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Immunocytochemical localization of nerve growth factor (NGF) in the submandibular gland of adult mice by light and electron microscopy

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Summary

Nerve growth factor (NGF) was localized in the submandibular gland of adult male mice by a direct immunocytochemical method using highly purified antibodies against NGF coupled to horseradish peroxidase. In light microscopic sections the reaction product was entirely confined to the cells of the secretory tubules. The acinar part of the gland was free of reaction product. This finding was confirmed by electron microscopy. Within the cells NGF was localized exclusively in the apical secretory granules.

No reaction was observed in the rough endoplasmic reticulum, the Golgi region or in the granules of the basal part of the cells. This observation favours the assumption that NGF is derived from a precursor molecule and that the precursor is transformed into immunologically active NGF within the secretory granules during their transport from the basal to the apical part of the tubular cells. Stimulation of the submandibular gland with carbachol (2 mg/kg) led to a massive release of the content of the secretory granules, including NGF, into the salivary duct.

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References

  • Amsterdam, A., Ohad, I., Schramm, M.: Dynamic changes in the ultrastructure of the acinar cell of the rat parotid gland during the secretion cycle. J. Cell Biol. 41, 753–773 (1969)

    Google Scholar 

  • Angeletti, R.H., Bradshaw, R.A.: Nerve growth factor from mouse submaxillary gland: Amino acid sequence. Proc. nat. Acad. Sci. (Wash.) 68, 2417–2420 (1971)

    Google Scholar 

  • Berger, E.A., Ishii, D.N., Server, A.C., Shooter, E.M.: Studies on the biosynthesis and structure of nerve growth factor from the mouse submandibular gland, p. 340. Sixth International Congress of Pharmacology, Helsinki, 1975

  • Bhoola, K.D., Dorey, G., Jones, C.W.: The influence of androgens on enzymes (chymotrypsin-, trypsin-like proteases, renin, kallikrein and amylase) and on cellular structure of the mouse submaxillary gland. J. Physiol. (Lond.) 235, 503–522 (1973)

    Google Scholar 

  • Bloom, G.D., Carlsöö, B., Danielsson, A., Marklund, S., Stigbrand, T.: Peroxidase and amylase activity of the guinea pig submandibular gland during the secretory cycle. Histochemie 38, 271–280 (1974)

    Google Scholar 

  • Bloom, G.D., Carlsöö, B., Kumlien, A.: Cytochemical localization of peroxidase activity in the submandibular salivary gland of the hamster. Histochemie 22, 294–301 (1970)

    Google Scholar 

  • Boyd, L.F., Bradshaw, R.A., Frazier, W.A., Hogue-Angeletti, R.A., Jeng, I., Pulliam, M.W., Szutowicz, A.: Nerve growth factor. Life Sci. 15, 1381–1391 (1974)

    Google Scholar 

  • Bueker, E.D.: Implantation of tumors in the hind limb field of the embryonic chick and developmental response of the lumbosacral nervous system. Anat. Rec. 102, 369–390 (1948)

    Google Scholar 

  • Burnham, P., Raiborn, C., Varon, S.: Replacement of nerve growth factor by ganglionic nonneuronal cells for the survival in vitro of dissociated ganglionic neurons. Proc. nat. Acad. Sci. (Wash.) 69, 3556–3560 (1972)

    Google Scholar 

  • Caramia, F.: Ultrastructure of mouse submaxillary gland. I. Sexual differences. J. Ultrastruct. Res. 16, 505–523 (1966a)

    Google Scholar 

  • Caramia, F.: Ultrastructure of mouse submaxillary gland. II. Effect of castration in the male. J. Ultrastruct. Res. 16, 524–536 (1966b)

    Google Scholar 

  • Caramia, F., Angeletti, P.U., Levi-Montalcini, R.: Experimental analysis of the mouse submaxillary salivary gland in relationship to its nerve growth factor content. Endocrinology 70, 915–922 (1962)

    Google Scholar 

  • Castle, J.D., Jamieson, J.D., Palade, G.E.: Radioautographic analysis of the secretory process in the parotid acinar cell of the rabbit. J. Cell Biol. 53, 290–311 (1972)

    Google Scholar 

  • Fenton, E.L.: Tissue culture assay of nerve growth factor and of the specific antiserum. Exp. Cell Res. 59, 383–392 (1970)

    Google Scholar 

  • Flickinger, C.J.: Synthesis, intracellular transport and release of secretory protein in the seminal vesicle of the rat, as studied by electron microscope autoradiography. Anat. Rec. 180, 407–426 (1974a)

    Google Scholar 

  • Flickinger, C.J.: Protein secretion in the rat ventral prostate and the relation of Golgi vesicles, cisternae and vacuoles, as studied by electron microscope radioautography. Anat. Rec. 180, 427–448 (1974b)

    Google Scholar 

  • Frazier, W.A., Angeletti, R.H., Bradshaw, R.A.: Nerve growth factor and insulin. Science 176, 482–488 (1972)

    Google Scholar 

  • Goldstein, M.N., Burdman, J.A.: Studies of the nerve growth factor in the submandibular glands of female mice treated with testosterone. Anat. Rec. 151, 199–209 (1965)

    Google Scholar 

  • Graham, R.C., Karnovsky, M.J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291–302 (1966)

    Google Scholar 

  • Greene, L.A., Shooter, E.M., Varon, S.: Enzymatic activities of mouse nerve growth factor and its subunits. Proc. nat. Acad. Sci. (Wash.) 60, 1383–1388 (1968)

    Google Scholar 

  • Hassell, J., Hand, A.R.: Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of crosslinking and ultrastructure obtained with dimethyl-suberimidate and glutaraldehyde. J. Histochem. Cytochem. 22, 223–239 (1974)

    Google Scholar 

  • Hendry, I.A., Iversen, L.L.: Reduction in the concentration of nerve growth factor in mice after sialectomy and castration. Nature (Lond.) 243, 500–505 (1973)

    Google Scholar 

  • Hendry, I.A., Stoeckel, K., Thoenen, H., Iversen, L.L.: The retrograde axonal transport of nerve growth factor. Brain Res. 68, 103–121 (1974)

    Google Scholar 

  • Herzog, V., Miller, F.: Die Lokalisation endogener Peroxidase in der Glandula parotis der Ratte. Z. Zellforsch. 107, 403–420 (1970)

    Google Scholar 

  • Hunt, L.T., Barker, W.C., Dayhoff, M.O.: Epidermal growth factor: Internal duplication and probable relationship to pancreatic secretory trypsin inhibitor. Biochem. biophys. Res. Commun. 60, 1020–1028 (1974)

    Google Scholar 

  • Jamieson, J.D., Palade, G.E.: Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34, 577–596 (1967a)

    Google Scholar 

  • Jamieson, J.D., Palade, G.E.: Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J. Cell Biol. 34, 597–615 (1967b)

    Google Scholar 

  • Kumar, S., Steward, J.K., Taylor, G., Waghe, M.A.: Fluorescence studies using anti-nerve growth factor. Exp. Cell Res. 74, 170–174 (1972)

    Google Scholar 

  • Levi-Montalcini, R.: Control mechanisms of the adrenergic neuron. In: Dynamics of degeneration and growth in neurons (K. Fuxe, L. Olson and Y. Zotterman, eds.), pp. 297–314. London: Pergamon Press 1974

    Google Scholar 

  • Levi-Montalcini, R., Angeletti, P.U.: Growth control of the sympathetic system by a specific protein factor. Quart. Rev. Biol. 36, 99–108 (1961)

    Google Scholar 

  • Levi-Montalcini, R., Angeletti, P.U.: Nerve growth factor. Physiol. Rev. 48, 534–569 (1968)

    Google Scholar 

  • Levi-Montalcini, R., Booker, B.: Excessive growth of the sympathetic ganglia evolved by a protein isolated from mouse salivary gland. Proc. nat. Acad. Sci. (Wash.) 46, 373–384 (1960)

    Google Scholar 

  • Levi-Montalcini, R., Hamburger, V.: Selective growth-stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J. exp. Zool. 116, 321–362 (1951)

    Google Scholar 

  • Lillie, J., Han, S.: Secretory protein synthesis in the stimulated rat parotid gland. J. Cell Biol. 59, 708–721 (1973)

    Google Scholar 

  • Longo, A.M., Penhoet, E.E.: Nerve growth factor in rat glioma cells. Proc. nat. Acad. Sci. (Wash.) 71, 2347–2349 (1974)

    Google Scholar 

  • Martinez-Hernandez, A., Nakane, P.K., Pierce, G.B.: The secretory granules of the acinar cells of the mouse submaxillary gland. Amer. J. Anat. 133, 259–268 (1972)

    Google Scholar 

  • Maurs, C., Herman, G., Busson, S., Ovtracht, L., Rossignol, B.: Regulation of protein secretion and metabolism in rat salivary glands. J. Microsc. (Paris) 20, 187–196 (1974)

    Google Scholar 

  • Mazurkiewicz, J.E., Nakane, P.K.: Light and EM localization of antigenes in tissues embedded in polyethylene glycol with a peroxidase-labeled antibody method. J. Histochem. Cytochem. 20, 969–974 (1972)

    Google Scholar 

  • Nakamura, T., Fujii, M., Kaiho, H., Kumegawa, M.: Sex difference in glucose-6-phosphate dehydrogenase activity in the submandibular gland of mice. Biochim. biophys. Acta (Amst.) 362, 110–120 (1974)

    Google Scholar 

  • Nakane, P.K., Kawaoi, A.: Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem. 22, 1084–1091 (1974)

    Google Scholar 

  • Oger, J., Arnason, B.G.W., Pantazis, N., Lehrich, J., Young, M.: Synthesis of nerve growth factor by L and 3T3 cells in culture. Proc. nat. Acad. Sci. (Wash.) 71, 1554–1558 (1974)

    Google Scholar 

  • Pasquini, F., Petris, A., Sparaglia, G., Scopelliti, R., Cenci, G., Frati, L.: Biological activities in the granules isolated from the mouse submaxillary gland. Exp. Cell Res. 86, 233–236 (1974)

    Google Scholar 

  • Perez-Polo, J.R., Shooter, E.M.: Subunit affinities in the 7 S nerve growth factor complex. Neurobiol. 4, 197–209 (1974)

    Google Scholar 

  • Reynolds, C.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963)

    Google Scholar 

  • Stoeckel, K., Schwab, M., Thoenen, H.: Specificity of retrograde transport of nerve growth factor (NGF) in sensory neurons: A biochemical and morphological study. Brain Res. 89, 1–14 (1975)

    Google Scholar 

  • Streefkerk, J.G., van der Ploeg, M.: Quantitative aspects of cytochemical peroxidase procedures investigated in a model system. J. Histochem. Cytochem. 21, 715–722 (1973)

    Google Scholar 

  • Strum, J.M., Karnovsky, M.J.: Ultrastructural localization of peroxidase in submaxillary acinar cells. J. Ultrastruct. Res. 31, 323–336 (1970)

    Google Scholar 

  • Thomson, J., Morell, D.B.: The structure, location and distribution of salivary gland peroxidase. J. Biochem. (Tokyo) 62, 483–486 (1967)

    Google Scholar 

  • Varon, S., Nomura, J., Shooter, E.M.: Subunit structure of a high-molecular-weight form of the nerve growth factor from mouse submaxillary gland. Proc. nat. Acad. Sci. (Wash.) 57, 1782–1789 (1967)

    Google Scholar 

  • Varon, S., Raiborn, Ch., Norr, S.: Association of antibody to nerve growth factor with ganglionic non-neurons (glia) and consequent interference with their neuron-supportive action. Exp. Cell Res. 88, 247–256 (1974)

    Google Scholar 

  • Young, M., Oger, J., Asdourian, H., Amos, H., Arnason, B.G.W.: Secretion of a nerve growth factor by primary chick fibroblast cultures. Science 187, 361–362 (1975)

    Google Scholar 

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We wish to thank Dr. C. Rufener, Geneva, for communicating to us a new method of antibodyperoxidase coupling and Mrs. M. Durand-Wenger for her excellent technical assistance. This study was supported by the Swiss National Foundation for Scientific Research (Grant Nr. 3.432.74)

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Schwab, M.E., Stöckel, K. & Thoenen, H. Immunocytochemical localization of nerve growth factor (NGF) in the submandibular gland of adult mice by light and electron microscopy. Cell Tissue Res. 169, 289–299 (1976). https://doi.org/10.1007/BF00219602

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  • DOI: https://doi.org/10.1007/BF00219602

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