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To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette., (© 2020 Blackwell Verlag GmbH.)

Melanoma cells are significantly resistance to the current treatments. Therefore, the best option for high-risk populations is prevention. Recently, many preventive cancer vaccines have been developed. In our previous study, several bioinformatic tools were employed for selection of the most immunodominant epitopes of acrosin binding protein (ACRBP) and synaptonemal complex protein 1 (SYCP1) antigens to design multiepitope DNA and peptide cancer vaccines. In the current study, the final construct of the multiepitope DNA vaccine was placed into a pcDNA3.1 vector and then, subcloned into a pET-28a (+) expression vector for transfecting BL21 E. coli strain. The recombinant multiepitope peptide vaccine, weighing 6.35 kDa, was purified by Fast protein liquid chromatography technique (FPLC) and detected by western blotting. Subsequently, C57BL/6 mice were immunized by a mixture of the peptide vaccine and incomplete Freund's adjuvant (IFA) (four vaccinations with one-week intervals). Two weeks after the last vaccination, the serum levels of the peptide-specific IgG total, IgG2a, and IgG1 were measured by enzyme-linked immunosorbent assays (ELISA). Also, the immunized mice splenocytes efficacy for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) after stimulation with the peptide vaccine was evaluated. At last, the prophylactic effect of the peptide vaccine immunization was evaluated in B16-F10 murine melanoma model. The peptide vaccine immunization caused a significant increase in the serum levels of IgG1, IgG2a, and IgG2a. Also, the immunized mice splenocytes exhibited significantly higher ability to produce IL-4 (10-fold) and IFN-γ (16-fold) after stimulation with the peptide vaccine, in comparison with the PBS and IFA groups. The peptide immunized mice exhibited 50.2% and 43% decrease in the mean tumors' volume in comparison with PBS and IFA groups. Also, the mean survival time for the peptide immunized mice was 33 ± 1.3 days which was 5 and 6 days more than the PBS and IFA groups, respectively. The obtained results exhibit high efficacy of the designed multiepitope peptide vaccine for the immune system activation and anti-tumor prophylactic effects in the murine melanoma model., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis., Methods: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry., Results: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids., Conclusions: An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.

We previously established an anti-mouse sperm auto-monoclonal antibody, Ts4, which shows immunoreactivity against several kinds of glycoproteins in the acrosomal region of epididymal spermatozoa, testicular germ cells, and early embryo, via binding to an epitope containing a common N-linked oligosaccharide (OS) chain on the molecules. In mice, we have already demonstrated that the OS chain in the epitope for Ts4 is a fucosylated agalacto-complex-type biantennary glycan carrying bisecting N-acetylglucosamine. In the testis, one of the specific OS chain-conjugated molecules is TEX101, a germ cell-marker glycoprotein, which is expressed in spermatocytes, spermatids, and testicular spermatozoa, but not in epididymal spermatozoa. In this study, we identified a Ts4-reactive glycoprotein in mouse cauda epididymal sperm. An immunoprecipitation method together with liquid chromatography-tandem mass spectrometry showed that alpha-N-acetylglucosaminidase (Naglu; a degradation enzyme of heparan sulfate) is one of the glycoproteins recognized by Ts4 in the epididymal spermatozoa. Western blot and immunohistochemical analyses revealed that mouse Naglu exists in two forms (82 and 77kDa) and is expressed in the acrosomal region and the flagellum of cauda epididymal sperm. Of the two Naglu-forms expressed in sperm, Ts4 immunoreacted against only the 82-kDa form located on the acrosomal region. The Ts4 mAb and anti-Naglu pAb negatively affected mouse fertilization in vitro. In addition, Ts4 inhibited sperm acrosome reaction induced by heparan sulfate. The Ts4-recognized fucosylated agalactobiantennary complex-type glycan with bisecting N-acetylglucosamine and Naglu on cauda epididymal spermatozoa may play a role in the process of fertilization., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization., (© 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.)

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization., (Copyright 2009. Published by Elsevier Ireland Ltd.)

The mammalian female reproductive tract has an abundance of complement components, which play a vital role in protection against genital pathogens. Sperm may be protected against complement-mediated damage by complement regulatory proteins, including membrane cofactor protein (CD46), decay accelerating factor (CD55) and CD59. However, sperm from Apodemus (field mice) do not express CD46 protein. The aim of the present study was to determine whether Apodemus sperm may be protected against complement-mediated damage by expression of CD55 and CD59 in the absence of CD46. We demonstrate here that, like Mus musculus mice (house mice), wild-caught Apodemus flavicollis, Apodemus microps and Apodemus sylvaticus mice express both glycosylphosphatidylinositol (GPI)- and transmembrane (TM)-anchored testicular CD55 mRNA transcripts. In Mus, testicular GPI- and TM-CD55 transcripts are generated by two distinct but closely related genes. We show that in contrast to Mus, CD55 isoforms in A. sylvaticus are generated by alternative splicing of a single copy gene. Testicular CD59 mRNA transcripts were also identified in A. flavicollis, A. microps, A. sylvaticus and M. musculus. CD55 and CD59 proteins are broadly distributed on epididymal sperm from wild-caught Apodemus and Mus mice as well as BALB/c mice, with expression on the acrosome, neck and tail. Thus, despite not expressing CD46 protein, Apodemus sperm may be protected against complement-mediated injury in the female genital tract by CD55 and CD59.

Problem: The aim of this study was to investigate seminal sperm-agglutinating antibodies, intra-acrosomal proteins, sperm head abnormalities, and cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70 TNF-alpha, and IFN-gamma) in men from infertile couples., Method of Study: The direct mixed anti-immunoglobulin reaction test for IgG, IgA, and IgE in semen, and immunocytochemical method using monoclonal antibodies and indirect immunofluorescence for the examination of intra-acrosomal proteins in the spermatozoa were used. Cytokines in seminal plasma were determined by multiplex immunoanalytic xMAP (LUMINEX) technology., Results: Sperm-agglutinating antibodies, IgG and IgA, in seminal plasma were found to be more in asthenospermatic and oligoasthenospermatic men than in normospermatic men. Sperm head pathology and very low amounts of acrosomal proteins were frequently detected in pathologic semen samples. Cytokine levels defined as 'high' (based on the 75 percentile for each cytokine in all groups) were obtained especially for IL-8, IL-5, IL-6, and IL-10. The high cellularity in semen was correlated with higher IL-5., Conclusion: Immunologic cause of male infertility is a very important risk factor in the pathogenesis of sperm cells. Sperm autoantibodies and the presence of intra-acrosomal factors must be studied together, cytokines according to accessory cellularity in the semen.

Identification of sperm antigens that elicit immunoglobulin (Ig) production and knowledge of their roles in sperm transport and fertilization may enhance diagnosis and treatment of immunologic infertility. Sperm antigens recognized by a female patient's serum anti-sperm antibodies were characterized using an indirect immunobead-binding test, immunoblot analysis, and immunochemical labeling. The anti-sperm antibodies' effect on sperm function was evaluated by acrosome induction by calcium ionophore. Immunobeads specific for IgG were bound to the head of 79% of motile donor sperm. Immunochemical labeling of antibody-binding sites was restricted to the plasma membrane over the acrosomal crescent. No labeling was observed on the inner acrosomal membrane of acrosome-reacted sperm. The antibodies reacted with 35-, 40-, 47-, and 65-kd proteins extracted from acrosome-intact donor sperm. Sperm incubated in 1:4, 1:8, 1:16, and 1:32 dilutions of anti-sperm antibody-positive serum had similar rates of spontaneous acrosome reaction and significantly lower rates of ionophore-induced acrosome reaction compared with sperm incubated in control serum. These results suggest that sperm antigens recognized by the patient's serum anti-sperm antibodies are restricted to the acrosomal region of the plasma membrane. The antibodies may impair fertility by compromising the sperm's ability to undergo capacitation and/or acrosome reaction.

Seminal sperm-agglutinating antibodies along with IgG antibodies against laminin-1 and intraacrosomal sperm proteins were examined in seventy-one men from infertile couples. The direct mixed antiimmunoglobulin reaction test for IgG, IgA, and the commercial ELISA method for detecting IgG antibodies against laminin-1 in seminal plasma were used. Intraacrosomal proteins in the sperm heads were detected by immunofluorescence using monoclonal antibodies. Cellular elements other than spermatozoa, collectively referred to as "round cells" were also examined. In association with a group of oligoasthenospermatic men, positive levels (44%) of antibodies against laminin-1 of the IgG isotype in seminal plasma were found in conjunction with increased cellularity in semen. Interestingly, the elevated levels of anti-laminin-l IgG and sperm agglutinating positivity were not correlated. The use of antibodies against sperm antigens targeted to adhesive molecules such as laminin-1 contributes to diagnosing reproductive failure. Detection of intraacrosomal proteins very often correlates to the state of semen pathology and inflammation.

A vaccine formula comprised of five recombinant human intra-acrosomal sperm proteins was inoculated into female monkeys to test whether specific antibodies to each component immunogen could be elicited in sera and whether antibodies elicited by the vaccine affected in vitro fertilization. Acrosomal proteins, ESP, SLLP-1, SAMP 32, SP-10 and SAMP 14, were expressed with his-tags, purified by nickel affinity chromatography and adsorbed to aluminum hydroxide. Five female cynomolgus monkeys were inoculated intramuscularly three times at monthly intervals. All five monkeys developed both IgG and IgA serum responses to each recombinant immunogen on Western blots. Each serum stained the acrosome of human sperm and bound to the cognate native protein on Western blots of human sperm extracts. By ELISA, all monkeys developed IgG to each immunogen, with the highest average absorbance values to ESP, SAMP 32 and SP-10, followed by lower values for SLLP-1 and SAMP 14. IgA was also generated to each component immunogen with the highest average absorbance values to SLLP-1 and SP-10. For antigens that induced an IgA response, the duration of the IgA response was longer than the IgG response to the same antigens. This study supports the concept that a multivalent contraceptive vaccine may be administered to female primates evoking both peripheral (IgG) and mucosal (IgA) responses to each component immunogen following an intramuscular route of inoculation with a mild adjuvant, aluminum hydroxide, approved for human use.

Human sperm lack major histocompatibility class I molecules, making them susceptible to lysis by natural killer (NK) cells. Major histocompatibility class I negative tumor cells block NK cell lysis by expressing sufficient amounts of bisecting type N-glycans on their surfaces. Therefore, sperm could employ the same strategy to evade NK cell lysis. The total N-glycans derived from sperm were sequenced using ultrasensitive mass spectrometric and conventional approaches. Three major classes of N-glycans were detected, (i) high mannose, (ii) biantennary bisecting type, and (iii) biantennary, triantennary, and tetraantennary oligosaccharides terminated with Lewisx and Lewisy sequences. Immunostaining of normal sperm showed that glycoproteins bearing Lewisy sequences are localized to the acrosome and not the plasma membrane. In contrast, defective sperm showed distinct surface labeling with anti-Lewisy antibody. The substantial expression of high mannose and complex type N-glycans terminated with Lewisx and Lewisy sequences suggests that sperm glycoproteins are highly decorated with ligands for DC-SIGN. Based on previous studies, the addition of such carbohydrate signals should inhibit antigen-specific responses directed against sperm glycoproteins in both the male and female reproductive systems. Thus, the major N-glycans of human sperm are associated with the inhibition of both innate and adaptive immune responses. These results provide more support for the eutherian fetoembryonic defense system hypothesis that links the expression of carbohydrate functional groups to the protection of gametes and the developing human in utero. This study also highlights the usefulness of glycomic profiling for revealing potential physiological functions of glycans expressed in specific cell types.

There is pronounced promiscuity and sperm competition in long-tailed field mice (Apodemus sylvaticus). These mice have evolved unusual sperm behaviour favouring rapid fertilisation, including dynamic formation of sperm trains and their subsequent dissociation. The cell surface complement regulatory (CReg) protein CD46 is broadly expressed in eutherian mammals other than rodents, in which it is expressed solely on the spermatozoal acrosomal membrane. Ablation of the CD46 gene has been associated with a faster acrosome reaction (AR) rate in inbred laboratory mice. Here, we demonstrate that wild-caught field mice of three species, A. sylvaticus, A. flavicollis and A. microps, exhibit a more rapid AR than wild-caught house mice Mus musculus or inbred laboratory BALB/c mice. We also demonstrate that wild-caught field mice of these three species, unlike house mice, produce alternatively spliced transcripts of testicular CD46 mRNA lacking exons 5-7 or 6-7, together with an extended 3' - and often truncated 5'-utr, leading to failure to express any sperm CD46 protein in both the testis and epididymis. Male field mice may therefore have traded expression of this CReg protein for acrosomal instability, providing a novel genus-specific strategy to favour rapid fertilisation and competitive advantage in the promiscuous reproductive behaviour of wild field mice.

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naïve females and female immune rats with naïve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.

CD55 is a key regulator of complement activation, expressed on most tissues and cells in man and other mammals. In the rat, alternative splicing in the gene encoding CD55 yields GPI-anchored (GPI-CD55) and transmembrane (TM-CD55) forms. Published Northern blot analysis indicated that while GPI-CD55 was broadly expressed, TM-CD55 was primarily expressed in the testis, although the precise site of expression was not identified. To clarify the distribution of CD55 isoforms in rat reproductive tissues, we first performed immunohistochemistry and Western blot analysis with an anti-rat CD55 mAb that recognized all reported CD55 isoforms, and a polyclonal immunoglobulin specific for TM-CD55. CD55 was absent in testis prior to puberty. Post-puberty, CD55 was expressed at high levels on all spermiogenic cells from step 6 spermatid onward, and on mature spermatozoa focussed on the acrosome, but was absent from support cells and early progenitors. Enzymatic digestion revealed that GPI-CD55 was predominant in testis and spermatozoa. Staining for TM-CD55 with specific immunoglobulin confirmed its absence from mature sperm and expression on spermatids only between steps 11 and 14 of development. GPI-CD55 on spermatozoa was of lower molecular weight than that in testis and other tissues; sequencing from spermatozoal mRNA identified a unique isoform of GPI-CD55 missing short consensus repeat 4. The predominant acrosome expression and presence of a unique, truncated isoform of CD55 on spermatozoa provides further support for the hypothesis that the acrosome is a highly specialized region in which closely regulated complement activation may contribute to reproductive function.

The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation-competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)-anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM-associated CD55 was localised within GM1-containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI-PLC, demonstrating them to be GPI-anchored. Analysis of acrosome-reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody-induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site.

Objective: To determine whether varied human spermatozoa, as detected with monoclonal antibodies against acrosomal proteins, have an influence on fertilization, transfer, pregnancy, and implantation rates when intracytoplasmic sperm injection is used., Design: A retrospective study., Setting: A private IVF center and academic research laboratory., Patient(s): One thousand two hundred forty men participating in the intracytoplasmic sperm injection program., Intervention(s): Sperm were divided into seven groups: oligozoospermia, oligoasthenozoospermia, and oligoasthenoteratozoospermia and fresh and frozen-thawed epididymal and fresh and frozen-thawed testicular sperm. Fertilization, transfer, pregnancy, and implantation rates were recorded in each category. Sperm were tested with antibodies for detection of the of the sperm acrosome., Main Outcome Measure(s): Fertilization, transfer, pregnancy and implantation rates, and percentage of acrosome-reacted cells., Result(s): The fertilization rate and statistical evaluation showed differences between morphologically normal and pathological sperm and other groups. The freezing-thawing procedure had no influence on the fertilization of testicular sperm, but epididymal frozen-thawed sperm had a higher fertilization rate. Immunofluorescence proved decreasing sperm quality in all groups compared with the control group. This difference is not manifested in other parameters (transfer, pregnancy, implantation rates)., Conclusion(s): The spermatozoa with varied semen characteristics and good quality, also detected with specific antibodies, gave the best fertilization rates. The paternal effect is not proved in other parameters.

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.

Immunocontraception has been proposed as an effective and humane means of controlling overabundant kangaroo populations in Australia. We have examined the feasibility of using a sperm-based vaccine for this purpose using a model macropod species, the tammar wallaby (Macropus eugenii). This study has demonstrated immunocontraception in a marsupial species following immunisation of males with homologous spermatozoa. Serum anti-sperm IgG titres were associated with a significant reduction in fertilisation rates following mating with superovulated female wallabies. Antigen-specific IgG penetrated the reproductive tract at the rete testis and bound spermatozoa in vivo. IgG was detected bound to the acrosome and midpiece regions of both epididymal and ejaculated spermatozoa. The absence of adverse testicular pathology and sperm movement effects suggests that contraception may have been achieved by antibody-mediated blocking of sperm surface antigens essential for fertilisation. This study demonstrates that a contraceptive vaccine targeting sperm antigens has potential for fertility control in male macropods.

The Mab 4B12 produced by us against capacitated boar spermatozoa was found to recognize a protein located in the acrosome portion of capacitated boar spermatozoa which is shared by different animal species, dogs included. It was shown that Mab 4B12 might affect fertilizing ability in vitro of boar spermatozoa. Using indirect immunofluorescence (IIF) test, we provide evidence here that Mab 4B12 stained the acrosome of the capacitated but not of the ejaculated and acrosome-reacted canine spermatozoa. The biological experiments using hemizona assay functional test in this study provide evidence supporting the involvement of Mab 4B12 corresponding antigen in the functional steps required for canine sperm-zona pellucida binding. These results together with the data on cell and tissue specificity of the 4B12 antigen suggest its contraceptive potential for canine fertilization.

Vasectomy results in the occlusion of testicular outflow, leading to autoimmunity characterized by the production of antisperm antibodies (ASA). Reports on the rise in ASA following vasectomy in several species are available; however, not much is known about the specific sperm autoantigens to which postvasectomy antibodies are directed. In the present study, monoclonal antibodies were generated using a vasectomized mouse. One of the monoclonal antibodies, D5E5, identified an approximately 70-kd antigen localized on the principal piece of the tail and also on the tip of the acrosome of mouse sperm. The cognate antigen was expressed postmeiotically in a stage-specific manner during spermiogenesis, starting from step 8 of elongating spermatids during spermiogenesis up to mature spermatozoa. The protein was conserved across the species, as observed by its presence in rat, bull, marmoset, and human sperm. Following capacitation, the antigen on the head was seen to shift to the acrosomal region and was lost after the acrosome reaction. However, the localization on tip of the acrosome still persisted, which indicates that the antigen may play a role post-acrosome reaction in sperm egg interaction. Resistance to Triton X-100 solubilization indicates that TSA70 could be an acrosomal matrix protein. In addition, we observed a significant reduction in forward progressive motility of mouse sperm treated in vitro with D5E5. In view of its testis specificity, acrosome and tail localization, and conserved nature, TSA70 is likely to play an important role in sperm function.

Problem: To determine the ability of monoclonal antibodies (MoAbs) against acrosomal antigens to detect physiology and pathology of human spermatozoa and to detect spermatids in ejaculates of infertile male with azoospermia., Method of Study: Sperm antigens detected with prepared MoAbs were partially characterized by biochemical and immunocytochemical methods. The acrosomal status of spermatozoa was compared in men with normal and pathological spermiograms and in sperm before and after induced acrosome reaction (AR). Ejaculates from patients were tested for the presence of spermatids., Results: MoAbs specifically bind to intra-acrosomal sperm antigens with quantitative difference between ejaculates with normal and pathological spermiograms. These antigens are released from the acrosome after induced AR. MoAbs labeled acrosomal proteins in round and elongated spermatids in the ejaculates of patients with azoospermia., Conclusion: MoAbs against intra-acrosomal sperm antigens are useful for human sperm diagnosis and prediction of spermatogenesis. The spermatids can be utilized in assisted reproduction.

The presence and role of the c-kit proto-oncogene protein was investigated in the mature sperm of the human. A polyclonal antibody against the c-kit peptide was used to perform immunohistochemical (IHC) staining, electron microscopy (EM) studies, and Western blot analysis. The acrosomal region of fresh sperm specifically stained with the antibody. No acrosomal staining or staining limited to the equatorial region was noted in the acrosome-reacted (AR) sperm. EM studies demonstrated immunogold label on the plasma membrane (PM) of the acrosome, and confirmed the lack of binding following the acrosome reaction. A 150 kDa band was detected by Western blot analysis. This protein was released from the sperm surface during sperm capacitation and the acrosome reaction. Antibody against the c-kit receptor significantly inhibited the acrosome reaction and increased sperm agglutination, but did not significantly inhibit sperm motility. These results suggest that the c-kit receptor protein is present in mature human sperm and is released during capacitation and/or the acrosome reaction. The assessment of the c-kit receptor may also be a useful assay for sperm function in male infertility., ((c) 2004 Wiley-Liss, Inc.)

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.

Problem: To determine the influence of an endocrine disruptor [bisphenol-A (BPA)] on the integrated reproductive process as well as on individual reproductive organs and gametes in order to select suitable markers for testing sperm damage., Method of Study: The effect of BPA on fertility in vivo in multigenerational studies in an outbred stock of mice was studied. Damage of reproductive organs was assessed by histochemical methods and damage of spermatozoa by means of a panel of monoclonal antibodies (MoAbs) against intra-acrosomal sperm proteins., Results: BPA had a negative influence on offspring born of mice, on reproductive organs, and on acrosome integrity of mice spermatozoa. Selected MoAbs against intra-acrosomal mammalian sperm proteins, cross-reacted with mouse spermatozoa, were used for determination of the acrosome integrity. BPA had no effect on body weight and testicle weight of males., Conclusions: The present results demonstrate that BPA has a negative effect on in vivo fertility of mice, with impact on spermatogenesis and sperm quality. Monoclonal antibodies against intra-acrosomal sperm proteins can be used for detecting sperm damage.

To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.

Problem: We recently cloned and sequenced a sperm-specific antigen, designated as testis-specific antigen-1 (TSA-1), from human testis. The present study was conducted to examine its expression and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects., Method of Study: The antibodies (Ab) were raised against purified human rTSA-1 in virgin female rabbits. The rTSA-1 was run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel containing the approximately 18 kDa band was cut, minced and used for immunization to obtain the specific Ab. The immunoglobulins from preimmune bleed and from animals injected with adjuvant alone served as control. These Ab were analysed in enzyme-linked immunosorbent assay (ELISA), Western blot procedure, immunoprecipitation procedure, immunocytochemical technique (ICT), immunobead binding technique (IBT), acrosome reaction and sperm-zona binding assay., Results: Active immunization of female rabbits with purified rTSA-1 protein of 18 kDa, produced high titer Ab against the recombinant antigen. These Ab to rTSA-1 were used in the present study. In Western blot procedure, rTSA-1 Ab recognized a specific protein band of approximately 24 +/- 3 kDa in murine sperm extract, the band similar to found in human sperm extract. In the immunoprecipitation procedure, rTSA-1 Ab immunoprecipitated the protein band of similar size from extracts of murine sperm and murine testis. The ICT and the IBT studies revealed the subcellular localization of TSA-1 on the surface of acrosome and tail regions of the non-capacitated and capacitated murine sperm cells. In functional bioassays, rTSA-1 Ab inhibited the acrosome reaction and sperm-egg binding in vitro., Conclusions: These data indicate that the TSA-1 is expressed in murine sperm and may have a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggests that the mouse can provide a suitable model to examine the immunocontraceptive effects of TSA-1 in actively immunized animals.

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.

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

Objective: To characterize immunocytochemically the antigens recognized by monoclonal antibodies (Mabs) in the library we have accumulated and to reveal their spatiotemporal distribution in testicular tissue in the course of testis development., Methods: Female BALB/c 2-month-old mice were immunized intraperitoneally with isolated immature Sertoli and germ cells obtained from 20 day old male Wistar rats. The obtained Mabs were characterized by its cell type-specific binding reaction using light immunocytochemistry (avidin-biotin-peroxidase complex technique, immunogold-silver staining, indirect immunofluorescence)., Results: On the basis of immunocystochemical results the selected Mabs were divided into four classes: 1. Mabs of class 1 recognized the differentiation specific antigens appearing during germ cell development, two of them revealing a stage-specific expression of nuclear antigens from preleptotene to early pachytene stage. Other Mabs of this class 1 detected the antigens in pachytene spermatocytes and acrosomes of round spermatids until their elongation; 2. the labeling of class 2 Mab was restricted only to Sertoli cell cytoplasm; 3. the binding of class 3 Mabs was observed in the cytoplasm of germ and Sertoli cells; 4. Mabs of class 4 reacted with antigens distributed in the cytoplasm of primary spermatocytes, Sertoli and Leydig cells., Conclusions: The Mabs from the library we have accumulated recognized the antigens in different cell types at various stages of testicular development and could be an useful tool for the characterization of cell- and development- specific molecules which may participate in germ cell differentiation and/or cell to cell interactions during testis development.

A new monoclonal antibody designated Hs-14 was generated after immunization of BALB/c mice with the acid extract of human sperm. In indirect immunofluorescence Hs-14 mAb binds to the acrosome of permeabilized sperm cells and consequently recognizes some intra-acrosomal protein. Western blotting analysis revealed that under non-reducing conditions the Hs-14 mAb detects a protein with a molecular mass of 220 kDa. Under reducing conditions the Hs-14 recognizes several peptide bands within the range from 55 kDa to 110 kDa. Beside human sperm the antibody positively reacts also with sperm of some other mammalian species. Using Hs-14 mAb it is possible to evaluate the acrosomal integrity of spermatozoa and to reveal sperm pathology.

Objective: Use of monoclonal antibodies against sperm proteins in human medicine., Design: Experimental and clinical studies., Setting: Dep. Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Prague, Laboratory IVF, Iscare IVF, Prague, Dep. of Immunobiology, Institute for the Care of Mother and Child, Prague., Methods: Monoclonal antibodies against human sperm intra-acrosomal and cell surface proteins were used for quantitative analysis of these proteins by the immunofluorescence test in samples of human sperm of good and poor qualities., Results: The detection of intra-acrosomal proteins was decreased and, on the other hand, detection of surface proteins was the same or higher in pathological spermatozoa., Conclusions: Monoclonal antibodies can be used for diagnostics of sperm pathology (quantitative detection of proteins) and for evaluation of the physiological state of sperm cells (state of acrosome before or after acrosome reaction). Finally, monoclonal antibodies could be useful for selection of a suitable method of fertilization (IUI, standard IVF, ICSI) in the laboratories of assisted reproduction.

Objective: To examine potential methods for distinguishing between the acrosome reaction and acrosomal loss., Design: Prospective randomized study., Setting: Department of Obstetrics and Gynecology, Osaka University Hospital, Suita, Japan., Patient(s): Five healthy volunteers and 34 patients with normozoospermia who were participating in an IVF program., Intervention(s): Semen samples were collected from the volunteers before the hamster egg penetration assay and from the patients at the time of IVF., Main Outcome Measure(s): The numbers of oocytes penetrated and spermatozoa bound were determined with the hamster egg penetration assay. Acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and MH61 (anti-CD46 monoclonal antibody) with Texas red-conjugated antimouse immunoglobulin G antiserum., Result(s): The MH61 monoclonal antibody inhibited the penetration of human spermatozoa into hamster oocytes but did not reduce the number of spermatozoa bound to the zona-free hamster oocytes. Two-color fluorescence staining revealed four staining patterns of the acrosomal region. The percentage of PSA-negative/CD46-positive spermatozoa increased to a greater extent than that of PSA-negative/CD46-negative spermatozoa with an increase in the incubation time., Conclusion(s): Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the acrosome reaction and acrosomal loss.

FITC-labelled sperm-specific antibodies against hamster spermatozoa were utilized as probes in acrosome reaction assays. An indirect immunofluorescence test demonstrated the localisation of two sperm proteins of 19 kDa and 23 kDa on the anterior acrosomal cap region of washed cauda epididymal sperm. These proteins were not detected in reacted acrosome or on immature or immotile sperm. Antisperm agglutinating antibodies specific to these two low molecular weight sperm antigens could be useful probes for evaluating the acrosomal status of mammalian spermatozoa.

During sperm/egg interaction, the sperm of Sicyonia ingentis undergoes a unique biphasic acrosome reaction. After acrosomal vesicle exocytosis, the sperm generates an acrosomal filament, over a period of 4-6 min, that is approximately 10 microns in length. Neither actin filaments nor normal microtubules have been demonstrated at the ultrastructural level of this unusual filament. Using a battery of cytoskeleton-directed antibodies the biochemical nature of this filament has been investigated. Antibodies to actin and tubulin do not label the subacrosome or acrosomal filament, but do recognise actin and tubulin in other shrimp tissues. Antibodies to tau, MAP2, and neurofilament medium and heavy subunits were all localised to the subacrosomal region of the sperm. It is interesting, however, that only the two clones of neurofilament monoclonal antibodies recognised the acrosomal filament. Electrophoretic and Western blot analysis in conjunction with amino acid sequencing revealed that the proteins localised to the acrosomal filament are of a unique sequence and may represent a new type of protein of centrosomal origin.

Limiting testicular biopsy for intracytoplasmic sperm injection (ICSI) to those with a high chance of having testicular spermatozoa has not been possible because of the poor predictive value of current clinical and laboratory methods. In order to predict testicular pathology and sperm extraction, we characterised the semen of 28 men with azoospermia due to gonadal failure in terms of the presence of spermatids using an immunological method. The results were compared with the assessment of testicular biopsies by histology and the extraction of spermatozoa into culture medium. Washed cellular elements in the ejaculate were smeared on microscope slides and fixed in 100% methanol, before incubation with acrosome-specific monoclonal antibody (18.6), fluorescein isothiocyanate-labelled anti-mouse goat IgG, and examination by epifluorescent microscopy. Semen from men with oligozoospermia and obstructive azoospermia served as positive and negative controls, respectively. Twelve patients who had positive immunofluorescence (one or more spermatids present) had spermatozoa retrieved from their testes (five hypospermatogenesis, seven focal spermatogenesis), and 16 patients with negative immunofluorescence (spermatids absent) had apparent Sertoli cell-only syndrome (12) or maturation arrest histological pattern (four). However, four patients with apparent Sertoli cell-only syndrome had testicular spermatozoa present after extraction from the biopsy. Plasma follicle stimulating hormone concentration and testicular volume did not predict retrieval of seminal spermatids or testicular spermatozoa. We conclude that the immunofluorescent localization of one or more spermatids in the ejaculate can be used to predict the likelihood of obtaining testicular spermatozoa for ICSI. However, in some patients with Sertoli cell-only syndrome, spermatozoa could still be recovered in the absence of apparent seminal spermatids.

The acrosome plays an important role in fertilization. This study was designed to examine the role and behavior of a molecule, equatorin (the antigenic molecule of the monoclonal antibody mMN9), localized at the equatorial segment of the acrosome. In vitro fertilization (IVF) investigation was conducted to examine the role of this molecule, by assessing the effect of mMN9 in TYH medium (a modified Krebs Ringer bicarbonate solution) containing mMN9 at 0 (control), 25, 50, and 100 microg/ml. Under these conditions, the IVF investigation was divided into two experiments: 1) the zona pellucida (zona)-intact experiment, in which capacitated sperm inseminated cumulus- and zona-intact oocytes; and 2) the zona-free experiment, in which acrosome-reacted sperm inseminated zona-free oocytes. It was found that mMN9 did not affect sperm motility, zona binding, or zona penetration, but it significantly inhibited fertilization, reducing the rates of pronucleus and two-cell embryo formation in both the zona-intact and zona-free oocyte experiments. In addition, when judged at 5 h after insemination in the zona-intact experiment, nearly half of the unfertilized oocytes had accumulated sperm in the perivitelline space (perivitelline sperm), and concurrently we confirmed by electron microscopy the presence of many unreleased cortical granules preserved beneath the oolemma, indicating no occurrence of sperm-oocyte fusion. Confocal laser scanning light microscopy with indirect immunofluorescence demonstrated that equatorin was localized at the equatorial segment in both capacitated and perivitelline sperm (acrosome-reacted sperm). These results suggest that equatorin that is preserved at the equatorial segment is involved in the process of sperm-oocyte fusion in mice.

Monoclonal antibody ACR.4 recognizing specifically the 28-kDa intra-acrosomal protein was prepared by immunization of mice with acetic acid extract of boar spermatozoa, but cross-reacted also with bull intra-acrosomal protein. This monoclonal antibody was used for immunostaining analysis of bull spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Immunostaining analysis showed changes of 28-kDa protein in the acrosome during capacitation and loss of this protein after induced acrosome reaction by ionophore A23187. Therefore, this monoclonal antibody can be used in the bull spermatozoa as an immunological test for detection of the acrosome state after manipulation with spermatozoa or after freezing/thawing. This test could be useful (apart from morphology and motility) for the selection of suitable spermatozoa for insemination or in vitro fertilization.

Among the numerous monoclonal antibodies generated against human sperm antigens, HS-13 monoclonal antibody was shown to react with an intra-acrosomal antigen from human, mouse and rat. In this study, HS-13 was used as the affinity ligand for the purification of the cognate antigen from human sperm by immunoaffinity chromatography. The purified cognate antigen from human sperm, designated as HSAg-13, was found to be a protein with a molecular weight of approximately 80 kDa on SDS-PAGE in the presence of reducing reagents. This monoclonal antibody was used as the probe to study the tissue distributions and developmental expression of the cognate antigen from human, mouse and rat by immunohistochemical assays. It was concluded that the antigen recognized by HS-13 antibody is highly sperm specific and found only in sperm and mature testis, but not in any other somatic tissues examined in human and mouse. The antigen was shown to be expressed at the postmeiotic stages of spermatogenesis in mouse and rat. By using indirect immunofluorescent staining assay, HS-13 was shown to react only with the methanol-fixed acrosome-intact sperm but not with the live sperm. Following calcium ionophore A23187 treatment, acrosome-reacted sperm showed either negative staining or residual staining in the equatorial region, suggesting the intra-acrosomal location of HSAg-13. The spontaneous acrosome reaction following overnight incubation in BWW medium resulted in a statistically significant decrease of antibody-stained human sperm. In view of excellent correlations for the scoring of acrosome-intact sperm with that of fluorescence-labeled Pisum sativum agglutinin (PSA) probe, HS-13 monoclonal antibody can be routinely used for monitoring sperm capacitation and acrosome reaction.

Objective: To determine whether antisperm antibodies can interfere with the induction of the acrosome reaction (AR) by the zona pellucida (ZP) and whether this interference also can occur in the absence of an inhibitory effect on ZP binding., Design: Prospective in vitro study., Setting: A tertiary care center, the Andrologic Clinic, University of L'Aquila., Patient(s): Sera from 12 infertile patients with high titers of circulating antibodies directed against the sperm head were studied., Intervention: None, Main Outcome Measure(s): The effect of antisperm antibodies on ZP binding was evaluated by matching antibody-exposed and nonexposed donor sperm suspensions labeled with fluorescein or rhodamine, respectively, and incubated with the same salt-stored human ZPs. The effect of antibodies on ZP-induced AR was determined by challenging antibody-exposed and nonexposed donor sperm suspensions with human ZPs disaggregated with acidic NaH2PO4. Acrosomal status was evaluated using fluorescein-labeled Pisum sativum agglutinin and supravital stain Hoechst 33258. In some selected cases, the effect of antisperm antibodies on the acrosomal status of sperm bound to intact ZP also was evaluated using transmission electron microscopy., Result(s): Five of 12 sera exhibited an inhibitory effect on ZP binding. An inhibition of AR induction by disaggregated ZPs (ranging from 64% to 98%) was produced by all 5 sera with an inhibitory effect on ZP binding and by 2 of 7 sera without an inhibitory effect on ZP binding. The different effects of antisperm antibodies on AR induction by disaggregated ZP were confirmed by comparing with ultrastructural evaluations on the acrosomal status of sperm bound to intact ZP., Conclusion(s): Antisperm antibodies can interfere with the induction of AR by ZP. This inhibition can occur even in the absence of an inhibitory effect on ZP binding. Neither effect may occur.

Three infertile patients fulfilling normal or subnormal criteria on routine semen analysis showed abnormal sperm CD46 (membrane cofactor protein of complement) by SDS-PAGE/immunoblotting analysis using a panel of monoclonal antibodies. The sperm CD46 isoform has been reported to be associated with sperm-egg interaction. These three patients expressed normal CD46 isoforms on their lymphocytes and granulocytes. Sperm-specific abnormalities in these proteins thus parallel male infertility, suggesting a new category of infertility, probably due to aberrations in the molecules related to sperm-egg interaction.

Problem: To develop simple and rapid assay procedures for determining human sperm acrosome reaction under various experimental conditions., Methods: Specific monoclonal antibodies against human sperm acrosome were generated and utilized as probes for acrosome reaction assays by direct labelling of antibodies with fluorescein-isothiocyanate (FITC)., Results: Among the generated monoclonal antibodies, HS-63 was shown to react with antigens in the acrosome content of permeabilized acrosome-intact human sperm, but not with those of the live ones. HSA-10 was found to recognize antigens on the surface of sperm inner acrosome and to react with acrosome-reacted human sperm in suspension. Following a swim-up procedure, highly motile sperm were recovered and incubated in BWW medium at 37 degrees C for 18 h. The percentages of acrosome-reacted sperm were determined at various incubation times by using FITC-labeled HS-63 or HSA-10 as the indicators in 10 min direct immunofluorescent assay. With the FITC-labeled HS-63 probe, the percentage of positively stained human sperm fixed in methanol decreased significantly after 18 h of incubation from > 90 to 70-80%. In contrast, positively stained live human sperm by HSA-10 increased significantly from < 1% to 15-30%. A high correlation was obtained between the use of monoclonal antibodies and PSA (Pissum sativum agglutinin) for direct assessment of human sperm acrosome reaction., Conclusion: In view of the simplicity in assay procedures and evaluations, these FITC-labeled monoclonal antibodies are reliable probes for direct assessment of acrosomal status of human sperm.

Problem: To determine whether the results of the Acrobeads test, which measures the expression of the complement regulator molecule CD46 on the inner acrosomal membrane following the acrosome reaction, accurately identifies semen specimens that will exhibit reduced or failed fertilization following conventional IVF insemination., Method: The Acrobeads test was performed on semen specimens from 97 consecutive patients preparing to undergo an IVF cycle utilizing a standardized insemination protocol. Motile sperm populations were examined at 6 h and 24 h post-isolation for sperm-bead agglutination. Results of the Acrobeads test were compared to that of TRITC-PSA staining in matched specimens to directly measure the spontaneous loss of acrosome content. The percentages of TRITC-PSA-negative sperm were determined in freshly isolated motile populations and in duplicate aliquots incubated 18 to 20 h under sperm capacitating conditions. The relationship between the results of both analyses estimating spontaneous acrosome reactions and the rate of fertilization of metaphase II oocytes was examined., Results: The Acrobeads score did not correlate significantly with the rate of fertilization by insemination at 6 h or at 24 h. The negative predictive value of this test was 21.4%. There was no correlation between the Acrobeads score and the percentage of sperm undergoing a spontaneous acrosome reaction as detected by TRITC-PSA labeling. In contrast, the increment increase in the percentage of spontaneous acrosome reactions as quantified by TRITC-PSA staining was correlated with the fertilization rate., Conclusions: Contrary to previous reports, our prospective, double-blinded study failed to demonstrate that the Acrobeads test can accurately predict fertilization outcome in IVF. Therefore, the routine use of this test to screen patients prior to an IVF cycle in order to select appropriate treatment (i.e., ICSI) cannot be recommended.

Sperm acrosome antigen-1 (SAA-1) is a molecule on the acrosomal cap of sperm from the human and a number of mammalian and lower species. SAA-1 was initially characterized by a monoclonal antibody (mab) AG7 directed against SAA-1. Previous studies indicate that SAA-1 may play an important role in the regulation of the acrosome reaction in the human and other species. Unselected couples seeking infertility treatment were subjected to an analysis of the amount of SAA-1 present on washed husband sperm. Using indirect immunofluorescence as well as radioimmunobinding assay, the expression of SAA-1 on patient spermatozoa was found to be significantly decreased compared to a group of healthy sperm donors. The decrease in SAA-1 did not correlate well with sperm morphology. Couples entered into the study were followed for an average of 12 months, while they received infertility treatment. Most couples conceived after a variable number of treatment cycles. It is concluded that a decrease of SAA-1 expression may contribute to subfertility, which can be overcome by the aid of assisted reproduction.

Problem: The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction., Methods: Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems., Results: The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 +/- 4%) and anti-CD46 (83.4 +/- 2%) binding to sperm. Both anti-CD15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD15. The mean (+/- SD of tightly bound sperm to hamster eggs were medium control 57 +/- 18%, irrelevant mAb 64 +/- 16%, anti-CD46 37 +/- 13%, and anti-CD15 19 +/- 10%. The corresponding values for human zona were: medium control 118 +/- 14%, irrelevant mAb 61 +/- 11%, anti-CD46 39 +/- 18%, and anti-CD15 99 +/- 19%., Conclusion: CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis(x)) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.

Objective: To study the effect of polyclonal/monospecific antisera on sperm agglutination versus capacitation as well as acrosome reaction., Materials and Methods: Swim-up spermatozoa from cauda epididymides of fertile male hamsters were incubated under liquid paraffin with polyclonal/monospecific antisera obtained from immunized BALB/C mice, as well as with normal serum from control BALB/C mice, at various dilutions., Results: The anti-sperm antibodies caused a significant (P < .05) sperm agglutination of various types of dilutions below 1:1000. Both capacitation and true acrosome reaction were inhibited significantly in the spermatozoa incubated with polyclonal/monospecific antisera. Capacitation in the spermatozoa with normal serum started earlier, i.e., at 2 hours of incubation compared to 3 hours of incubation in controls., Conclusion: The data differentiate the sperm agglutinating activity from anticapacitation and antiacrosome reaction activity of antisperm antisera at 1:1000 dilution.

Among the numerous anti-sperm monoclonal antibodies generated in our laboratory, HS-33 was shown to react with a conserved antigen on the acrosome of spermatozoa from human and mouse. By using indirect immunofluorescent assay, it was demonstrated that HS-33 did not bind to live human sperm. However, this antibody was found to react with the methanol-fixed acrosome-intact, but not with acrosome-reacted sperm. The human sperm antigen recognized by this antibody was purified from human sperm extract by immunoaffinity chromatography. The purified cognate human sperm antigen designated as HSAg-33 was found to be a protein with a molecular weight of approximately 72 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. The tissue-specificity and the developmental expression of this sperm antigen were examined using frozen sections of various human and mouse tissues. The antigen was shown to be expressed specifically in the testicular sperm at the postmeiotic stages of spermatogenesis but not in any other somatic tissues. "Spontaneous" acrosome reaction was determined following 18 hours of incubation in Biggers, Whitten, and Whittingham (BWW) medium by using HS-33 monoclonal antibody and Pisum sativum agglutinin (PSA) as probes. The number of sperm stained positively with this antibody decreased significantly following overnight incubation, indicating the occurrence of an acrosome reaction. The results of this study suggest that HSAg-33 is a potentially useful sperm-specific acrosome marker for studies of sperm capacitation and acrosome reaction.

The study was set up to determine the relationship between the human sperm acrosome reaction and fertilization in couples undergoing routine in-vitro fertilization (IVF) treatment. Prospective data analysis was carried out on all IVF patients during a 6 month period. Exceptions were those patients having insufficient sperm concentration to allow both acrosome reaction determination and insemination. The main outcome measures were the prediction of fertilization in IVF patients using flow cytometric analysis of the spontaneous and ionophore-induced acrosome reaction [giving the acrosomal response to ionophore challenge (ARIC) score] in the male partner's spermatozoa versus standard analytical methods of sperm motion parameters and morphology. Stepwise logistic regression indicated only two independent factors predictive of fertilization: ARIC score (chi 2 = 109.6, P < 0.0001) and post-Percoll % motility (chi 2 = 8.8, P < 0.003). Of patients with an ARIC score of > 10, 92% had > 30% of oocytes fertilized; 100% of patients with an ARIC score of < 10 had < 30% fertilization of oocytes. The sensitivity and specificity of the assay system were 1.00 and 0.82 respectively. The results would indicate that the ARIC test as measured by flow cytometric analysis of CD46 binding is a sensitive and specific assay for use in the prediction of fertilization in IVF patients, thus enabling direct channelling of those patients with ARIC scores of < 10 into the more invasive micro-assisted fertilization schemes.

Problem: The purpose of this study was to examine whether the products of activated immune cells influence spontaneous and ionophore-induced sperm acrosome reaction., Method: The spontaneous and ionophore-induced acrosome reaction were evaluated by staining with fluorescein isothiocyanate (FITC) Pisum sativam agglutinin after incubation in capacitating media supplemented with either supernatants from Con-A activated leukocyte cultures or human recombinant (r) IL-1 beta, TNF-alpha, and INF-gamma., Results: The supernatants from Con A-activated peripheral blood leukocyte cultures at 1:1 and 1:10 dilution significantly increased the rate of spontaneous acrosome reaction (P < 0.001 and P < 0.01). Along with displayed abnormally elevated levels of spontaneous acrosome loss, sperm cells showed an insufficient ability to undergo acrosome reaction in response to the ionophore treatment. Recombinant IL-1 beta at increasing concentrations from 30 to 3 x 10(4) U/ml did not have an effect on spontaneous and ionophore-induced acrosome reaction. In contrast, spermatozoa that underwent capacitation in media with 7 x 10(3), 7 x 10(4), and 7 x 10(5) U/ml of rINF-gamma showed a significant increase in spontaneous and induced acrosome reaction compared to the control (P < 0.001). Recombinant TNF-alpha at concentrations of 3.5 x 10(3) U/ml and 3.5 x 10(4) U/ml significantly inhibited ionophore-induced acrosome reaction (P < 0.001). Both rINF-gamma and rTNF-alpha together revealed an effect on the acrosome reaction similar to Con-A generated supernatants (1:1 and 1:10 dilution) only at the highest concentrations., Conclusions: Some cases of infertility may result from a defective acrosome reaction (premature acrosome loss or insufficient acrosome response to the stimulants) caused by products of activated lymphocytes and macrophages that are released into the male and female reproductive tracts.