Immune infertility: towards a better understanding of sperm (auto)-immunity

The value of proteomic analysis

Claudia Bohring and Walter Krause1

Department of Andrology and Venerology, University Hospital, Philipp University, D-35033 Marburg, Germany

1 To whom correspondence should be addressed. e-mail: krause{at}mailer.uni-marburg.de


    Abstract
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
Antisperm antibodies (ASA) in the male cause an autoimmune disease ‘immune infertility’. It has to be clarified whether each antibody binding to an antigen, which is identified on the sperm surface, also influences sperm function. In the past, the clinical interest in ASA was hampered by the fact that a standardized assay for the detection of ASA was lacking. There are several methods to characterize the cognate antigens of ASA. In the following article, reports from the recent literature of immunologically characterized sperm proteins—as cognate antigens of naturally occurring ASA or of artificially produced antibodies—will be quoted with respect to different sperm functions. As a practical consequence of the research on ASA-related sperm proteomics, those ASA that decrease male fertility by inhibiting sperm functions essential for fertilization will be identified.

Key words: antisperm antibodies/autoimmunity/proteomics/sperm antigens


    Immune infertility—an autoimmune disease
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
The term immune infertility is used if spontaneously occurring antibodies binding to antigens of the gametes impair sperm– oocyte interaction. Antisperm antibodies (ASA) are far more frequent than oocyte antibodies.

ASA in the male may fulfil the criteria of an autoimmune disease. The first definition of autoimmune diseases was published in 1957, consciously modelled on Koch postulates: specific autoantigens and autoantibodies or T-cells exist, the disease occurs also in animals, and passive transfer of antibodies induces the disease in experimental animals or in the human. All these points are fulfilled in male immune infertility.

The definition of autoimmune diseases as revised by Rose and Bona (1993Go) includes three types of evidence to establish that a human disease is actually of autoimmune origin: direct proof, indirect evidence and circumstantial evidence. The direct proof is easy to achieve for ASA, because they can be transferred to the sperm of healthy persons. This is a great advantage in comparison with autoantibodies in diseases of other organs. The impairment of functions (e.g. decrease of motility) can be demonstrated in the receiving cells. Since the direct proof of ASA specificity is easy to achieve, experimental models giving indirect proof, in particular in animals, are rare. Also circumstantial evidence is rare, in particular the ‘favourable response to immunosuppression’ was not demonstrated in ASA. This may be because it is impossible to treat patients seeking fertility with cytotoxic drugs, but not because of the ineffectiveness of the treatment. The observation that the expression of ASA is associated with certain HLA classes (Omu et al., 1999Go) supports the suggestion that immune infertility represents an autoimmune disease.

Taking the arguments of Rose and Bona (1993Go) into consideration, the definition of an autoimmune disease was extended to the facts summarized in Table I (Storch, 1998Go).


View this table:
[in this window]
[in a new window]
 
Table I. Criteria for the presence of an autoimmune disease (Storch, 1998)
 
In order to define the autoimmune disease ‘immune infertility’ it has to be clarified whether each antibody binding to an antigen of the sperm surface also influences sperm function. In general, most autoantibodies occurring in biological fluids do not cause autoimmune diseases, but they are physiological phenomena and are without pathological relevance, i.e. they do not alter the function associated with the cognate antigen. This means that not all sperm autoantibodies will alter sperm function, either because the cognate antigen is not involved in the process of fertilization or because the antibodies do not bind to the functional domain of the antigen. It is well known from monoclonal antibodies that the antibody-binding region of the antigen may be different from the region being active in metabolic processes.

The development of ASA in the male depends on the sequestration of antigens on germ cells by the presence of the blood–testis barrier. During maturation of spermatogenesis, new antigens are expressed on developing spermatocytes and spermatids. When these antigens come into contact with immunocompetent cells, ASA formation occurs. Develop mental abnormalities of the formation of the blood–testis barrier, traumatic disruption or unilateral focal cryptic obstructions could lead to ASA formation. Also gastrointestinal exposure to sperm has been associated with the development of ASA both in animal experiments as well as in homosexual men. There are also results suggesting active local immuno-regulatory mechanisms being operative within the testis (Bronson, 1999aGo).


    Tests for ASA
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
It appears to be accepted that only ASA binding to sperm membrane antigens are of relevance. In vital sperm, ASA are not able to penetrate the outer cell membrane. Supporting this is the fact that ASA from patients were not found to produce abnormal results of the hypo-osmotic swelling (HOS) test in donor sperm in the study of Jairaj et al. (2000)Go, which indicates no alteration to the outer sperm membrane per se by ASA.

Thus the antigen analysis should be restricted to antigens of the outer sperm membrane. The exception to this rule concerns acrosomal antigens. Physiologically, during the acrosome reaction, the inner acrosomal membranes of the spermatozoon become externalized and antigens are presented, which are otherwise not recognized.

In the past, the clinical interest in ASA was hampered by the fact that a standardized and universally accepted assay for the detection of ASA was lacking (Helmerhorst et al., 1999Go). A survey of UK reproductive medicine centres in 1998 revealed that most centres used one or two tests to detect antibodies (Krapez et al., 1998Go). The tests—such as the mixed antiglobulin reaction (MAR) or the immunobead test (IBT)—use beads coated with antiglobulins. They are not able to identify specific antigens because of the relatively large ‘labels’ (erythrocytes, latex beads, polystyrol beads). This is also true for immunfluorescence assays. The methods are also unable to detect the number of antibody molecules or antigens involved in the binding. They can only detect (i) the immunoglobulin class of the antibody concerned and (ii) the proportion of sperm binding antibodies, thus forming ASA ‘titres’. The discrimination, however, of which antibodies are of clinical relevance and which are not, is not possible.

Discrimination also concerns flow cytometry, which was used for the determination of the numbers of sperm binding ASA, giving comparable results with the IBT or MAR test (Räsänen et al., 1996Go). Although Hjort (1999Go) quoted..."flow cytometry seems to be a promising technique which may be able to determine the exact amount of IgA and IgG on individual sperm"..., it has to be considered that this method does not always provide objective and reproducible results (Nikolaeva et al., 2000Go).

Trials were published that used sperm extracts as antigens binding naturally occurring ASA in a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). They were hampered by the fact that the antigens were not biochemically defined and those relevant for the process of fertilization might not be contained in the antigen mixture (Helmerhorst et al., 1999Go). In addition, seminal plasma itself interferes with the antigen-antibody reaction in such an immunoassay (Lu and Zha, 2000Go). Bronson (1999aGo) quoted: "There is urgent need for tests that allow one to determine the antigenic moieties. ASA have been shown to be directed against several different antigens, and each would be expected to have different effects on sperm functions".

Antigen specific tests will facilitate statements on the clinical relevance of the presence of ASA and the discrimination of ASA as the cause of infertility from other pathologies of sperm parameters (Hjort 1999Go). The tests will also improve the decision for treatment options. Currently, the primary choice of treatment is ICSI to overcome ASA effects inhibiting fertilization (Nagy, 1999Go; Check et al., 2000Go; Mardesic et al., 2000; Lombardo et al., 2001Go). Once it is proven that a patient presents ASA, which interfere with the fertilizing ability of sperm, it will be possible to offer ICSI only to these patients, while the others may undergo less invasive treatment procedures.


    Analysis of cognate antigens of ASA
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
Table II summarizes sperm functions, which the impairment by ASA have previously been demonstrated.


View this table:
[in this window]
[in a new window]
 
Table II. ASA effects on sperm functions
 
There are several methods to characterize the cognate antigens of ASA. The antigen analysis should be restricted to antigens of the outer sperm membrane, since in vital sperm ASA are not able to penetrate the outer cell membrane. The exception from this rule concerns acrosomal antigens. As previously mentioned, during the acrosome reaction the inner acrosomal membrane of the spermatozoon becomes externalized and antigens are presented, which are otherwise not recognized.

Firstly, the localization of ASA binding on the surface of live sperm of a healthy donor may be visualized by one of the methods of immune staining. If the results are compared with the sperm function in the patient from whom the ASA are derived, some insights into the functional relevance of ASA are possible.

Another method is the comparison of the localization of ASA binding with the binding of sperm-specific monoclonal antibodies with a known cognate antigen. This also gives an approximate estimation for the localization of the antigen concerned.

A third approach to the analysis of antigen functionality is the analysis of proteins derived from DNA libraries of the testis. This approach, however, will lead to the identification of only part of ASA-inducing sperm antigens, since a number of sperm membrane antigens are secreted or altered during the epididymal passage. Poulton et al. (1996Go) described an antigen of ASA in infertile or vasectomized men that was present on epididymal sperm but absent in testicular sperm. The lectin-binding glycoproteins GP-83 and GP-39 present on the membranes of mature sperm were found to be secreted by the principal cells of the epididymis, and conjugated to sperm during their transit in the epididymis (Liu et al., 2000Go).

The exact identification of the biochemical nature of the cognate antigens of ASA is possible only after reliable separation of the sperm membrane proteins and confirmation of their potency to bind ASA. This is achieved using two-dimensional electrophoresis of isolated membrane (Görg et al., 1998Go), blotting of the proteins to a polyvinylidone difluoride (PVDF) membrane and in-loco binding of ASA, visualized by electrochemiluminescence (ECL) (immunoblotting). After this, the proteins isolated may be sequenced or analysed using matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and subsequent peptide matching.

The comparison with known proteins in a protein database reveals the biochemical nature of the protein or identifies it as a novel, so far unknown, protein. Care has to be taken that the antigens are not altered during analysis. Currently, this is the only accepted method to identify the nature of antigens, giving rise to the identification of the proteomics of sperm with respect to their function.

Shetty et al. (1999Go) reported on their results achieved with this technique: 98 sperm auto- and iso-antigenic protein spots were recognized by antibodies from sera from infertile males and females but not from fertile subjects. Based on vectorial labelling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility. Also Bohring and Krause (1999Go) reported on 18 antigens from isolated sperm membranes, which could be identified after 2D electrophoresis by ASA from seminal plasma of infertile or vasectomized men. In 2001, we reported on the isolation of six of the recognized proteins and their analysis by means of mass spectrometry and peptide matching (Bohring et al., 2001aGo). They were identified as heat shock proteins HSP70 and HSP70-2, the disulphide isomerase ER60, the inactive form of caspase-3 and two subunits of the proteasome complex (component 2 and zeta chain). The spermatid specific HSP70 antigen is highly conserved in mammals (Tsunekawa et al., 1999Go). The human homologue HSPA2 gene of the murine Hsp70-2 gene shows 91.7% identity in the nucleotide coding sequence. It is also highly expressed in male germ cells, suggesting a specific role in meiosis (Son et al., 1999Go).

Domagala et al. (2000Go) performed immunoblotting of a repertoire of sperm antigens reacting with ASA present in sera. Sperm antigens with molecular weights of 57, 58, 62, 63 and 66 kDa were the most immunodominant entities recognized by ASA in sera of infertile men and women, also prepubertal boys with testicular failure.

Diekman et al. (2000Go) obtained a monoclonal antibody, H6-3C4, from the lymphocytes of an infertile woman who exhibited sperm-immobilizing titres in her serum. The H6-3C4 cognate antigen, designated sperm agglutination antigen-1 (SAGA-1), was characterized as a polymorphic, highly acidic, glycosylphosphatidylinositol (GPI)-anchored glycoprotein on the surface of human sperm. Microsequencing demonstrated that the SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. The two peptides represented glycoproteins with the same core peptide but with different carbohydrate structures. This peptide is one of the few well-defined sperm surface glycoproteins in human antibody-mediated infertility. It appears to undergo changes in the antigenic characteristics during sperm maturation and capacitation, possibly by increasing accessibility of some sialic acid residues and of the core peptide, particularly the GPI anchor (Yeung et al., 2000Go; 2001)


    Sperm proteins characterized by immunological methods
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
Reports from the recent literature of immunologically characterized sperm proteins—as cognate antigens of naturally occurring ASA or of artificially produced antibodies—will be quoted below with respect to different sperm functions. The number of identified proteins has increased since about 1997.

Agglutination
Koide et al. (2000Go) investigated the cognate antigens of agglutinating ASA, which were obtained from the blood serum of infertile women or were monoclonal antibodies (mAb) raised in the mouse against human sperm proteins. They identified:

(a) YWK-II mAb with a cognate antigen of a molecular mass of 60 and 72 kDa, immunolocalized in the equatorial sector of the human sperm head. The cDNA of YWK-II antigen was identified, the polypeptide belongs to the amyloid precursor protein (APP) family (also found in plaques of Alzheimer disease).

(b) BE-20, an epididymal specific protein, whose ASA blocked the ability of human sperm to penetrate hamster oocytes. The cDNA consisted of 585 base pairs, encoding a 13 kDa polypeptide. The nucleotide sequence has high similarity to the proteinase inhibitors of the human epididymis, designated as HEA-4, HE-5 and HE-6. mRNA was measurable only in the epididymis. As a proteinase inhibitior it may assist in maintaining the integrity of acrosomes.

(c) rSMP-B, a sperm tail component of 72 kDa, recognized by ASA from the serum of infertile women. These antibodies may immobilize sperm and block their interaction with the oocyte. The gene was expressed only in spermatids. The human analogue (hSMP-1) is coded by the HSD-I gene, which is located on human chromosome 9, region p12-p13 (Kuang et al., 2000Go).

(d) BS-17 (calpastatin) is a 17.5 kDa protein. Immuno staining with polyclonal antibodies was seen over the acrosomal region and slightly on the tail. Antibodies blocked the penetration of hamster oocytes, but not the attachment to human ova or the sperm motility. The cDNA consisted of 758 base pairs, having 99.7% homology with the gene coding calpastatin. The gene was found to be transcribed only in spermatids. Calpastatin binds calpain, a calcium-dependent cysteine endopeptidase.

(e) HED-2 (zyxin), a Sertoli-cell component similar to EP-20. EP-20, was isolated from the caudal epididymis. Antibodies block motility and penetration of hamster oocytes. The gene consists of 1908 base pairs. Activation of a kinase might be the mechanism by which the HED-2 may trigger the signal transduction system of germ cells to undergo differentiation.

(f) BS-63 cDNA contained an open reading frame consisting of 1765 codons and XFXFG or GLFG repetitive sequence motifs. These repetitive motifs are structural characteristic of nucleoporins. It is hypothesized that BS-63 is a testis-specific nucleoporin and possibly acts as a docking site and a co-transporter of Ran and transportin (Cai et al., 2002Go).

A 75 kDa protein was found with no homology of the nucleotide sequence to other DNA.

Interestingly, most of the ASA investigated, bound to multiple antigens, thus the authors conclude that immune infertility is the consequence of the combined actions of ASA in immobilizing and/or agglutinating sperm, blocking sperm– oocyte interaction, preventing implantation, and/or arresting embryo development.

A mouse monoclonal antibody A36 induced extensive ‘tangled’ sperm agglutination. The cDNAs encoding its cognate antigen displayed >99% homology to glucose phosphate isomerase/neuroleukin (GPI/NLK) mRNA. The mAb A36 cognate sperm surface antigen is a GPI/NLK-like protein involved in sperm agglutination (Yakirevich and Naot, 2000Go). A monoclonal antibody that immobilized human sperm was shown to react also with seminal plasma, indicating that the cognate antigen on the sperm surface might stem from the epididymis (Komori et al., 1997Go).

Sperm agglutination is closely related to immune contraception. Several studies describe antibodies to sperm proteins, which lead to intense sperm agglutination, being candidates for immune contraception. The YLP(12), a 50 ± 5 kDa membrane protein of human sperm extracts may have applications in contraceptive vaccine development (Naz and Chauhan, 2001Go). A polyclonal antibody, raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm, showed extensive agglutination both in mouse and human sperm. Passive immunization of female mice with this antibody caused 67% reduction in fertility (Singh et al., 2001Go).

Norton et al. (2001Go) engineered a recombinant single-chain variable fragment (scFv) antibody binding to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1, the sperm glycoform of CD52, which has already been mentioned above. The recombinant anti-sperm antibody (RASA) was expressed in E.coli HB2151 cells. RASA aggregated human sperm in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination.

Motility
Since it is likely that ASA bind to antigens of sperm membranes, while subcellular structures will not be reached by ASA in the living cell, it is difficult to explain how ASA will interfere with sperm motility. It may be speculated that the function of proteins with intra-and extra-membranaceous parts may be altered by the ASA concerned. Another possible explanation is via a complement-mediated membrane damage (see cervical mucus penetration).

Munuce et al. (2000Go) determined sperm motility parameters by computer-aided semen analysis (CASA). They found no differences between samples containing ASA, as detected using the MAR test and IBT, and those free of ASA. ASA were present in 15% of normozoospermic samples and in 9.5% of abnormal semen samples.

Neilson et al. (1999Go) used serum from an infertile male with high titres of ASA to identify novel human sperm antigens by screening a testis-expression library. The human gene encodes 1.8- and 2.8-kb mRNAs highly expressed in testis but not in other tissues tested. The deduced amino acid sequence of the full-length cDNA revealed striking homology to the product of the Chlamydomonas reinhardtii PF16 locus, which encodes a protein localized to the central pair of the flagellar axoneme. The gene could be mapped to chromosome 10p11.2-p12. Antibodies raised against the peptide sequences localized the protein to the tails of permeabilized human sperm. Obviously the protein was not part of the sperm membranes. Previously, Munoz et al. (1996)Go had indicated the possibility that HSP60 from chlamydia might induce ASA production owing to an antigen similarity.

Various tubulins are involved in the functional organization of the mammalian sperm flagellum and head. Monoclonal antibodies directed against epitopes on the C-terminal end of neuron-specific class III beta-tubulin that is widely used as a neuronal marker, stained the flagella (Peknicova et al., 2001bGo). Another family of filament-forming proteins are the tektins that are co-assembled with tubulins to form ciliary and flagellar microtubules. Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature sperm, perhaps being required to assemble the basal body (Larsson et al., 2000Go).

The results of Inaba et al. (1998)Go suggested on the basis of immunoelectron microscopy that flagellar movements of sperm is also modulated by proteasomes, which regulate the activity of outer dynein arm by cAMP-dependent phosphorylation of the 22 kDa dynein light chain.

Complement regulatory proteins such as C1-INH, CD55, CD46, and CD59 were expressed on sperm (Jiang and Pillai, 1998Go). IgG fraction of antibodies to these proteins significantly reduced sperm motility in general and other motion parameters.

Chloride/bicarbonate (Cl-/HCO3-) exchangers, a family of proteins [anion exchanger (AE) gene family] that regulate many vital cellular processes such as intracellular pH, cell volume, and Cl- concentration, are also involved in the regulation of sperm motility. Holappa et al. (1999Go) identified sperm cell anion exchanger as the AE2 isoform of this gene family. They showed a polypeptide immunologically related to erythrocyte band 3 to be expressed in mammalian sperm.

Cervical mucus penetration
Impairment of sperm penetration into the cervical mucus appears—in contrast with the other antibody effects quoted—to be independent from the cognate antigens of ASA. It appears to be a consequence of two mechanisms: firstly the activation of the complement cascade by immunoglobulins attached to the sperm surface, at the end of which cell lysis and initiation of the phagocytotic process may take place. The complement-induced cell lysis depends on the immunglobulin class of the antibody, IgM is far more effective than IgG, while some IgA subclasses are unable to interact with the early complement components.

Activation of complement components in cervical mucus has been carefully studied. Complement activating ASA may be effective only in the mucus, because the seminal plasma contains complement inhibitors. During their residence in the cervical mucus, sperm are exposed to complement activity. The complement activity in cervical mucus is ~12% of that of serum (Haas, 1987Go). Thus it may take longer time for sperm immobilization to occur. This is the rationale for performing post-coital tests only after 6–7 h.

The other mechanism explaining the impairment of cervical mucus penetrating ability and the induction of the shaking phenomenon by ASA, in particular of the IgA class, appears to be mediated through the Fc portion of the IgA (Jager et al., 1981Go; Clarke, 1985Go). Sperm recovered after mucus penetration displayed a reduced binding to IgA immunobeads (Wang et al., 1985Go). Experimentally, Bronson (1987Go) showed that IgA bound to the sperm surface, which was degraded by an IgA protease from Neisseria gonorrhoeae did no longer inhibit mucus permeation. This kind of infertility may be overcome by intrauterine insemination (Kutteh et al. 1996Go).

Acrosome reaction
There is a large data pool on antigens involved in acrosome reaction and antibodies to these antigens. A number of spontaneously occurring ASA was shown to enhance the number of acrosome reacted sperm (Bohring et al., 2001aGo), but none of them was able to inhibit acrosome reaction in vitro. An anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium (Liu et al., 2002Go).

However, there are also reports on ASA or antigens inhibiting the spontaneous acrosome reaction: Santhanam et al. (2001Go) demonstrated a novel cDNA encoding for a sperm antigen, designated TSA-1, in a human testis cDNA library. TSA-1 was specifically expressed only in the human testis. When antibodies against the computer generated translated protein were produced in the rabbit, they were found to bind to acrosomal, equatorial, mid-piece, and tail regions of human sperm and caused a significant and concentration-dependent inhibition of human sperm acrosome reaction. When seminal plasma samples containing ASA or sperm loaded with ASA were adsorbed with fertilization antigen-1, the percentage of immunobead-free swimming sperm increased on an average of 50% (Menge et al., 1999Go). The rate of sperm undergoing acrosome reaction as induced by the calcium ionophore A23187 showed improvement in 78% of the sperm samples after FA-1 adsorption.

Antisera against a porcine liver endomembrane progesterone-binding protein inhibited the progesterone-initiated acrosome reaction of human sperm (Buddhikot et al., 1999Go). Indirect immunofluorescence studies detected antigens in the sperm head that moved during capacitation from a posterior head region to a midhead region. This observation suggested that a sperm protein with at least partial homology to the liver endomembrane progesterone-binding protein, is a progesterone-receptor on the sperm plasma membrane.

The two intra-acrosomal molecules acrin 1 (MN7) and acrin 2 (MC41) are essential for distinct events before sperm penetration of the zona pellucida (ZP) in mice. The monoclonal antibody mMN7 prevented completion of acrosomal matrix dispersal, whereas mMC41 did not affect the acrosome reaction. mMC41 appeared to inhibit secondary binding or some biochemical steps on the ZP after the acrosome reaction but before penetration of the ZP. (Saxena et al., 1999Go).

The acrosome reaction involves different calcium-dependent requiring activities. The elevation of intracellular calcium and bicarbonate concentrations stimulates adenyl cyclases to produce cyclic-AMP, which activates phosphorylation of certain proteins by the protein kinase A (PKA). For continuation of the acrosome reaction, further activation of cAMP/PKA and protein kinase C (PKC) is necessary. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. (Breitbart., 2002aGo).

Calpastatin, a 17.5 kDa protein, is an integral part of the acrosomal cytoplasma. Using polyclonal antibodies to calpastatin, immunostaining was seen over the acrosomal region and slightly on the tail. The cDNA consists of 758 base pairs. The gene was found to be transcribed only in spermatids. The inhibition of calpastatin lead to a premature acrosome reaction (Koide et al., 2000Go). Calpastatin binds calpain, a calcium-dependent cysteine endopeptidase. This protease system may be functional in (cynomolgus macaque) sperm during capacitation, the acrosome reaction, or both (Yudin et al., 2000Go). Antibodies to calpains bound to the region between the plasma membrane and the outer acrosomal membrane of sperm. After the acrosome reaction, all of the anti-calpain antibodies labelled the acrosomal shroud presenting acrosomal contents, suggesting that calpains are located throughout the cytoplasmic area between the two outer sperm membranes.

Two forms of clusterin have been detected in the human male reproductive tract: the conventional heterodimeric form and a novel acrosomal form. Specific immunogold labelling showed the antigen present over the surface mainly of the acrosomal contents exposed by the loss of the plasma lemma and outer acrosomal membrane (Atlas-White et al., 2000Go).

The mammalian glucosamine 6-phosphate deaminase (GNPDA) is present in spermatids and sperm close to the acrosomal region and might play a role in the acrosome reaction (Montag et al., 1999Go).

A neuronal glycine receptor/Cl(-) channel (GlyR) was detected on the plasma membrane of mammalian sperm (Sato et al., 2000Go). Pharmacological studies suggested that this receptor/channel is important for initiation of acrosome reaction by the ZP. A monoclonal antibody against GlyR completely blocked ZP initiation of AR in normal mouse sperm. These findings indicate that sperm GlyR plays an essential role in the AR as initiated by the ZP (Sato et al., 2000Go).

Zona binding
The binding of the sperm to the ZP occurs via specific receptors localized over the head region of the sperm. ZP binds at two different receptors in the sperm membrane. One is a Gi-coupled receptor that activates {beta}1-phosphorlipase C, the other one is a tyrosine kinase receptor coupled to {gamma}-phosphorlipase C (Breitbart, 2002aGo).

Welch et al. (1998Go) identified three nucleotide sequences encoding SP22, a protein originally identified in detergent extracts of cauda epididymal sperm, from a rat testis cDNA library. There was evidence that SP22 was a member of a highly conserved and widely expressed gene family found in organisms as diverse as human and Escherichia coli. An antibody to SP22 peptide bound to the anterior-ventral surface of the equatorial segment of the sperm head. Although no conclusive function has been attributed to any members of the SP22 gene family, the localization of SP22 over a discrete region of the sperm head suggested a role in sperm–oocyte interactions.

Mollova et al. (1999Go) described a protein (Ag 1F10) composed of a single peptide chain with a relative molecular mass of 68/70 kDa and an isoelectric point of 3.5 human sperm membranes. The zona binding activity of sperm pre-incubated in the presence of an mAb to this protein was significantly inhibited both in porcine and human IVF systems. The authors assumed that the protein bearing the epitope recognized by the mAb might be one of the molecules with receptor function in sperm–ZP interaction.

Naz et al. (2000Go) described a dodecamer sequence, designated as YLP(12) that is involved in sperm–ZP recognition/binding. Anti-YLP(12) Fab' antibodies of natural occurring ASA recognized a protein band of ~72 ± 2 kDa only in the lane of testis homogenates. The peptide sequence was localized on the acrosomal region of the human sperm cell.

Some other monoclonal antibodies (ACR.2, Hs-8) against boar intra-acrosomal proteins (ACR 2: 55, 53, 45 and 38 kDa; Hs-8: 230, 110, 88, 60, 48 kDa) reduced the secondary sperm–ZP-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization; however, they were not biochemically characterized in this study (Peknicova et al., 2001aGo).

Zona penetration
Mahony et al. (1991Go) observed patients who expressed ASA in their sera that bound to the sperm surface, most specifically the head region, and that reduced ZP tight binding of sperm as assessed by the hemizona assay (HZA). Sperm motion characteristics or development of hyperactivated motility were not affected. The responsible protein was not characterized. Liu et al. (1991Go) described similar results. In their study they confirmed that ASA interfered predominantly with sperm–ZP binding. They concluded from their observations that the inhibition of oolemma binding may not be the major cause of failed fertilization with sperm autoimmunity. However, only seven patients were studied.

One of the possible antigens involved in zona penetration is PH-20, a glycerolphosphatidylinositol-linked hyaluronidase. In the guinea pig, two regions of this enzyme (res. 94–119 and res. 424–444) were highly immunogenic. These regions are accessible on the sperm surface when native gpPH-20 is in solution or anchored on sperm surface. Since PH-20 is present in many species and also in the human, it may be a cognate antigen of ASA in the human (Chan et al., 1999Go).

In mice, a sperm antigen designated as fertilization antigen 1 (FA-1) was identified (Zhu and Naz, 1997Go). The authors cloned and sequenced the cDNA and were able to translate a protein, which was a novel protein not included in protein databases up to that time. The protein specifically reacted with zona protein 3 (ZP3) of oocyte ZP. When polyclonal antibodies were generated, they completely blocked sperm-ZP interaction in mice. Similar results were found in the human system.

Recombinant antibodies to SP22, a sperm membrane protein (Klinefelter et al., 2002Go) significantly inhibited the fertility of cauda epididymal sperm from the rat in vitro as well as the fertilization of both zona-intact and zona-free hamster oocytes. The characterization of SP22, however, is lacking (see below).

Oolemma binding
A 20 kDa glycoprotein (GP20) was isolated from human sperm by Focarelli et al., (1998Go) An anti-GP20 antibody intensely stained the head and midpiece; however, in acrosome reacted sperm the antibody binding was restricted to a small band in the equatorial region. The antibody did not bind to sperm precursor cells in the testis but to epididymal epithelial cells. Thus it seems to be a protein added to the sperm membrane in the epididymis and represents one of the sperm coating antigens. Anti-GP20 exerted a blocking effect in a test for sperm penetration of zona-free hamster oocytes. This concerns also an abundant epididymal gene product that has been identified as lymphocyte surface antigen CD52.

Gabriele et al. (1998Go) described a human sperm protein with a molecular mass of 65 kDa, which bound D-mannose coupled to albumin (DMA) in presence of cations and a neutral pH. The binding of human sperm to zona-free hamster oocytes was reduced by DMA in a dose-dependent manner, suggesting that DMA-binding sites in human sperm are involved in sperm– oocyte fusion.

Francavilla et al. (1991Go) studied the effect of ASA on the hamster oocyte penetration assay (HEPA). They added ASA from patients to motile donor sperm, but they did not find ASA with the ability to reduce the rate of acrosome reacted sperm as well as ASA with the ability to reduce the hamster oocyte penetration rate. At that time, the authors concluded that interference of ASA with the acrosome reaction or with oolemma binding could not be advocated as an explanation for the impairment of the interaction of human sperm with the oocyte.

Noor et al. (1999Go) constructed monoclonal antibodies that bound to various regions of the sperm head and inhibited fertilization. One of these specifically inhibited sperm–oocyte fusion in a concentration-dependent manner, while sperm– oolemma binding and sperm motility remained unaffected. This mAb exclusively recognized an epitope in the equatorial segment, the expression of which increased after capacitation and the acrosome reaction. The antigen was preliminary characterized as two protein bands of 37.5 and 34.0 kDa.

The attachment of human sperm to hamster oocytes may not be a valuable model of human sperm–oocyte interaction. The inhibition of calpain, either by calpain inhibitor-I (calpastatin) or by specific antibodies, impaired the hamster oocyte penetration rate in a dose dependent manner (Rojas et al., 2000Go). The effects did not involve the oocyte, nor did the inhibitor alter sperm motility. Consequently, it was shown that antibodies to calpastatin blocked the penetration of hamster oocytes, but not the attachment to human ova (Koide et al., 2000Go)

Cryopreserved rooster sperm were found to bind to the perivitelline membrane of a chicken egg, when a protein isolated from the supernatant of cryopreserved rooster sperm was added. The partial purification of this activity demonstrated a fragment of prosaposin, which is present also in human semen. This observation may be of relevance also for human fertilization and its impairment by ASA (Hammerstedt et al., 2001Go).

Hamatani et al. (2000Go) isolated and characterized SP-10, a sperm intra-acrosomal protein, which is produced specifically in the testis, but expressed in human sperm only after acrosome reaction. A mAb to this protein inhibited sperm–oolemma binding in the zona-free hamster oocyte penetration test, but it did not inhibit sperm–zona binding in the hemizona assay. Human SP-10 was found to mediate sperm–oolemma binding in an integrin-independent manner, but not sperm–zona binding.

A CD 46 (membrane co-factor protein of complement) isoform is found on human sperm. Experiments showed that it is associated with the sperm–oocyte interaction (Nomura et al., 2001Go). The expression of CD46 in other cells confers resistance to complement-mediated injury. Nomura et al. (2001Go) found three infertile subjects with no expression of CD46 isoform on their sperm when screening 542 idiopathic male infertile patients. All three patients, however, expressed normal CD46 isoforms on their lymphocytes and granulocytes. Thus, the loss of CD46 was sperm-specific, probably due to testicular germ cell-specific regulation of CD46 production. The analysis of the CD46 gene in the patients revealed no abnormality in 3' and 5' regions of the CD46 genome. Thus, in these infertile patients sperm-specific depletion of CD46 was not governed by the regulators in the CD46 gene as identified up to now. Obviously factors outside the known regulatory regions play a role in the regulation of sperm-specific CD46 expression.

Pronucleus formation
At gamete fusion the sperm tail is incorporated into the ooplasm, and the centriolar region forms the sperm aster. While the sperm head is decondensing, the aster guides the female pronucleus towards the male pronucleus. ASA against proteins of the centrioles may be responsible for mitotic arrest (Palermo et al., 1997Go).

The human nuclear autoantigenic sperm protein, (NASP), is a testicular histone-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In a study recombinant deletion mutants spanning the entire protein coding sequence were screened with vasectomy patients’ sera (Batova et al., 2000Go). The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Multiple continuous autoimmune epitopes in NASP involving sequences comprised the histone-binding sites. These may be the cognate antigens of autoantibodies in vasectomized men. The clinical relevance of this antigen as well of their antibodies remains unclear.

Other antibody effects
Klinefelter et al. (2002Go) described extensive investigations of a rat protein of 22 kDa (SP22), a testis-specific transcript of which is expressed in postmeiotic germ cells. It was immunolocalized on spermatids and over the equatorial segment of the sperm head of epididymal sperm, but also on clear cells of the epididymal epithelium, possibly due to the phagocytosis of cytoplasmic droplets. When the expression of SP22 was decreased as a consequence of the treatment of the animals with several toxicants or when normal epididymal sperm were incubated with antibodies against SP22, the in-vivo or in-vitro fertility was reduced. The authors concluded that SP22 plays a pivotal role in fertilization (at least in the rat). Indeed it appears to be a highly immunogenic compound. Unfortunately, however, despite the extensive experiments, the authors did not present attempts on amino-sequencing of SP22, which would allow its comparison with known proteins such as sperm adhesins or other adhesion molecules. They also did not present experiments that allow the determination of the particular function in the fertilization process, which would be inhibited by SP22.

There are sex-chromosome specific proteins (SCSP) on the sperm membrane. Sex-specific antibodies (SSAbs) raised against these SCSPs appear to bind to these proteins and make possible a sperm-sexing procedure. SCSPs are evolutionarily more highly conserved than non-SCSPs (Blecher et al., 1999Go). The role in infertility is unclear.

An unanswered question is that of sperm apoptosis. Several proteins of the signal transduction pathways of apoptosis are present on the sperm surface, e.g. the externalization of phosphatidylserin, CD 95, and some caspases (Paasch et al., 2002Go). It is questionable, however, whether these proteins are functionally active. Sakkas et al. (2002)Go suggest that the presence of DNA damage is not directly linked to an apoptotic process occurring in sperm. The presence of apoptotic proteins in ejaculated sperm may be linked to defects in cytoplasmic remodelling during the later stages of spermatogenesis. ASA binding to the inactive form of caspase-3 as a cognate antigen were demonstrated in our group (Bohring et al., 2001aGo). The pathophysiologic significance of these ASA is unclear at time.


    Conclusions and treatment options
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
As practical consequences of the research on ASA related sperm proteomics those ASA will be identified, which decrease male fertility by inhibiting sperm functions that are essential for fertilization. The presence of antibodies in a biological substrate (serum, seminal plasma) that bind to specific antigens may be visualized by an ELISA or a RIA. In contrast with the earlier immunoassays, however, the sperm antigens used will be known proteins or peptides. Since ASA of an individual patient bind to up to 10 different proteins, in a patient with a significantly positive MAR test or IBT, up to 10 different ELISA’s have to be performed in order to decide whether the patient suffers from immune infertility.

The identification of functionally relevant antigens is a prerequisite for treatment options. Currently, no antibody-specific treatment of autoimmune diseases is possible, but the treatment is based on the suppression of antibody production in general. Increasingly, however, the use of monoclonal antibodies in autoimmune diseases is described. It may be speculated that this procedure may be adapted also to the treatment of autoimmune infertility.

The analysis of the cognate antigens of ASA involved in the process of fertilization is important from another point of view: it improves the identification of immunogenic proteins being candidates for immune contraception, i.e. which allow the artificial induction of antibodies in either male or female thereby inhibiting fertilization. Many groups have described approaches to this topic, but their inclusion is outside the scope of this paper.


    References
 Top
 Abstract
 Immune infertility—an...
 Tests for ASA
 Analysis of cognate antigens...
 Sperm proteins characterized by...
 Conclusions and treatment...
 References
 
Atlas-White, M., Murphy, B.F. and Baker, H.W. (2000) Localisation of clusterin in normal human sperm by immunogold electron microscopy. Pathology, 32, 258–261.[ISI][Medline]

Batova, I.N., Richardson, R.T., Widgren, E.E. and O’Rand, M.G. (2000) Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides. Clin. Exp. Immunol., 121, 201–209.[CrossRef][ISI][Medline]

Blasco, L. (1984) Clinical tests of sperm fertilizing ability. Fertil. Steril., 41, 177–192.[ISI][Medline]

Blecher, S.R., Howie, R., Li, S., Detmar, J. and Blahut, L.M. (1999) A new approach to immunological sexing of sperm. Theriogenology, 52, 1309–1321.[CrossRef][ISI][Medline]

Bohring, C. and Krause, W. (1999) The characterization of human spermatozoa membrane proteins–surface antigens and immunological infertility. Electrophoresis, 20, 971–976.[CrossRef][ISI][Medline]

Bohring, C., Krause, E., Habermann, B. and Krause, W. (2001a) Isolation and identification of sperm membrane antigens recognized by antisperm antibodies, and their possible role in immunological infertility disease. Mol. Hum. Reprod., 7, 113–118.[Abstract/Free Full Text]

Bohring, C., Skrzypek, J. and Krause, W. (2001b) Influence of antisperm antibodies on the acrosome reaction as determined by flow cytometry. Fertil. Steril., 76, 275–280.[CrossRef][ISI][Medline]

Breitbart, H. (2002a) Role and regulation of intracellular calcium in acrosomal exocytosis. J. Reprod. Immunol., 53, 151–159.[CrossRef][ISI][Medline]

Breitbart, H. (2002b) Intracellular calcium regulation in sperm capacitation and acrosomal reaction. Mol. Cell. Endocrinol., 187, 139–144.[CrossRef][ISI][Medline]

Bronson, R. (1999a) Detection of antisperm antibodies: an argument against therapeutic nihilism. Hum. Reprod., 14, 1671–1673.[Free Full Text]

Bronson, R. (1999b) Antisperm antibodies: a critical evaluation and clinical guidelines. J. Reprod. Immunol., 45, 159–183.[ISI][Medline]

Bronson, R.A., Cooper G.W., Rosenfeld, D.L., Gilbert J.V. and Plaut A.G. (1987) the effect of an IgA1 protease on immunoglobulins bound to the sperm surface and sperm cervical mucus penetrating ability. Fertil. Steril., 47, 985–991.[ISI][Medline]

Buddhikot, M., Falkenstein, E., Wehling, M. and Meizel, S. (1999) Recognition of a human sperm surface protein involved in the progesterone-initiated acrosome reaction by antisera against an endomembrane progesterone binding protein from porcine liver. Mol. Cell. Endocrinol., 158, 187–193.[CrossRef][ISI][Medline]

Cai, Y., Gao, Y., Sheng, Q., Miao, S., Cui, X., Wang, L., Zong, S. and Koide, S.S. (2002) Characterization and potential function of a novel testis-specific nucleoporin BS-63. Mol. Reprod. Dev., 61, 126–134.[CrossRef][ISI][Medline]

Chan, C.P., Gupta, S. and Mark, G.E, (1999) Identification of linear surface epitopes on the guinea pig sperm membrane protein PH-20. Life Sci., 64, 1989–2000.[CrossRef][ISI][Medline]

Check, M.L., Check, J.H., Katsoff, D. and Summers-Chase, D. (2000) ICSI as an effective therapy for male factor with antisperm antibodies. Arch. Androl., 45, 125–130.[CrossRef][ISI][Medline]

Clarke G.N. (1985) Induction of the shaking phenomenon by IgA class antispermatozoal antibodies from serum. Am. J. Reprod. Immunol. Microbiol., 9, 12–14[Medline]

Diekman, A.B., Norton, E.J., Westbrook, V.A., Klotz, K.L., Naaby-Hansen, S. and Herr, J.C. (2000) Anti-sperm antibodies from infertile patients and their cognate sperm antigens: a review. Identity between SAGA-1, the H6-3C4 antigen, and CD52. Am. J. Reprod. Immunol., 43, 134–143.[CrossRef][ISI][Medline]

Domagala, A., Kamieniczna, M. and Kurpisz, M. (2000) Sperm antigens recognized by antisperm antibodies present in sera of infertile adults and prepubertal boys with testicular failure. Int. J. Androl., 23, 150–155.[CrossRef]

Focarelli, R., Giuffrida, A., Capparelli, S., Scibona, M., Fabris, F.M., Francavilla, F., Francavilla, S., Giovampaola, C.D. and Rosati, F. (1998) Specific localization in the equatorial region of gp20, a 20 kDa sialylglycoprotein of the capacitated human spermatozoon acquired during epididymal transit which is necessary to penetrate zona-free hamster eggs. Mol. Hum. Reprod., 4, 119–125.[Abstract]

Francavilla, F., Romano, R. and Santucci, R. (1991) Effect of sperm-antibodies on acrosome reaction of human sperm used for the hamster egg penetration assay. Am. J. Reprod. Immunol., 25, 77–80.[ISI][Medline]

Gabriele, A., D’Andrea, G., Cordeschi, G., Properzi, G., Giammatteo, M., De Stefano, C., Romano, R., Francavilla, F. and Francavilla, S. (1998) Carbohydrate binding activity in human spermatozoa: localization, specificity, and involvement in sperm-egg fusion. Mol. Hum. Reprod., 4, 543–553.[Abstract]

Görg, A., Postel, W. and Günther, S. (1998) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 9, 531–546.

Haas, G.G. Jr (1987) Immunologic infertility. Obstet. Gynecol. Clin. North Am., 14, 1069–1085.[ISI][Medline]

Hamatani, T., Tanabe, K., Kamei, K., Sakai, N., Yamamoto, Y. and Yoshimura, Y. (2000) A monoclonal antibody to human SP-10 inhibits in vitro the binding of human sperm to hamster oolemma but not to human zona pellucida. Biol. Reprod., 62, 1201–1208.[Abstract/Free Full Text]

Hammerstedt, R.H., Cramer, P.G., Barbato, G.F., Amann, R.P., O’Brien, J.S. and Griswold, M.D. (2001) A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens. J. Androl., 22, 361–375.[Abstract/Free Full Text]

Helmerhorst, F.M., Finken, M.J. and Erwich, J.J. (1999) Antisperm antibodies: detection assays for antisperm antibodies: what do they test? Hum. Reprod., 14, 1669–1671.[Free Full Text]

Hjort, T. (1999) Antisperm antibodies. Antisperm antibodies and infertility: an unsolvable question? Hum. Reprod., 14, 2423–2426.[Free Full Text]

Holappa, K., Mustonen, M., Parvinen, M., Vihko, P., Rajaniemi, H. and Kellokumpu, S. (1999) Primary structure of a sperm cell anion exchanger and its messenger ribonucleic acid expression during spermatogenesis. Biol. Reprod., 61, 981–986.[Abstract/Free Full Text]

Inaba, K., Morisawa, S. and Morisawa, M. (1998) Proteasomes regulate the motility of salmonid fish sperm through modulation of cAMP-dependent phosphorylation of an outer arm dynein light chain. J. Cell Sci., 111, 1105–1115.[Abstract/Free Full Text]

Jager, S., Kremer, J., Kuiken, J. and Mulder, I. (1981) The significance of the Fc part of antispermatozoal antibodies for the shaking phenomenon in the sperm-cervical mucus contact test. Fertil. Steril., 36, 792–797.[ISI][Medline]

Jairaj, S., Check, J.H. and Bollendorf, A. (2000) Do antisperm antibodies cause functional impairment of the sperm membrane as manifested by a low hypo-osmotic swelling test score? Arch. Androl., 44, 231–235.[CrossRef][ISI][Medline]

Jiang, H. and Pillai, S. (1998) Complement regulatory proteins on the sperm surface: relevance to sperm motility. Am. J. Reprod. Immunol., 39, 243–248.[ISI][Medline]

Klinefelter, G.R., Welch, J.E., Perreault, S.D., Moore, H.D., Zucker, R.M., Suarez, J.D., Roberts, N.L., Bobseine, K. and Jeffay, S. (2002) Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro. J. Androl., 23, 48–63.[Abstract/Free Full Text]

Koide, S.S., Wang, L. and Kamada, M. (2000) Antisperm antibodies associated with infertility: properties and encoding genes of target antigens. Proc. Soc. Exp. Biol. Med., 224, 123–132.[Abstract/Free Full Text]

Komori, S., Kameda, K., Sakata, K., Hasegawa, A., Toji, H., Tsuji, Y., Shibahara, H., Koyama, K. and Isojima, S. (1997) Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity. Clin. Exp. Immunol., 109, 547–554.[CrossRef][ISI][Medline]

Krapez, J.A., Hayden, C.J., Rutherford, A.J. and Balen, A.H. (1998) Survey of the diagnosis and management of antisperm antibodies. Hum. Reprod., 13, 3363–3367.[Abstract]

Kuang, Y., Yan, Y.C., Gao, A.W., Zhai, Y.M., Miao, S.Y., Wang, L.F. and Koide, S.S. (2000) Immune responses in rats following oral immunization with attenuated Salmonella typhimurium expressing human sperm antigen. Arch. Androl., 45, 169–180.[CrossRef][ISI][Medline]

Kutteh, W.H., Byrd, W., Blankenship, L., Kutteh, C.C. and Carr, B.R. (1996) Cervical mucus anti-sperm antibodies: treatment with intrauterine insemination. Am. J. Reprod. Immunol., 35, 429–433.[ISI][Medline]

Larsson, M., Norrander, J., Graslund, S., Brundell, E., Linck, R., Stahl, S. and Hoog, C. (2000) The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme. Eur. J. Cell Biol., 79, 718–725.[ISI][Medline]

Liu, D.Y., Clarke, G.N. and Baker, H.W. (1991) Inhibition of human sperm-zona pellucida and sperm-oolemma binding by antisperm antibodies. Fertil. Steril., 55, 440–442.[ISI][Medline]

Liu, H.W., Lin, Y.C., Chao, C.F., Chang, S.Y. and Sun, G.H. (2000) GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation. Mol. Hum. Reprod., 6, 422–428.[Abstract/Free Full Text]

Liu, D.Y., Martic, M., Clarke, G.N., Grkovic, I., Garrett, C., Dunlop, M.E. and Baker, H.W. (2002) An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm. Mol. Hum. Reprod., 8, 37–47.[Abstract/Free Full Text]

Lombardo, F., Gandini, L., Dondero, F. and Lenzi, A. (2001 Immunology and immunopathology of the male genital tract. Hum. Reprod. Update, 7, 450–456.

Lu, N.Q. and Zha, S.W. (2000) Inhibitory effects of human seminal plasma on an ELISA used to detect anti-sperm antibodies: implications for the determination of sperm quality. J. Reprod. Immunol., 47, 33–40.[CrossRef][ISI][Medline]

Mahony, M.C., Blackmore, P.F., Bronson, R.A. and Alexander, N.J. (1991) Inhibition of human sperm-zona pellucida tight binding in the presence of antisperm antibody positive polyclonal patient sera. J. Reprod. Immunol., 19, 287–301.[CrossRef][ISI][Medline]

Mardesic, T., Ulcova-Gallova, Z., Huttelova, R., Muller, P., Voboril, J., Mikova, M. and Hulvert, J. (2000) The influence of different types of antibodies on in vitro fertilization results. Am. J. Reprod. Immunol., 43, 1–5.[CrossRef][ISI][Medline]

Menge, A.C., Christman, G.M., Ohl, D.A. and Naz, R.K. (1999) Fertilization antigen-1 removes antisperm autoantibodies from spermatozoa of infertile men and results in increased rates of acrosome reaction. Fertil. Steril., 71, 256–260.[CrossRef][ISI][Medline]

Mollova, M., Djarkova, T., Ivanova, M., Stamenova, M. and Kyurkchiev, S. (1999) Isolation and biological characterization of boar sperm capacitation-related antigen. Am. J. Reprod. Immunol., 42, 254–262.[ISI][Medline]

Montag, M., van der Ven, K., Dorbecker, C. and van der Ven, H. (1999) Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA). FEBS. Lett., 458, 141–144.[CrossRef][ISI][Medline]

Munoz, M.G., Jeremias, J. and Witkin, S.S. (1996) The 60 kDa heat shock protein in human semen: relationship with antibodies to spermatozoa and Chlamydia trachomatis. Hum. Reprod., 11, 2600–2603.[Abstract]

Munuce, M.J., Berta, C.L., Pauluzzi, F. and Caille, A.M. (2000) Relationship between antisperm antibodies, sperm movement, and semen quality. Urol. Int., 65, 200–203.[CrossRef][ISI][Medline]

Nagy, Z.P., Aragona, C. and Greco, E. (1999) Results of ICSI in the treatment of male immunological infertility. Andrologia, 31, 316–317.[ISI][Medline]

Naz, R.K., (1992) Effects of antisperm antibodies on early cleavage of fertilized ova. Biol. Reprod., 46, 130–139.[Abstract]

Naz, R.K., Zhu, X. and Kadam, A.L. (2000) Identification of human sperm peptide sequence involved in egg binding for immunocontraception. Biol. Reprod., 62, 318–324.[Abstract/Free Full Text]

Naz, R.K. and Chauhan, S.C. (2001) Presence of antibodies to sperm YLP(12) synthetic peptide in sera and seminal plasma of immunoinfertile men. Mol. Hum. Reprod., 7, 21–26.[Abstract/Free Full Text]

Neilson, L.I., Schneider, P.A., Van Deerlin, P.G., Kiriakidou, M., Driscoll, D.A., Pellegrini, M.C., Millinder, S., Yamamoto, K.K., French, C.K. and Strauss, J.F. III. (1999) cDNA cloning and characterization of a human sperm antigen (SPAG6) with homology to the product of the Chlamydomonas PF16 locus. Genomics, 60, 272–280.[CrossRef][ISI][Medline]

Nikolaeva, M.A., Kulakov, V.I., Korotkova, I.V., Golubeva, E.L., Kuyavskaya, D.V. and Sukhikh, G.T. (2000) Antisperm antibodies detection by flow cytometry is affected by aggregation of antigen-antibody complexes on the surface of spermatozoa. Hum. Reprod., 15, 2545–2553.[Abstract/Free Full Text]

Nomura, M., Kitamura, M., Matsumiya, K., Tsujimura, A., Okuyama, A., Matsumoto, M., Toyoshima, K. and Seya, T. (2001) Genomic analysis of idiopathic infertile patients with sperm-specific depletion of CD46. Exp. Clin. Immunogenet., 18, 42–50.[CrossRef][ISI][Medline]

Noor, M.M. and Moore, H.D. (1999) Monoclonal antibody that recognizes an epitope of the sperm equatorial region and specifically inhibits sperm-oolemma fusion but not binding. J. Reprod. Fertil., 115, 215–224.[Abstract]

Norton, E.J., Diekman, A.B., Westbrook, V.A., Flickinger, C.J. and Herr, J.C. (2001) RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives. Hum. Reprod., 16, 1854–1860.[Abstract/Free Full Text]

Omu, A.E., al-Qattan, F., Ismail, A.A., al-Taher, S. and al-Busiri, N. (1999) Relationship between unexplained infertility and human leukocyte antigens and expression of circulating autogeneic and allogeneic antisperm antibodies. Clin. Exp. Obstet. Gynecol., 26, 199–202.[CrossRef]

Paasch, U., Grunewald, S. and Glander, H.J. (2002) Presence of up- and downstream caspases in relation to impairment of human spermatogenesis. Andrologia, 34, 279–280.

Palermo, G.D., Colombero, L.T. and Rosenwaks, Z. (1997) The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev. Reprod., 2, 19–27.[Abstract/Free Full Text]

Peknicova, J., Capkova, J., Geussova, G., Ivanova, M. and Mollova, M. (2001a) Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro. Theriogenology., 56, 211–223.[CrossRef][ISI][Medline]

Peknicova, J., Kubatova, A., Sulimenko, V., Draberova, E., Viklicky, V., Hozak, P. and Draber, P. (2001b) Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail. Biol. Reprod., 65, 672–679.[Abstract/Free Full Text]

Poulton, T.A., Everard, D., Baxby, K. and Parslow, J.M. (1996) Characterisation of a sperm coating auto-antigen reacting with antisperm antibodies of infertile males using monoclonal antibodies. Br. J. Obstet. Gynecol., 103, 463–467.[ISI][Medline]

Räsänen, M., Agrawal, Y.P. and Saarikoski, S. (1996) Seminal fluid antisperm antibodies measured by direct flow cytometry do not correlate with those measured by indirect flow cytometry, the indirect immunobead test, and the indirect mixed antiglobulin reaction. Fertil. Steril., 65, 170–175.[ISI][Medline]

Rojas, F.J. and Moretti-Rojas, I. (2000) Involvement of the calcium-specific protease, calpain, in the fertilizing capacity of human spermatozoa. Int. J. Androl., 23, 163–168.[CrossRef]

Rose, N.R. and Bona, C. (1993) Defining criteria for autoimmune diseases (Witebsky’s postulates revised) Immunol. Today, 14; 421–426.

Sakkas, D., Moffat, O., Manicardi, G.C. Mariethoz, E., Tarozzi, N. and Bizzaro, D. (2002) Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol. Reprod., 66, 1061–1067.[Abstract/Free Full Text]

Santhanam, R. and Naz, R.K. (2001) Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein. Mol. Reprod. Dev., 60, 1–12.[CrossRef][ISI][Medline]

Sato, Y., Son, J.H., Tucker, R.P. and Meizel, S. (2000) The zona pellucida-initiated acrosome reaction: defect due to mutations in the sperm glycine receptor/Cl(-) channel. Dev. Biol., 227, 211–218.[CrossRef][ISI][Medline]

Saxena, D.K., Tanii, I., Yoshinaga, K. and Toshimori, K. (1999) Role of intra-acrosomal antigenic molecules acrin 1 (MN7) and acrin 2 (MC41) in penetration of the zona pellucida in fertilization in mice. J. Reprod. Fertil., 11, 17–25.[CrossRef]

Shetty, J., Naaby-Hansen, S., Shibahara, H., Bronson, R., Flickinger, C.J. and Herr, J.C, (1999) Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women. Biol. Reprod., 61, 61–69.[Abstract/Free Full Text]

Singh, S., Joshi, S. and Khole, V. (2001) Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen. Indian. J. Exp. Biol., 39, 209–217.

Son, W.Y., Hwang, S.H., Han, C.T., Lee, J.H., Kim, S. and Kim, Y.C. (1999) Specific expression of heat shock protein HspA2 in human male germ cells. Mol. Hum. Reprod., 5, 1122–1126.[Abstract/Free Full Text]

Storch, W.B. (1998) Autoantikörper und ihre diagnostische Bedeutung. Deutsch. Med. Wschr., 123, 1213–1216.[Medline]

Tsunekawa, N., Nishida, T. and Fujimoto, H. (1999) Expression of the spermatid-specific Hsp70 antigen is conserved in mammals including marsupials. J. Vet. Med. Sci., 61, 381–388.[CrossRef][ISI][Medline]

Wang, C., Baker, H.W., Jennings, M.G., Burger, H.G. and Lutjen, P. (1985) Interaction between human cervical mucus and sperm surface antibodies. Fertil. Steril., 44, 484–488.[ISI][Medline]

Welch, J.E., Barbee, R.R., Roberts, N.L., Suarez, J.D. and Klinefelter, G.R. (1998) SP22: a novel fertility protein from a highly conserved gene family. J. Androl., 19, 385–393.[Abstract/Free Full Text]

Yakirevich, E. and Naot, Y. (2000) Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination. Biol. Reprod., 62, 1016–1023.[Abstract/Free Full Text]

Yeung, C.H., Schroter, S., Kirchhoff, C. and Cooper, T.G. (2000) Maturational changes of the CD52-like epididymal glycoprotein on cynomolgus monkey sperm and their apparent reversal in capacitation conditions. Mol. Reprod. Dev., 57, 280–289.[CrossRef][ISI][Medline]

Yeung, C.H., Perez-Sanchez, F., Schroter, S., Kirchhoff, C. and Cooper T.G. (2001) Changes of the major sperm maturation-associated epididymal protein HE5 (CD52) on human ejaculated spermatozoa during incubation. Mol. Hum. Reprod., 7, 617–624.[Abstract/Free Full Text]

Yudin, A.I., Goldberg, E., Robertson, K.R. and Overstreet, J.W. (2000) Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa. J. Androl., 21, 721–729.[Abstract/Free Full Text]

Zhu, X. and Naz, R.K. (1997) Fertilization antigen-1: cDNA cloning, testis-specific expression, and immunocontraceptive effects. Proc. Natl Acad. Sci. USA, 944, 704–709.

Zouari, R. and De Almeida, M. (1993) Effect of sperm-associated antibodies on human sperm ability to bind to zona pellucida and to penetrate zona-free hamster oocytes. J. Reprod. Immunol., 24, 175–186.[CrossRef][ISI][Medline]