infertility is a new approach. The characterization and identification of these antigens are important for under-standing the mechanism by which ASA might impair sperm fertilization capacity because of the close interactions that prostasomes and spermatozoa are capable of. The uneven occurrence of these antigens as targets for ASA with a predominance of PIP and clusterin is of interest. Both are glycoproteins, which means that they might display different posttranslational modification patterns, perhaps giving rise to different isoforms and antibody responses. This, in turn, increases the possibility of finding unique antibodies against these prostasome glycoproteins relevant to infertility. The recognition of such antigens could also be important for potential immunocontraception (Frayne and Hall, 1999).

	Footnotes

 
^? This investigation received financial support from the Swedish Medical Research Council, project K2004-73PD-15161-01A.

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