Influence of ASA on sperm agglutination seems feasible, since observation of agglutination is a proven method of ASA detection. A first investigation of cognate antigens binding sperm agglutinating ASA was published by Koide et al. [31]. The ASA were obtained from the blood serum of infertile women. Among the antigens identified were:

Domagala et al. [17] described agglutination between sperm tail tips by antibodies from an infertile female patient suffering from systemic lupus erythematosus. Using proteomic analysis, the cognate antigen was identified as the heavy chain of clathrin, the main structural coat protein of coated vesicles which play a key role in the intracellular transport between membranous organelles. By immunofluorescence, it was localized in the principal piece and the cytoplasmatic droplets.

A polyclonal antibody from the rabbit against the human c-kit peptide was able to inhibit acrosome reaction in human sperm and to increase sperm agglutination. By immune fluorescence, the localization of the c-kit peptide in the acrosomal region was demonstrated, but the staining was absent in acrosome reacted sperm. Thus the c-kit peptide may be involved in acrosome reaction ([19]; Fig. 4.1).

Norton et al. [50] engineered a recombinant single-chain variable fragment (scFv) antibody binding to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), the sperm glycoform of CD52. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells. RASA aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination [64]. For further details of CD52, see Chap. 11.

Bandivdekar et al. [2] described antibodies binding to a human sperm-specific antigen of about 80 kDa, which agglutinated epididymal spermatozoa. The partial N-terminal amino acid sequence of 80 kDa HSA (peptide NT) and its peptides obtained by enzymatic digestion with endoproteinase Lys-C (peptides 1, 2, 3 and 4) and Glu-C (peptides 5 and 6) did not show sequence homology with any of the proteins in Gene database. In a further study, the authors showed that antibodies from the rabbit against this protein could cause infertility in mice [3].

Fu et al. [21] demonstrated a marked reduction of fertility in female mice by autoantibodies to ACTL7a from vasectomized men. The protein ACTL7a plays an important role in spermiogenesis, in particular in the morphogenesis of spermatozoa, but its functional role was not yet described. In spermatids, it forms a complex with other components of the cytoskeleton. In human spermatozoa, this protein has been located in the acrosome. The antibodies caused a marked agglutination of sperm in vitro.

Antibodies to Catsper1, one of the proteins of the cationic channel of sperm, experimentally induced sperm agglutination and inhibited fertility in the mouse [38]. Since Catsper1 is clearly associated with sperm functions, ASA against Catsper1 might be able to impair fertility. Evidence for this mechanism, however, is lacking up to now [55].

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 [51]. On the other hand, spermatozoa do not stain with Fas protein antibodies [12], thus it is questionable whether the complete instruments of apoptosis are present in spermatozoa and whether these proteins are functionally active.

Inflammasome components and end-product cytokines are present in semen. Caspase-1 in sperm fractions and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) in seminal plasma and sperm fractions could be identified. Immunocytochemistry revealed that ASC was located in the acrosome, equatorial segment, and midpiece, and caspase-1 in the midpiece [67].

Reports on ASA binding to functional proteins involved in apoptosis in the literature are scarce. A binding of ASA to the inactive form of caspase-3 and to HSP70 as cognate antigens were demonstrated in our group [8]. Naaby-Hansen and Herr [45] described antibodies to HSP70 that blocked fertilization in vitro.

A special feature concerns sperm immobilizing ASA. They were demonstrated exclusively in the sera of infertile women. They appear to activate the complement system; their presence is frequently associated with impaired penetration of the cervical mucus. The antigen was identified as human CD52 antigen, which is inserted into the sperm membrane during the epididymal passage. Details on this topic are discussed in Chap. 11.

In general, it is hard to explain how other ASA will interfere with sperm motility, since it is likely that ASA bind to antigens of sperm membranes, while sub-cellular structures will not be reached by ASA in the living cell.

Neilson et al. [49] used serum from an infertile male with high titers of ASA to identify a novel human sperm antigen (SPAG6) by screening of 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. Antibodies raised against the peptide sequences localized the protein to the tails of permeabilized human sperm.

The results of Inaba et al. [26] using immune electron microscopy suggested that flagellar movement 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. In our group, we were able to demonstrate ASA binding to the component 2 and to the zeta chain of the proteasome complex [7]. Complement regulatory proteins such as C1-INH, CD55, CD46, and CD59 has been found to be expressed on sperms [29]. IgG antibodies to these proteins significantly reduced sperm motility in general and other parameters of motility.

Applying Catsper1-antibodies to spermatozoa, as already mentioned in the previous chapter, was able to inhibit total motility and progressive motility. The mechanism of this inhibition remained unclear. CatSper1 expression has been found to be positively related to progressive and hyperactivated (HA) motility, men with asthenozoospermia showed a reduced expression of CatSper1 in the spermatozoa [39]. Also antibodies binding to the voltage-gated anion channel protein (VDAC) showed impact on sperm motility, possibly by influencing the Ca+ influx into the cells [40]. However, there is no evidence of ASA binding to the two channel proteins up to now.

Immunglobulin concentrations in the cervix mucus are generally low, thus ASA are rarely detected. Kamieniczna et al. [30] described a frequency of 3.2 % in infertile women, compared to 10.4 % of seminal samples of infertile men. In particular, women with immobilizing antibodies may display different titers also in the cervix mucus, which inhibit sperm migration [54] and result in poor post-coital test and reduced fertility.

However, the impairment of sperm penetration into the ovulatory cervical mucus is largely independent from ASA. During their residence in the cervical mucus, spermatozoa are exposed to complement activity, although the complement activity in cervical mucus amounts only to approximately 12 % of that in serum [23]. Immunoglobulins attached to the sperm surface activate the complement cascade, initiating cell lysis and a phagocytotic process. The complement-induced cell lysis depends on the immunoglobulin class of the antibody concerned, IgM is far more effective than IgG, while some IgA subclasses are unable to interact with the early complement components.

Another mechanism explaining the impairment of cervical mucus penetrating ability and the induction of the shaking phenomenon by ASA, in particular those of the IgA class, appears to be mediated through the Fc portion of the IgA [16], [28]. Sperm recovered after mucus penetration displayed a reduced binding to IgA immunobeads [61]. Experimentally, Bronson et al. [9] showed that IgA bound to the sperm surface, which was degraded by an IgA protease from Neisseria gonorrhoeae did no longer inhibit mucus permeation.

The loss of the acrosome including the release of the acrosomal content in order to enable the spermatozoa to permeate through the zona pellucida is called acrosome reaction. There is a large data pool on antigens involved in acrosome reaction and antibodies to these antigens.

In general, the majority of ASA increase the number of acrosome-reacted spermatozoa. In our group we showed that a number of spontaneous occurring ASA was able to enhance the number of acrosome reacted sperm [7], but none of them was able to inhibit acrosome reaction in vitro. In our study all patients, whose ASA bound to the acrosome region of the donor sperm, showed abnormal acrosin activity in their own spermatozoa, indicating a functional relevance of the cognate antigens. In contrast, Feng et al. [18] could not demonstrate an increase in the rate of acrosome reacted spermatozoa after incubation with ASA-containing serum.

When seminal plasma samples containing ASA or spermatozoa loaded with ASA were adsorbed with fertilization antigen-1 (FA-1), the percentage of immunobead-free swimming sperm increased on an average of 50 % [44]. The rate of spermatozoa undergoing acrosome reaction as induced by the calcium ionophore A23187 showed improvement in 78 % of the sperm samples after FA-1 adsorption.

Calpastatin, a 17.5 kDa protein, is an integral part of the acrosomal cytoplasma. Using polyclonal antibodies to calpastatin, immunstaining was seen over the acrosomal region and slightly on the tail. The calpastatin gene was found to be transcribed only in spermatids. The inhibition of calpastatin leads to a premature acrosome reaction [31]. Calpastatin binds calpain, a Ca-dependent cysteine endopeptidase, fromwhich at least two isotypes exist. Antibodies to calpain bound to the region between the plasma membrane and the outer acrosomal membrane of sperm. Following the acrosome reaction, the anti-calpain antibodies labeled the acrosomal shroud presenting acrosomal contents, suggesting that calpain is located in the cytoplasmic area between the two outer sperm membranes. Calpain is relocated from cytoplasm to plasma membrane, where it cleaves spectrin, one of the proteins of the cytoskeleton, and thus facilitating the acrosome reaction [4].

Auer et al. [1] isolated a protein P36 as a cognate antigen of ASA, which was identified as a glycolytic enzyme. P36 was not detectable at the surface of live non acrosome-reacted sperm cells. It was characterized as human triosephosphate isomerase (TPI), which catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. Its functional role is unclear, but may be independent of the catalytic activity, as demonstrated already for other sperm enzymes (moonlighting proteins, see following section).

Cheng et al. [14] found ASA from an infertile female patient being specific for a human sperm membrane protein (hSMP-1, PubMed locus U12978), a testis-specific protein. Polyclonal antibodies against a fragment of the mouse protein homologue showed intense hSMP-1 immune reactivity on the acrosome of human sperm. hSMP-1 is also active in the zona binding (see below).

Wang et al. [60] described the sperm lysozyme-like protein 1 (SLLP-1), which is a unique nonbacteriolytic, c-lysozyme–like protein and is present in the acrosome of human spermatozoa. Antisera to SLLP1 were shown to block binding of sperm to hamster oocytes. The occurrence of ASA binding to this antigen was not described up to now.

Chiu et al. [15] described two men with high concentrations of ASA, which bound to a novel protein localized in the acrosome called SPRASA. They were able to determine the peptide sequence of the protein by MALDI-MS and could show that it was a theoretical protein, XP-085564 encoded by the lysozyme/alpha-lactalbumine gene family. Only ASA from infertile men reacted with SPRASA, suggesting that this novel protein may be important in the processes of fertility. Later, it was demonstrated that SPRASA is also expressed in ovarian follicles and corpora lutea. Spontaneous antibodies to SPRASA were found only in infertile women, but not in fertile women, indicating its role also in female immune infertility [58].