ARTICLE |
Correspondence to: Denise Escalier, INSERM U25, Hôpital Necker, 149-161, rue de Sèvres, 75743 Paris Cedex 15, France.
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Summary |
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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 Mr 95 x 103 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, KI). 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 co-localizes 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. (J Histochem Cytochem 45:909-922, 1997)
Key Words: actin, cytoskeleton, flagellum, intermediate filaments, spermatogenesis, human spermatozoa
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Introduction |
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In most vertebrates, the flagellar axoneme of spermatozoa is surrounded by unique cytoskeletal structures. Two such structures are the fibrous sheath (FS) and the outer dense fibers of the spermatozoa of reptiles and mammals (
FS biogenesis occurs in late spermiogenesis (
Both human (
Disulfide content and resistance to solubilization have long suggested that the FS may be related to proteins of the intermediate filament (IF) family (
Data on the mouse FS therefore reveal that spermatogenic cells contain unique cytoskeletal protein(s) (
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Materials and Methods |
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Production of Monoclonal Antibodies
Balb/c mice were immunized with the insoluble fraction from homogenates of human spermatozoa performed in 0.1 M HEPES, 1 mM MgSO4, 2 mM EGTA, 2 M glycerol, pH 6.9, and 1% Nodinet P-40 (
Sperm Samples and Testicular Biopsies
Human semen samples were collected after 3 days of sexual abstinence. They were from eight donors whose sperm parameters were considered normal according to standard criteria of semen analysis and from four infertile patients with spermatozoa exhibiting FS dysgenesis whose characteristics were defined by electron microscopy as previously described (
Testis biopsies were obtained from two men with obstructive azoospermia and a normal germinal epithelium as revealed by light microscopy. Spermatozoa from rodent species (rat, mouse, hamster, and guinea pig) and from the monkey Macaca fascicularis were obtained by perfusion of caudae epididymes with PBS at pH 7.2. Spermatozoa from horse, bull, and boar were obtained by sexual stimulation, then washed in PBS.
Sperm Treatments for Indirect Immunofluorescence
Washed motile sperm cells were air-dried onto microscope slides. Some samples were directly immunolabeled, and others were treated with the following procedures before immunostaining: (a) fixation with acetone or methanol or 5% acetic acid in ethanol at -20C for 7 min; (b) permeabilization with 0.1% (w/v) Triton X-100 in PBS at 4C for 10 min; (c) incubation in PBS containing 2 M NaCl or 2 M KCl or 1 M potassium iodide or 3 M guanidine hydrochloride (grade I; Sigma) or 6 M urea (Bio-Rad Laboratories; Richmond, CA) at 4C for 1 hr; and (d) three-step FS extractions at 4C (see below). FS extraction on slides was performed according to
Sperm cells from infertile patients were air-dried onto slides and treated with 0.1% (w/v) Triton X-100 in PBS for 10 min, then washed with PBS. A sample of each biopsy was Bouin-fixed and paraffin-embedded and another was frozen as previously described (
Indirect Immunostaining
Sperm samples processed as above were preincubated in PBS containing 1% (w/v) BSA (grade V; Sigma) for 15 min and then with MAb 4F7 (undiluted culture supernatant) for 45 min at room temperature (RT). After extensive washing in PBS, fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins (GAM/FITC; Nordic, Tilburg, The Netherlands) diluted 1:35 were added for another 45 min. After extensive washing in PBS, cells were fixed with 3% formaldehyde in PBS. Slides were counterstained with 0.02% Evans blue and mounted in Citifluor-PBS-glycerol solution (Citifluor; London, UK). Samples from infertile men were also labeled with an anti-actin MAb (N 350; Amersham, Poole, UK) diluted 1:100 in PBS/BSA. The secondary antibody was an anti-mouse IgM (Biosys SA; Compiègne, France) diluted 1:40 in PBS. Testis sections were labeled by the three-step immunoperoxidase technique using the biotin-avidin system (Vectastain ABC kit PK-4002; Vector Laboratories, Burlingame, CA) as previously described (
Immunoelectron microscopy
Before pre-embedding immunolabeling, pellets of sperm cells from donors were treated by one of the three following methods: (a) one cycle of freezing-thawing in liquid nitrogen; (b) 0.05% (w/v) Triton X-100 in PBS for 10 min; and (c) 0.1 % (w/v) Triton X-100 in PBS for 15 min, then 6 M urea and 100 µg/ml DNAse I (grade II; Boehringer, Mannheim, Germany) in PBS for 2 hr. In this latter method, a cocktail of protease inhibitors was added to the incubating solutions (see below). After each treatment, sperm cells were washed in PBS. Pre-embedding immunolabelings were performed as previously reported (
Sperm Extraction Procedures for Immunoblotting
Extraction of sperm proteins from motile spermatozoa was performed at 4C after several methods of sequential extraction. Protease inhibitors were added: 2 mM phenyl-methyl-sulfonyl-fluoride (PMSF), 10 mM aprotinin, 5 mM soybean trypsin inhibitor (Type I-S), 1 mM benzamidine hydrochloride, 7 mM 2 macroglobulin (Sigma). In addition, 100 µg/ml DNAse I (grade II, Boehringer) was also present in solutions. The first method for sperm cell extraction was the three-step method of FS isolation according to
Electrophoresis and Immunoblotting
Samples were electrophoresed in 8.5% (w/v) polyacrylamide gels according to
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Results |
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MAb 4F7 Recognizes a Flagellar Component of Primate Spermatozoa
The hybridoma MAb 4F7 secreted an Ig (belonging to the IgG1 class) that stained the flagellar principal piece of permeabilized human spermatozoa (Figure 1). Unrelated 4D4 and 1B41 MAbs did not reveal any staining of the flagellum. Unfixed and unpermeabilized spermatozoa remained unstained, indicating that MAb 4F7 did not react with the cell surface but with an intracellular antigen. MAb 4F7 did not stain the blood cells and desquamated epithelial cells present in some ejaculates. Permeabilized spermatozoa from M. fascicularis were stained with MAb 4F7 in a pattern similar to that observed for human spermatozoa. By contrast, spermatozoa from horse, boar, bull, rat, mouse, hamster, and guinea pig were not stained with MAb 4F7, regardless of the chemical treatment used before labeling (see Materials and Methods) (data not shown).
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MAb 4F7 Recognizes an Mr 95 x 103 Polypeptide Doublet
From sperm polypeptides separated by electrophoresis, a band at about Mr 95 x 103 could be observed in the FS-containing fraction isolated after treatment with 8 M urea and 1% Triton X-100 (Figure 2A, Lane 1). This band was absent in the soluble sperm fraction (Figure 2A, Lane 2) and was poorly distinguishable in the whole sperm extract (Figure 2A, Lane 3). In Western blotting, this Mr 95 x 103 doublet was also present in the pellet obtained after treatment with 8 M urea and 1% Triton X-100 (Figure 2B, Lane 1). Total sperm cell extracts directly boiled in SDS sample buffer also showed that MAb 4F7 reacted with a polypeptide doublet of about Mr 95 x 103 (Figure 2B, Lane 3). Pellets obtained after treatment with 6 M urea and 2 mM DTT, or after the IF extraction method, exhibited the labeled Mr 95 x 103 doublet and an additional fainter doublet of about Mr 82 x 103 (Figure 2B, Lane 4). A similar result was obtained after the three-step extraction method (data not shown). Soluble fractions obtained after each of these various treatments did not exhibit MAb 4F7 labeling except for small amounts after potassium thiocyanate treatment for the three-step extraction method (data not shown). By contrast, the insoluble fraction obtained by the two-step method of extraction with 1.5 M KCl (Step 2) showed predominantly the lower band of the Mr 95 x 103 doublet and also several labeled bands at about Mr 82 x 103, 74 x 103, and 45 x 103, and minor bands at Mr 68 x 103, 59 x 103 and 49 x 103 (Figure 2B, Lane 5). Increasing KCl concentration to 2 M led to very faint or no labeling (data not shown). Together, these data suggest that the additional bands corresponded to degraded products of the FS polypeptide studied.
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7-Related Polypeptides Are Not Recognized by Anti-intermediate Filament Antibodies
Known crossreaction of FS proteins with IF antibodies (
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The 4F7-immunoreactive Protein Is Resistant to Denaturing Solutions but Not to High Ionic Strength Treatments
Characterization of the somatic IF after biochemical treatments has established that IFs are resistant to high-salt buffers (see review in
Moderate and variable 4F7 labeling was obtained with air-dried spermatozoa permeabilized by acetone or methanol. The 4F7 labeling was increased after treatment with 5% acetic acid in ethanol (Figure 1). Very intense labeling was found after treatment with 6 M urea (Figure 4A) or 3 M guanidine (Figure 4B) or with 0.1% Triton X-100 alone. Spermatozoa were poorly stained after treatment with 2 M KCl (Figure 4C) or 1 M potassium iodide, and they lost their staining after 2 M NaCl treatment (Figure 4D). Immunolabeling with MAb 4F7 after the three-step FS extraction method (Figure 5) showed that sperm cells were (a) weakly stained after 50 mM sodium borate incubation (Step 1), (b) intensely stained after 0.6 M potassium thiocyanate incubation (Step 2) (Figure 5A), and (c) strongly stained after 4 M urea treatment (Step 3) (Figure 5B). For all the above treatments, no staining was observed in regions of the sperm cells other than the principal piece of the flagellum.
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MAb 4F7 Binds Specifically to the Flagellar Fibrous Sheath
Immunogold labeling performed on pre-embedded human sperm samples showed that the epitope recognized by MAb 4F7 was located on the FS (Figure 6A). The other flagellar components such as mitochondria, the connecting piece, the outer dense fibers, and the axoneme were unstained by MAb 4F7. FS isolated by 6 M urea showed many gold particles on both the ribs and columns of the FS (Figure 6B) and on outer and inner FS surfaces (Figure 6C). This staining restricted to the FS surfaces could be due to the tight packing of FS filaments, forming a barrier to the gold particles. Indeed, sections exhibiting FS packaging alteration due to prolonged urea treatment showed that the 4F7-related polypeptides are structural components of the FS (Figure 6D). Controls omitting MAb 4F7 did not show any staining. Samples permeabilized by either Triton X-100 or freeze-thawing showed very sparse labeling (data not shown).
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Sequential Expression of 4F7-Immunoreactive Protein During Fibrous Sheath Biogenesis
4F7 labeling was obtained in frozen sections from testis biopsies. The avidin-biotin-peroxidase labeling procedure revealed different labeling patterns and allowed visualization of the cell maturation steps (Figure 7).
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Only spermatids with a condensed nucleus and an acrosomal cap were labeled with MAb 4F7 at the flagellar level, whereas the flagellar spermatid cytoplasm was free of labeling. The labeling was restricted to the principal piece (Figure 7A). Several labeling patterns were observed in distinct epithelial seminiferous regions: (a) an intense staining of the proximal part of the principal piece and, in addition, enlarged staining beneath the middle piece whose location and size corresponded to the spindle-shaped body (0.8 µm wide vs 0.4 µm for the FS) (Figure 7B); (b) staining extending over the principal piece but with either a granular (Figure 7C and Figure 7D) or a striated pattern (Figure 7E and Figure 7F); and (c) more homogeneous staining extending to the whole flagellar principal piece (Figure 7A). Staining of only the distal principal region of the flagella (i.e., of the flagellar part emerging into the tubule lumen) was never seen, indicating that human FS assembly occurs first in the proximal flagellar region.
Comparative Distribution of the 4F7-Immunoreactive Protein and Actin
The N 350 anti-actin MAb has revealed the presence of actin in FS in normal human spermatozoa (
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Discussion |
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The MAb 4F7 selected among MAbs directed against human sperm extracts recognizes an Mr 95 x 103 polypeptide doublet in the human FS. This FS component is insoluble in urea but is degraded by high ionic strength treatments. MAb 4F7 crossreacts with the sperm flagella of M. fascicularis but not in many other mammal species. A failure of cross-species reactivity of FS antibodies has been frequently observed, even among rodent species (
Until now, immuno-analysis of the FS has been studied mainly in rodents (
Western blotting reveals that MAb 4F7 labels two bands around Mr 95 x 103 in human FS extracts. These two polypeptides are found after various conditions of sperm extraction. Because the two bands are of equal intensity, the 4F7-labeled polypeptides should correspond to a dimer whose peptide chain monomers were separated. The presence of a dimeric protein in the FS has already been suggested for the mouse (
The MAb 4F7 epitope is accessible only after treatment with detergents or dissociating solutions, such as urea or guanidine. The recognized FS protein may be associated with other flagellar proteins, such as those of the cross-filaments of the cytoplasmic matrix which link the FS to the other flagellar components (
The FS polypeptides recognized by MAb 4F7 do not crossreact with an antibody recognizing a large panel of cytokeratins in somatic cells nor with two antibodies recognizing IF common domains: the IFA, at the carboxy terminal end of the rod domain (
The N 350 anti-actin antibody labels the connecting piece and the fibrous sheath of the human spermatozoon. As for the MAb 4F7, the N 350 MAb stains both the inner and the outer FS sides (
The FS appears after initiation of condensation of the spermatid nucleus. This is also the case for the FS component detected by MAb 4F7. It has been suggested that mRNAs coding for FS proteins are first stored, to be translated later in spermiogenesis (
Therefore, the location of FS protein synthesis and the mechanism of their eventual transport from a cytoplasmic compartment and/or along the flagellum remain to be elucidated. However, an as yet unknown step in FS development has been revealed by MAb 4F7, which shows that the FS arises from granules regularly distributed along the flagellum. In addition, MAb 4F7 reveals that in the course of FS biogenesis the region proximal to the FS exhibits a transient accumulation of the 4F7-related FS component, in a size, form, and location that could correspond to the spindle-shaped body (
In conclusion, the protein of the sperm fibrous sheath recognized by MAb 4F7 could represent a spermatogenic-specific type of cytoskeletal proteins. A cDNA clone for a gene encoding the FS has been obtained only in the mouse (
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Acknowledgments |
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We thank Dr M.M. Portier (Collège France) for the gift of anti-IF antibodies. We also thank Dr J. Selva (CHU Bicêtre, France), Dr J.L. Dacheux (INRA, Nouzilly, France), Dr D. Goffaux (Ecole Vétérinaire, Maison Alfort, France), and Dr M. Albert (UER Biomédicale des Saints Pères, Paris, France) for providing non-human semen samples.
Received for publication October 14, 1996; accepted January 13, 1997.
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