Sperm-zona pellucida interaction and immunological infertility

Immune reactions against gametes appear to be physiologically important for the maintenance of homeostasis in reproduction. In contrast, aberration of the immune homeostasis might give rise to ‘immunological infertility’. Antisperm antibodies cause infertility by blocking fertilization. The mechanism can be explained as inhibiting the acrosome reaction of sperm by their blocking effect on capacitation through inhibiting an increase of fluidity of the sperm membrane. Autoantibodies against zona pellucida also cause infertility by blocking sperm-zona pellucida interaction, though the definitive mechanism has not been elucidated. Pretreatment of spermatozoa with D-mannnose completely inhibited sperm penetration through, but not binding to, the zona pellucida. Furthermore, very rapid kinetics between sperm extracts and D-mannnose by a BIAcore apparatus suggest that a D-mannose ligand of the sperm surface is easy to bind to and dissociate from a D-mannose residue in the sperm receptor site on the zona pellucida. Thus, D-mannnose on the human zona pellucida might be an essential molecule acting as a second sperm receptor, through which sperm penetrate into the zona pellucida. Because these antibodies appear to not cause any deleterious clinical symptoms, sperm and zona pellucida antigens are promising candidates in the development of an immunocontraceptive.     

Inhibition of fertilization by a monoclonal antibody recognizing the oligosaccharide sequence GalNAc beta 1----4Gal beta 1----4 on the mouse zona pellucida.

Mouse eggs and pre-implantation stage embryos express on their surfaces a carbohydrate epitope, TEC-2, defined by an IgM monoclonal antibody, TEC-02. The TEC-2 epitope involves the oligosaccharide sequence GalNAc beta 1----4Gal beta 1----4 that is expressed on the plasma membrane and zona pellucida of mouse eggs and on a very limited number of other cell types. In this study we addressed the question whether or not the binding of TEC-02 antibody to the mouse eggs would interfere with their fertilization. Our data showed that the TEC-2 epitope is carried by two zona pellucida glycoproteins, ZP2 and ZP3. Binding of TEC-02 antibody to mouse eggs inhibited specifically and in a dose-dependent manner their fertilization in vitro. The inhibitory effect of TEC-02 antibody was dependent on the presence of an intact zona pellucida. Direct radioantibody binding assays indicated that the TEC-02 antibody completely inhibited fertilization at a concentration at which one quarter of all available TEC-2 binding sites was occupied. Binding of TEC-02 antibody to an egg did not interfere with initial attachment of the sperm to the egg but inhibited maintenance of sperm binding to the zona pellucida, the secondary binding. The combined data indicate that TEC-2, which is a well-defined zona pellucida specific carbohydrate epitope, might be a part of the secondary sperm receptor.     

Effects of antibodies to sperm surface fertilization antigen-1 on human sperm-zona pellucida interaction.

OBJECTIVE: To investigate the effects of antibodies to well-defined sperm surface antigens (the fertilization antigen [FA-1] and germ-cell antigen [GA-1]) and nuclear antigen (protamine) on human sperm-zona interaction. DESIGN: Number of total and acrosome-reacted human sperm bound to the human zona pellucida and the sperm movement characteristics assessed by computer-aided sperm analysis were evaluated after incubation of sperm with the antibodies. SETTING: Academic research environment approved by the Institute Review Board. PATIENTS: Human oocytes were obtained from ovaries removed at surgery. Semen from fertile donors was used in all assays. INTERVENTIONS: Human oocytes were stored in salt solution at -80 degrees C until used. Spermatozoa were treated with the antibodies to various sperm antigens. MAIN OUTCOME MEASURES: Total and acrosome-reacted sperm bound to zona pellucida and sperm movement characteristics were evaluated after 3 to 5 hours of incubation of the antibodies with human sperm. RESULTS: Anti-FA-1 antibodies significantly reduced human sperm fusion with zona-free hamster oocytes and sperm binding to the human zona pellucida but did not affect binding of acrosome-reacted sperm and sperm movement characteristics. Anti-GA-1 and antiprotamine antibodies did not affect sperm-oocyte interaction, acrosomal reaction, or sperm motility. CONCLUSIONS: Antibodies to FA-1 but not to GA-1 and protamine inhibit human sperm-zona interaction.     

Immunological approach to male contraception

Sperm surface antigens may induce an immune response in mammals. In humans, the presence of antisperm antibodies has been noted in the blood, seminal plasma, cervical mucus, and follicular liquid. Because they provoke immobilization and/or agglutination of sperm and a diminution of the rate of fertilization in vitro, these antibodies are believed to be a factor in some unexplained infertility, as for example after surgical reversal of vasectomy. These observations have led to research oriented toward development of a nonhormonal contraceptive method based on the immunological capacities of some sperm antigens. Possible secondary effects and the modes of action remain poorly understood. Antisperm immunoglobulins in the male should be induced by epitopes that are specific to sperm excluding the proteins of the sperm membrane, so that they will be without effect on functions other than fertilization. The epitopes should be located on the sperm surface and should be of post-testicular origin because of the risk of orchitis posed by testicular antigens. The antisperm immunoglobulins should also be present in the area surrounding he sperm in the male genital tract because of the limited permeability of the tract to immunoglobulins and other large molecules. The antisperm immunoglobulins should not be inhibited in the male by the immunosuppressive activities of the seminal plasma or in the female by capacitation. In vivo and in vitro observations indicate that various functions may be altered by antisperm antibodies. The antibodies may immobilize the sperm, block interaction with the oocyte at the level of the zona pellucida, block adherence to the vitelline membrane, or cause anomalies in embryonic development. In rats, isoimmunization against epididymal cofactor of the sperm zona pellucida receptor results in reduced sperm mobility and reduced fertility. The same study was conducted in 12 rams selected for homogeneity of sperm characteristics. Isoimmunization of rams induced transient asthenospermia. Most sperm antigens involved in fertilization are poorly understood. Observations in the ram suggest that embryonic mortality following isoimmunization should be studied.     

抗精子单克隆抗体抑制小鼠体外受精

抗人精子单克隆抗体YWKⅠ和YWK-Ⅱ及抗兔精子单克隆抗体rSMP-B均能显著抑制人精子穿入去透明带金黄地鼠卵。在本文的小鼠体外受精实验中,获能精子先与10倍稀释的抗精子单克隆抗体作用10min后再与卵子混合。结果表明:三种抗精子单克隆抗体对小鼠体外受精均有明显的抑制,其中YWM-Ⅰ使受精率下降至16.9%。此外还发现YWK-Ⅱ及rSMP-B主要抑制小鼠精子穿入透明带,而YWK-Ⅰ除此之外还抑制穿入透明带后的精子进一步完成受精。     

Generation of mouse oocyte monoclonal isoantibodies: their effects and those of antisperm monoclonal antibodies on in vitro fertilization

Monoclonal isoantibodies to mouse oocyte antigens were generated by modified hybridoma techniques similar to those described for mouse sperm monoclonals. Following isoimmunization with mouse oocytes and cell fusion, hybrid cells were cultured initially in a semi-solid medium containing methylcellulose. Seven to ten days after cell fusion about 350 hybrid clones were recovered for subculture. By an indirect immunofluorescence assay using frozen or fresh mouse oocytes, twenty hybridomas were shown to produce antibodies that bind to various oocyte components including antigens of the zona pellucida. However, they did not cross-react with mouse spermatozoa or lymphocytes.A system was established to evaluate whether monoclonal antibodies to gamete-specific antigens have any inhibitory effects on the fertilization of mouse oocytes in vitro. A monoclonal antibody against zona antigen(s), ME 56, was shown to block fertilization of mouse oocytes via the inhibition of sperm binding to the zona pellucida. On the other hand, three out of four antibodies reacting with mouse sperm acrosomes were also inhibitory to mouse in vitro fertilization, perhaps mainly due to the inhibition of sperm acrosomal reactions. Using a sodium dodecylsulfate gel/protein blot radioimmunobinding method, the molecular weight of zona antigen(s) that react with ME 56 was determined to be in the range of 95,000, whereas that of the acrosomal antigen(s) reacting with the fertilization-inhibiting antibody, MS 207, was about 30,000. The results of this preliminary study suggest that monoclonal antibodies to certain gamete antigens can be a valuable tool for the analysis of sperm-egg interactions during the fertilization processes.     

Studies on biological actions of sperm immobilizing antibody on human fertilization in vitro

Effects of sperm immobilizing antibodies on sperm penetration through human zonae pellucidae have been studied. Exposure of human spermatozoa obtained from fertile donors to seven serum samples with sperm immobilizing antibody impaired sperm penetration completely in six cases and incompletely in one case. During the course of treatment of a patient with circulating sperm immobilizing antibody by means of an in vitro fertilization and embryo transfer program, it was found that fertilization was completely blocked in the presence of the patient's serum, but three matured ova fertilized successfully when umbilical cord serum was used instead of autoserum from the patient. Furthermore, when spermatozoa were exposed to an IgG fraction of sera containing sperm immobilizing antibody, sperm binding and penetration were markedly inhibited. The spermatozoa, preincubated with sperm immobilizing antibody, showed penetrability across the zona pellucida. However, exposure of possibly capacitated sperm to the antibody completely blocked sperm binding to and penetration through the zona pellucida. These results suggest that sperm immobilizing antibodies cause infertility by preventina sperm binding to and penetration through the zona pellucida, possibly by interfering with the step of fertilization beyond sperm capacitation.     

Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.     

Biochemical and functional characterization of the human zona pellucida

A successful interaction between spermatozoa and the zona pellucida is critical for fertilization. This biological step reflects multiple sperm functions, including the acquisition and completion of capacitation, recognition and binding to specific zona pellucida receptors, and induction of the physiological acrosome reaction. The recognition of carbohydrate sequences by complimentary receptors has been demonstrated in gamete interaction in different animal species. It has been proposed that, in the human, sperm binding to the zona pellucida requires a 'selectin-like' interaction. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of human spermatozoa to the homologous zona pellucida) and advanced methods of carbohydrate analysis have been used to test this hypothesis. Compelling evidence exists to demonstrate that oligosaccharide recognition is also required for specific, tight human gamete binding. The induction of the acrosome reaction using the physiological inducers, i.e. the zona pellucida and progesterone, was also examined. It has also been demonstrated that there is a priming effect of the steroid on the acrosome reaction inducing capacity of the zona pellucida. These studies may allow for a better understanding of human gamete interaction in physiological and pathological situations.     

Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.     

Macrophage secretory products and sperm zona pellucida binding

OBJECTIVE: To determine if exposure of human gametes to macrophage secretory products reduces sperm binding to the zona pellucida, and to determine which cytokine(s) may be responsible for this effect. METHODS: A human macrophage cell line was cultured and either activated with lipopolysaccharide for 2 hours and then washed or left unactivated. Culture-conditioned media from activated or unactivated cells was used in hemizona assay. Hemizonae were incubated with sperm suspended in culture medium from either unactivated macrophages or activated macrophages, with the matching hemizona incubated with sperm suspended in control medium. Matching hemizonae were incubated with sperm suspended in unactivated macrophage medium paired with sperm suspended in activated macrophage culture medium. Conditioned medium from activated macrophages was found to have elevated levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, and transforming growth factor-beta, therefore, gametes were also exposed to these cytokines followed by the hemizona assay. After each incubation, the number of sperm tightly bound to the outer surface of each hemizona was determined. RESULTS: Exposure of gametes to activated and unactivated macrophage culture-conditioned media significantly decreases sperm binding to the zona pellucida, with medium from activated macrophages inducing the greatest effect (P < .05). Exposure of sperm to TNF-alpha significantly impaired sperm binding (P < .05), whereas other cytokines tested had no effect. CONCLUSION: These results suggest that macrophage secretory products in the basal and activated state may be a factor in endometriosis-associated infertility through the interference of sperm binding to the zona pellucida, and that TNF-alpha is a key cytokine responsible for this effect.     

Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization

The role of monosaccharides in human fertilization was studied by testing their effects on penetration of spermatozoa into mature human oocytes (zona penetration test). When oocytes were pretreated with concanavalin A, wheat germ agglutinin, or Ricinus communis agglutinin-I at a concentration of 100 micrograms/ml, no spermatozoa were found to bind to or penetrate through the zona pellucida. Penetration of spermatozoa was restored when the zona pellucida pretreated with wheat germ agglutinin and Ricinus communis agglutinin-I were rinsed with N-acetyl-D-glucosamine (wheat germ agglutinin inhibitor) and D-galactose (Ricinus communis agglutinin inhibitor), respectively. Conversely, the blocking effect of concanavalin A on sperm penetration was not reversed by treatment with D-mannose (concanavalin A inhibitor). Furthermore, pretreatment of spermatozoa with D-mannose (50 mmol/L) completely inhibited sperm penetration through the zona pellucida. However, sperm penetration was clearly demonstrated when the zona pellucida was pretreated with D-mannose. These data suggest that D-mannose residues are essential in, or sterically closely related to, the sperm receptor site on the human zona pellucida.     

Inhibition of human spermatozoa-zona pellucida binding by a combinatorially derived peptide from a synthetic target

Intact zona-free human oocytes were screened using a combinatorial peptide library selection protocol. Pieczenik Peptide Sequence 1 (PPS1) HEHRKRG binds human spermatozoa. A complementary and unique binding sequence HNSSLSPLATPA (PPS2) was developed from the first PPS1 ligand that binds to the human zona pellucida or oolemma. Cytoplasm-free zonae from unfertilized eggs were obtained and used as an assay system to test the effects of exposure to these two ligands. Spermatozoa were inserted into evacuated zonae and their behaviour and binding activity were assessed at regular intervals. The behaviour of spermatozoa exposed to PPS1 and unlabelled spermatozoa injected into unexposed zonae was similar as far as binding was concerned (50 and 54% binding), but PPS1 exposed spermatozoa had higher motility and displacement, marked by their escape from the zona pellucida. Zonae exposed to PPS2 inhibited the interaction between injected spermatozoa and the inside of the zona when compared with controls (8.3 and 53.8% attached respectively, P < 0.001). The sperm-zona pellucida interaction described in this paper is applied as a functional assay for molecular interactions of sperm binding and can be used to assess function for potential surface markers on gametes. It is shown here that a unique binding ligand (PPS2) can be synthesized from another complimentary ligand (PPS1) without the need for a known intermediate substrate. PPS1 and PPS2 may have properties that can be used to target processes involved in conception and assisted reproduction. A movie sequence taken approximately 30 min after injection of spermatozoa into empty human zonae pellucidae shows behaviour of non-manipulated spermatozoa into zonae not exposed or exposed to ligand. This may be purchased for viewing on the Internet at www.rbmonline.com/Article/2159 (free to web subscribers).     

Immunocontraception

The immunological contraceptive methods whose development is described in this work appear to inhibit the action of antigenic molecules necessary for fertilization. Antigens in the gametes or their envelopes that intervene in reconnaissance or fusion of the gametes appear to be more promising targets than those at the level of gamete production in the gonads. Clinical examples show that infertility may be spontaneously acquired in both sexes through active immunization. Contraceptive action can only be sought if the gametes carry specific antigens, so that other physiological functions will not be disturbed, and if the antigens play a determining role in fertilization. Research currently is oriented toward 3 complementary targets, the sperm and the 2 envelopes of the oocyte, the zona pellucida and the cumulus oophorus. Possibilities appear to exist of preventing the intervention of several different molecules in fertilization, although most of them are still poorly understood. In the past 10 years, various monoclonal antibodies have been produced against sperm of different animal species, some of which are capable of inhibiting fertilization in the same species and also in human beings. But in vivo effects of these antibodies have not been valuated, even in the same species. Active immunization of male or female guinea pigs with a sperm surface antigen has led to sterility, through inhibition of attachment of sperm to the zona pellucida, but the monoclonal antibody against the protein does not recognize the human sperm. Polyclonal antibodies against the same protein might be possible for human contraception. The biochemical and physiological study of monoclonal antibodies against human sperm is facilitated if the antibodies cross with rodent sperm. 2 such antibodies directed against proteins secreted by the human testicles are capable of inhibiting murine and human fertilization in vitro. Attempts to achieve active or passive immunization by targeting antigens of the zona pellucida have been underway for 2 decades in different animal species using ever more selective antigenic material. But in vivo animal studies caused serious ovarian disorders that would be unacceptable in contraception. A polyclonal antibody against the intercellular matrix of the human cumulus oophorus is capable of inhibiting fertilization in vitro, with the action resulting from a strong reduction in the number of sperm attached to the zona pellucida. Numerous aspects of immunocontraception are still at the research stage. Apart from the choice of the moist appropriate antigens, active immunization in human subjects must be preceded by massive production of purified antigens. Research is needed on the adjuvant, the possibility of maintaining high levels of antibodies, and the return of fertility. Despite the obvious public health need, few laboratories are engaged in this type of research.     

Nature of the inhibitory effect of complex saccharide moieties on the tight binding of human spermatozoa to the human zona pellucida.

Fucoidin and heparin sulfate inhibit binding of human sperm to the human zona pellucida under hemizona assay (HZA) conditions. Here we used the HZA to further assess tight sperm binding with/without preincubation of the sperm with other sulfated and nonsulfated glycoconjugates and charged polymers. Fucoidin significantly inhibited binding compared with controls (greater than 75% inhibition), even if sperm were washed after preincubation with the saccharide. Dextran sulfate also produced significant inhibition, although to a lesser extent (54% inhibition). Chondroitin sulfates A and B, heparin, and dextran did not affect binding. Sodium sulfate and polyglutamic acid did not affect HZA results; polyphosphates produced only moderate inhibition. The potent inhibitory effect of the sulfated carbohydrates fucoidin and dextran is probably competitive (receptor-ligand type) in nature. However, the lack of significant effects of simple charged molecules (nonspecific effects) suggests that the degree of sulfation (charge) may not be crucial to its inhibitory action.     

Horse and marmoset monkey sperm bind to the zona pellucida of salt-stored human oocytes

The present study demonstrates that horse and marmoset monkey sperm can bind to the human zona of salt-stored oocytes that failed to fertilize in vitro. Marmoset monkey sperm are also able to penetrate the salt-stored human zona. In contrast, human sperm do not bind to the zona of either horse or marmoset monkey oocytes. These results suggest that human sperm binding to the zona pellucida is more strictly species-specific than it is for horse and marmoset monkey sperm. In contrast, horse and marmoset monkey sperm contain receptors recognized by the human zona.     

Defining bioassay conditions to evaluate sperm/zona interaction: Inhibition of zona binding mediated by solubilized human zona pellucida

Objective: Our goal was to study the influence of solubilized human zonae pellucidae on zona binding potential and acrosome reaction. Materials and Methods: Zona pellucida (ZP) solutions were prepared by dissolving zona in acidic buffer, NaH₂PO₄ (pH 2.5), to obtain 0.1 and 0.5 zona pellucida/µl. Zona binding capacity was evaluated by the addition of oocytes (10-fold) to sperm/zona pellucida solution droplets. The number of sperm bound to each oocyte was recorded. Zona pellucida-mediated acrosome activity was evaluated after 60 min of coincubation of sperm and 0.5 ZP/µl. Results: The mean (±SE) number of sperm bound for control, 0.1 ZP/µl, and 0.5 ZP/µl was 181.2±12, 79.6±5, and 38.8±3, respectively. Zona pellucida-exposed sperm populations showed significant more acrosome-reacted sperm compared to control sperm, namely, 78 versus 32%, respectively (P=0.001). Conclusions: The observed zona binding inhibition might be ascribed to zona receptor blocking on the sperm surface or the inability of acrosome-reacted sperm to bind to the zona pellucida.     

Antagonistic and agonistic properties of saccharide moieties in the hemizona assay

The hemizona assay (HZA) was used to evaluate the effects of different sugars on human sperm-zona pellucida interactions. In each controlled assay, sperm binding from men of known fertility with/without preincubation with different carbohydrate constituents was compared. We used human prophase I oocytes obtained from surgically removed ovaries; these oocytes were stored in salt solution and later bisected by micromanipulation. Preincubation of sperm (1 hour) with fucoidin (1.0 mg/mL) significantly inhibited binding by more than 85% as compared with controls. Similarly, lesser fucoidin concentrations of 0.5, 0.25, and 0.01 mg/mL were markedly inhibitory. In addition, preincubation times of 60, 30, and 15 minutes were not different in their degree of inhibitory effect. On the other hand, preincubation of the hemizonae with fucoidin before HZA inhibited binding by 43% compared with controls. Here, the fucoidin effect was significantly less inhibitory than the sperm preincubation effect of fucoidin. Preincubation of sperm with d-fucose had no measurable effect on binding, whereas d-glucose (1.0 mg/mL) and 1-fucose (1.0 mg/mL) increased binding. These results indicate: (1) that the HZA provides a unique internally controlled homologous bioassay to evaluate the effects of simple and complex carbohydrates on sperm zona interactions; (2) that fucoidin is an immediate (less than 15 minutes) inhibitor of human sperm binding to the human zona pellucida under HZA conditions.     

The cellular immune response to immunization with zona pellucida antigens.

The cellular immune response of mice to porcine and rat zona pellucida and cynomolgus macaques to porcine zona pellucida antigens was evaluated. Mice mounted a vigorous cellular response to both antigens, as determined by the T cell proliferation response in vitro. There was poor cross-reactivity to murine zonae by T cells or serum antibodies from mice immunized with rat zona pellucida. Nevertheless, ovaries from the mice immunized with rat zona had significantly fewer antral follicles than adjuvant-treated controls, suggesting that the immune response to the zona antigen disrupted follicular development. T cells from two macaques that had been immunized with porcine zona pelludica proteins proliferated in vitro in response to this antigen. Both macaques also had strong antibody responses. The patterns of urinary steroid metabolites in these animals provided clear evidence of ovarian malfunction following immunization. The data indicate that a significant cellular immune response is generated upon immunization of animals with zona pellucida antigens regardless of whether the antigens are cross reactive with the host zona antigens. Whether impaired ovarian function and follicular development are related to the cellular response must be determined in future studies.