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Reproductive Cytotoxicity Is Predicted by Magnetic Resonance Microscopy and Confirmed by Ubiquitin–Proteasome Immunohistochemistry in a Theophylline-Induced Model of Rat Testicular and Epididymal Toxicity

  • M.W. Tengowski (a1), P. Sutovsky (a2), L.W. Hedlund (a3), D.J. Guyot (a1), J.E. Burkhardt (a1), W.E. Thompson (a4), M. Sutovsky (a2) and G.A. Johnson (a3)...

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques. In vivo T1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring. In vivo MRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting that in vivo MRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use of in vivo MRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

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Callaghan, P.T. (1993). Spatially heterogeneous motion and dynamic NMR microscopy. In Principles of Nuclear Magnetic Resonance Microscopy, Callaghan, P.T. (Ed.), pp. 492501. Oxford, UK: Clarendon Press.
Costabile, R.A., Choyke, P.L., Frank, J.A., Girton, M.E., Diggs, R., Billups, K.L., Moonen, C., & Desjardins, C. (1993). Dynamic enhanced magnetic resonance imaging of testicular perfusion in the rat. J Urol 149, 11951197.
Daston, G.P., Neubecker, T.A., Yonker, J.E., Busse, L.J., Pratt, R.G., Samaratunga, R.C., & Thomas, S.R. (1987). Magnetic resonance imaging of congenital hydrocephalus in the rat. Fundam Appl Toxicol 9, 415422.
Dixon, D., Johnson, G.A., Cofer, G.P., Hedlund, L.W., & Maronpot, R.R. (1988). Magnetic resonance imaging (MRI): A new tool in experimental toxicologic pathology. Toxicol Pathol 16, 386391.
Farghali, H., Williams, D.S., Gavaler, J., & Van Thiel, D.H. (1991). Effect of short-term ethanol feeding on rat testes as assessed by 31P NMR spectroscopy, 1H NMR imaging, and biochemical methods. Alcohol Clin Exp Res 15, 10181023.
Friedman, L., Weinberger, M.A., Farber, T.M., Moreland, F.M., Peters, E.L., Gilmore, C.E., & Khan, M.A. (1979). Testicular atrophy and impaired spermatogenesis in rats fed high levels of the methylxanthines caffeine, theobromine, or theophylline. J Environ Pathol Toxicol 2, 687706.
Groettrup, M., Kraft, R., Kostka, S., Standera, S., Stohwasser, R., & Kloetzel, P.M. (1996). A third interferon-gamma-induced subunit exchange in the 20S proteasome. Eur J Immunol 26, 863869.
Hermo, L., Dworkin, J., & Oko, R. (1988). Role of epithelial clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa. Am J Anat 183, 107124.
Hermo, L. & Jacks, D. (2002). Nature's ingenuity: Bypassing the classical secretory route via apocrine secretion. Mol Reprod Dev 63, 394410.
Hershko, A. (1998). The ubiquitin system: Past, present and future perspectives. In Ubiquitin and the Biology of the Cell, Peters, J.-M., Harris, J.R. & Finley, D. (Eds.), pp. 117. New York: Plenum Press.
Hochstrasser, M. (2002). Molecular biology. New proteases in a ubiquitin stew. Science 298, 549552.
Lauterbur, P.C. (1973). Image formation by induced local interactions: Examples employing nuclear magnetic resonance. Nature 242, 190191.
Lippert, T.H., Seeger, H., Schieferstein, G., & Voelter, W. (1993). Immunoreactive ubiquitin in human seminal plasma. J Androl 14, 130131.
Maeda, M., Maley, J.E., Crosby, D.L., Quets, J.P., Zhu, M.W., Lee, G.J., Lawler, G.J., Ueda, T., Bendixen, B.H., & Yuh, W.T. (1997). Application of contrast agents in the evaluation of stroke: Conventional MR and echo-planar MR imaging. J Magn Reson Imaging 7, 2328.
Morrissey, R.E., Collins, J.J., Lamb, J.C., Manus, A.G., & Gulati, D.K. (1988). Reproductive effects of theophylline in mice and rats. Fundam Appl Toxicol 10, 525536.
Pickart, C.M. (1998). Polyubiquitin chains. In Ubiquitin and the Biology of the Cell, Peters, J.-M., Harris, J.R. & Finley, D. (Eds.), pp. 1963. New York: Plenum Press.
Santamaria, L., Martin, R., Paniagua, R., Fraile, B., Nistal, M., Terenghi, G., & Polak, J.M. (1993). Protein gene product 9.5 and ubiquitin immunoreactivities in rat epididymis epithelium. Histochemistry 100, 131138.
Schluter, G. (1989). Ciprofloxacin: Toxicologic evaluation of additional safety data. Am J Med 87, 37S39S.
Strous, G.J. & Govers, R. (1999). The ubiquitin-proteasome system and endocytosis. J Cell Sci 112, 14171423.
Sutovsky, P., Moreno, R., Ramalho-Santos, J., Dominko, T., Thompson, W.E., & Schatten, G. (2001a). A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis. J Cell Sci 114, 975.
Sutovsky, P., Neuber, E., & Schatten, G. (2002). Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay. Mol Reprod Dev 61, 406413.
Sutovsky, P., Terada, Y., & Schatten, G. (2001b). Ubiquitin-based sperm assay for the diagnosis of male factor infertility. Hum Reprod 16, 250258.
Tanaka, K. & Tsurumi, C. (1997). The 26S proteasome: Subunits and functions. Mol Biol Rep 24, 311.
Tantibhedhyangkul, J., Weerachatyanukul, W., Carmona, E., Xu, H., Anupriwan, A., Michaud, D., & Tanphaichitr, N. (2002). Role of sperm surface Arylsulfatase A in mouse sperm-zona pellucida binding. Biol Reprod 67, 212219.
Thompson, W.E., Sutovsky, M., Fischer, K.A., Safranski, T., Moreno, R., Ramalho-Santos, J., & Sutovsky, P. (2002). Differential ubiquitination of prohibitin, p64/lillie and tubulin in mammalian spermatozoa isolated from testis, epididymis and ejaculate (Abstract). Biol Reprod 66, 161.
Wehrli, F.W., Shaw, D., & Kneeland, J.B. (1988). Biomedical Magnetic Resonance Imaging. New York: VCH Publishers.
Weinberger, M.A., Friedman, L., Farber, T.M., Moreland, F.M., Peters, E.L., Gilmore, C.E., & Khan, M.A. (1978). Testicular atrophy and impaired spermatogenesis in rats fed high levels of the methylxanthines caffeine, theobromine, or theophylline. J Environ Pathol Toxicol 1, 669688.
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Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
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