BRIEF REPORT |
Correspondence to: Fabio Grizzi, Istituto Clinico Humanitas, Scientific Direction, Via Manzoni 56, 20089 Rozzano MI, Italy. E-mail: fabio.grizzi@humanitas.it
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Summary |
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Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.
(J Histochem Cytochem 51:12451248, 2003)
Key Words: sperm protein 17, testis, spermatogenesis, immunohistochemistry
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Introduction |
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THE INTERACTION of capacitated spermatozoa with the zona pellucida of the oocyte is a complex process involving a number of spermatic molecules (
A family of low molecular weight sperm autoantigens (RSA) has been detected in rabbit spermatogenic cells and spermatozoa and was shown to bind the carbohydrate components of the zona pellucida (
Because no data are yet available concerning the immunolocalization of HSp17 in human primate testis and ejaculated spermatozoa, and the suggested potential role of Sp17 as a key-target in human immunotherapy (
The study was carried out in accordance with the guidelines of the Ethics Committee of the hospital treating the patients, all of whom were fully informed of the possible discomfort and risks of surgical treatment. None of the specimens (n=10) represented benign or malignant tumors or damaged tissues, as confirmed by the histological analyses independently performed by two pathologists.
The specimens were fixed in 10% neutral buffered formalin (12 hr) and embedded in paraffin. The histological analysis was performed on 2-µm hematoxylin and eosin (H&E)-stained sections under a light microscope (Leica DMLA; Milan, Italy). The immunolocalization of HSp17 in human testis and ejaculated spermatozoa was investigated by using a self-produced recombinant Sp17 protein to immunize a BALB/c mouse to produce mouse Sp17 monoclonal antibodies (MAbs) from the immunized splenocytes. The successful generation of mouse Sp17 MAbs was first confirmed by Western blotting analyses demonstrating their ability to bind the recombinant Sp17 but not a control recombinant protein produced in an identical manner (
A further Western blot using this antibody was made on a testis homogenate and sperm lysate to demonstrate the specificity of this antibody on the labeled tissues. Testis homogenate and sperm lysate were separated under reducing and denaturing conditions on a 10% polyacrylamide gel, and transferred onto nitrocellulose membranes (
Consecutive 2-µm sections were cut and processed for IHC. After dewaxing and rehydration, the sections were autoclaved for antigen retrieval for 15 min at 121C in a freshly made 1 mM EDTA solution, incubated with 3% H2O2 for 30 min to quench endogenous peroxidase activity, and then treated with either primary antibody raised against HSp17 at room temperature for 2 hr (monoclonal mouse anti-HSp17, dilution 1:100 in PBS) or with 1 mg/ml mouse IgG1 (DAKO; Milan, Italy) as a negative control. This was followed by 30-min incubation with the DAKO Envision system, which is based on a unique enzyme-conjugated polymer backbone that also carries secondary antibody molecules. 3,3'-Diaminobenzidine tetrahydrochloride (Sigma; St Louis, MO) 12.5 mg and 500 µl H2O2 in 50 ml TRIS-buffered saline was used as a chromogen to yield brown reaction products. The nuclei were lightly counterstained with hematoxylin solution (Medite; Bergamo, Italy). Experiments were performed independently three times to confirm the findings. Cytological samples of acrosome-intact ejaculated spermatozoa obtained from four healthy donors with a mean age of 27 ± 5.19 years were independently analyzed by two expert cytologists.
To investigate the immunocytochemical expression of HSp17, washed spermatozoa were cytocentrifuged for 1 min at 800 rpm on a glass slide using a Cytospin-2 centrifuge (ALC pk130; Milan, Italy) and then fixed with Biofix (Bio-Optica; Milan, Italy). The cells were permeabilized with 0.5% Triton X-100 (Sigma), 0.1% sodium citrate in PBS at 4C for 15 min, followed by treatment with either a primary antibody at RT for 2 hr or with 1 µg/ml mouse IgG1 (DAKO) as a negative control. This was followed by 30-min incubation with the DAKO Envision System. 3,3'-Diaminobenzidine tetrahydrochloride (Sigma) 12.5 mg and 500 µl H2O2 in 50 ml of TRIS-buffered saline was used as a chromogen to yield brown reaction products. Experiments were performed three times to confirm the results. Almost 1000 spermatozoa were counted under a light microscope and the number of those immunopositive for HSp17 was expressed as the mean percent (±SD) of all spermatozoa.
Western blotting analysis has confirmed the successful generation of mouse Sp17 MAbs by showing their binding to a recombinant Sp17 but not to a control recombinant protein (data not shown). Fig 1 shows the Western blotting analysis on a testis homogenate and spermatozoa lysate, which demonstrates the specificity of the antibody on the tissue being labeled.
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Although a large number of tubules were highly immunopositive for HSp17 (Fig 2A), some seminiferous tubules were negative (Fig 2B). At higher magnification (Fig 2C and Fig 2D), all of the spermatogonia and Sertoli cells were immunonegative for HSp17, but it was weakly observed in the cytoplasm of some spermatocytes (Fig 2E) and strongly present in the cytoplasm of early and late spermatids (Fig 2F). All tails of the spermatozoa identified in the lumen of the seminiferous tubules were also strongly positive. Although all of the observed Leydig cells were immunonegative, some interstitial cells were immunopositive for HSp17 (Fig 2A). The IgG controls were always negative.
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Two distinct HSp17 populations, one immunopositive and the other immunonegative (Fig 3A), were recognized in the ejaculated spermatozoa obtained from healthy donors. The immunopositive spermatozoa accounted for 90 ± 5% of all spermatozoa.
Observed using light microscopy (final magnification x1000), HSp17 was clearly detected throughout the principal piece of the flagellum, but the intermediate piece, head, and acrosome vesicle appeared to be immunonegative (Fig 3A, inset). Control IgG was always negative (Fig 3B).
To achieve fertilization, human spermatozoa and oocytes are characterized by the presence of specific molecules that mediate the complex steps of their interactions (
Although
In conclusion, previous investigation in baboons (
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Footnotes |
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1 These authors contributed equally to this study.
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Acknowledgments |
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Supported by the National Institute of Health/National Cancer Institute (RO1 CA 88434), the Cancer Treatment Research Foundation, the Mary Kay Ash Charitable Foundation, and the Fondazione "Michele Rodriguez" Istituto Scientifico per le Misure Quantitative in Medicina.
We are very grateful to Prof Massimo Roncalli of the Pathology Unit, the core facility of the Laboratory for Reproductive Biology of the Istituto Clinico Humanitas, Dr Nicoletta Gagliano of the Department of Human Anatomy, University of Milan, and Dr Ruth Tapparo for their help and expertise.
Received for publication September 10, 2002; accepted March 26, 2003.
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