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Biology of Reproduction 65, 672-679 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Differential Subcellular Distribution of Tubulin Epitopes in Boar Spermatozoa: Recognition of Class III ß-Tubulin Epitope in Sperm Tail1

Jana Peknicová2,a, Alena Kubátováa, Vadym Sulimenkob, Eduarda Dráberováb, Vladimír Viklickyb, Pavel Hozákc, and Pavel Dráberb

a Department of Biology and Biochemistry of Fertilization b Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic c Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic

ABSTRACT

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III ß-tubulin. The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III ß-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of ß-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization. Three monoclonal antibodies, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III ß-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all ß-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.

acrosome reaction, gamete biology, sperm

INTRODUCTION

Microtubules are cytoskeletal structures essential for a wide variety of cellular functions. They are assembled from {alpha}ß-tubulin heterodimers and a collection of microtubule-associated proteins [1]. Tubulin is a highly heterogeneous protein, and more than 20 charge variants (isoforms) can be distinguished by isoelectric focusing. Some of the isoforms on both subunits result from the expression of multiple tubulin genes because {alpha}-tubulin as well as ß-tubulin are encoded by multigene families. The gene products differ primarily in the C-terminal variable domains that consist of 15 amino acids. In vertebrates, seven ß-tubulin isotypes were identified on the basis of these variable domains and cell-type distribution [2]. In contrast, the N-terminal structural domains are less heterogeneous and contain GTP-binding sites essential for assembly into microtubules [3]. It is still an unresolved issue whether all ß-tubulin isotype classes are functionally equivalent or whether some are unique in specifying the functional properties of microtubules in particular tissues. Tubulin heterogeneity is further increased by post-translational modifications [4].

The spermatozoon is the end product of the process of gametogenesis in the male and represents a highly specialized mammalian cell with specific morphology and motility. The mature spermatozoon exhibits extraordinary structural compartmentalization that is related to the presence of cytoskeletal proteins [5–8] and also has a functional role in connection with fertilization and motility [9]. The major compartments of a mature spermatozoon are the head, the neck, and the tail. The sperm head contains the nucleus and acrosome surrounded by cytoplasm. The acrosome contains enzymes required for the sperm binding to enable sperm to penetrate through the investments of eggs and to achieve fertilization [10]. Cytoskeletal proteins were detected in subacrosomal, para-acrosomal, and postacrosomal regions of the head [11]. The tail can be subdivided into the middle piece, principal piece, and end piece. The main structural component within the flagellum of the sperm is the axoneme that is composed of a complex of microtubule arrays [12]. Although the organization of microtubules in the neck and tail is known in great detail [11], data on differential distribution of the antigenic determinants of tubulin in mammalian sperm cells are limited. Using well-characterized monoclonal antitubulin antibodies, it was possible to distinguish different structural regions immunologically in mature mammalian spermatozoa [13–16], indicating functional heterogeneity of tubulins.

In the present study we report on differential subcellular distribution of tubulin epitopes on boar spermatozoa, detectable by a panel of well-characterized monoclonal antibodies against different structural domains of tubulin and antibodies specific to class III ß-tubulin. We show that antibodies discriminate between various parts of the spermatozoon and that epitopes of class III ß-tubulin are present in the flagellum. A tubulin epitope was also detected in the head, and its distribution changed after the acrosome reaction.

MATERIALS AND METHODS

Materials

Analytical-grade chemicals were utilized. Ionophore A 23187, Percoll, and standards of molecular weights were obtained from Sigma-Aldrich (Prague, Czech Republic). Vecta-Shield mounting medium was obtained from Scandic (Prague, Czech Republic). Bio-Lyte 5/7 and 3/10 were obtained from BioRad Laboratories (Hercules, CA). Supersignal WestPico Chemiluminescent reagents were bought from Pierce (Rockford, IL). Autoradiography films X-Omat AR were from Eastman Kodak (Rochester, NY). Ejaculated spermatozoa were suspended in KARE I dilutor (Plebo, Brno, Czech Republic).

Antibodies

The following monoclonal antibodies were used: TU-01 (IgG1) [17, 18] and TU-04 (IgM) [19] are directed against the N-terminal and C-terminal domain of {alpha}-tubulin, respectively [17]. Antibodies TU-06 (IgM) [20] and TU-12 (IgM) [14] are directed against the N-terminal domain or C-terminal domain of ß-tubulin, respectively. Antibodies TU-20 (IgG1) [21], SDL.3D10 (IgG2b) (Sigma-Aldrich), and TUJ1 (IgG2a) (BAbCO, Richmond, CA) were raised against the neuron-specific class III ß-tubulin. Antibodies TU-20 [21] and SDL.3D10 [22] were prepared against the synthetic eight-amino acid peptide, ESESQGPK, corresponding to the human class III ß-tubulin sequence 441–448 [23]. Antibody TUJ1 was prepared against chicken tubulin [24]; its epitope is located in the peptide covering the last 14 amino acids on the C-terminal end of chicken class III ß-tubulin [25]. The quality of ejaculates was evaluated by monoclonal antibody ACR.2 (IgG) against acrosin [26]. Monoclonal antibodies Prog.12 (IgM) and Prog.13 (IgG) against progesterone [27] were used as negative controls. Anti-mouse Ig antibody conjugated with horseradish peroxidase was purchased from Promega Biotech (Madison, WI). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin (SwAM-FITC) was obtained from SEVAC (Prague, Czech Republic) and 10-nm gold-conjugated anti-mouse antibody was from British BioCell International (Cardiff, UK).

Cells

Fresh boar ejaculates (Insemination Station, Klimetice, Czech Republic), diluted in KARE I dilutor (14.3 mM sodium bicarbonate, 12.25 mM sodium citrate, 364 mM glucose, and 12.3 mM EDTA, pH 7.5), were centrifuged at 400 x g for 10 min at room temperature. The sperm pellet was washed with Tris-buffered medium (TBM; 113.1 mM NaCl, 3 mM KCl, 10 mM CaCl2, 20 mM Tris, 1 µM glucose, 5 mM natrium pyruvate, pH 7.7) [28] to remove the dilutor components, layered on a 40–80% discontinuous Percoll gradient in TBM, and centrifuged at 200 x g for 45 min at room temperature. After centrifugation, the 80% layer was diluted in TBM, and the cells were washed again in the same medium. After washing, the spermatozoa were diluted in TBM to a final concentration of 2 x 107 cells/ml.

Ionophore Treatment

The acrosome reaction was induced in freshly ejaculated boar spermatozoa (2 x 107 cells/ml) after gradient centrifugation by addition of the calcium ionophore A-23187 to a final concentration of 2 µM, followed by incubation for 30 min at 37°C in 5% CO2.

Immunofluorescence

For indirect immunofluorescence the washed sperm cells (2 x 107 cells/ml) were resuspended in phosphate-buffered saline (PBS; 0.15 M NaCl, 0.02 M NaH2PO4, pH 7.4), and 10-µl drops were loaded on glass slides and air-dried. Slides were fixed 12 min with methanol precooled to -20°C and then incubated for 6 min in acetone at -20°C [14]. To detect the intra-acrosomal protein acrosin, the air-dried cells were fixed 10 min with acetone at room temperature [26]. The cells on slides were incubated with monoclonal antibodies, in the form of ascitic fluids diluted in PBS. The antibodies TU-01, SDL.3D10, and TUJ1 were diluted 1:80; TU-04, TU-06, TU-12, and TU-20 were diluted 1:20. The incubation with primary antibodies took place at 37°C for 90 min. After three washings in PBS (10 min each), incubation with the secondary FITC-conjugated antimouse antibody diluted 1:20 in PBS took 60 min at 37°C. Afterward, slides were washed again in PBS (3 x 10 min) and the cells covered with mounting medium. For appropriate controls, smears were incubated with a nonspecific monoclonal antibody, with the supernatant of myeloma cells, and with the FITC-conjugate alone. Samples were examined with a Nikon Labophot-2 fluorescent microscope equipped with 40x Nikon Plan 40/0.65 lenses and photographed with a COHU4 charge-coupled device camera (Japan) with the aid of Lucia imaging software (Laboratory Imaging, a.s., Prague, Czech Republic) or with a confocal microscope Leica TCS-SP (Leica, Micro, Prague, Czech Republic) with Ar (488, 458 nm) and HeNe (543, 633 nm) lasers equipped with a 60x objective. Neither the negative control antibodies nor the conjugate alone gave any detectable staining.

In some immunofluorescence experiments antibodies TU-20 and SDL.3D10 were preabsorbed with the peptide used for immunization; i.e., an eight-amino acid peptide, ESESQGPK, corresponding to the human class III ß-tubulin sequence 441–448. An eight-amino acid peptide GEEEGEEY corresponding to the porcine {alpha}-tubulin sequence 444–451 [29] was used as control. Two molar ratios of antibody:peptide were used, 1:10 and 1:100. Mixtures of antibody and peptides were incubated for 30 min at room temperature.

Immunoelectron Microscopy

Sperm cells (2 x 107/ml) were pelleted, fixed (40 min, 0°C) in 3% paraformaldehyde, 0.1% glutaraldehyde in Sörensen buffer (SB; 0.1 M Na/K phosphate buffer, pH 7.3) and washed in SB (2 x 10 min). After centrifugation into 1% agarose, blocks were incubated in 0.02 M glycine in SB (20 min), washed in SB, and dehydrated in ethanol. Ethanol was replaced in two steps by LR White resin, and the blocks were polymerized by UV light (20 h, 4°C). Eighty-nanometer sections were prepared using a diamond knife. Nonspecific labelling was blocked for 30 min by normal 10% goat serum in PBT (PBS with 0.05% Tween 20). Next, the sections were incubated 45 min with individual undiluted monoclonal antibody supernatants, washed three times with PBT, and incubated with 10-nm gold-conjugated goat antimouse antibody (1:30 in PBT, 30 min). After washing in PBT and water, the sections were stained 5 min with a saturated solution of uranyl acetate in water and observed in a Philips CM 100 electron microscope (Eidhoven, Holland). Control samples were developed without primary antibody.

Gel Electrophoresis and Immunoblotting

Sodium dodecyl sulfate PAGE was performed according to Laemmli method. Washed isolated sperm cells (5 x 106 cells/ml) were pelleted and resuspended in 100 µl of twice concentrated SDS-sample buffer. After heating in a boiling water bath (3 min), cooling, and centrifuging (15 000 x g, 3 min, 4°C), the samples were divided in aliquots and stored at -70°C. Aliquots of the extract corresponding to 0.5 x 106 cells were loaded per lane. Samples were run on 7.5% polyacrylamide gels, and separated proteins were then electrophoretically transferred onto nitrocellulose [31]. Details of the immunostaining procedure are described elsewhere [26]. The antibodies TU-01, TU-06, and TU-12, in the form of ascitic fluids were diluted 1:1000; the antibody SDL.3D10 was diluted 1:500. Blots were incubated with secondary antibody conjugated with horseradish peroxidase and diluted 1:5000, and the bound antibodies were detected after washing with enhanced chemiluminescence reagents according to the manufacturer's directions using autoradiography films X-Omat AR.

Two-dimensional electrophoresis was performed as described [32]. Washed isolated sperm cells (1 x 108 cells) were pelleted and extracted for 30 min at 21°C in 100 µl of the extraction buffer consisting of 9.5 M urea, 1% (v:v) Triton X-100. After centrifugation (15 000 x g, 3 min, 4°C), supernatants were collected and stored in aliquots at -70°C. Isoelectric focusing was performed in tube gels (Mini 2-D electrophoresis cell; Bio-Rad) containing 4% (w:v) acrylamide, 0.1% (w:v) N,N'-methylenebisacrylamide, 9.2 M urea, 2% Triton X-100, 1.6% (w:v) Bio-Lyte 5/7, and 0.4% (w:v) Bio-Lyte 3/10. Samples in extraction buffer were mixed (1:1) with the loading buffer containing 9.5 M urea, 2% (v:v) Triton X-100, 5% (v:v) 2-mercaptoethanol, 1.6% (w:v) Bio-Lyte 5/7, and 0.4% (w:v) Bio-Lyte 3/10. The second dimension was performed on 12% SDS-PAGE. Separated proteins were electrophoretically transferred onto nitrocellulose [31].

The antibodies TU-01, TU-12, and TU-20 in the form of ascitic fluids were diluted 1:400. Bound antibodies were detected by autoradiography as described in the preceding paragraph.

RESULTS

Subcellular Distribution of Tubulin Epitopes

In the first set of experiments, tubulin epitopes were detected in boar spermatozoa with a panel of monoclonal antibodies directed against antigenic determinants located in different structural domains of tubulin subunits. The quality of cells isolated from ejaculates was evaluated by immunostaining the tested spermatozoa with antiacrosin antibody ACR.2 that served as a marker of the acrosome state. Only samples showing more than 80% spermatozoa with intact acrosomes were used. The antitubulin antibodies gave a differential staining pattern and differed in labeling of individual structural parts of spermatozoa. Antibodies TU-01 and TU-06 against epitopes in the N-terminal structural domains of {alpha}- and ß-tubulin, respectively, and TU-04 against an epitope in the C-terminal structural domain of {alpha}-tubulin stained different regions of the tail brightly, while the head was unstained. Monoclonal antibodies TU-01 and TU-04 stained the neck, principal piece, and end piece of the tail intensively (Fig. 1, A and B). On the other hand, monoclonal antibody TU-06 labeled the middle piece of the tail most intensely (Fig. 1C). The monoclonal antibody TU-12 against an epitope in the C-terminal structural domain of ß-tubulin intensely decorated the neck, principal piece, and end piece of the tail. Interestingly, the TU-12 antibody gave bright staining of the head. The examination of samples at higher magnification in confocal microscopy clearly showed that the corresponding epitope was located in the triangular postacrosomal segment that was not decorated with other tested antibodies (Fig. 1D). This reaction was clearly distinguishable from the labeling of the posterior part of the head that we irregularly observed with different antitubulin antibodies. When three different monoclonal antibodies, TU-20, SDL.3D10, and TUJ1, directed against the neuron-specific class III ß-tubulin were used for immunofluorescence, staining along the whole length of the tail was observed. The intensity of labeling was comparable with antibodies TU-20 and SDL.3D10 (Fig. 2, A and B), whereas the TUJ1 antibody stained the tail regions with lower intensity (not shown). The antibody SDL.3D10 also gave bright staining of the neck. Negative control antibodies of IgG and IgM classes and the conjugate alone gave no staining. Results of several immunofluorescence experiments are summarized in Table 1. When antibodies TU-20 and SDL.3D10 were absorbed with the peptide used for immunization (an eight-amino acid peptide ESESQGPK, corresponding to the human class III ß-tubulin sequence 441–448), no staining of spermatozoa was observed. On the other hand, absorption of antibodies with the control peptide (an eight-amino acid peptide GEEEGEEY corresponding to the porcine {alpha}-tubulin sequence) caused no reduction of immunofluorescence staining. The same results were obtained regardless of whether the molar ratio of antibody:peptide was 1:10 or 1:100.



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  		FIG. 1. Immunofluorescence staining of boar spermatozoa with antitubulin monoclonal antibodies TU-01 (A) and TU-04 (B) against {alpha}-tubulin, TU-06 (C) and TU-12 (D) against ß-tubulin. Bar = 10 µm. Insert in D, immunofluorescence labeling in laser screening confocal microscopy, magnification x100



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  		FIG. 2. Immunofluorescence staining of boar spermatozoa with antitubulin monoclonal antibodies TU-20 (A) and SDL.3D10 (B) against class III ß-tubulin. Bar = 10 µm


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  		TABLE 1. Immunostaining of boar spermatozoa with antibodies against {{alpha}}- and {ß}-tubulins

To rule out that the observed immunostaining reactions reflect the cross-reactivity of antitubulin antibodies with other boar spermatozoa proteins, immunoblotting on the whole cell lysate was performed after one-dimensional electrophoresis. The antibodies reacted specifically with proteins with relative electrophoretic mobilities corresponding to the positions of the {alpha}-tubulin (TU-01) and ß-tubulin (TU-06, TU-12, TU-20, and SDL.3D10) subunits (Fig. 3). The antibody TU-04 also reacted only with the {alpha}-subunit of tubulin (not shown). A weak labeling of the lower molecular weight polypetide with antibody TU-06 possibly reflected the generation of a proteolytic fragment of tubulin during sample preparation (Fig. 3, lane 3). Secondary antibodies alone gave no staining. Antibody TUJ1 applied at various dilutions did not react on immunoblots. Immunoblotting after two-dimensional electrophoresis revealed that the antibodies TU-01 and TU12 reacted with multiple {alpha}- or ß-tubulin spots, reflecting a charge heterogeneity of tubulin subunits. The antibody TU-20 reacted only with the basic spot of ß-tubulin. The results of immunoblotting are documented in Figure 4, in which only the tubulin region is shown. No reactivity with other proteins was detected.



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  		FIG. 3. Immunoblot of boar sperm total protein extract with antitubulin antibodies. 1) Coomassie blue staining. 2–6) Immunostaining with antibodies: 2) TU-01, 3) TU-06, 4) TU-12, 5) TU-20, 6) SDL.3D10. The same amounts of proteins were loaded. Position of molecular mass markers (Mr x 10-3) are shown on the left margin



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  		FIG. 4. Immunoblot of boar sperm total extract, separated by two-dimensional electrophoresis, with antitubulin antibodies. Antibodies TU-01 (A), TU-12 (B), and TU-20 (C)

To evaluate the subcellular location of the class III ß-tubulin epitope in sperm tail more precisely, the TU-20 antibody was applied to sectioned and fixed sperm cells. Bound antibody was detected with a secondary antibody conjugated with 10-nm gold particles. Electron microscopy examination revealed that the TU-20 antibody specifically decorated only microtubules. The electron micrograph in Figure 5 shows positive immunolabeling of longitudinal sections of principal parts of the flagella. Different sperm samples from three boars were tested, and the labeling patterns were identical. The negative control antibody gave no staining of the tail.



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  		FIG. 5. Electron micrograph showing immunostaining of the sperm tail with antibody TU-20 recognizing class III ß-tubulin. Bar = 100 nm

Changes in Localization of Tubulin Epitopes Before and after Induced Acrosome Reaction

As the TU-12 antibody clearly detected the postacrosomal segment in boar sperm head (Fig. 1D), we have followed the distribution of the correponding epitope after acrosomal exocytosis (acrosome reaction) that represents the early phase of fertilization. The monoclonal antibody ACR.2 against acrosin was used for the control of the acrosome state. Acrosome-intact spermatozoa showed intensive and uniform fluorescence in the acrosome of sperm head, and this was confirmed by phase contrast microscopy (Fig. 6, A and B). After induction of the acrosome reaction with calcium ionophore A-23187, acrosin was released from the acrosome to the medium, and acrosome-reacted spermatozoa were not stained with antiacrosin antibody (Fig. 6, C and D). Under the same condition, TU-12 antibody labeled the head with substantially lower intensity as demonstrated also at high magnification by confocal microscopy (inserts in Figs. 1D and 6E). Staining of the flagellum remained basically unchanged and was similar as in untreated cells (Figs. 1D and 6E). No substantial changes in the labeling pattern were detected with the other antibodies applied after an induced acrosome reaction.



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  		FIG. 6. Immunofluorescence staining and phase contrast of boar spermatozoa before (A, B) and after (C–E) induced acrosome reaction. Staining with antibody ACR.2 against acrosin (A, C) and staining with antibody TU-12 against ß-tubulin (E). Insert in E, immunofluorescence labeling in laser screening confocal microscopy, magnification x100. Phase contrasts of A and C are in B and D, respectively. Bar = 10 µm

DISCUSSION

Mammalian sperm tubulins are characterized by a large number of tubulin variants that are based mainly on several posttranslational modifications as well as on the expression of multiple tubulin genes, some of them testes specific [4, 7, 15]. In the flagellum of mammalian spermatozoa, post-translationally modified tubulins were detected in longitudinal gradients and on different axoneme structures. These findings suggested a role for tubulin isoforms in the regulation of flagellar beating [33, 34]. Here we demonstrate a differential subcellular distribution of tubulin epitopes in boar spermatozoa detected by two sets of antibodies. Antibodies (TU-01, TU-04, and TU-12) discriminated between structural domain tubulin subunits, and antibodies (TU-20, SDL.3D10, and TUJ1) were prepared against neuron-specific class III ß-tubulin.

The monoclonal antibodies against {alpha}-tubulin (TU-01 and TU-04) and ß-tubulin (TU-06, TU-12) used in this study were chosen from a panel of antibodies prepared against porcine brain tubulin and have been shown by immunoblotting to react with boar sperm tubulin. The antibodies discriminate between the structural domains on both subunits. The TU-01 and TU-04 are specific for the N-terminal and C-terminal domains of ß-tubulin, respectively; the TU-06 and TU-12 for the N-terminal and C-terminal domains of ß-tubulin, respectively [14, 20]. The peptide reacting with TU-01 corresponds to positions {alpha} (65–97) [17]. The epitopes are phylogenetically conserved, and the recognized antigenic determinants do not contain, as far as we know, any post-translationally modified amino acids. Two of the three monoclonal antibodies against the neuron-specific class III ß-tubulin sequence, the TU-20 [21] and the SDL.3D10 [22], were prepared against the synthetic eight-amino acid peptide, ESESQGPK, corresponding to the human class III ß-tubulin sequence 441–448 [23]. The TUJ1 antibody was raised against chicken brain tubulin [24]. Its epitope is located in the peptide covering the last 14 amino acids on the C-terminal end of chicken class III ß-tubulin [35].

All antibodies reacted with various intensities with the middle, principal, and end pieces of the tail. The antibodies differed in the labeling of the neck that was decorated with TU-01, TU-04, TU-12, and SDL.3D10. The observed nonreactivity with this part of the spermatozoa is not caused by any steric hindrance due to the antibody class, as strong staining was observed under the same conditions with the antibody TU-04 (IgM). Because the TU-06 antibody stained the neck in human spermatozoa [14] and recognized all tubulin isoforms tested so far [36, 37], we suppose that the TU-06 epitope resides in the neck of boar spermatozoa and is specifically masked.

Out of the set of antibodies used in this study, only the TU-12 reacted with the triangular postacrosomal segment of the head (Fig. 1D). As the corresponding epitope is not located in the isotype-defining carboxy-terminal extreme of ß-tubulin, which is also highly modified post-translationally, the observed staining probably does not result from the detection of specific tubulin isotype in the sperm head or from post-translational modification [14]. It is, however, possible that the epitope is in a place outside the isotype-defining region, in which the tubulin isotypes differ [38]. A more detailed knowledge of the chemical nature of the epitope is needed to rule out this possibility; there is also an alternative possible explanation that the antibody recognizes another, still unknown modification of tubulin. Immunoblotting after two-dimensional electrophoresis revealed the staining of multiple ß-tubulin spots, including acidic isotubulins, which seems to indicate that the epitope is located on post-translationally modified tubulin species [15]. The TU-12 epitope was previously detected in the equatorial segment of human spermatozoa; the relevant antibody also stained the postacrosomal segment in dog spermatozoa (unpublished results). This suggests that the corresponding epitope could be widely expressed in the head of mammalian spermatozoa. In induced acrosome reactions, verified by the absence of staining with antiacrosin antibody, the immunoreactivity with TU-12 was not detected in the postacrosomal segment but was preserved in the tail. The other tested antitubulin antibodies showed the same staining pattern before and after acrosome reaction. The labeling of the sperm head could be explained either by the presence of unpolymerized tubulin or by the presence of manchete remnants in immature or abnormal cells [15]. Due to the observed changes during the acrosome reaction, we would rather expect the former explanation to be more plausible. In the preceding studies we have already found a relocation of cytoskeletal proteins ({alpha}-tubulin, actin, and spectrin) during capacitation and induced acrosome reaction [39, 40], and on this basis we proposed that cytoskeletal proteins could participate in the process of capacitation and acrosome reaction of mammalian spermatozoa [8, 41].

When the three monoclonal antibodies against class III ß-tubulin were used in immunofluorescence microscopy, they decorated all parts of the tail. The weaker staining of microtubules with the TUJ1 antibody, in comparison with TU-20 and SDL.3D10, and no reactivity in immunoblotting could reflect the fact that the chicken and human class III ß-tubulin differ in 2 amino acid residues in the last 14-amino acid region on the C-terminal end [42]. Highly specific labeling of neuronal tissues [21] and cells of neuronal origin [43, 44] were documented previously for TU-20. The specificity of spermatozoa staining with this antibody was confirmed by absorption of the antibody with the peptide used for immunization, by immunoblotting after one- and two-dimensional electrophoresis, and by immunoelectron microscopy. The staining was not confined to boar spermatozoa, as the tail staining was also observed on isolated human and dog spermatozoa (unpublished results). The limited reactivity of TU-20 observed only with the basic ß-tubulin spot on two-dimensional electrophoresis indicates that in spermatozoa this epitope is not present on all tubulin iosoforms. The class III ß-tubulin was already immunodetected in mouse testes by immunoblotting [24, 45] as well as by immunofluorescence in murine Sertoli cells. Although class III ß-tubulin was detected by immunoblotting in bovine cilia, specific immunolabeling of axoneme was not found [46]. The class III ß-tubulin epitope was detected by immunofluorecence with TU-20 in the axoneme of insect spermatozoa (A. Taddei, personal communication). Here we show for the first time that the class-III ß-tubulin corresponding sequence is localized in the mammalian sperm axoneme.

In conclusion, the presented data demonstrate differential exposition of tubulin epitopes, including class III ß-tubulin epitopes, in the stable arrays of flagellar microtubules in boar spermatozoa. The unique exposure of the ß-tubulin epitope in the postacrosomal segment of the head and its changes during the acrosome reaction indicate that tubulin is involved in functional organization of the sperm head and could play a role in fertilization.

FOOTNOTES

First decision: 5 January 2001.

1 This work was supported by grant nos. 524/96/K162 and 204/98/1054 from the Grant Agency of the Czech Republic, grant no. NJ 5851-3 from the Grant Agency of the Ministry of Health of the Czech Republic, and Eureka 1985-NFDK-MOAB. Back

2 Correspondence: Jana Peknicová, Department of Biology and Biochemistry of Fertilization, Academy of Sciences at the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. FAX: 420 2 44471707;jpeknic{at}biomed.cas.cz Back

Accepted: April 5, 2001.

Received: November 28, 2000.

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