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Plasma membrane of trout spermatozoa: I. Isolation and partial characterization

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Abstract

The plasma membrane from spermatozoa of rainbow trout was isolated by four techniques: sonication, hypotonic shock, mechanical homogenization after freeze-thawing, and nitrogen cavitation, in combination with continuous sucrose gradient centrifugation. Nitrogen cavitation (900 PSI, 20 min equilibration at 4°C) was the most effective technique.

Following nitrogen cavitation, four bands were recovered in the sucrose gradient at densities ≈ 1.03, 1.05, 1.09 and 1.15 g/ml. Electron microscopy revealed membrane vesicles of various sizes in bands 1 to 3, while enzyme analysis revealed a 3.9 to 5.5-fold enrichment in 5'-nucleotidase and little contamination by lactate dehydrogenase (cytosol) and succinic dehydrogenase (mitochondria). Lipid analysis of bands 1 and 2 indicated a 6 to 7-fold enrichment in cholesterol and a cholesterol: phospholipid ratio of 0.59–0.70. Seven classes of phospholipids were present in bands 1–3 with no significant differences observed among bands. These data indicate that the vesicles (in bands 1 and 2) obtained after nitrogen cavitation are primarily plasma membranes. Membranes in band 3 appear to be slightly contaminated with nuclear membranes.

Most of the plasma membrane proteins were acidic to neutral. The 2 main membrane proteins were 42 and 30 Kilodaltons.

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References cited

  • Ablett, R.F. and Selivonchick, D.P. 1983. The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 76B: 107–111.

    Google Scholar 

  • Agrawal, P., Magargee, S.F. and Hammerstedt, R.H. 1988. Isolation and characterization of the plasma membrane of rat cauda epididymal spermatozoa. J. Androl. 9: 178–189.

    Google Scholar 

  • Bartlett, G.R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466–468.

    Google Scholar 

  • Baynes, S.M. and Scott, A.P. 1982. Cryopreservation of rainbow trout spermatozoa: variation in membrane composition may influence spermatozoan survival.In Proc. Int. Symp. Reproductive Physiol. Fish. pp. 128. Edited by C.J.J. Richter and H.J.T. Goos. Pudoc, Wageningen.

    Google Scholar 

  • Billard, R. and Jalabert, B. 1974. L'insémination artificielle de la truite (Salmo gairdneri Richardson). II. Comparaison des effets des différents dilueurs sur la conservation de la fertilité des gamètes avant et après insémination. Ann. Biol. Anim. Bioch. Biophys. 14: 601–610.

    Google Scholar 

  • Cosson, M.P., Billard, R. and Letellier, L. 1989. Rise of internal Ca2+ accompanies the initiation of trout sperm motility. Cell Motil. Cytoskeleton 14: 424–434.

    Google Scholar 

  • Dacheux, J.L., Dacheux, F. and Paquignon, M. 1989. Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar. Biol. Reprod. 40: 635–651.

    Google Scholar 

  • Damerval, C., Le Guilloux, M., Blaisonneau, J. and de Vienne, D. 1986. Coloration des proteines au nitrate d'argent: une modification de la méthode de Heukeshoven et Dernick.In Proc. 4eme Coll. Soc. Franç. Electro., Rouen.

  • Folch, J.M., Lees, M. and SloaneStanley, G.H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497–506.

    Google Scholar 

  • Gillis, G., Peterson, R. and Russell, L. 1978. Isolation and characterization of membrane vesicles from human and boar spermatozoa: Methods using nitrogen cavitation and ionophore induced vesiculation. Prep. Biochem. 8: 363–378.

    Google Scholar 

  • Green, D.E., Mii, S. and Kohout, P.M. 1955. Studies on the terminal electron transport system: I Succinic dehydrogenase. J. Biol. Chem. 217: 551–557.

    Google Scholar 

  • Hazel, J.R. 1984. Effects of temperature on the structure and metabolism of cell membranes in fish. Am. J. Physiol. 246: 460–470.

    Google Scholar 

  • Holt, W.V. and North, R.D. 1985. Determination of lipid composition and thermal phase transition temperature in an enriched plasma membrane fraction from ram spermatozoa. J. Reprod. Fert. 73: 285–294.

    Google Scholar 

  • Jaana, H. and Yamamoto, T.S. 1984. Local variation of the cell surface in chum salmon sperm as revealed by their agglutination reaction. J. Exp. Zool. 230: 449–463.

    Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227: 680–685.

    Google Scholar 

  • Lanzetta, P.A., Alvarez, L.J., Reinach, P.S. and Candia, O.A. 1979. An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100: 95–97.

    Google Scholar 

  • Loir, M., Labbé, C. and Vizzano, D. 1990a. Steroids in seminal fluid in the rainbow trout.In Proc. 15th Conf. Europ. Comp. Endocrinol. pp. 15.

  • Loir, M., Labbé, C., Maisse, G., Pinson, A., Boulard, G., Mourot, B. and Chambeyron, F. 1990b. Proteins of seminal fluid and spermatozoa in the trout (Onchorhynchus mykiss): Partial characterization and variations. Fish Physiol. Biochem. 8: 485–495.

    Google Scholar 

  • Lou, Y.H., Yamauchi, K. and Takahashi, H. 1990. Isolation and partial characterization of sperm plasm membrane of masu salmon,Onchorhynchus masou. Comp. Biochem. Physiol. 95B: 187–192.

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275.

    Google Scholar 

  • Mack, S.R. and Szuchet, S. 1981. Synthesis of myelin glycosphingolipids by isolated oligodendrocytes in tissue culture. Brain Res. 214: 180–185.

    Google Scholar 

  • Mack, S.R., Everingham, J. and Zaneveld, L.J.D. 1986. Isolation and partial characterization of the plasma membrane from human spermatozoa. J. Exp. Zool. 240: 127–136.

    Google Scholar 

  • Maisse, G., Pinson, A. and Loir, M. 1988. Caractérisation de l'aptitude à la congélation du sperme de truite arc-en-ciel (Salmo gairdneri) par des critéres physico-chimiques. Aquat. Living Res. 1: 45–51.

    Google Scholar 

  • Malejac, M.L., Loir, M. and Maisse, G. 1990. Qualité de la membrane des spermatozoides de truite arc-en-ciel (Onchorhynchus mykiss). Relation avec l'aptitude du sperme á la congélation. Aquat. Living Res. 3: 43–54.

    Google Scholar 

  • McNamee, M.G. 1989. Isolation and characterization of cell membrane. Biotechniques 7: 466–475.

    Google Scholar 

  • Michell, R.H. and Hawthorne, J.N. 1965. The site of diphosphoinositoside synthesis in rat liver. Biochem. Biophys. Res. Commun. 21: 333–346.

    Google Scholar 

  • Morisawa, S. and Morisawa, M. 1988. Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon. J. Exp. Biol. 136: 13–22.

    Google Scholar 

  • Noland, T.D., Olson, G.E. and Garbers, D.L. 1983. Purification and partial characterization of plasma membranes from bovine spermatozoa. Biol. Reprod. 29: 987–998.

    Google Scholar 

  • O'Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007–4021.

    Google Scholar 

  • Parks, J.E. and Hammerstedt, R.H. 1985. Developmental changes occurring in the lipids of ram epididymal spermatozoa plasma membrane. Biol. Reprod. 32: 653–668.

    Google Scholar 

  • Peterson, R., Russell, L., Bundman, D. and Freund, M. 1980. Evaluation of the purity of boar sperm plasma membranes prepared by nitrogen cavitation. Biol. Reprod. 23: 637–645.

    Google Scholar 

  • Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76–85.

    Google Scholar 

  • Watanabe, T., Takeuchi, T., Saito, M. and Nishimura, K. 1984. Effect of low protein-high calory or essential fatty acid deficiency diet on reproduction of rainbow trout. Bull. Jap. Soc. Scient. Fish. 50: 1207–1215.

    Google Scholar 

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  1. INRA, Physiologie des Poissons, Campus de Beaulieu, 35042, Rennes Cedex, France

    C. Labbé & M. Loir

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  1. C. Labbé
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  2. M. Loir
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Labbé, C., Loir, M. Plasma membrane of trout spermatozoa: I. Isolation and partial characterization. Fish Physiol Biochem 9, 325–338 (1991). https://doi.org/10.1007/BF02265153

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