Characteristics and Possible Role of Bovine Sperm Head-to-Head Agglutination.

Although sperm head-to-head agglutination has been reported in many mammalian species, the biological significance of this unique sperm-sperm interaction remains largely unknown. Here, we aimed to examine the functional characteristics of agglutinated bovine sperm to determine the possible role of sperm agglutination in the fertilization process. We initially examined temporal changes to the degree of head-to-head agglutination in culture, and found that bovine sperm agglutinated despite the lack of sperm agglutination inducers in medium. Sperm viability and motility were evaluated by SYBR14/PI and JC-1 staining, respectively, to identify the relationship between sperm agglutination and fertilizing ability. Agglutinated sperm had increased motility, viability, and intact mitochondrial function compared with unagglutinated sperm. Furthermore, we found that heparin significantly increased the percentage of unagglutinated sperm, but did not affect viability of both agglutinated and unagglutinated sperm, suggesting that sperm agglutination dictated the viability. In conclusion, agglutinated bovine sperm maintained viability and motility for a longer time than unagglutinated sperm. Thus, we propose that the head-to-head agglutination is a crucial sperm-sperm interaction to ensure the fertilizing ability of sperm.

Obtaining and characterization of anti-testis monoclonal antibodies: Invaluable tools toward the identification of testis antigens involved in fertilization.

BACKGROUND: Generation and utilization of the specific monoclonal antibodies against testis antigens is reported to identify the antigens that are important in reproductive field. OBJECTIVE: Current study aimed to introduce a hybridoma that producing a specific anti-testis monoclonal antibody to identify the testis antigens that can be important in the reproduction field. METHODS: To make hybridoma against testis antigens, mice were immunized with testis cell lysate. After cell fusion, resulted hybridomas were screened by indirect ELISA, then cloned by limiting dilution and finally the produced monoclonal antibody were characterized by some of the molecular laboratory techniques such as immunohistochemistry, immunocytochemistry and western blot. RESULTS: By using hybridoma technique, cell fusion was performed and ten (8A11, 8D6, 8D7, 9F6, 9G11, 10C3, 10B3, 10B2, 10C2 and 10H7) antibodies specific to the testis antigens were produced finally. Among the produced antibodies, 10C3 was found to cross-react with testis, but not detected in other tissues. mAb 10C3 recognized the sperm and testis antigens, specifically the intertestitial tissue of testis, spermatogonia, primary and secondary spermatocyte antigens, so they were most likely the target of generated mAb. Also our mAb could totally detect the mouse sperm antigens and the specific antigens of head and tail of human sperm. In western blotting analysis, mAb 10C3 could recognize the specific protein bands of sperm and testis extracts. Also in this study the testis specific genes that were target of generated mAb, were selected according to the mouse EST profile available at UniGene part of NCBI. CONCLUSIONS: So this stable anti-testis mAb has a potential for laboratory researches and also for diagnostic procedures in fertilization. Thus it could be exploited as a suitable tool for target-specific diagnosis and research in several diseases.

Sperm cephalic vacuoles: new arguments for their non acrosomal origin in two cases of total globozoospermia.

To add new arguments concerning the origin of the sperm-head vacuoles observed under high magnification with interference contrast microscopy, we carried out in two patients with total globozoospermia confirmed using transmission electron microscopy (TEM), a detailed sperm morphometric analysis with high magnification (×6000) under Nomarski contrast, an acrosomal status analysis (using fluorescent labelling with peanut agglutinin (PNA) lectins and anti-CD46 antibodies) and a nuclear status analysis (using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay TUNEL, sperm chromatin structure assay SCSA and aniline blue staining). Our two patients with globozoospermia had relative sperm vacuole areas of 6.3% and 5%, similar to those observed in a reference population of 12 fertile men (5.9%). TUNEL and SCSA assays gave normal results in both patients, although the percentage of immature nuclei using aniline blue staining was increased (27 and 46% for patient 1 and 2 respectively). Cytofluorescence and TEM analysis evidence differences between the two patients: although no acrosomal neither Golgi residue could be detected in patient 1, patient 2 had positive PNA lectin labelling for 9% of spermatozoa and Golgi residues were seen using electron microscopy. Unlike patient 1, a live birth could be obtained after intracytoplasmic sperm injection (ICSI) for patient 2. This descriptive study of two patients with total globozoospermia confirmed using TEM argue in favour of a deep analysis of total globozoospermia before assisted reproductive technology and provides further information on the non-acrosomal origin of the sperm-head vacuoles observed under high magnification.

Standardization of a method to detect bovine sperm-bound antisperm antibodies by flow cytometry.

The objectives were to standardize some methodological and analytical aspects of a direct technique to detect sperm-bound antisperm antibodies (ASAs) in bovine semen using flow cytometry, including the effects of prefixation of sperm membranes with formalin buffer solution and inclusion of dead cells in the analysis. Fourteen Angus bulls, including ASA-positive (experimentally induced ASAs) and 10 reproductively normal ASA-negative bulls, were used. Fixation of sperm membranes had no significant effect on the percentage of IgG- or IgA-bound spermatozoa detected by flow cytometry. However, including dead cells in the analysis increased the percentage of IgG-bound spermatozoa in fixed (live and dead 18.6 ± 9.7% and live 1.3 ± 0.5%; median ± SEM) and nonfixed samples (live and dead 18.8 ± 9.2%, live 1.5 ± 0.6%; P = 0.0029), as well as IgA-bound spermatozoa in fixed (live and dead 16.3 ± 6.4%, live 0.3 ± 0.5%) and nonfixed samples (live and dead 21.4 ± 4.6%, live 1.0 ± 0.5%; P = 0.0041) in semen from ASA-negative bulls. Intrasample, intra-assay, and interassay coefficients of variation (CV) were 0.8%, 4.6%, and 5.3%, respectively, for determination of sperm-bound IgG, and were 2.8%, 8.4%, and 40.3% for determination of sperm-bound IgA. Despite the high interassay CV for IgA determination, all ASA-positive bulls consistently had high percentages of IgA-bound spermatozoa. Flow cytometry correctly identified ASA-positive bulls. Confocal laser microscopy confirmed binding of ASAs to sperm heads and cytoplasmic droplets, and less frequently to midpieces and principal piece. In conclusion, although fixation was not necessary, dead cells should be excluded from the analysis, because ejaculates with a large proportion of dead cells can yield false-positive results. Flow cytometry was accurate and reliable for detection of sperm-bound IgG and IgA and discrimination between ASA-positive and ASA-negative bulls.

Engulfment of spermatozoa by cumulus cells and the role of CD36.

The function of cumulus cells after sperm penetration is not well understood. The present study examined the phagocytic action of cumulus cells on sperm after dispersion of cumulus-oocyte complexes. In a co-incubation system of cauda sperm and cumulus cells, the sperm heads were beginning to vanish after 2.5h and 77%±1.34 of sperm heads had disappeared at 30 h. Most of the sperm heads were engulfed by cumulus cells. Immunofluorescent studies showed that cumulus cells were expressing the CD36 molecule, and sperm were exposing phosphatidylserine (PS). Anti-CD36 antibody and annexin V inhibited the engulfment of sperm by cumulus cells by 26.0% and 40.5%, respectively. These results suggested that the cumulus cells recognized the PS molecules on sperm via CD36 and this molecular interaction possibly triggered the phagocytosis of sperm by cumulus cells. These results suggest that cumulus cells might play a role in inhibiting undesired immune reactions induced by sperm antigens.

Lipopolysaccharide-binding protein is produced in the epididymis and associated with spermatozoa and prostasomes.

Lipopolysaccharide-binding protein (LBP) is an acute phase protein known to play a central role in the defense against Gram-negative bacteria. It binds lipopolysaccharides of Gram-negative bacteria and, after binding to CD14, the complex signals through Toll-like receptor (TLR)-4, eliciting host-defense responses, such as cytokine production, in inflammatory cells. The present study demonstrates constitutive expression of the gene encoding lipopolysaccharide-binding protein in the epithelium of the human epididymis by in situ hybridization. Using immunohistochemistry lipopolysaccharide-binding protein was shown to be present in the same cells and also attached to the heads and tails of spermatozoa. Cell-free seminal plasma, lysed spermatozoa and lysed prostasomes were subjected to Western blot; all showed immunoreactive bands corresponding to the size of lipopolysaccharide-binding protein. Gel filtration demonstrated that lipopolysaccharide-binding protein colocalizes with prostasomes. The concentration of lipopolysacharide-binding protein in seminal plasma was 127+/-42ng/mL (mean+/-S.D.; range 73-215ng/mL). Taken together, our results suggest roles for lipopolysaccharide-binding protein during human reproduction.

Validity of a rapid assay for antisperm antibodies in semen.

OBJECTIVE: To determine the validity of a rapid assay for antisperm antibodies in semen. DESIGN: Prospective comparison of the results of standard and rapid antisperm antibody assays performed simultaneously. SETTING: Tertiary care infertility center. PATIENT(S): Couples who presented for infertility evaluation. INTERVENTION(S): Semen analysis and measurement of antisperm antibodies in semen using a standard and a rapid immunobead binding test (IBT). MAIN OUTCOME MEASURE(S): [1] Comparison of sperm parameters between semen-containing antisperm antibodies and semen free of antisperm antibodies. [2] Validation of the rapid test by calculation of sensitivity, specificity, positive and negative predictive values of the rapid assay using the standard assay as a gold standard. [3] Cost comparison of the standard and rapid test. RESULT(S): [1] Nine semen specimens with antisperm antibodies had a significantly lower sperm concentration, motility, and total motile fraction compared to 44 specimens without antisperm antibodies. Also, specimens with antisperm antibodies had a significantly higher percentage of vibratory sperm and percent of bound antisperm antibodies. The strict morphology, liquefaction time, semen volume, and white blood cell concentration were no different between the two groups. [2] Using a threshold of > or =12% of bound antisperm antibodies in the rapid assay, the sensitivity, specificity, positive and negative predictive values of the test are 100% when correlated with a threshold of > or =20% in the standard assay. Increasing the threshold in the standard assay decreases the specificity and positive predictive value of the rapid assay but not the sensitivity and the negative predictive value. [3] The cost of the rapid assay was 16% that of the standard test and its performance took 20% of the time it took to set and perform the standard test. CONCLUSION(S): A rapid test for antisperm antibodies is valid, reliable, and more cost and labor effective than a standard IBT.

Sulfogalactosylglycerolipid is involved in human gamete interaction.

Recent results from our laboratory have revealed the role of sulfogalactosylglycerolipid (SGG) in mouse sperm-zona pellucida (ZP) binding. In this report, we demonstrated the presence of SGG in Percoll-gradient centrifuged (PGC) human sperm by high performance thin layer chromatography with orcinol and Azure A staining, specific for glycolipids and sulfolipids, respectively. SGG in human PGC sperm was quantified by its affinity to Azure A to be 12-15 mol% of sperm lipids. Indirect immunofluorescence revealed that SGG existed on both live and aldehyde fixed human sperm in the head region. Pretreatment of human PGC sperm with affinity purified antiSGG Fab markedly inhibited sperm binding to the ZP in a concentration dependent manner, without any changes in the spontaneous acrosome rate or sperm motility parameters. Fluorescently labeled SGG liposomes also bound uniformly to isolated human ZP, while fluorescently labeled galactosylglycerolipid (GG, SGG's parental lipid) or phosphatidylserine (PS, negatively charged like SGG) liposomes did not. All of these results suggested the role of human sperm SGG in ZP binding.

Identificación de proteínas espermáticas humanas involucradas en la interacción espermatozoide-zona pellucida / aaa

La interacción inicial entre las gametas es mediada por proteínas de superficie de la cabeza del espermatozoide y de la matriz extracelular del ovocito, la zona pellucida (ZP). El presente trabajo tuvo como objetivo la identificación de antígenos de superficie de espermatozoides humanos potencialmente involucrados en el reconocimiento de la ZP. Para ello se obtuvo un extracto de proteínas periféricas de espermatozoides humanos por tratamiento de las células con solución de alta fuerza iónica (HSE - High Salt Extract) (Buffer Pipes 100mM, pH 7,4; NaCl1M, sacarosa 0,25 M). Se desarrollaron anticuerpos policlonales (anti-HSE) que reconocieron numerosas proteínas en HSE (9-200 KDa). Las proteínas fueron separadas por electroforesis en geles de poliacrilamida, transferidas a membranas de nitrocelulosa y utilizadas para adsorber inmunoglobulinas de anti-HSE. Con éste método se obtuvieron anticuerpos contra dos polipéptidos mayoritarios de 49 (p49) y 66 (p66) kDa. Ambos anticuerpos (anti-p49 y anti-p66) reconocieron epitopes localizados en la cabeza y el flagelo de espermatozoides eyaculados y capacitados...(AU)

Systematic search and molecular characterization of the antigenic targets of myeloma immunoglobulins: a monoclonal IgA from a female patient targeting sperm-specific cylicin II.

The identification of the antigenic stimuli of B-cell neoplasms might be of considerable importance since a causal relationship between these neoplasms and antigenic stimulation has been suggested. To date the identification of such antigens has been erratic and accidental. For a systematic search and molecular characterization of human proteins that are antigenic target structures of myeloma-associated immunoglobulins, we applied SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using a testis cDNA expression library and myeloma proteins from 42 patients. A monoclonal IgA from a female patient was shown to target sperm-specific cylicin II. The specificity of the reaction was confirmed by the characteristic staining of the equatorial belt of human sperm heads by the patient's myeloma protein. Serological analysis of recombinantly expressed cDNAs is a straightforward and high throughput approach for the molecular characterization of the targets of myeloma-associated immunoglobulins. The analysis of the antigenic spectrum of immunoglobulins associated with B-cell neoplasms will provide valuable information for the understanding of the pathogenesis of these diseases.

Monoclonal antibody that recognizes an epitope of the sperm equatorial region and specifically inhibits sperm-oolemma fusion but not binding.

Balb/c mice were immunized with purified hamster sperm heads for induction of antisera and the production of monoclonal antibodies that recognize preferentially the equatorial segment. Twenty-six hybridoma clones secreted monoclonal antibodies with strong affinity for spermatozoa. The supernatants of 16 clones contained antibodies against the equatorial segment, of which 11 were specific to this region. Five supernatants (M1-M5) containing antibodies that bind to various regions of the sperm head were selected and assessed for the ability to inhibit hamster fertilization in vitro using intact and zona-free oocytes. All the supernatants inhibited fertilization compared with the control. However, M1 supernatant specifically inhibited sperm-egg fusion in a concentration-dependent manner, while sperm-oolemma binding and sperm motility remained unaffected. M1 supernatant recognized an epitope that is exclusive to the equatorial segment and expression of this epitope increased after capacitation and the acrosome reaction. Preliminary immunoblot analysis indicated that M1 monoclonal antibody recognized two protein bands of 37.5 and 34.0 kDa.

An innate natural antibody is reactive with a cryptic sequence of lactoferrin exposed on sperm head surface.

Lactoferrin (LF), an 80-kDa glycoprotein of ubiquitous occurrence in body fluids, is multifunctional and capable of assuming different configurations to serve those functions. The capacity of LF to undergo endocytosis and the recent demonstration of LF binding to sequence specific DNA indicate that a function or capability of LF, in addition to iron chelation, bacteriostasis, and receptor-specific lymphocyte binding, may be that of gene activation or silencing. The data of this report present a human physiological system, that of sperm entry into the oocyte in performance of fertilization in which, since LF is a component of the sperm protein coat, that capability could be expressed. However, the configuration of LF in that locus is one in which a revealed cryptic sequence provides the specific binding site for a natural antibody present in the fertilization milieu. The presence of that antibody suggests that a system of control of the potential interaction of LF with the intra-ooplasmic DNA, that of gametes or pronuclei, is operative. The configuration of LF on the sperm surface and designation of the reactive site for the natural antibody were enabled by a monoclonal antibody secreted by a hybridoma derived from a human cord blood B cell. Thus, in addition to information concerning the molecular flexibility of LF, these observations support the proposition that the repertoire of natural antibodies provides an innate homeostatic system, with each antibody serving a specific role.

Distribution of 105-kDa sperm unique antigen on goat epididymal mature spermatozoa.

The sperm surface antigens are organized into topographic domains, specifically formed into the membrane during the sperm maturation in the epididymis. The present study was directed to determine the distribution of 105 KD sperm membrane antigen (SMA2) into the cell by affinity purified antibody, generated by 105 KD sperm antigen. The cytoimmunofluorescence study of the integrated sperm cell reveal that the antigen is localized in the anterior head overlying the acrosome. On immunoblotting of isolated head and flagellum polypeptides it has been demonstrated that this antigen is selectively present in the head membrane and absent in the flagellum. Because of its selective presence in the anterior head membrane overlying the region of acrosome on mature spermatozoa, the 105 KD sperm unique glyco-antigen may have a role in events leading to sperm-egg recognition. It is a first report of a high molecular mass membrane antigen being localized in the anterior head membrane of goat mature epididymal spermatozoa.

Cell specific nuclear antigens of boar spermatozoa.

Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.

Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies cross-reactivity.

The overall objective of this series of experiments is to generate immunological markers that may elucidate bull sperm surface changes in vitro. Here we report the initial experiments of the study, involving the production and characterization of monoclonal antibodies (mAbs) again bull sperm. BALB/c mice were immunized with phosphate-buffered saline (PBS)-washed whole bull sperm, and their spleen cells were fused with NS-1 myeloma cells in two separate cell fusion experiments, resulting in the generation of 15 mAbs. The mAbs were specific to antigens of either the posterior tail or the head regions of bull sperm and detected five major domains of antigen localization in the bull sperm (apical crescent, equatorial band, principal acrosomal, whole head, and posterior tail). Eleven of the 13 head-specific mAbs recognized intra-acrosomal antigens, whereas 2 mAbs recognized antigens that were localized in the plasma membrane. One mAb specific to the tail region was of the IgM class; the remaining 14 mAbs were of the IgG class. They were all sperm specific, with no cross-reactivity to bovine oocytes or to any of the 12 bovine somatic tissues tested. The mAbs were not species specific, however, because 11, 10, 2, and 1 of the 15 mAbs cross-reacted with sheep, pig, mouse, and human sperm, respectively. None of the mAbs cross-reacted with rooster sperm. The cognate antigens of the 11 tested mAbs were of testicular origin, but several of them showed enhanced binding to epididymal sperm. In western blot analysis, 3 of the 13 mAbs tested identified more than one protein band (40-200 kDa). Seven others recognized proteins of > or = 200 kDa, whereas three mAbs recognized no proteins.

Can the immunobead assay for detecting sperm antibodies in fresh samples be reproduced in cryopreserved/thawed human spermatozoa?

PROBLEM: To evaluate the reproducibility of the Immunobead Assay (IBA) on sperm samples before and after cryopreservation. METHOD: Sperm samples (fresh and post-thaw) from known antibody negative donors (N = 20) were evaluated for percent immunobead binding by IBA following incubation with known antibody-positive serum. RESULTS: In both fresh and thawed negative samples, the mean sperm head binding was 0.5% +/- 0.5, the mean sperm tail binding was 2.0% +/- 2.0 and the mean sperm head-tail binding was 3.0% +/- 2.0 for IgG, IgA and IgM type antibodies, respectively. The same samples exposed to positive sera showed 40.0% +/- 10.0 mean head binding, 7.0% +/- 8.0 mean tail binding and 47.0% +/- 11.0 mean head-tail binding. CONCLUSIONS: IBA is highly reproducible for detecting sperm antibodies in both fresh and cryopreserved/thawed samples of human spermatozoa.

Monoclonal antibody against a 52 K sperm surface protein inhibits sperm-zona pellucida interactions in the rat.

A major regulatory site for species specificity of fertilization in mammals lies at the level of sperm binding to the zona pellucida. This implies a high degree of complementarity between gamete and receptor molecules. These molecules support species-specific interactions between sperm and oocyte that lead to gamete fusion (fertilization). We identified a rat sperm head surface antigen using an IgG1 monoclonal antibody (HD1) against rat sperm of epididymis cauda. By electron microscopy the antigen was shown to be present on the plasma membrane surface of the sperm dorsal head. One- and two-dimensional immunoblotting analysis of sperm proteins demonstrated that HD1 reacted only with a 52 K molecule with a pI ranging from 6.6 to 7.2. The 52 K protein was first detected in situ by indirect immunofluorescence and showed to be underlining few elongated spermatids in testis. However, when the sperm reached the epididymis caput, the antigen was seen to be expressed on the dorsal surface of spermatozoa head. A similar fluorescence reaction was detected on sperms in the epididymis corpus and cauda. The specific spermatozoa-zona pellucida interaction was inhibited in the presence of monoclonal antibody HD1 in a sperm binding assay of in vitro fertilization.

Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium.

A quantitative immunohistochemical study was performed of the distribution of protein gene product 9.5 (PGP, a soluble protein localized in neurons and neuroendocrine cells as well as in some non-nervous cells) and ubiquitin along the rat epididymis. In the ductuli efferents, PGP immunoreaction was observed in the whole cytoplasm of some columnar cells; a smaller number of columnar cells showed ubiquitin immunoreactivity with limited apical and basal cytoplasmic localization. In the proximal caput epididymidis, the whole cytoplasm of all columnar cells showed PGP immunoreactivity, ubiquitin immunostaining was negative in this region. In the middle and distal caput epididymidis and the distal cauda, the apical cytoplasm of some columnar cells and the whole cytoplasm of some basal cells showed immunoreactivity to PGP. In these regions, immunoreactivity to ubiquitin was positive in the supranuclear cytoplasm of some columnar cells but not in the basal cells. No immunoreactivity to PGP or ubiquitin was detected in the corpus epididymis and the proximal cauda. Double immunostaining revealed that all the epididymal ubiquitin immunoreactive cells were also PGP immunoreactive, whereas most PGP immunoreactive cells did not immunoreact to ubiquitin. In ubiquitin-PGP immunoreactive cells, the site of the PGP immunoreaction differed from that of the ubiquitin immunoreaction. PGP-ubiquitin immunoreactive cells also seemed to be immunoreactive to anti-AE1/AE3 keratin antibodies. The spermatozoal heads were immunoreactive to PGP antibodies in the epididymal regions from proximal caput to distal cauda but not in the ductuli efferents. The findings suggest that non-ubiquitinated PGP immunoreactive proteins are secreted in the epididymis, mainly in the proximal caput, and attach to spermatozoa.

Expression of decay-accelerating factor (CD55) of the complement system on human spermatozoa.

Human spermatozoa were analyzed for their expression of decay-accelerating factor (DAF, CD55), a glycolipid-anchored regulatory protein of the C casade. Morphologic data showed that DAF was localized at the acrosomal region of the sperm head. Analysis with anti-DAF antibody-immunoprecipitated proteins of acrosome-reached spermatozoa revealed a 44- to 54-kDa protein. Carbohydrate analysis of sperm DAF indicated that it contains nonsialated N- and O-linked sugars. The absence of mature oligosaccharides on this protein appears to account for the difference in molecular mass between sperm DAF and the 70-kDa DAF expressed on other human tissues. Sperm DAF reinserted into sheep E and inhibited C-mediated lysis. This effect was reversed by mAb, which block DAF function. These results indicate that sperm DAF also possesses a glycolipid anchor. The expression of DAF on acrosome-reacted spermatozoa suggests that it may act concomitantly with other C regulators such as membrane cofactor protein to modulate the activation of C in the immunocompetent female genital tract and protect acrosome-reacted spermatozoa from C-mediated attack.

Characterization of the antigen recognized by a monoclonal antibody MN9: unique transport pathway to the equatorial segment of sperm head during spermiogenesis.

MN9, a monoclonal antibody raised against mouse spermatozoa, specifically recognizes the equatorial segment of sperm head in several mammalian species, including humans. Colloidal gold-immuno-electron microscopy of mouse spermatozoa has shown that the antigen is localized in the space between the outer and inner acrosome membranes and on the acrosome membranes at the equatorial segment. Immunoblotting after electrophoresis of spermatozoa from the cauda epididymidis has identified two immunoreactive bands: 38 kDa and 48 kDa in mouse, and 48 kDa in rat. During spermiogenesis in rat, this antigen is transported to the equatorial segment via a unique pathway, first appearing in some cisternae of the endoplasmic reticulum and in the Golgi apparatus of spermatids at around step 3. The antigen can further be found on the vesicles at the trans-side of the Golgi apparatus, in the matrix of the head cap, and on the head cap membrane in step-4 to step-7 spermatids. The antigen appears to be concentrated at the equatorial segment during late spermiogenesis. Neither the (pro-)acrosomic granule nor the surrounding membrane are required in this pathway. This pathway can be termed the 'Golgi-head cap tract'.