Canine-specific tail-in, head-out sperm agglutination.

An interesting pattern of tail-in, head-out sperm agglutination was identified in a Brucella canis seronegative subfertile dog. Centrifuged seminal plasma from this dog could induce a similar pattern of agglutination in six other dogs, but not in ejaculates from a single stallion and two rams. The agglutination pattern was short-lived and appeared to depend on motility of spermatozoa, although intensity of agglutination may have been affected by concentration of agglutinating factor.

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.

GalNAcß1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121.

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with ß-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcß-nLc4Cer² sequence.

Sperm fibrous sheath proteins: a potential new class of target antigens for use in human therapeutic cancer vaccines.

Cancer vaccines have been demonstrated to be a promising strategy for treating human neoplastic disease, but one of the limitations of these vaccines remains the paucity of target antigens to which to direct an effective immune response. We hypothesize that sperm fibrous sheath proteins may be a new class of useful antigens for developing successful cancer vaccines. This hypothesis is supported by the expression of two sperm fibrous sheath proteins, called sperm protein 17 and calcium-binding tyrosine-phosphorylation regulated protein, in tumors of unrelated histological origin and their capability to induce T cell-based immune responses.

Monoclonal antibody from vasectomized mouse identifies a conserved testis-specific antigen TSA70.

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.

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.

Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence.

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.

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.

Testis-specific antigen (TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization.

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.

Production and characterization of monoclonal antibodies to the major protein of boar outer dense fibers.

Outer dense fibers (ODF) are structural elements in the mammalian sperm tail which surround the axoneme in the midpiece and principal piece and probably may help to maintain the elastic structures and elastic recoil of the sperm tail. In the present study, we have generated and characterized and describe a series of monoclonal antibodies (mAbs) against the 30 kDa major protein from boar ODF. For antibody screening an ELISA was developed using a newly developed method to fix the ODF proteins to the solid phase. A total of seven mAbs were selected and characterized by ELISA, Western blot and immunofluorescence. The mAbs recognize the major protein component of boar ODF on preparative Western blot and mark the mid- and principal piece of demembranated flagella. These mAbs also recognize the mid- and principal piece of demembranated human spermatozoa from normozoospermic patients, but not from those with asthenozoospermia. For the first time, we succeeded in obtaining hybridoma cell lines that secrete mAbs of class IgM, which react with the 30 kDa protein of boar ODF.

Immunochemical characterization of a human sperm fibrous sheath protein, its developmental expression pattern, and morphogenetic relationships with actin.

Among the monoclonal antibodies (MAbs) prepared against human sperm extracts, MAb 4F7 was found to be specific to the human and Macaca fascicularis sperm cytoskeletal fibrous sheath (FS). In Western blotting, MAb 4F7 stains a doublet of polypeptides of about M(r) 95 x 10(3) in extracts of human sperm cells. These polypeptides are not recognized by the KL1 anti-cytokeratin MAb, nor by the MAbs known to bind to the carboxy terminal (IFA) and to the amino terminal (ME101) rod domain of intermediate filaments. Sequential extraction procedures shows that the FS polypeptides recognized by MAb 4F7 are exposed after treatment with 8 M urea 4F7 immunoreactivity is lost after treatment with high ionic solutions (NaCl; KCl, Kl). Immunogold electron microscopy reveals that this protein is present throughout the FS. This FS antigenic determinant first accumulates in an FS proximal body in late spermatids, then in granules extending distally along the flagellum. Staining of spermatozoa with flagellar dysgenesis reveals that this FS protein colocalizes with actin no matter what the location of their abnormal assembly. These data suggest that the transient microtubule-like spindle-shaped body of as yet unknown function could be involved in FS protein deposition and that the assembly of the FS and actin could be under the control of some common morphogenetical factor(s). MAb 4F7 should allow further investigations of this peri-axonemal structure in both normal and pathological conditions.

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.

The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility.

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: unique distribution of glutamylated tubulin.

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti alpha and beta-tubulin, 6-11B-1 anti-acetylated alpha-tubulin, and GT335 anti-glutamylated alpha and beta-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated alpha-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated alpha and beta-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum.

Inhibition of flagellar beat frequency by a new anti-beta-tubulin antibody.

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

Immunological cross-reaction between sperm dynein heavy chains from different species.

The 19S outer arm dynein of trout sperm flagella is a complex of proteins composed of two heavy chains, five intermediate chains and at least six light chains. After dialysis against a low ionic strength buffer, its beta subunit was purified and used to generate a rabbit polyclonal antibody. This polyclonal antibody reacted strongly with the trout beta dynein heavy chain (DHC) but not with the trout alpha dynein heavy chain; it also recognised the dynein intermediate chains and tubulins. A specific antibody directed against the beta DHC was obtained by blot-affinity purification. This specific anti-trout beta DHC reacted with the beta DHC from the sea-urchin sperm 21S dynein and also with one heavy chain (> 400 kDa) of demembranated ram sperm. This anti-beta DHC antibody, and also the whole polyclonal, did not react with heavy chains in trout brain or liver extracts, sheep brain extract or purified brain and testicular cytoplasmic dyneins. These results suggest that a specific epitope of one of the sperm outer arm dynein heavy chains is conserved throughout evolution and that this epitope is not present on cytoplasmic dynein.

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.

Identification of a 53-kDa antigen in the fibrous sheath of avian spermatozoa.

The intracellular fibrous sheath that surrounds the proximal part of the sperm flagellar axoneme in non-passerine birds is structurally different from that of mammals. We raised a monoclonal antibody against the fibrous sheath of cockerel spermatozoa by in vitro immunisation. Indirect immunofluorescence and immunogold labelling showed that the antibody bound specifically to the fibrous sheath. It also labelled the fibrous sheath in quail spermatozoa. In both species the antibody bound an antigen that had a molecular weight of about 53 kDa. In tissue sections from adult cockerel testis the antigen was located in spermatids and spermatozoa with little cross-reactivity with the basal region of the seminiferous epithelium or interstitial tissue. The antibody may prove to be a useful tool in studies of avian spermiogenesis.

Isolation and partial characterization of rat sperm tail fibrous sheath proteins and comparison with rabbit and human spermatozoa using a polyclonal antiserum.

Rat sperm tail fibrous sheath was isolated using mechanical and chemical dissection methods from spermatozoa collected from the cauda epididymis. The procedures used to isolate the fibrous sheath were monitored by phase-contrast microscopy and purity was verified by electron microscopy. SDS-PAGE of isolated total fibrous sheath revealed at least 17 bands when stained with Coomassie brilliant blue and 20 bands with silver stain. The most intensely staining proteins, using both staining methods, were a double band at 80-87 kDa, and a band at 28.5 kDa, whereas with silver staining, bands at 66.2 kDa and kDa were also intensely stained. Electroelution following SDS-PAGE was used to isolate 11 of these proteins (116.4, 87.5, 80.9, 66.2, 57.2, 49.7, 46.8, 37.3, 32.7, 28.5 and 15.5 kDa). Amino acid analysis revealed that these proteins were abundant in aspartic and glutamic acid, glycine, serine and leucine, while histidine and phenylalanine were of low abundance. The content of cystine varied widely from 9.4% to 1.4%. The amino termini of the 80.9 kDa, 32.7 kDa, 28.5 kDa and 15.5 kDa proteins were blocked. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat fibrous sheath was localized to the principal piece of the rat, rabbit and human spermatozoa, but in the rabbit it also labelled the equatorial region of the head. Western blotting detected all protein bands in isolated fibrous sheath and a similar range of proteins in the spermatozoa of rat and rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral steroid therapy for subfertile males with antisperm antibodies in the semen: prediction of the responders.

This study was performed to examine the effectiveness of steroid therapy in subfertile men with antisperm antibodies and infertility lasting > 1 year, to predict those who would respond positively, and to evaluate the effect of the therapy on semen parameters and antisperm antibodies. The patients included 48 subfertile couples in whom the male partner had > or = 20% motile spermatozoa with bound antibodies of immunoglobulin (Ig)G, IgA or a combination of both, and were treated with prednisolone, 40 mg a day, for the first 10 days, then 5 mg on days 11 and 12 of the partner's cycle for 9 months. Twelve couples became pregnant; a cumulative conception rate of 30.2% was achieved at 9 months. The pregnant group started with significantly higher concentrations of IgG (tail) and grade I motility (P = 0.03 and P = 0.02 respectively). Multi-covariate discrete logistic regression analysis on the initial screening semen samples predicted a higher chance of conception for those with high levels of IgG (tail) (P = 0.006, sensitivity = 33%, specificity = 93%, correct = 75%, false positive = 33% and false negative = 24%). In the pregnant group, prednisolone caused a significant increase in grade I motility (P = 0.03). In the non-pregnant group, there was a significant increase in grade I motility (P = 0.0002), amplitude of lateral head displacement (P = 0.03), curvilinear velocity (P = 0.02) and decrease in grade IV motility (P = 0.03) following prednisolone treatment. In both groups there was suppression of the total antisperm antibody concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)