Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand
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Abstract |
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Key words: antisperm antibodies/IgG/infertility/spermatozoa/vasectomy
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Introduction |
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A mainstay of treatment for ASA has previously been immunosuppression of antibodies (Hendry, 1992). However, immunosuppression has had limited success in controlled clinical trials and patients frequently suffered from treatment-related complications (Grigoriou et al., 1996
). Part of the reason for the poor performance of immunosuppression for ASA may be that subjects were recruited into trials based on the presence of ASA as detected by diagnostic tests that were unable to distinguish between those antibodies that cause infertility and those that do not. Thus, developing a diagnostic test that can distinguish between ASA would be particularly valuable. In order to develop such a test, we examined the specificities of ASA by Western blot from men with vasectomy reversal. When vasectomies are reversed, ~50% of men with sperm in their ejaculates will be infertile, while the remaining 50% will regain fertility. Both groups are known to have antisperm antibodies (Royle et al., 1981
; Parslow et al., 1983
). Therefore, by comparing and contrasting the antigenic specificities of ASA from men following vasectomy reversal, we hoped to identify the sperm antigens that could be used to develop new and more specific diagnostic tests for ASA.
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Materials and methods |
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Preparation of normal sperm and leukocyte extracts for Western blotting
Normal semen (World Health Organization, 1999) was collected from donors by masturbation. The semen was allowed to liquefy at room temperature for 30 min and divided into aliquots containing 50x106 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; pH7.4) and centrifuged at 1500 g at 4°C. The supernatant was discarded and the washing procedure repeated three times.
For experiments where proteins from motile sperm were needed, they were prepared from semen samples using a discontinuous 4090% PureSperm® (Arctech, Australia) gradient centrifugation at 300 g. The sperm pellet was resuspended and adjusted to 50x106 then subjected to washing as above.
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).
Sperm protein extracts (SPE) were prepared freshly before use as follows. Cryopreserved sperm aliquots were thawed and the sperm were solubilized by mixing with Triton X114 (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.
Leukocytes were separated from 5 ml of freshly drawn heparinized blood by Ficoll gradient centrifugation (Lymphoprep-Nycomed®; Life Technologies, Auckland, New Zealand) according to the manufacturer's instructions. The cells were washed twice with PBS, pH 7.4, and the leukocytic proteins were extracted using the method described above for sperm.
Isolation of immunoglobulin G from normal sperm extracts
Normal sperm extracts, prepared from 200x106 sperm, pooled from six donors as above, were passed through a 5 ml protein A-AffiGel® column (Biorad, Auckland, New Zealand) that had been equilibrated with PBS, pH 7.4. Passage of proteins was monitored at optical density 280 nm with a UVcord II® flow cell (Pharmacia, Auckland, New Zealand). When protein had ceased to wash through the column, bound proteins were eluted with 0.1 mol/l glycine, pH 2.5. Fractions containing eluted proteins were pooled, neutralized by drop addition of a 1 mol/l Tris solution, concentrated in a centrifugal concentrator (Vivaspin 500®; Sartorius, Auckland, New Zealand), and equilibrated with PBS, pH 7.4, by dialysis. The proteins were then analysed by sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.
PAGE and Western blotting
Normal sperm protein extracts and lymphocyte extract (LE) were boiled for 4 min with sample buffer (0.0625 mol/l Tris, 2% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue dye). A total of 400 µg of proteins were then separated by electrophoresis on a preparatory, precast 415% gradient polyacrylamide gel with an 11 cm wide well using a Criterion® system (BioRad). The separated proteins were transferred to nitrocellulose membranes (Hybond-C extra; Amersham, Auckland, New Zealand) using a semi-dry blotting apparatus according to the manufacturer's instructions (Gelman Science, Life Technologies). The membranes were then stained with a Ponceau-S solution [0.1% (w/v) Ponceau-S in 5% (v/v) acetic acid] to confirm the transfer and destained with PBS, pH 7.4, containing 0.05% (v/v) Tween 20 (PBST). The membranes were then blocked overnight at 4°C in 5% (w/v) non-fat milk powder (Pams, Auckland, New Zealand) dissolved in PBST (blocking buffer). They were cut into strips and incubated with patient sera, mouse anti-CD46 (Serotec, Oxford, UK), mouse anti-CD 45 (Dako, Auckland, New Zealand) or rabbit anti-CD55 (gift from Dr A.R.A.Blanco, University of Liverpool, UK), all diluted 1/500 in blocking buffer under gentle rocking at room temperature for 1 h. The membranes were then washed three times with PBST and incubated for an additional 1 h, at room temperature, with horseradish peroxidase (HRP)-conjugated goat anti-human chain, anti-mouse immunoglobulin (Ig)G, or anti-rabbit IgG serum (Caltag, Auckland, New Zealand), as appropriate, diluted 1/5000 in blocking buffer. The membranes were again washed three times with PBST and incubated with enhanced chemiluminescence reagent (New England Nuclear, Life Science Technologies, Auckland, New Zealand) according to the manufacturer's instructions. Finally, the membranes were placed in a heat-sealed polythene envelope and exposed to Hyperfilm® ECL (Amersham) for varying time periods ranging from 30 s to 30 min and developed in an automated film processor (Agfa Curix 60, Germany).
Molecular weights of detected sperm proteins were deduced by comparison with prestained recombinant molecular weight standards (BioRad).
Western blot of normal sperm extract with protein A-purified IgG from normal sperm extract
Normal sperm extract was prepared as above and resolved on a 10% SDSPAGE using a BioRad MiniProtean II system (BioRad). The proteins were then transferred to a nitrocellulose membrane (Hybond-C®; Amersham) as above. The membrane was then blocked overnight at 4°C in blocking buffer and followed by probing either HRP-conjugated goat anti-human chain or with 100 µg of the IgG purified from sperm in blocking buffer, followed by goat anti-human
chain HRP conjugate. The binding of the proteins on the blot was visualized by adding ECL reagent as above.
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Results |
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Isolation and characterization of IgG bound to normal sperm
Protein A affinity chromatography was used to isolate the antibody from normal (MAR negative) sperm extracts. The isolated antibody, which proved to be IgG, was then used to probe normal sperm extracts by Western blot. This analysis demonstrated that the IgG isolated from normal sperm did not react with specific sperm proteins, suggesting the antibody is not bound to sperm via an antigen/antibody reaction (Figure 3).
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Discussion |
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The presence of the sperm-specific isoforms of proteins CD46 and CD55 in our sperm protein extract confirm that these extracts contain membrane proteins but it is also likely that the extracts contain cytoplasmic proteins. We plan to determine the sub-cellular localization of these sperm protein(s) once they are further characterized.
Some previous Western blotting studies have employed denaturing conditions (Lehmann et al., 1985; Naaby-Hansen and Bjerrum, 1985
; Mathur et al., 1988
; Auer et al., 1995
) which may have caused the loss of conformation-dependent epitopes on sperm proteins (Shai and Naot, 1992
). We have tried to minimize antigen denaturation by employing a non-ionic detergent, Triton X114 (Helenius and Simons, 1975
), for sperm protein extraction and non-reducing electrophoresis. We also used a very sensitive detection system, enhanced chemilluminescence (Bronstein et al., 1990
), as opposed to the more traditional colorimetric methods used by others (Lehmann et al., 1985
; Naaby-Hansen and Bjerrum, 1985
; Parslow et al., 1987
).
Our analysis demonstrated a complex pattern of antibody reactivity with sperm proteins regardless of whether we used serum derived from fertile or infertile patients. However, there were several striking features.
Firstly, sperm protein(s) of molecular weights 919 kDa were reactive with antibodies from three of the infertile men but not with antibodies from their fertile counterparts. Sperm protein(s) of similar molecular weights have been found by others in males with unexplained infertility (Lehmann et al., 1985; Auer et al., 1997
) and we are currently investigating the identities of these proteins.
Secondly, IgG was found to be present in the normal sperm extracts. These extracts were prepared from MAR-negative sperm; therefore, this antibody was not detected by a conventional ASA test. The washing procedure used in the preparation of the sperm protein extracts provides us with reasonable certainty that this IgG is not simply a carry-over from seminal plasma. Dead sperm have been known to be non-specifically coated with IgG (Rasanen et al., 1992). Our findings indicated that protein extracts prepared from highly motile sperm by density gradient centrifugation also contained IgG. That IgG is present on sperm is not a new finding (Allen and Bourne, 1978
; Hjort, 1996
). It is believed that the binding of this antibody is not via a specific antigen/antibody interaction but rather by some form of non-antigenic, possibly Ig Fc segment-mediated, interaction. In order to test this question, we purified the IgG from normal sperm and used the purified antibodies to probe a Western blot of sperm proteins. Our results confirm that the antibody present on normal sperm does not react with sperm antigens. However, the mechanism by which these antibodies bind to sperm remains unresolved. It has been suggested that direct disulphide bonding of the antibody to sperm may occur (Richards and Witkin, 1984
). Alternatively, it has been suggested that there is an Fc receptor on sperm. A 16/20 kDa antibody-binding protein, suggested to be related to Fc
RIII on the basis of monoclonal antibody reactivity, was reported (Kamada et al., 1991
). However, we (unpublished data) and others have not found Fc
RIII immunoreactivity on sperm (Bronson et al., 1992
). If the IgG we have shown is associated with sperm by an Fc-mediated mechanism, this may explain why the sperm-associated antibody is not detected by the MAR test, as steric hindrance may prevent the MAR beads reacting with the sperm-bound antibodies.
The biological significance of having IgG attached to sperm is not clear. One could speculate that the presence of IgG on sperm could facilitate binding to macrophage or neutrophil Fc receptors to mediate disposal of excess sperm from the female reproductive tract (Overstreet and Mahi-Brown, 1993). On the other hand, the presence of IgG on the spermatozoal surface may be of benefit to the process of fertilization. For example, hamster oocyte plasma membranes are reported to express Fc
receptors and immunoglobulins on the surface of sperm, which may therefore act as a ligand to facilitate their binding or penetration of oocytes (Bronson et al., 1990
).
A notable feature of our analysis is the demonstration that serum from all men studied contained sperm-reactive antibodies. In most instances antibodies from fertile men reacted with apparently the same sperm proteins as antibodies from infertile men. At face value one might then draw the conclusion that there is no difference in the antigenic specificity of sperm antibodies from fertile and infertile men. However, it is quite possible that antibodies from infertile men are reacting with a protein of given molecular weight while antibodies from fertile men are reacting with a different protein of the same molecular weight. Furthermore, even if antibodies from fertile and infertile men are reacting with the same protein(s), it possible that the antibodies from these two groups react with different epitopes within the protein(s). It is well known that antibodies that react with biologically active proteins can be either neutralizing or non-neutralizing depending upon the epitope with the protein with which the antibody reacts. For example, antibodies reactive with the first short consensus repeat (SCR) domain of CD46 inhibit the ability of sperm to interact with oocytes, whereas antibodies reacting with other domains of CD46 do not inhibit spermoocyte interaction (Taylor et al., 1994; Taylor and Johnson, 1996
). Thus, where antibodies from fertile and infertile men react with the same protein, it is possible that fertile men produce non-neutralizing antibodies while infertile men produce neutralizing antibodies. Furthermore, quantitative differences in the amount of antibody may also be important in determining whether a given antibody disrupts fertility, as is believed to be the case with MAR tests. Our results show that some individuals have substantially more antibodies reactive with particular proteins than other individuals who have antibodies reactive with the same proteins. These quantitative differences in antibody levels may be very important in determining the effects of ASA on fertility.
The complications discussed above make the interpretation of Western blots for ASA difficult, but our use of differential Western blot analysis of fertile and infertile men has enabled identification of candidate proteins that may be involved in fertility/infertility and we are continuing our investigations to identify these proteins.
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Acknowledgements |
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Notes |
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References |
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Allen, G.J. and Bourne, F.J. (1978) Interaction of immunoglobulin fragments with the mammalian sperm acrosome. J. Exp. Zool., 203, 271276.[ISI][Medline]
Auer, J., Pignot-Paintrand, I. and De Almeida, M. (1995) Identification of human sperm surface glycoproteins by sperm membrane-specific autoantibodies. Hum. Reprod., 10, 551557.[Abstract]
Auer, J., Senechal, H. and De Almeida, M. (1997) Sperm-associated and circulating IgA and IgG classes of antibodies recognise different antigens on the human sperm plasma membrane. J. Reprod. Immunol., 34, 121136.[ISI][Medline]
Ayvaliotis, B., Bronson, R., Rosenfeld, D. et al. (1985) Conception rates in couples where autoimmunity to sperm is detected. Fertil. Steril., 43, 739742.[ISI][Medline]
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248254.[ISI][Medline]
Bronson, R.A. (1999) Antisperm antibodies: a critical evaluation and clinical guidelines. J. Reprod. Immunol., 45, 159183.[ISI][Medline]
Bronson, R.A., Fleit, H.B. and Fusi, F. (1990) Identification of an oolemmal IgG Fc receptor: its role in promoting binding of antibody-labelled human sperm to zona-free hamster eggs. Am. J. Reprod. Immunol., 23, 8792.[ISI][Medline]
Bronson, R.A., Fusi, F.M. and Fleit, H.B. (1992) Monoclonal antibodies identify Fc gamma receptors on unfertilized human oocytes but not spermatozoa. J. Reprod. Immunol., 21, 293307.[ISI][Medline]
Bronstein, I., Voyta, J.C., Lazzari, K.G. et al. (1990) Improved chemiluminescent detection of alkaline phosphatase. Biotechniques, 9, 160161.[ISI][Medline]
Collins, J.A., Burrows, E.A., Yeo, J. et al. (1993) Frequency and predictive value of antisperm antibodies among infertile couples. Hum. Reprod., 8, 592598.[Abstract]
Grigoriou, O., Konidaris, S., Antonaki, V. et al. (1996) Corticosteroid treatment does not improve the results of intrauterine insemination in male subfertility caused by antisperm antibodies. Eur. J. Obs. Reprod. Biol., 65, 227230.
Heidenreich, A., Bonfig, R., Wilbert, D.M. et al. (1994) Risk factors for antisperm antibodies in infertile men. Am. J. Reprod. Immunol., 31, 6976.[ISI][Medline]
Helenius, A. and Simons, K. (1975) Solubilization of membranes by detergents. Biochem. Biophys. Acta, 415, 2979.[ISI][Medline]
Hendry, W.F. (1992) The significance of antisperm antibodies: measurement and management. Clin. Endocrinol. (Oxf.), 36, 219221.[ISI][Medline]
Hjort, T. (1996) Quantitative determination of IgG and IgA on sperm from infertile patients with and without antisperm antibodies. Am. J. Reprod. Immunol., 36, 211215.[ISI][Medline]
Kamada, M., Liang, Z. and Koide, S.S. (1991) Identification of IgG and Fc-binding proteins in human seminal plasma and sperm. Arch. Androl., 27, 17.[ISI][Medline]
Lehmann, D., Temminck, B., Da Rugna, D. et al. (1985) Blot-immunobinding test for the detection of anti-sperm antibodies. J. Reprod. Immunol., 8, 329336.[ISI][Medline]
Mathur, S., Chao, L., Goust, J.M. et al. (1988) Special antigens on sperm from autoimmune infertile men. Am. J. Reprod. Immunol. Microbiol., 17, 513.[Medline]
Meinertz, H., Linnet, L., Fogh-Andersen, P. et al. (1990) Antisperm antibodies and fertility after vasovasostomy: a follow-up study of 216 men. Fertil. Steril., 54, 315321.[ISI][Medline]
Mosher, W.D. and Pratt, W.F. (1991) Fecundity and infertility in the United States: incidence and trends (editorial; comment). Fertil. Steril., 56, 192193.[ISI][Medline]
Naaby-Hansen, S. and Bjerrum, O.J. (1985) Auto- and iso-antigens of human spermatozoa detected by immunoblotting with human sera after SDS-PAGE. J. Reprod. Immunol., 7, 4157.[ISI][Medline]
Overstreet, J.W. and Mahi-Brown, C.A. (1993) Sperm processing in the female reproductive tract. In Griffin, P.D. and Johnson, P.M. (eds) Scientific Basis of Fertility Regulation, Local Immunity, in Reproductive Tract Tissues. World Health Organization, Oxford University Press, Delhi, pp. 321334.
Parslow, J.M., Royle, M.G., Kingscott, M.M. et al. (1983) The effects of sperm antibodies on fertility after vasectomy reversal. Am. J. Reprod. Immunol., 3, 2831.[ISI][Medline]
Parslow, J.M., Poulton, T.A. and Hay, F.C. (1987) Characterization of sperm antigens reacting with human antisperm antibodies. Clin. Exp. Immunol., 69, 179187.[ISI][Medline]
Rasanen, M.L., Hovatta, O.L., Penttila, I.M. et al. (1992) Detection and quantitation of sperm-bound antibodies by flow cytometry of human semen. J. Androl., 13, 5564.
Richards, J.M. and Witkin, S.S. (1984) Non-immune IgG binding to the surface of spermatozoa by disulphide rearrangement. Clin. Exp. Immunol, 58, 493501.[ISI][Medline]
Royle, M.G., Parslow, J.M., Kingscott, M.M. et al. (1981) Reversal of vasectomy: the effects of sperm antibodies on subsequent fertility. Br. J. Urol., 53, 654659.[ISI][Medline]
Seya, T., Hara, T., Matsumoto, M. et al. (1993) Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and `sterile' subjects. Eur. J. Immunol., 23, 13221327.[ISI][Medline]
Shai, S. and Naot, Y. (1992) Identification of human sperm antigens reacting with antisperm antibodies from sera and genital tract secretions. Fertil. Steril., 58, 593598.[ISI][Medline]
Simpson, K.L. and Holmes, C.H. (1994) Presence of the complement-regulatory protein membrane cofactor protein (MCP, CD46) as a membrane-associated product in seminal plasma. J. Reprod. Fertil., 102, 419424.[Abstract]
Sinisi, A.A., Di Finizio, B., Pasquali, D. et al. (1993) Prevalence of antisperm antibodies by SpermMARtest in subjects undergoing a routine sperm analysis for infertility. Int. J. Androl., 16, 311314.[ISI][Medline]
Taylor, C.T. and Johnson, P.M. (1996) Complement-binding proteins are strongly expressed by human preimplantation blastocysts and cumulus cells as well as gametes. Mol. Hum. Reprod., 2, 5259.[Abstract]
Taylor, C.T., Biljan, M.M., Kingsland, C.R. et al. (1994) Inhibition of human spermatozoonoocyte interaction in vitro by monoclonal antibodies to CD46 (membrane cofactor protein). Hum. Reprod., 9, 907911.[Abstract]
Templeton, A., Fraser, C. and Thompson, B. (1990) The epidemiology of infertility in Aberdeen. Brit. Med. J., 301, 148152.[ISI][Medline]
Wolff, H. (1995) The biologic significance of white blood cells in semen. Fertil. Steril., 63, 11431157.[ISI][Medline]
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 4th edn. Press Syndicate of the University of Cambridge, Australia.
Submitted on June 29, 2001; resubmitted on October 22, 2001; accepted on November 28, 2001.