Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

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.

Production, purification, and in vivo evaluation of a novel multiepitope peptide vaccine consisted of immunodominant epitopes of SYCP1 and ACRBP antigens as a prophylactic melanoma vaccine.

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 ±â€¯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.

[Preparation of a polyclonal antibody against human LYZL4 and its expression in the testis].

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.

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro.

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.

Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

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.

Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro.

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.

CD55 and CD59 protein expression by Apodemus (field mice) sperm in the absence of CD46.

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.

Sperm antibodies, intra-acrosomal sperm proteins, and cytokines in semen in men from infertile couples.

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.

Antibodies against laminin-1 and sperm, intraacrosomal proteins in semen from infertile couples.

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.

Characterization of human sperm antigens reacting with anti-sperm antibodies from an infertile female patient's serum.

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.

Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.

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.

Rapid sperm acrosome reaction in the absence of acrosomal CD46 expression in promiscuous field mice (Apodemus).

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.

Expression of bisecting type and Lewisx/Lewisy terminated N-glycans on human sperm.

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.

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa.

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 in rat male reproductive tissue: differential expression in testis and expression of a unique truncated isoform on spermatozoa.

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 (decay accelerating factor) and CD59 are expressed on the inner acrosomal membrane of human spermatozoa as well as CD46 (membrane cofactor protein).

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.

Evaluation of reproductive potential after intracytoplasmic sperm injection of varied human semen tested by antiacrosomal antibodies.

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.

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.

Immunisation of the male tammar wallaby (Macropus eugenii) with spermatozoa elicits epididymal antigen-specific antibody secretion and compromised fertilisation rate.

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.

Study on the role of a sperm membrane-associated antigen in canine fertilization.

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.