Fertilization Antigen Research Articles

Influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency

Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends: a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 (HE2), fertilization antigen-1 (FA-1), clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency (P<0.05). Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent. Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.

Do men with prostate abnormalities (prostatitis/benign prostatic hyperplasia/prostate cancer) develop immunity to spermatozoa or seminal plasma?

Prostate is an immunocompetent and not an immunoprivileged organ. It has an active immunologic armamentarium. There are three major prostate abnormalities namely, prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer. In all these abnormalities, infection/inflammation has been implicated. As infection/inflammation of the male genital tract can also be involved in induction of antisperm antibodies (ASA), this study was conducted to examine if these prostate abnormalities lead to the formation of ASA. Sera were obtained from normal healthy men (n = 20), men with chronic prostatitis (n = 20), men with BPH (n = 25), men with prostate cancer (n = 25) and immunoinfertile men (n = 10). The presence of antisperm antibodies against lithium diiodosalicylate (LIS)-solubilized human sperm extract (HSE), seminal plasma and synthetic peptides based upon sperm-specific antigens namely fertilization antigen (FA-1) and YLP(12), were analysed using the sperm immobilization technique (SIT), tray agglutination technique (TAT), enzyme-linked immunosorbent assay (ELISA) and indirect immunobead binding technique (IBT). All the sera from normal men and men with prostate abnormalities (chronic prostatitis/BPH/prostate cancer) were found to be negative in SIT and TAT. In ELISA, a few sera from men having prostate abnormalities (4-24%) showed a weak positive immunoreactivity (2-3 SD units) with some of the spermatozoa/seminal plasma antigens. Majority of the samples did not show any immunoreactivity (<2 SD units) in ELISA. Even the samples that showed a weak positive immunoreactivity in ELISA did not bind to live human sperm in IBT, indicating lack of sperm binding antibodies in these sera. In all these assays, the sera from immunoinfertile men were positive. Our findings indicate that chronic prostatitis, BPH and prostate cancer do not induce antibodies to spermatozoa, sperm-specific antigens and seminal plasma components. Although prostate is an immunologically competent organ, and its abnormalities cause a rise in circulating prostate-specific antigen (PSA), it appears that there is no concomitant induction of immunity to spermatozoa/seminal components including sperm-specific fertility-related antigens, thus not causing ASA-induced immunoinfertlity. This is the first study to our knowledge reporting the absence of ASA in men with BPH and prostate cancer.

ORIGINAL ARTICLE: Presence of Antisperm Antibodies Reactive with Peptide Epitopes of FA‐1 and YLP12 in Sera of Immunoinfertile Women

ProblemRecent studies in several laboratories are focused on delineating sperm antigens that are relevant to fertility and examining involvement of antibodies to these antigens in human immunoinfertility. Our laboratory has characterized two such antigens, namely fertilization antigen (FA‐1) and YLP12 dodecamer sequence that are involved in sperm‐oocyte binding. The present study was conducted to examine the occurrence of isoantibodies to various peptide epitopes of human and murine FA‐1 antigen and YLP12 peptide in sera of immunoinfertile and fertile women.Method of studySera from 67 immunoinfertile and 19 fertile women were collected. Various peptides based up on human and murine FA‐1 antigen and YLP12 were synthesized, and examined for immunoreactivity with these sera by using ELISA. Four immunodominant sequences, two each from human (hFA‐182‐97aa and hFA‐1200‐219aa) and mouse (mFA‐12‐19aa and mFA‐1117‐136aa) FA‐1 antigen, were selected for the present study. Another human FA‐1 sequence, hFA‐1220‐240aa, that was not in the immunodominant region was used as a control.ResultsFor human FA‐1 peptides, 41.8% of the immunoinfertile sera reacted positively (≥2 SD units) with hFA‐182‐97aa, 24.6% (16/65) with hFA‐1200‐219aa, and 3% (2/66) with hFA‐1220‐240aa peptide. For two murine FA‐1 peptides, 41.7% (25/60) of the immunoinfertile sera reacted positively with mFA‐12‐19aa, and 41.5% (27/65) with mFA‐1117‐136aa peptide. For the YLP12 dodecamer peptide, 43.3% (29/67) of the immunoinfertile sera reacted positively. None of the sera from fertile women reacted positively with any of these peptides.ConclusionIn conclusion, our data indicate that the immunoinfertile women have circulating isoantibodies against at least two immunodominant peptide epitopes of human and murine FA‐1 antigen and YLP12 peptide sequence. These peptides may find clinical application in the specific diagnosis and treatment of female infertility and contraceptive vaccine development.

Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development

Contraceptive vaccines can provide valuable alternatives to current methods of contraception. We describe here the development of sperm-reactive human single chain variable fragment (scFv) antibodies of defined sperm specificity for immunocontraception. Peripheral blood leukocytes (PBL) from antisperm antibody-positive immunoinfertile and vasectomized men were activated with human sperm antigens in vitro, and the complementary DNA prepared and PCR-amplified using primers based on all the variable regions of heavy and light chains of immunoglobulins. The scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. Panning of the library against specific sperm antigens yielded several clones, and the four strongest reactive were selected for further analysis. These clones had novel sequences with unique complementarity-determining regions. ScFv antibodies were expressed, purified and analyzed for human sperm reactivity and effect on human sperm function. AFA-1 and FAB-7 scFv antibodies both reacted with fertilization antigen-1 antigen, but against different epitopes. YLP20 antibody reacted with the expected human sperm protein of 48 +/- 5 kDa. The fourth antibody, AS16, reacted with an 18 kDa sperm protein and seems to be a human homologue of the mouse monoclonal recombinant antisperm antibody that causes sperm agglutination. All these antibodies inhibited human sperm function. This is the first study to report the use of phage display technology to obtain antisperm scFv antibodies of defined antigen specificity. These antibodies will find clinical applications in the development of novel immunocontraceptives, and specific diagnostics for immunoinfertility.

Immunocontraceptive effect of Izumo and enhancement by combination vaccination

Sperm proteins are being investigated for their applications in the development of contraceptive vaccines (CV) in several laboratories. In the present study, various synthetic peptides based upon four sperm proteins, namely Izumo, fertilization antigen-1 (FA-1), YLP(12), and SP56 that are involved in various steps of the fertilization cascade were examined for their immunocontraceptive effect. The synthetic peptides were conjugated to four carrier proteins namely keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken gamma-globulin, and chicken ovalbumin. Female mice were immunized with various peptide vaccines and each booster injection was given with the peptide conjugated to a different carrier protein. Two different fertility trials with different doses of the peptide vaccines were conducted to examine the contraceptive effect. Injection of 150 microg of the peptides (Trial II) caused a significantly higher immune response in serum as well as in the vaginal tract causing enhanced contraceptive effect than those observed after injection with 75 microg of the peptides (Trial I). Immunization with the peptide vaccines based upon Izumo, which is involved in sperm-egg plasma membrane fusion, caused a significant reduction in fertility. The contraceptive effect was enhanced by immunizing with the peptides based upon other antigens (FA-1, YLP(12), and SP56), resulting in an overall 73.33% reduction in fertility. When the antibodies against the peptides disappeared after >9-10 months from circulation and genital tract, all the animals regained fertility. These findings indicate for the first time that the immunization with Izumo and other sperm peptides namely FA-1, YLP(12), and SP56 induces antibodies in serum and genital tract that cause a reversible long-term contraceptive effect in female mice. The data further indicate that the proteins involved in sperm-egg fusion can also be used for contraceptive vaccine development. The contraceptive effects are enhanced by immunizing with the multipeptide vaccines.

Effect of fertilization antigen (FA‐1) DNA vaccine on fertility of female mice

Vaccination of female mice with recombinant fertilization antigen (FA-1) causes a long-term reversible contraceptive effect. Also, a DNA vaccine based upon a dodecamer sequence YLP(12) present in sperm causes a reduction in fertility. In the present study, the effects of FA-1 DNA vaccine alone, and FA-1 and YLP(12) DNA vaccines together were examined. FA-1 495-bp DNA was cloned into pVAX1 vector to prepare the DNA vaccine. Four groups of female mice were immunized intradermally by using a gene gun with FA-1 DNA, FA-1 DNA + YLP(12) DNA, FA-1 DNA + YLP(12) DNA mixed with exogenous synthetic CpG oliogodeoxynucleotide (ODN), or vector DNA alone, respectively. Vaccination with all three formulations caused a significant reduction in fertility, with FA-1 DNA + YLP(12) DNA mixed with exogenous synthetic CpG ODN showing the highest reduction. Vaccination with all three formulations raised antibody response in both the sera as well as locally in the vaginal tract, with ODN mixed group demonstrating the highest titers. There was no antibody response in the mice injected with the vector alone. In sera, the highest titers were obtained for the IgG class for all vaccine formulations followed by the IgA class. In vaginal washing, the highest titers were obtained by the IgA class followed by the IgG class. Within the IgG class, the titers for the IgG2a subclass were significantly greater than the IgG1 subclass. The immunocontraceptive effects were long-lasting over 1 year of the observation period and increased with time. These novel findings indicate that the intradermal immunization with a sperm-specific FA-1 DNA vaccine causes a long-term circulating and local immune response resulting in immunocontraceptive effects in female mice. The anti-fertility effects were enhanced when FA-1 DNA vaccine was combined with YLP(12) DNA vaccine and injected with ODN.

Molecular and immunological characteristics of sperm antigens involved in egg binding

Development of a vaccine(s) based on sperm antigens represents a promising approach to contraception. The utility of an antigen in immunocontraception is contingent upon sperm/testis-specificity and its involvement in the fertilization process. Since, the sperm–zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, molecules involved at this site are attractive candidates for immunocontraception. Using hybridoma technology, subtractive hybridization, and differential display technology, our laboratory has delineated several sperm antigens. These antigens have testis-specific expression and have a role in the fertilization process. The cDNAs encoding for the antigens have been cloned and sequenced. Among these, the fertilization antigen (FA-1) is particularly interesting, because it is involved in immuno-infertility in humans. Using the phage peptide display technique, a novel dodecamer sequence of a ∼72±5 kD antigen, designated as YLP 12, that is testis-specific and involved in human sperm-ZP recognition/binding, was identified. A synthetic 12-mer peptide was generated based on this sequence. In the hemizona assay, YLP 12 peptide and its monovalent Fab′ antibodies specifically and significantly inhibited human sperm-ZP binding. Furthermore, the presence of specific antibodies reactive with YLP 12 peptide, were identified in the serum and seminal plasma of immuno-infertile men. Thus, FA-1 and YLP 12 are promising target antigens for the development of contraceptive vaccines as well as for specific diagnosis and treatment of male infertility.

Fertilization-related sperm antigens and their immunocontraceptive potentials.

Vaccine(s) for contraception will provide an attractive alternative to contraception. There has been a renewed interest in defining fertilization-related sperm antigens that can be used for the development of a contraceptive vaccine. The sperm-zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, and the molecules involved at this site are attractive candidates for immunocontraception. Extensive research indicates that there are four sperm surface proteins that bind to oocyte ZP3 in humans and they belong to the four molecular regions of 95, 63, 51, and 14-18 kDa, respectively. Our laboratory is actively engaged in cloning and sequencing of cDNAs encoding for these sperm proteins and investigating the applications of recombinant (r) proteins in immunocontraception and infertility. Presently, the cDNAs encoding for the 14-18-kDa proteins, designated the NZ-1 and NZ-2, respectively, and the 51-kDa protein, designated fertilization antigen, have been cloned and sequenced. Active immunization with the rFA-1 antigen caused a reversible block/inhibition in fertility of female mice by raising a sperm/testis-specific immune response. FA-1 antigen is also involved in human immunoinfertility, and a recent clinical trial indicates that it will have a clinical application in the treatment of immunoinfertile men. The long-term goal is to employ various recombinant fertilization antigens and/or their bioeffective peptide epitopes in a single formulation, to generate an anti-sperm vaccine that will be strongly immunogenic and highly efficacious for regulation of fertility.

Vaccine for contraception targeting sperm.

Development of a vaccine(s) based on sperm antigens represents a promising approach to contraception. The utility of a sperm antigen in immunocontraception is contingent upon its tissue specificity, involvement in fertility and on raising high antibody titer, especially locally in the genital tract, that is capable of inducing reversible infertility. Several sperm antigens, such as lactate dehydrogenase C4, PH-20, sperm protein (SP)-10, fertilization antigen (FA)-1, FA-2, cleavage signal (CS)-1, NZ-1, and NZ-2 have been proposed as potential candidates for the vaccine development. Spermzona pellucida (ZP) binding is a pivotal tissue- and mostly species-specific event in the fertilization process, and the molecules involved in this site constitute the most exciting candidates for immuno-contraception. FA-1 is a sperm-specific glycoprotein having receptor activity for ZP recognition and binding. Complementary DNA encoding for FA-1 antigen has been cloned and sequenced. Active immunization of animals with recombinant FA-1 antigen causes a long-lasting reversible inhibition in fertility by raising a sperm-specific immune response. This antigen is also involved in human immunoinfertility. The exciting findings from the recent trial in immunoinfertile couples indicate that the FA-1 antigen may have clinical application in the treatment of male infertility. A vaccine having most appropriate tissue-specific and effective recombinant and/or synthetic epitopes of various sperm antigens, such as the FA-1 antigen, in a single formulation may provide a highly immunogenic and efficacious antisperm vaccine for contraception. The advances made during the last 5 years suggest that it may be a realistic proposition.

Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.

Fertilization antigen-1 removes antisperm autoantibodies from spermatozoa of infertile men and results in increased rates of acrosome reaction

Objective: To determine if fertilization antigen (FA)-1 will remove autoantibodies from the surface of sperm cells of immunoinfertile men by immune adsorption and permit an increased acrosome reaction (AR). Design: Prospective analytic study. Setting: University medical center. Patient(s): Men from 18 infertile couples with autoantibodies present on their spermatozoa. Intervention(s): Sperm samples after processing were examined for antibody binding and AR before and after adsorption with control medium or FA-1. Main Outcome Measure(s): Sperm-bound antibody was assessed by the immunobead assay (immunoglobulin [Ig] A and IgG) and the AR by induction with ionophore A23187. Result(s): Adsorption with FA-1 compared with control medium increased immunobead-free swimming sperm an average of 50% and 76% for IgA and IgG antisperm antibodies, respectively, with 78% and 100% of the 18 semen specimens increasing significantly. The AR rate increased an average of 10.3% compared with control medium and showed improvement in 78% of the sperm samples after FA-1 adsorption. Conclusion(s): The FA-1 sperm antigen appears to significantly free sperm cells coated with autoantibodies in the semen of most infertile men examined. Reducing sperm-bound antibodies that inhibited the AR allowed the sperm cells to undergo successful AR induction by calcium ionophore.

Recombinant fertilization antigen-1 causes a contraceptive effect in actively immunized mice.

Recombinant (r) fertilization antigen (FA)-1 was investigated for its immunocontraceptive effect using the mouse as a model. Active immunization with the murine rFA-1 antigen raised high antibody titers in all the immunized mice (n = 16 in two trials); these titers were long lasting and reached preimmunization levels by the 255th day. There was a significant (p < 0.0001) effect (64% reduction in trial I and 70% reduction in trial II) in fertility of immunized animals compared to PBS-control animals (n = 22 in two trials). The effect on fertility was reversible. When the antibody titers declined to control levels, all the animals conceived and delivered healthy babies without a significant (p > 0.05) effect on the litter size compared to that of controls. There was a significant (p = 0.025) correlation (r = 0.76) between the reduction in fertility and the circulating rFA-1 antibody titers. Anti-rFA-1 antibodies from immunized mice, and not the immunoglobulins from the PBS-control mice, significantly (p < 0.001) blocked murine sperm binding to zona pellucida and in vitro fertilization of murine oocytes. In a Western blot procedure, the anti-rFA-1 antibodies specifically recognized the protein band of approximately 47 kDa (dimeric form of cognate FA-1 antigen) only in the protein extract of testes and not in the extracts of somatic tissues tested, namely kidney, liver, intestine, spleen, muscle, heart, lung, brain, and ovary. In conclusion, our data indicate that active immunization with rFA-1 antigen induces a strong and sperm/testis-specific antibody response that causes a reversible inhibition of fertility by affecting sperm-zona binding and the fertilization process. These findings suggest that rFA-1 antigen is an exciting candidate for the development of a contraceptive vaccine.

Fertilization antigen-1: cDNA cloning, testis-specific expression, and immunocontraceptive effects.

cDNA encoding for a sperm antigen, designated fertilization antigen (FA-1), was cloned and sequenced from murine testis cDNA-lambdagt11 expression library using FA-1 mAb. Computer-generated translation analysis of 649-bp cDNA yielded an ORF of 164 amino acids with the first ATG Met start codon at nucleotide 81 and the stop codon TAA at nucleotide 577 and a polyadenylylation tail following the stop codon. The translated protein has a calculated molecular mass of 18.2 kDa and a potential N-linked glycosylation site at amino acids 158-160, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced amino acid sequence indicated it to be a membrane-anchored peptide. Extensive computer search in the GenBank, National Biomedical Research Foundation, and Swiss sequence banks did not identify any known nucleotide/amino acid sequence having homology with FA-1 cDNA or deduced amino acids, indicating it to be a novel protein. Northern blot analysis and reverse transcription-PCR indicated testis-specific expression of FA-1 antigen. The FA-1 cDNA was subcloned into pGEX-2T vector and expressed in glutathione S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with ZP3 of oocyte zona pellucida and its affinity-purified antibodies completely blocked sperm-zona pellucida interaction in mice. These findings suggest that the sperm-specific recombinant FA-1 antigen is an attractive candidate for the development of a contraceptive vaccine.

Fertilization antigen (FA-1) completely blocks human sperm binding to human zona pellucida: FA-1 antigen may be a sperm receptor for zona pellucida in humans

The effects of purified human sperm fertilization antigen-1 (FA-1), affinity-purified monoclonal Fab' antibody to FA-1, and monoclonal Fab' antibody to phosphotyrosine residues on human sperm-zona interaction were investigated. The purified FA-1 antigen completely blocked sperm binding to zona pellucida ( P < 0.0001). Also, the monoclonal Fab' antibodies to FA-1 antigen and phosphotyrosine residues significantly ( P < 0.05) reduced sperm-zona pellucida interaction. Effects were observed when FA-1 antigen was preincubated with zonae pellucidae and the antibodies were preincubated with sperm before insemination and not vice versa. These results suggest that the tyrosine phosphorylation especially of FA-1 antigen has an important role in zona pellucida receptor recognition and binding. These findings also suggest that FA-1 antigen may be the sperm receptor involved in zona pellucida binding in humans.

Monoclonal antibody to human fertilization antigen-1 (FA-1) inhibits bovine fertilization in vitro: application in immunocontraception.

A monoclonal antibody (mAb) to the human sperm plasma membrane protein, fertilization antigen-1 (FA-1), was tested for its reactivity with bovine spermatozoa and its effects on bovine fertilization in vitro. Western blot analysis revealed that the FA-1 mAb reacted with proteins of similar molecular mass (53 +/- 2 kDa) in human and bovine sodium deoxycholate (DOC)-solubilized sperm extracts. Indirect immunofluorescence, using epifluorescence microscopy and laser scanning confocal microscopy, revealed that the FA-1 antigen is present in the post-acrosomal region of bovine spermatozoa, which is similar to human FA-1 localization. In bovine in vitro fertilization (IVF) trials, using oocytes obtained from slaughterhouse ovaries, addition of 20, 40, or 80 micrograms/ml of FA-1 mAb to the IVF medium resulted in a linear decrease in the fertilization rate from 86.3% in the controls to 54.6%, 21.6%, and 1.8% in the respective experimental groups (p < 0.01). There was no inhibitory effect (p > 0.10) of the FA-1 mAb on percent sperm motility or other motility characteristics tested, suggesting that human FA-1 mAb inhibits bovine sperm cell function at some point after capacitation. In conclusion, the evolutionarily conserved antigen FA-1 has a molecular identity in bovine sperm similar to that in human sperm, and mAb to human sperm FA-1 inhibits fertilization of bovine oocytes. These results indicate that FA-1 is a promising candidate for the development of a contraceptive vaccine. The research also suggests that bovine species could be used as a model for investigating the use of FA-1 as an immunovaccine in ruminants.

Significance of antibodies against human sperm FA-1 antigen in immunoinfertility.

A fertilization antigen (FA-1) has been identified on human sperm cells that is involved in human fertilization. To better explain the participation of FA-1 in human infertility, the relationship of anti-FA-1 antibodies with the presence of general antisperm antibodies and with sperm function was examined in the sera of 30 men and women. None of the sera from fertile men and women (n = 10) reacted with FA-1. In contrast, 55% of the immunoinfertile sera (n = 20) that were positive for antisperm antibodies detected by OPTI-BEAD (immune-labeled microsphere) tested positive with FA-1 (p < .05). In these sera, penetration of hamster oocytes was reduced and was inversely related to monospecific anti-FA-1 absorbance values determined by an ELISA and with heterospecific general antisperm antibody binding to sperm in the OPTI-BEAD test (p < .05). Anti-FA-1 activity approached significance with antisperm antibody binding to the sperm head (p = .052). It is concluded that immunoinfertile sera have antibodies reacting with the FA-1 sperm antigen and their presence is significantly related to results of other functional tests, such as the SPA and the OPTI-BEAD test. The association of anti-FA-1 antibodies with antibody binding site (sperm head) may be relevant to the diagnosis and treatment of immunoinfertility.

Immunoglobulin (Ig) G, IgA, and IgA subclass antibodies against fertilization antigen-1 in cervical secretions and sera of women of infertile couples

To assess the occurrence of immunoglobulin (Ig) G, IgA, and IgA subclass antibodies against human sperm fertilization antigen-1 (FA-1) in cervical mucus (CM) and serum of women of infertile couples. Enzyme-linked immunosorbent assay methodology was used to detect anti-FA-1 antibodies. Antisperm antibodies were detected by agglutinating, immobilizing, and indirect immunobead (IB) methods. Control samples for the ELISA were from 10 women negative in the antisperm antibody assays. Samples were from women of 32 infertile couples undergoing antisperm antibody analysis. One of 10 control CM samples was slightly positive for IgG anti-FA-1 and none for IgA. Of the 22 CM samples from antisperm antibody-positive women, 9 were positive for IgG antibodies, 9 for IgA, 7 for IgA1, and 6 for IgA2. Cervical mucus samples from eight women were positive for both IgA and IgG antibodies. Assay of 19 serum samples, including 8 controls, by ELISA, indicated 9 of 11 from antisperm antibody-positive women and none from controls were positive for IgA and IgG (7 of 9 identical women). In addition, of the nine IgA-positive sera, seven were of the A1 subclass and five were of the A2 subclass. Positive IB assays occurred more frequently in CM and serum samples positive for anti-FA-1 antibodies than in negative samples. The results suggest that cervical secretions and sera of antisperm antibody-positive women contain IgA and IgG antibodies against sperm antigen FA-1 that may be involved in antifertility effects.

The fertilization antigen-1 does not have proteolytic/acrosin activity, but its monoclonal antibody inhibits sperm capacitation and acrosome reaction

To determine if human sperm surface fertilization antigen exhibits proteolytic or acrosin activity and to investigate the mechanism(s) whereby monoclonal antibody (mAb) to fertilization antigen inhibits human sperm penetration of zona-free hamster ova. Proteolytic and acrosin activities of human fertilization antigen were determined. Acrosomal status, acrosin activity, and motion characteristics were evaluated after incubation of human sperm with immunoaffinity-purified mAb to fertilization antigen. Academic research environment. Fertile donors used as controls for infertile patients for fertility evaluation. Human spermatozoa were treated with mAb to fertilization antigen and induced to undergo acrosome reaction using calcium ionophore A23187. Proteolytic and acrosin activities of fertilization antigen. Sperm penetration assay, acrosomal status, and motion parameters. Fertilization antigen does not exhibit proteolytic or acrosin activity; however, its mAb completely blocks human sperm penetration of zona-free hamster ova. The mAb to fertilization antigen inhibits ionophore-induced acrosome reaction and blocks development of the hyperactivated state of human sperm cells. Monoclonal antibody to fertilization antigen blocks fertilization by inhibiting capacitation and acrosome reaction.

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

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. 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. Academic research environment approved by the Institute Review Board. Human oocytes were obtained from ovaries removed at surgery. Semen from fertile donors was used in all assays. Human oocytes were stored in salt solution at -80 degrees C until used. Spermatozoa were treated with the antibodies to various sperm antigens. 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. 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. Antibodies to FA-1 but not to GA-1 and protamine inhibit human sperm-zona interaction.

Antigenic differences in human sperm samples related to various morphological abnormalities.

Sperm samples were collected from male partners of fertile and infertile couples having unexplained and male factor infertility and analyzed for morphological abnormalities. After a staining procedure, sperm samples of these infertile men showed a variety of abnormalities. The sperm samples were also studied for their binding with the Abs directed against protamine (the nuclear protein), fertilization antigen (FA-1, sperm surface antigen), and the lithium diiodosalicylate- (LIS-) solubilized sperm preparation, and the binding was correlated with the percentage of morphologically abnormal sperm in the sample. Abs used in the present study were raised in female rabbits and had high titers (ELISA titers, 1:4096 to 1:8196), and recognized no band (anti-protamine), one 49,000- +/- 2000-dalton band (anti-FA-1), and at least 8 bands of various molecular identities in the 14,000- to 92,000-dalton range (anti-LIS sperm) on the Western blot of LIS-solubilized human sperm. The affinity-purified Fab' antibodies were iodinated and used in a radioimmunobindingassay for testing their binding with the sperm cells. Anti-protamine Fab's showed minimal binding and the binding activity (cpm bound/10 x 10(6) sperm cells) did not correlate (head defect, r = .12; midpiece defect, r = .07; tail defect, r = .11) significantly with percentage of abnormal cells in the sample. The binding of the anti-FA-1 Fab's also did not show a significant correlation (head defect, r = .29; midpiece defect, r = .48; tail defect, r = .23), though a positive trend (p = .05) was observed with the midpiece defect. In contrast, the binding of anti-LIS sperm Fab's demonstrated a significant correlation with the percentage of abnormal sperm cells in the sample (head defect, r = .51, p = .04; midpiece defect, r = .80, p less than .0001; tail defect, r = .23, p = .039). With an increased percentage of abnormal sperm cells, especially those with midpiece defect, there was an increased binding of anti-LIS sperm Fab's. These results suggest that with an increase in percentage of abnormal sperm, there is a change in antigenicity of the sperm cells. An increase in structural abnormality may enhance antibody binding to an abnormal sperm cell, thus causing a further decrease in its fertilizing capacity.