GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies

GDA-J/F3 monoclonal antibody (MoAb) recognized an antigen in the fibrous sheath (FS) of human spermatozoa. This was based on: (i) intracellular localization of the antigen which was limited to the principal piece of the sperm tail; (ii) its absence from the cilia of trachea and nasal mucosa which lack FS; (iii) immunogold electron microscopy (IEM) which confirmed its ultrastructural localization to the FS. The antibody was used for the detection of abnormal germ cells in human semen. Nucleated cells other than spermatozoa (NCOS) obtained from oligozoospermic donors were screened with GDA-J/F3 MoAb using the indirect immunofluorescence test. The antibody which stains only the tails in normal cells, produced diffuse cytoplasmic immunofluorescence inside some spermatids. Using phase contrast microscopy, the tails in these spermatids were either present or undetectable. Electron microscopy studies of the NCOS showed the lack of the FS in those which had the tails (afibrous tail), or the presence of disorganized tails (tail dysgenesis) in the others. This antibody therefore provides a useful analytical tool for probing the FS as well as for the easy identification of certain abnormal germ cells in human semen.     

Isolation and biochemical characterization of the human sperm tail fibrous sheath.

The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament.     

Andrology: The use of GDA-J/F3 monoclonal antibody for the detection of dead spermatozoa (necrosperm) during semen analysis

The distinction between immotile necrosperm (dead spermatozoa)and those with immotility due to other causes is of the utmostclinical importance. The supravital dyes currently used forthe identification of necrosperm are not highly reliable oraccurate. In this study, GDA-J/F3 monoclonal antibody (MoAb)which reacts with the fibrous sheath (FS) was used as a specificprobe for the detection of necrosperm using indirect immunofluorescence(IIF). Previously, several lines of evidence indicated the reactionof the antibody with necrosperm. This was confirmed in the currentstudy where GDA-J/F3 MoAb failed to react with viable swim-upseparated spermatozoa; such cells were only stained followingsperm demembranation with 1% Triton X-100. Furthermore, by usingimmunogold electron microscopy of a normozoospermic sperm sample,all the spermatozoa which reacted with GDA-J/F3 MoAb showeddamaged cytoplasmic membranes. Following these initial studies,sperm samples were obtained from 42 men attending infertilityclinics and assessed by conventional semen analysis and GDA-J/F3MoAb screening using IIF. The results showed a wide variationin sperm immotility and GDA-J/F3 reactivity; the ranges were19–99 and 0–50% respectively. This novel immunologicalapproach provides a simple and specific method of necrospermenumeration for the investigation of male infertility.     

The target antigen for GDA-J/F3 monoclonal antibody in the human sperm tail fibrous sheath is a non-collagenous asialo-glycoprotein: implications and significance

Enzymes and chemicals were used to analyse the biochemical structureof the antigenic epitope recognized by GDA-J/3 monoclonal antibody(MoAb) in the human sperm tail fibrous sheath. Treatment ofsperm dried onto slides with trypsin or dispase enzymes abolishedtheir immunofluorescence staining with GDA-J%3 MoAb, thus indicatingthe proteinaceous nature of the antigen. The proteolytic cleavageof GDA-J%F3 protein by trypsin, which also caused sperm decapitation,indicated the presence of peptide bonds involving the carboxylgroups of the basic amino acids, arginine and%or lysine. Theepitope was also glycosylated as demonstrated by its sensitivityto sodium metaperiodate treatment which was dose–dependent.The GDA-J%F3 antigenic epitope lacked sialic acid since pre–treatmentof spermatozoa with sialidase enzyme (neur–aminidase)had no effect on their reactivity with the antibody. The lackof collagenous domains in the GDA-J%F3 antigen was demonstratedby the failure of collagenase to abrogate sperm immunostainingwith the MoAb. Furthermore, type VII collagen of the skin basementmembrane (BM) was previously thought of as a potential targetantigen for GDA-J%F3 MoAb. This was ruled out since severalmonoclonal and polyclonal antibodies failed to detect the antigenin the spermatozoa using immunofluorescence and Western blotting.These data, therefore, show that the target antigen for GDA-J%F3MoAb is a non-collagenous asialo–glycoprotein, and byinference provide the first evidence for the glycosylation ofthe sheath proteins as another step of post–translationalmodification occurring during sperm tail development.     

AJ-FSI monoclonal antibody detects a novel group of non-glycostlated antigens within the human sperm tail fibrous sheath

AJ-FS1 is one of a new series of monoclonal antibodies (MoAbs)raised by immunizing mice with isolated human sperm tail fibroussheath. Usinjg indirect immunofluorescence (IIF), the AJ-FS1MoAb didnot react with the surface antigens of viable sperm,but didstain the falgellar principal piece of sperm dried ontoslides or those demenbranated with 1% Triton X-100. The specificityof the antibody for the fibrous sheatht was confirmed by immunogoldelectron microscopy which showed the distrobution of gold particleson the outer fibrous sheath surface, and its reaction with theWestern blots of purified fibrous sheath preparations wheremultiple protein bands with mol. wt ranging between 97 and 28kDa were identified. The same peptides were alsod detected inurea-dithiothreitol (DTT) fraction of sequentially extractedspermatozoa but not in Triton or Triton-DTT sperm lysates. Thefailure of sodium metaperiodate to abrogate the antobody reactionin both Western blotting and IIf indicated on the non-glycosylatednature of the antigens. IIF screening of human testicular cryosatatsections with AJ-FS1 MoAb showed its reactivity with the assembledfibrous sheath of maturing sperm tails only; thus indicatingthe late apperance of the antigens during spermatogensis. Theanti-body did not react with skin, oesophagus, tongue, liver,kidney, placenta, uterus, cervix or their blood vessels. Thesignificance of these results is discussedtogether with theimportnce of AJ-FS1 MoAb as a specific probe for the characterizationof the fibrous sheath antigens in both normal and abnormal falgella.     

Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa

Dysplasia of the fibrous sheath (DFS) is an anomaly found in spermatozoa of severe asthenozoospermic patients. Marked hypertrophy and hyperplasia of the fibrous sheath is the common characteristic. Immunocytochemistry allowed us to visualize the distortions and incidence of tail structure abnormalities associated with this phenotype in six patients; four with a complete form and two with an incomplete form of this pathology previously diagnosed and studied by electron microscopy. Microtubules and fibrous sheaths were studied using monoclonal antibodies against alpha-acetylated tubulin and anti-FSC1 (the major protein component of the fibrous sheath). Mitochondrial sheaths were visualized using the mitochondrion-specific vital dye MitoTracker green FM(TM). Phase contrast and fluorescent microscopy of semen samples showed large numbers of spermatozoa with short, rigid, thick and irregular tails. As expected, anomalous and completely distorted fibrous sheaths, severe alterations of the axonemal microtubules and different patterns of mitochondrial sheath configurations were found. While ultrastructural studies of thin sections allow an in-depth knowledge of the internal organization of the sperm tail, fluorescence labelling of selected sperm components affords a unique view of the whole flagellum including topographical relationships of various organelles. The combination of these different approaches is essential for a comprehensive understanding of this particular pathology.     

Immunology: AJ-FS9 monoclonal antibody detects masked antigens within the human sperm tail fibrous sheath

AJ-FS9 is one of a new series of monoclonal antibodies (mAbs)raised by immunizing mice with isolated human sperm tail fibroussheath (FS). Using indirect immunofluorescence (IIF) of humanspermatozoa dried onto slides, the AJ-FS9 mAb reacted with theprincipal piece of occasional spermatozoa. Following their enzymatictreatment with trypsin, dispase or collagenase, but not sulphatase,all the spermatozoa were stained at their principal piece. Theultrastructural localization of the antigens to the FS was establishedby immunogold electron microscopy, which showed the distributionof gold particles on the FS outer surface of spermatozoa sequentiallytreated with 1% Triton and dispase; spermatozoa demembranatedby Triton alone showed no reaction. For biochemical characterization,spermatozoa were lysed with 1% Triton, and the sperm pelietwas run through a reducing sodium dodecyl sulphate-polyacrylamidegel electrophoreesis, Western blotted and immunostained withAJ-FS9. The results showed the reaction of the antibody withthree protein bands with molecular masses ranging between 46and 56 kDa. IIF screening of human testicular cryostat sectionswith AJ-FS9 mAb showed its reactivity with occasional spermtails; but following their dispase treatment, all spermatozoawere stained. The restricted staining of the assembled FS ofmaturing sperm tails indicated the late appearance of the antigensduring spermatogenesis. The antibody did not react with spermcell precursors or other cell types within/without the seminiferoustubules. Untreated and dispase-treated frozen sections of skin,oesophagus, tongue, liver, kidney, stomach, ileum or their bloodvessels showed no reaction. These data provide the first evidencefor the presence of masked antigens within the human sperm tailFS, and their significance is discussed.     

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

Immunochemical localization and characterization of proteins from mouse cauda epididymis using human sperm specific monoclonal antibody

Monoclonal antibodies (mAbs) to sperm specific antigens were generated by fusion of mouse myeloma cells (SP2/0) with splenocytes of female BALB/c mice hyperimmunized with washed human spermatozoa. The resultant hybridomas producing antisperm antibodies were first screened by ELISA. Out of these, one of the mAbs (D2G4) which recognized target antigens restricted to the acrosomal cap was chosen for these studies. The mAb D2G4 was found to be an agglutinating antibody and was also found to cross-react with mouse epididymal spermatozoa in ELISA and indirect immunofluorescence. The origin of antigens reacting with monoclonal antibody D2G4 was investigated. When frozen sections of murine testis and various regions of epididymis were reacted with mAb D2G4, only the cauda epididymal region was stained. Western blot of proteins from the epididymal spermatozoa and fluid indicated the presence of two bands of mol. wt 45 and 26 kd. These bands were identical under reducing and non-reducing conditions. These observations suggest that the two proteins are structurally similar or at least have a common epitope. These data indicate that the proteins recognized by D2G4 are acquired by spermatozoa during their passage and storage in the cauda epididymis.     

Sperm autoantigens and fertilization. III. Ultrastructural localization of guinea pig autoantigens

Guinea pig sperm autoantigens have been localized by direct and indirect immunoferritin techniques in (1) plasma membrane over the entire sperm head, (2) acrosomal contents, (3) fibrous sheath and outer dense fibers of the tail filament, and (4) the inner acrosomal membrane of 50% of acrosome reacted spermatozoa. These cellular structures are known to be involved in guinea pig sperm rouleaux formation, acrosome reaction, interaction of acrosome-reacted sperm with zona pellucida and with the vitellus of guinea pig ova. Since IgG and Fab of autoantiserum to guinea pig spermatozoa have been shown to interfere with these cellular reactions, this study provides further evidence, albeit indirect, that sperm autoantigens are involved in these cellular events.     

Gene deletions in an infertile man with sperm fibrous sheath dysplasia

BACKGROUND: Asthenozoospermia may sometimes be related to geneticstructural defects of the sperm tail detectable by transmissionelectron microscopy. Dysplasia of the fibrous sheath (DFS) isa genetic sperm defect, characterized by dysplastic developmentof the axonemal and periaxonemal cytoskeleton. We report thecase of an infertile man with normal sperm count and total spermimmotility in which dysplasia of the fibrous sheath, Akap3,Akap4 gene deletions, meiotic segregation of chromosomes 18,X and Y and Y microdeletions were investigated. METHODS: A 32-year-oldman with a 3-year history of primary infertility presented atour Regional Referral Center for Male Infertility. Family medicalhistory, lymphocyte karyotype, PCR analysis, physical examination,hormone assays and semen analysis were performed. RESULTS: Ultrastructuralsperm evaluation showed dysplasia of the fibrous sheath. Immunostainingof AKAP4 protein was negative in sperm tails. PCR analysis revealedintragenic deletions of the Akap3 and Akap4 genes. Fluorescencein situ hybridization on sperm showed a high frequency of XYdisomy. CONCLUSION: In this infertile patient, our results suggesta possible relationship between dysplasia of the fibrous sheath,partial deletions in the Akap3 and Akap4 genes and absence ofAKAP4 protein in the fibrous sheath. These findings, however,were not detected in another four patients with dysplasia ofthe fibrous sheath. Our results require future confirmatorymolecular analyses.     

SHORT COMMUNICATION: Origin, development and ultrastructure of boar spermatozoa with folded tails and with two tails

Speramtozoa from the three epididymal regions (head, body andtail) of healthy and sexually mature boars been examined bylight miroscopy, and scanning and transmission elctron and internalmorphologies of aberrant spermatozoa with folded tails and spermatozoawith one or two heads and two fused tails and spermatozoa withone or two heads and two fused tails have been established.A count carried out in each region of the epididymis indicatedthat significant differences(P >0.01) exist in the frequenciesof each type of malformation and the epididymal region fromwhich the spermatozoa in the cauda of the epididymis from immaturespermatozoa that have not ejected the distal cytoplasmic droplet.The plasma membrane which covers the main piece is fused withthe membranes of the midpiece, the connecting piece and thehead. The fibrous sheath deforms the connectiong piece and thehead. the fibrous sheath deforms the mitochondrial sheath andisplaced between the plasma membrane and the postacrosomal denselamina. Spermatozoa with one head and two fused tails originatein the epididyaml body from spermatozoa with one head and twounfused tails coming from the cephalic region of the edididymis.Spermatozoa with two heads and two fused tails originate inthe cephalic region of the epididymis by head-to-head agglutinationof two spermatozoa with two fused tails increases as they progressthrough the epidididymal duct. Their tails, parallel in monocephalicspermatozoa and helicoid in bicephalic spermatozoa, have twocomplete axonemal axes. In their midpiece, the mitochondrialsheaths of the two axes are fused, producing an 8-shapedsheath.     

Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15–25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg–sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.     

Origin, development and ultrastructure of boar spermatozoa with folded tails and with two tails.

Spermatozoa from the three epididymal regions (head, body and tail) of healthy and sexually mature boars have been examined by light microscopy, and scanning and transmission electron microscopy. The origin, development and external and internal morphologies of aberrant spermatozoa with folded tails and spermatozoa with one or two heads and two fused tails have been established. A count carried out in each region of the epididymis indicated that significant differences (P less than 0.01) exist in the frequencies of each type of malformation and the epididymal region from which the spermatozoa come. Spermatozoa with folded tails at Jensen's ring originate in the cauda of the epididymis from immature spermatozoa that have not ejected the distal cytoplasmic droplet. The plasma membrane which covers the main piece is fused with the membranes of the midpiece, the connecting piece and the head. The fibrous sheath deforms the mitochondrial sheath and is placed between the plasma membrane and the postacrosomal dense lamina. Spermatozoa with one head and two fused tails originate in the epididymal body from spermatozoa with one head and two unfused tails coming from the cephalic region of the epididymis. Spermatozoa with two heads and two fused tails originate in the cephalic region of the epididymis by head-to-head agglutination of two spermatozoa and later fusion of their tails. The frequency of spermatozoa with two fused tails increases as they progress through the epididymal duct. Their tails, parallel in monocephalic spermatozoa and helicoid in bicephalic spermatozoa, have two complete axonemal axes. In their midpiece, the mitochondrial sheaths of the two axes are fused, producing an 8-shaped sheath.     

Nuclear immunofluorescence in porcine kidney cells infected with Japanese encephalitis virus.

Acetone-fixed porcine stable kidney (PS) cells infected with Japanese encephalitis (JE) virus were stained in indirect fluorescent antibody (FA) assay with anti-JE virus monoclonal (MoAb) and polyclonal (immune PF) antibodies. First positive immunofluorescence (IF) occurred in the cytoplasm with MoAb Hs-1 (anti-envelope, JE-specific) and immune PF after 7 hr post-infection (p. i.); it became prominent by 15 hr to 48 hr (maximum) when cells reacted strongly also with MoAb Hx-3 (flavivirus crossreactive epitope). In addition, 15 to 20% of the infected cells, which revealed positive cytoplasmic IF, showed intranuclear IF with Hs-1, Hx-3, and immune PF by 20 to 24 hr p.i. By 48 hr, the intranuclear IF was not observed or became diminished. These observations indicate that the JE virus specific epitope Hs-1 appeared first followed by the flavivirus cross-reactive epitope Hx-3. Nuclei of the infected cells seem to play some role in the replication of JE virus.     

RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives

BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.     

Sperm ubiquitination in patients with dysplasia of the fibrous sheath

BACKGROUND: Human sperm with structural abnormalities display an increased content of the cellular proteolytic marker peptide, ubiquitin. We investigated whether dysplasia of the fibrous sheath (DFS), a severe structural anomaly found in the sperm of some asthenozoospermic patients, is accompanied by (i) increased ubiquitination of the sperm surface and (ii) by increased ubiquitination of the sperm mitochondria. METHODS AND RESULTS: Five DFS patients and eight fertile donors were studied by immunocytochemistry with anti-ubiquitin antibodies. Increased cross-reactivity of the ubiquitinated mitochondrial epitopes was seen in 32-50% of DFS sperm, but only 2-4.1% of sperm from fertile donors. Sperm surface ubiquitination assessed by sperm-ubiquitin tag immunoassay (SUTI) and immunofluorescence demonstrated an increased sperm ubiquitination in all DFS patients. The average median value of ubiquitin-induced fluorescence in DFS patients was 25.8 counts (range 19.8-37.9), as opposed to 13.4 counts range (9.3-16.6) in fertile men. Sperm with 'stump tails', coiled tails, twin and triplet sperm, and clusters of immature spermatogenic cells were common. CONCLUSIONS: DFS sperm have increased cross-reactivity to anti-ubiquitin antibodies, a finding consistent with the ubiquitination of defective sperm shown in animal models. These results justify the use of ubiquitin-based assays for objective semen analysis in infertile men with heritable defects.     

Antisperm antibodies detection by flow cytometry is affected by aggregation of antigen-antibody complexes on the surface of spermatozoa

Flow cytometry (FCM) analysis of live antibody-coated spermatozoa subjected to immunofluorescence staining (FCM test) is considered an objective method for the quantitative detection of antisperm antibodies (ASA). But the cross-linking of cell surface antigen (Ag) with bivalent antibodies and/or antigen-antibody (Ag-Ab) complexes with second antibodies may induce the reorganization of surface components (patching and capping) and result in their shedding from the sperm surface. The present study estimates the relationship between aggregation of Ag-Ab complexes on the sperm surface and the results of indirect FCM test. Swim-up spermatozoa of normozoospermic men were incubated with ASA-positive sera from infertile patients and with second antibodies fluorescein isothiocyanate (FITC)-labelled goat anti-human IgG polyclonal antiserum under different conditions and then analysed by FCM and fluorescence microscopy. It was shown that low temperature, cytochalasin B, excess or lack of the primary and/or secondary antibodies and sperm fixation by paraformaldehyde may inhibit aggregation and shedding of Ag-Ab complexes and dramatically increase ASA quantity determined on the sperm surface. However, inhibition of aggregation on the live sperm surface was observed only in a minority of ASA-positive samples and was poorly reproducible using semen of different donors. A high probability of Ag-Ab complex shedding from the sperm surface during experimental manipulation limits the use of indirect FCM test for quantitative ASA determination.     

Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82.

Sperm motility is regulated by the cAMP-dependent protein kinase (protein kinase-A)-mediated phosphorylation of a group of largely unidentified flagellar proteins. Human AKAP82 (hAKAP82) and its precursor protein, pro-hAKAP82, are members of the A-kinase anchor protein (AKAP) family. These proteins tether protein kinase-A to the fibrous sheath of human spermatozoa and presumably localize the activity of the kinase near specific targets in the sperm flagellum. In this way, pro-hAKAP82 and hAKAP82 may be involved in regulating sperm motility. Similar to its homologues in other species, pro-hAKAP82 is proteolytically processed to hAKAP82. However, the amount of processing of pro-hAKAP82 in human spermatozoa is less than the amount of processing of the precursor in other species. We postulated that this lower extent of processing may be related to lower percentages of human sperm motility. In addition, both pro-hAKAP82 and hAKAP82 are tyrosine phosphorylated in a capacitation-dependent manner. Since capacitation is associated with hyperactivated motility, we postulated that tyrosine phosphorylation of pro-hAKAP82/hAKAP82 is associated with changes in motility. However, using a combination of immunofluorescence and immunoblotting approaches, we found no evidence for an association between either processing or tyrosine phosphorylation of pro-hAKAP82/hAKAP82 and significant differences in motility in spermatozoa from normal men.     

Localization of actin, alpha-actinin, and tropomyosin in bovine spermatozoa and epididymal epithelium.

Actin, alpha-actinin, and tropomyosin were localized in the testicular, epididymal, and ejaculated spermatozoa and in the epithelium of the bovine epididymis by means of specific antibodies using an indirect immunofluorescence technique. Immunocytochemical results were confirmed by the western blot analysis. Independent of the method of fixation, washing, or sonication, actin, alpha-actinin, and tropomyosin were all consistently localized in the neck of the spermatozoa. Actin and tropomyosin present in the postacrosomal area could be removed by sonication, whereas alpha-actinin in the basal plate appeared to be resistant to the treatment. In the unwashed spermatozoa alpha-actinin-specific immunofluorescence was seen over the acrosomal area, whereas in the washed sperm it appeared as a narrow cap at the margin of the head. In the latter location, its distribution was similar to that of tropomyosin. In the majority of preparations, tropomyosin could be localized in the principal piece of the tail. Even though some actin-specific immunofluorescence could be identified in the principal piece of the tail of the testicular and epididymal spermatozoa, a strong immunoreaction appeared only in the ejaculated spermatozoa. In the principal cells of the epididymal epithelium, specific fluorescence for actin, alpha-actinin, and tropomyosin occurred in the apical junctional complex. Basal bodies of the solitary cilia of the epididymal epithelium were labelled with antitropomyosin and anti-alpha-actinin antibodies. Besides offering new information about the cytoskeletal composition of the mammalian sperm, the present results support the hypothesized homology between the connecting piece of the sperm neck and the basal body of the cilia.