Antisperm antibody: a monkey wrench in conception / magic bullet of contraception?

Antisperm antibody: a monkey wrench in conception / magic bullet of contraception?

Masaharu Kamada^a, Masahiko Maegawa^a, Yuan-chang Yan^b, Samuel S. Koide^c,and Toshihiro Aono^a

^aDepartment of Obstetrics and Gynecology, The University of Tokushima School of Medicine, Tokushima, Japan;^bShanghai Institute of Cell Biology, Academia Sinica, Shanghai, China;and ^cCenter for Biomedical Research, The Population Council, New York, USA

Abstract:Antisperm antibodies can cause infertility by interacting with spermatozoa through immunoglobulin binding protein thereby blocking their penetrance of cervical mucus and / or by interfering with sperm-egg interaction. However, these antibodies appear not to be cytotoxic to embryos since a high implantation rate and consequently high pregnancy rate were achieved by IVF-ET treatment of women with antisperm antibodies. Also the finding that these antibodies do not appear to cause any deleterious clinical symptoms and have yet be associated with infertility suggested that sperm antigens are promising candidates in the development of immunocontraceptives. Some synthetic peptides corresponding to segments of human sperm antigens have effectively induced infertility in female rats when administered as an immunogen. Different peptides, adjuvants and routes of administration should be studied to determine the optimum conditions for inducing high antisperm antibody titers in the host. Moreover, identification of various steps and factors that are involved in regulating the production of antisperm antibodies such as immunoglobulin binding factor may open new paths in the treatment of immunological infertility and at the same time lead to a more effective immunocontraceptive. J. Med. Invest. 46:19-28, 1999

Keywords:antisperm antibody, infertility, contraception, immunoglobulin binding factor, sperm antigen

INTRODUCTION
Nonspecific and specific immune reactions against gamete or embryo appear to be physiologically important for the maintenance of homeostasis in reproduction (1). For example, in the male and fe-male reproductive tracts, activation of the complement system by immature and aged spermatozoa can enhance their destruction and clearance during the selection of active motile sperm. This mechanism of sperm selection is physiologically important, in increasing the opportunity of healthy sperm to fer-tilize the ova (2). Furthermore, we have demon-strated that the occurrence of cellular and/or humoral immune reaction against sperm antigens expressed on the surface of embryos appears to augment their receptivity by the uterus (3).
On the other hand, aberration of the immune homeostasis may give rise to "immunological infer-tility". As a matter of fact, the majority of subjects with asthenozoospermia and/or oligozoospermia have a higher incidence of low complement-inhibiting activity and a reduced level of complement regula-tory proteins, such as membrane cofactor protein (MCP, CD46) and decay accelerating factor (DAF, CD55) in their seminal plasma, such that nonspecific activation of the alternative complement pathway may occur, inflicting injury to sperm (4).
An immune reaction acting adversely against gametes is a mechanism that contributes to the development of "immunological infertility". Auto-antibodies against zona pellucida may cause infer-tility by blocking sperm-zona pellucida interaction(5, 6). These antibodies are found in approximately6% of infertile women with indeterminate etiology(6);whereas the incidence of infertility with asso-ciated antisperm antibodies is higher (approximately 18%).
The presence of antibodies in blood, follicular and uterine fluids and cervical mucus can impair reproductive processes that are crucial for a success-ful pregnancy. Since these antibodies appear not to cause any deleterious clinical symptoms and have yet be associated with infertility, sperm antigens are promising candidates in the development of an immunocontraceptive. Here, we review the signifi-cance of antisperm antibodies as a causative factor of infertility and their potential as immunogens in the development of a contraceptive vaccine.

ANTIFERTILITY EFFECT OF ANTISPERM ANTIBODY
Spermatozoa can elicit an immune response in women as an alloantigen and in men as an autoanti-gen. Antisperm antibodies may cause infertility by1) impairing sperm motility; 2) blocking penetrance of cervical mucus by spermatozoa; 3) interfering with sperm-egg interaction; 4) interfering with the implantation of blastocysts; or 5) inhibiting growth and development of embryos.

Impairment of sperm motility
Antibodies capable of agglutinating or immobilizing spermatozoa may impair their motility in the female reproductive tract, thereby preventing fertilization. However, to be effective biologically the titer of the agglutinating antibodies should be sufficiently high. In the case of immobilizing antibodies, high com-plement activity is required.

Blocking sperm penetrance of cervical mucus
Blockage of sperm penetrance of cervical mucus is a common cause of infertility. Fjallbrant (7) showed that the ability of antibody-bound sperm to penetrate normal cervical mucus and the capacity of active motile sperm to traverse cervical mucus containing antisperm antibody are markedly reduced or com-pletely abolished. Kremer and Jager (8) described the"shaking phenomenon"of antibody-bound sper-matozoa entrapped in cervical mucus. They hypoth-esized that the Fc portion of the antibodies interact nonspecifically with the glycoprotein micelles of the cervical mucus (9). A similar observation was made earlier by Isojima et al. (10). The shaking phenom-enon observed with spermatozoa found in cervical mucus is induced only by intact complete anti-bodies;whereas Fab fragments are inactive. These findings suggest that cervical mucus contains an Fc receptor-like component.
In analyzing cervical mucus for antisperm factors, we have isolated and identified a 15kDa protein that binds IgA, IgM and all subclasses of human IgG, determined by Western blot (11). The amino acid sequence of the N-terminus was found to be identical to secretory leukocyte protease inhibitor (SLPI). The capacity of SLPI to bind IgG was validated by Western blot using two recombinant SLPI preparations. SLPI has been considered to be an important factor in the defense against in-flammatory stimuli by the mucosal epithelial cells (12, 13). Thus SLPI has an important physiological role in the local cellular defense system. Moreover, it is also involved in the pathogenesis of immuno-logical infertility by entrapping sperm in cervical mucus.

Blocking of sperm-zona interaction
Naturally occurring antisperm antibodies, espe-cially sperm immobilizing antibodies can block in vitro sperm-zona interaction (Table1). We were the first to demonstrate that sperm immobilizing anti-bodies can block penetration of the sperm through the human zona pellucida (14). This observation has been confirmed subsequently by many investigators (15) and further substantiated by our clinical data(16). Although the definitive mechanism, whereby antisperm antibodies block sperm-zona interaction has not been elucidated, the capacity of antisperm antibodies to inhibit the acrosome reaction of sperm, an indispensable process for successful fertilization, is well established (15, 17)
It is noteworthy that the inhibition is reversible as shown in Fig. 1. Spermatozoa preincubated with antisperm antibody recover their ability to penetrate the zona pellucida upon subsequent washing in antibody-free medium (1). Recovery from the blocking effect of antisperm antibodies on the acrosome reaction is also observed after incubation of the antibody-bound sperm in antibody-free medium (17).
Benoff et al. (18) reported that antisperm anti-bodies inhibit the acrosome reaction and prevent capacitation by blocking the release of cholesterol from sperm membrane;thereby lowering the mem-brane fluidity. Finally, antibody-bound spermatozoa failed to express mannose-binding sites, essential for sperm-zona interaction (19, 20).

Inhibition of implantation of blastocyst and embryo growth
Since embryos are capable of expressing genes encoding some sperm antigens, specific antisperm antibodies can be embryotoxic and inhibit their growth (21, 22). We have previously identified the human sperm membrane component, YWK-II, as the target antigen of a monoclonal antibody (mAb) that agglutinates human sperm in vitro (23, 24).Passive immunization of pregnant female mice with anti-YWK-II mAb impeded the growth of the embryos (24). Furthermore, the antibody raised against the synthetic peptide, YAL-198, residue21-36 of the YWK-II polypeptide interfered with the growth of the mouse zygotes beyond the 2-cell stage (Takikawa, in preparation).
To date, reports on the cytotoxic effects of anti-sperm antibodies on embryonic growth have been restricted to studies with experimental animals. On the other hand, naturally occurring antisperm antibodies found in the sera of immunologically infertile women do not appear to inhibit embryo growth or implantation of blastocysts. The clinical data clearly show that high implantation rates and subsequently high pregnancy rates are achieved by in vitro fertilization and embryo transfer treat-ment of infertile women possessing sperm immobi-lizing antibodies in their circulation (13, 16).

MECHANISM OF ANTIBODY PRODUCTION AGAINST SPERM IN THE REPRODUCTIVE TRACT
Immunoglobulin binding factor family
Antisperm antibodies are rarely found in the sera of sexually active women who are constantly exposed to allogeneic sperm or in normal men who are in contact with autoantigenic sperm. This lack of antisperm antibodies production in fertile women and men suggests a possible suppression of immuno-reactivity against sperm and the existence of some factors that prevent an immunological response against sperm. Components of seminal plasma may play a role in the immunomodulating network of the female and male reproductive tracts (25).
We have identified in human seminal plasma a component that binds IgGs of several species and interacts with anti-Leu-11b monoclonal antibody (mAb) raised against FcγRIII/CD16 (26 -28). The protein is designated as "Immunoglobulin Binding Factor (IgBF)" This designation is based on the findings that it failed to interact with another mAb raised against FcγRIII/CD16 (3G8), showing that the interaction was not dependent on the presence of a specific antigenic determinant, but rather due to a common structure of immunoglobulins.
Three components having IgBF activity were separated by HPLC (Fig. 2) (29). The amino acid sequence of the major IgBF had structural identity to β-microseminoprotein (β-MSP)/prostatic secre-tory protein composed of 94amino acids (PSP94)and β-inhibin. Furthermore, identity in the structure and molecular mass of the major IgBF and β-MSP/PSP94 was confirmed by mass spectrometry. In addition a large IgBF and β-MSP consisting of 93amino acids were detected. The ability of β-MSP to bind human IgG and anti-Leu-11b antibody were demonstrated by Western blot analysis. Thus, this family of proteins is composed of at least three isoforms with β-MSP/PSP94 as the principal mem-ber, and should be designated as the IgBF family(30, 31).

Immunosuppressive activity of immunoglobulin binding factor
The in vitro effects of IgBF purified from human seminal plasma on mitogen-induced lymphocyte blastogenesis are depicted in Fig. 3. IgBF specifi-cally inhibited PWM-stimulated lymphocyte blasto-genesis (32), but did not influence PHA-stimulated blastogenesis and had a slightly inhibitory effect on Con A-stimulated blastogenesis. Furthermore, IgBF may not be directly involved with the immunosur-veillance system, since it does not influence natural killer cell activity, antibody-dependent cell-mediated cytotoxicity or complement activity (32). These findings support the hypothesis that seminal plas-ma IgBF suppresses B cell activation directly or represses the function of helper T cells, thereby preventing antibody production against sperm in the reproductive tracts.

Localization of immunoglobulin binding factor in the female reproductive tract
IgBF is a sperm-coating component. This con-tention is supported by the finding that anti-IgBF antibodies agglutinate and immobilize human sperm and is validated by its localization on the sperm surface by immunofluorescence staining (33).
Production of IgBF by cervical epithelial cells of the uterus was recently demonstrated by reverse transcription-polymerase chain reaction and in situ hybridization (Kamada, unpublished data) and high levels were determined in the cervical mucus by enzyme-linked immunosorbent assay (34).

Activation of immunoglobulin binding factor
IgBF exists in two forms:a 16kDa monomer under reducing condition and a27kDa homodimer under non-reducing condition (28). The two forms can be differentiated in that the 16 kDa form of IgBF binds IgG and anti-Leu-11b antibody;whereas the 27 kDa homodimer form does not (28). Further-more, the binding of IgG by the monomer is lost following carboxymethylation (28). In summary, native IgBF is present as the inactive dimer and is converted to the active monomer with free thiol groups required for the binding of IgG. To validate the above hypothesis that IgBF prevents the pro-duction of antisperm antibodies in vivo, the presence of an activating system capable of converting the native inactive dimer to the active form under physiological conditions has been demonstrated.
During the process of immunization with IgBF the dimer is transformed to the active monomer. This contention is supported by the finding that when rabbits are immunized with native IgBF, the raised antibodies interact predominantly with the active monomer rather than with the dimer (35, 36).
As shown in Fig. 4, native IgBF treated with re-duced glutathione (GSH) migrated as a 16kDa band in SDS-PAGE under non-reducing conditions and was found to react with anti-Leu-11b antibody and human IgG assayed by Western blotting (Fig. 4, lane e and f, respectively). Fig. 4 also shows that native IgBF treated with protein disulfide isomerase (PDI), a molecular chaperone, is converted to active monomers that bind anti-Leu-11b antibody (lane g) and human IgG (lane h) under non-reducing condi-tions. The present finding suggests that one of the activating systems of IgBF is PDI, which mediates the rearrangement of intramolecular disulfide bonds of IgBF. Auxiliary activation systems are the cleavage of IgBF by20S proteasomes and arginylendopepti-dase producing active fragments (37).
GSH and proteasome are widely distributed as cellular enzymes in vivo. Also we demonstrated the production of PDI mRNAs in the uterine cervix, endometrium and fallopian tube by reverse transcription-polymerase chain reaction (Fig. 5). It is important to note that all these activating systems are related to the immune network. Proteasome is a threonine protease produced abundantly in activated human T cells (38). It is reasonable to assume that allogeneic sperm in the female reproductive tract attract and activate the T cells to produce consider-able amounts of proteasomes. During the activation of macrophages, the intracellular GSH level increases greatly. Also the synthesis of DNA in mitogenically stimulated lymphocytes is accompanied by the generation of acid-soluble thiol as cysteine (39).Interferon-β, which is secreted by fibroblasts and white blood cells, e. g. macrophages, B cells and granulocytes, induces expression of PDI in fibro-blasts (40).

Suppression of antibody production against sperm by immunoglobulin binding factor and its breakdown
A schema depicting a hypothesis of antibody suppression by IgBF is presented in Fig. 6. Immuno-competent cells activated by their interaction with allogeneic sperm generate GSH and PDI or induce their production in uterine tissue. Thus, in the female genital tract, intracellular or secreted GSH, PDI and20S proteasome can activate IgBF, which in turn inhibit proliferation of B cells. The mechanism of the latter process is unclear.
A decrease in the secretion of IgBF by the cervical glands and/or a failure of the activation systems may result in a stimulation of antibody production. Inflammation such as cervicitis may decrease IgBF secretion, thereby promoting antibody formation. As a corollary finding, significantly high levels of antisperm antibodies are found in the sera of pro-stitutes, the majority of whom are afflicted with a variety of sexually transmitted diseases (41). Addi-tional factors such as cytokines may be involved in regulating this process, and need to be identified.

SPERM ANTIGENS AS AN IMMUNOCONTRA-CEPTIVE VACCINE rSMP-B and YWK-II
Several laboratories have identified sperm antigens and their encoding genes by using monoclonal anti-sperm antibodies (42) or naturally occurring antisperm antibodies found in the sera of immunologically infertile women (43). With the use of mAb, we have identified two sperm antigens;rabbit and human membrane proteins designated as rSMP-B and YWK-II, respectively. rSMP-B is an antigen located on the surface of the tail and midpiece of human sperm (44, 45). It has been tested as an antifertility immunogen in rats (44). The YWK-II protein was identified as the target antigen of an mAb raised against human sperm (23, 24). These two antigens appear to be involved in the fertilization process since both mAb blocked in vitro fertilization of zona-free hamster eggs by human sperm (45, 46). The cDNAs encoding rSMP and YWK-II proteins were isolated from the rat testis λgt11 expression library and found to contain open reading frames encoding polypeptides composed of 146 and 191 amino acids residues, respectively (23, 47).

ACTIVE IMMUNIZATION WITH SYNTHE-TIC PEPTIDES CORRESPONDING TO SEG-MENTS OF SPERM ANTIGENS
The use of peptides as a contraceptive vaccine offers distinct advantages since they can be synthe-sized readily and in sufficient amounts for biological testing. Also a region of the polypeptide specific to germ cells can be selected, thereby avoiding poten-tial unexpected immunological cross-reactivity of induced antibodies with somatic cells of the testis and other tissues. Goldberg and associates (48) suc-cessfully tested a synthetic peptide corresponding to the immunodominant B cell epitope of human sperm-specific lactate dehydrogenase (LDH-C4) as a contraceptive vaccine in female baboons. A 75%reduction in fertility was observed compared to the controls. We have attempted to develop an immuno-contraceptive by using small synthetic peptides whose sequences correspond to the hydrophilic extracellu-lar domain of the above two sperm antigens (Table 2). Three peptide segments corresponding to the extracellular domain were synthesized as multiple antigen peptides. Female rats were actively immu-nized with the synthetic peptides with MDP-P-T as an adjuvant (49). Only YAL-198, corresponding to residues 21-36 of the YWK-II polypeptide significant-ly (9/12) reduced fertility (49). By the same method, active immunization with rSMP-230, residues1-28of the rSMP-B protein, administered with complete Freunds'adjuvant induced infertility in 83% of treated female rats (50). As the next step, before testing in primates, female rats were immunized with the synthetic peptides without adjuvant. The peptides were conjugated to tetanus toxoid as a car-rier protein. Fertilization was reduced by 38%with YAL-198 and 45% with rSMP-230 as compared to the controls (51). It is noteworthy that antibody titers against each peptide were higher in the non-pregnant groups than in the pregnant groups.
Another approach is mucosal immunization using recombinant Salmonella dublin expressing YAL-198. The microorganism was administered orally or vagi-nally to female rats. Both routes of immunization induced significant levels of antibody titers in sera and vaginal secretions associated with infertility in60% (3/5) and 80% (4/5) of treated animals (Shan et al., in preparation).

CONCLUSION
Antisperm antibodies can cause infertility by blocking reproductive processes including sperm penetration of the cervical mucus and the acrosome reaction, which are essential steps for a successful fertilization. Although these antibodies disrupt sperm function, they appear not to be systematically det-rimental. These two tenets are the basis for the development of an immunocontraceptive using sperm antigens. Nonetheless, identification of sperm-specific antigens is crucial. Our recent studies show that synthetic peptides or recombinant proteins and the method of mucosal immunization are promising breakthroughs in the strategy of developing an effective antifertility vaccine. Moreover, identification of various steps and factors involved in regulating the production of antisperm antibodies may open new paths in the treatment of immunological in-fertility and at the same time may lead to a more effective immunocontraceptive.

ACKNOWLEDGEMENTS
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Educa-tion, Science, Sports and Culture, Japan, and by a grant from the Rockefeller Foundation.

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Received for publication December 22, 1998;accepted January 20, 1999.

Address correspondence and reprint requests to Masaharu Kamada, M.D., Ph.D., Department of Obstetrics and Gynecology, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-631-2630.