Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm.

STUDY QUESTION: Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility? SUMMARY ANSWER: Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. WHAT IS KNOWN ALREADY: Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLCζ. PLCζ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLCζ antibody specificity and detection protocols. STUDY DESIGN, SIZE DURATION: Two PLCζ polyclonal antibodies, with confirmed PLCζ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCζ visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1-0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. MAIN RESULTS AND THE ROLE OF CHANCE: Despite high specificity for native PLCζ following immunoblotting using epitope-specific polyclonal PLCζ antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLCζ compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLCζ visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLCζ visualization efficacy may be due to steric or conformational occlusion of native PLCζ, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLCζ fluorescence, and the proportion of sperm exhibiting detectable PLCζ fluorescence in sperm from different males. LIMITATIONS, REASONS FOR CAUTION: Direct correlation of fertility outcomes with the level of PLCζ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCζ visualization we propose would indeed reflect fertility status. WIDER IMPLICATIONS OF THE FINDINGS: We propose that AUM alters conformational interactions to enhance PLCζ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCζ-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCζ and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLCζ appears to be kept in an inactive form in the sperm. LARGE SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTERESTS: J.K. is supported by a Health Fellowship award from the National Institute for Social Care and Health Research (NISCHR). M.N. is supported by a Marie Curie Intra-European Research Fellowship award. This work was also partly funded by a research grant from Cook Medical Technologies LLC. There are no competing financial interests to declare.

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa.

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.

[Progress in the study of immunocontraceptive antigens].

Fertility management is a global issue of medical, economic, and social consequence. Although many methods have been devised to inhibit reproduction, more acceptable alternatives are still needed. Regulation by immune intervention is a promising technology as applied to human beings. The objective of this review is to indicate several immunocontraceptive antigens.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin.

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-ß1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.

The effect on fertility of immunizing female sheep with ram sperm acrosin and hyaluronidase.

Female sheep were injected with highly purified and partially purified preparations of ram sperm acrosin and hyaluronidase. The fertility and immune response of the sheep were monitored. Fertility was not significantly reduced in any single group, though a positive correlation was observed between high antibody titres against acrosin and reduced fertility. Studies on the direct action of sera from the ewes on ejaculated ram spermatozoa did not show any evidence of sperm agglutination or immobilization. Similar studies with denuded spermatozoa (detergent induced 'acrosome reaction') sometimes resulted in agglutination and enzyme inhibition was also seen; there was no correlation between any of these parameters and pregnancy.

Biochemical and immunological comparisons between the human and boar proacrosin-acrosin proteinase systems.

Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55,000), alpha-acrosin (45,000-49,000) and beta-acrosin (34,000-38,000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60 min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological differences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34,000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm.

Guinea pig testicular proacrosin-acrosin system: preliminary immunological characterization.

In this study, immunochemical techniques were employed to examine the guinea pig (GP) testicular proacrosin-acrosin system. Monospecific polyclonal antibodies to the highly stable enzymatically active 34,000 molecular weight form of GP testicular acrosin were generated. Western blot analysis of acid extracts prepared from snap-frozen freshly excised GP testes revealed two major immunoreactive bands with mol. wts of approximately 62,000 and 48,000 and one minor band with an approximate mol. wt of 54-56,000. The 62,000 mol. wt molecule identified is in close agreement with the previously reported mol. wt for purified GP testicular proacrosin. Western blot analysis of different species of testicular acid extracts demonstrated the evolutionary relatedness of the proacrosin-acrosin systems since immunoreactivity was observed primarily with acid extracts from rodent species (rat, mouse and hamster) and not with extracts from evolutionarily less-related species (goat, ram and bovine). The majority of the cross-reactivity observed was characterized by immunoreactivity with the 62,000 and 48,000 mol wt molecules. The only species which exhibited cross-reactivity with the 54-56,000 mol. wt protein was rat. In addition, the iso-immunogenic and aspermatogenic properties of the 34,000 mol. wt form of GP testicular acrosin were examined. One out of five Hartley and one out of seven Strain 2 female GPs immunized and boosted with a total of 200 micrograms of purified protein exhibited increased levels of circulating anti-acrosin iso-antibodies. The antigenic specificity of the iso-antibodies observed in the two animals was verified by Western blot analysis. All other female animals, including two strain 13 GPs, exhibited very low or undetectable levels of such antibodies following immunization. One out of three male Hartley GPs immunized with 50 micrograms of the purified protein exhibited typical lesions of experimental allergic orchitis while none of a group of three animals developed lesions at a 5 micrograms dose. Taken together, these results suggest that the 34,000 mol. wt form of GP testicular acrosin is neither a highly potent iso-immunogen nor aspermatogenic autoantigen.

The interaction of ram proacrosin and acrosin forms with antiserum raised against ram m beta-acrosin.

An antiserum was raised in rabbits to pure m beta-acrosin, the stable form of active acrosin from ram spermatozoa. By use of immunoprecipitation with Protein A-bearing Staphylococcus aureus cells, so as to react antigen with antibody and isolate the complex rapidly, it could be demonstrated that the anti-m beta-acrosin cross-reacted strongly with native proacrosin, with a degenerate proacrosin form, and with at least one form of active acrosin other than m beta-acrosin. Sperm proteins unrelated to acrosin were not recognized. It is concluded that rabbit anti-m beta-acrosin will react specifically with all the major ram acrosin forms; the antiserum can therefore be used with confidence in immunocytochemical studies to locate both proacrosin and acrosin in ram spermatozoa.

Antisperm antibody binding to human acrosin: a study of patients with unexplained infertility.

OBJECTIVE: Antisperm antibody binding to acrosin was investigated by Western Blotting. The clinical significance of this binding specificity was assessed in a 2-year clinical follow-up. DESIGN: Consecutive serum samples positive for antisperm antibodies by both enzyme-linked immunosorbent assay and immunobead testing were evaluated for acrosin-binding specificity. SETTING: The patients were followed in an outpatient setting by private infertility specialists. PATIENTS: Sixty-five consecutive infertile referral patients with positive antisperm antibody were evaluated. Clinical follow-up was obtained on 8 of 9 females with evidence of antibody binding to acrosin and 19 of 26 females with no specific binding to acrosin. INTERVENTIONS: Prednisone therapy was given during six courses of intrauterine insemination with husband's sperm. All treatment decisions were made by private physicians independent of the acrosin-binding result. MAIN OUTCOME MEASURES: Pregnancy status was obtained as part of a 2-year follow-up. RESULTS: Acrosin-binding specificity was demonstrated in 10 (15%) of the 65 patients. Two of the 8 women (25%) with antibody binding to acrosin and 6 of the 19 women (32%) with antisperm antibodies but no specific binding to acrosin delivered normal children. CONCLUSIONS: Although antibody-binding specificity to acrosin could be demonstrated, a 2-year clinical follow-up showed no difference in pregnancy rates when compared with women with antisperm antibodies showing no binding specificity to acrosin.

Active immunization of female rabbits with purified rabbit acrosin and effect on fertility.

Acrosin immunogen was purified from rabbit testes by sequential acid extraction, ammonium sulfate fractionation, cation-exchange, and affinity chromatography. Twelve females received intradermal injections of purified acrosin in Freund's complete adjuvant followed by a booster injection 6 weeks later. A radioimmunoassay for rabbit acrosin was developed and used to monitor the immune response of the recipients. The females were mated at the time when serum titers of acrosin antibodies were maximal. Four of the animals did not become pregnant, and three of these had the highest antibody titers in the total group. The remaining eight rabbits delivered normal litters at term. Of four control females (immunized with bovine serum albumin), one did not become pregnant. The pregnancy rates for the control and acrosin-immunized rabbits were 75% and 67%, respectively. It is concluded that, although active immunization with acrosin had no significant effect on fertility, the antibody titer produced may be a factor.

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin.

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.

Evidence of proacrosin molecule abnormality as a possible cause of low acrosin activity and unexplained failure of fertilization in vitro.

The aim of this study was to investigate whether the molecular abnormality of proacrosin is associated with an unexplained failure of in vitro fertilization (IVF) and low acrosin activity in human infertility. Seventeen infertile men, whose spermatozoa appeared to be normal but did not fertilize using a conventional IVF technique, were included in this study. Proacrosin molecules from each patient were screened with electrophoresis and Western blotting using either anti-human proacrosin or anti-boar acrosin polyclonal antibodies. The bands of proacrosin appeared equally on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at an identical site and reacted equally with human proacrosin antibodies in all patients studied. The anti-boar acrosin antibodies cross-reacted with the proacrosin bands of the patients in all but two cases. The two patients also demonstrated lower levels of total acrosin activity and different peptide maps of the isolated proacrosin. In conclusion, the molecular abnormality of proacrosin, which is reflected by a lack of cross-reactivity to the anti-boar acrosin antibodies, is associated with low acrosin activity and may explain some cases of unexplained failure of human IVF treatment.

Preparation and characterization of a monoclonal antibody against boar acrosin.

BALB/c mice were immunized with the crude fraction of boar acrosin. Immune spleen cells were fused with myeloma cells SP2/0-Ag14. One of the 6 hybridomas produced was cloned and characterized by ELISA and SDS-PAGE. Possible uses of the monoclonal antibody against acrosin for immunological detection of the amount of acrosin liberated after manipulations with spermatozoa and for selection of undamaged spermatozoa for insemination are discussed.

Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro.

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.

Antisperm antibodies: invaluable tools toward the identification of sperm proteins involved in fertilization.

The identification of sperm proteins involved in fertilization has been the subject of numerous investigations. Much interest has been dedicated to naturally occurring antisperm antibodies (ASA) and their impact in fertility. Their presence in men and women has been associated with 2-50% of infertility cases. ASA may impair pre- and post-fertilization steps. Experimental models have been developed using sperm proteins as immunogens to evaluate their involvement in sperm function. Our team has pursued investigations to assess ASA presence in biological fluids from patients consulting for infertility and their effect on fertilization. We found ASA in follicular fluids with ability of inducing the acrosome reaction and blocking sperm-zona pellucida interaction and used them to identify sperm entities involved in these events. We generated and utilized antibodies against proacrosin/acrosin to characterize the sperm protease system. We implemented an ELISA to detect proacrosin/acrosin antibodies in human sera and evaluated their impact upon fertility by developing in vitro assays and a gene immunization model. This review presents a summary of ASA history, etiology, current approaches for detection and effects upon fertility. ASA (naturally occurring, generated by animal immunization and/or of commercial origin) are invaluable tools to understand the molecular basis of fertilization, better diagnose/treat immunoinfertility and develop immunocontraceptive methods.

DNA immunization against proacrosin impairs fertility in male mice.

PROBLEM: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility. METHOD OF STUDY: Mice received 50 µg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied. RESULTS: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05). CONCLUSION: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility.

Anti-human proacrosin antibody inhibits the zona pellucida (ZP)-induced acrosome reaction of ZP-bound spermatozoa.

The anti-acrosin monoclonal antibody AcrC5F10 inhibited proacrosin activation, proacrosin-human zona pellucida glycoprotein A (ZPA) binding, and the zona pellucida (ZP)-induced acrosome reaction of the ZP-bound spermatozoa but had no significant effect on sperm-ZP binding. These results suggest that proacrosin-acrosin may play an important role in the ZP-induced acrosome reaction of spermatozoa after primary binding to the ZP.