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Histochemistry of 3β-hydroxysteroid dehydrogenase in rat ovary

I. A methodological study

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Summary

By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3β-HSD was investigated:

  1. 1.

    Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced.

  2. 2.

    Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% w/v for 5 min) thereby enhancing the Nothing dehydrogenase reaction.

  3. 3.

    Preextraction of lipids with acetone for 20 min at −30° C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells).

  4. 4.

    No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20° C.

  5. 5.

    By using the double-section incubation method no diffusion of 3β-HSD or rediffusion of NADH or PMSH could be noticed within 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0 mg/ml).

  6. 6.

    The utility of different inhibitors of alkaline phosphomonoesterase was tested and discussed.

  7. 7.

    By inhibiting alkaline phosphomonoesterase with 0.1 mM ofl-p-bromotetramisole or 16 mM ofβ-glycerophosphate, 3β-HSD was shown to be exclusively NAD-linked.

  8. 8.

    Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3β-HSD, but not of NADPH-tetrazolium reductase.

  9. 9.

    3β-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited byn-ethyl maleimide.

  10. 10.

    Whether alcohol dehydrogenases may use steroids as substrate is discussed. It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed.

  11. 11.

    Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase.

  12. 12.

    Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF).

  13. 13.

    The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone.

  14. 4.

    If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.

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References

  • Altman, F.P.: The use of a new grade of polyvinyl alcohol for stabilishing tissue sections during histochemical incubations. Histochemie28, 236–242 (1971)

    Google Scholar 

  • Altman, F.P.: Studies on the reduction of tetrazolium salts. III. The products of chemical and enzymic reduction. Histochemistry38, 155–171 (1974)

    Google Scholar 

  • Andersen, H., Høyer, P.E.: Studies in succinate dehydrogenase histochemistry. Histochemie35, 173–188 (1973)

    Google Scholar 

  • Andersen, H., Høyer, P.E.: Simplified control experiments in the histochemical study of coenzymelinked dehydrogenases. Histochemistry38, 71–83 (1974)

    Google Scholar 

  • Arvy, L.: The female gonalds. In: Histoenzymology of the endocrine glands (Arvy, L., ed.), pp. 418–476. Oxford-New York: Pergamon Press 1971

    Google Scholar 

  • Baillie, A.H., Ferguson, M.M., Hart, D.Mck.: Developments in steroid histochemistry. London-New York: Academic Press 1966

    Google Scholar 

  • Bennett, H.S., Watts, R.M.: The cytochemical demonstration and measurement of sulphydryl groups by azo-aryl mercaptide coupling, with special reference to mercury orange. In: General cytochemical methods (Danielli, J.F., ed.), Vol. 1. New York: Academic Press 1958

    Google Scholar 

  • Bjersing, L.: Histochemical demonstration of Δ5-3β- and 17β-hydroxysteroid dehydrogenase activities in porcine ovary. Histochemie10, 295–304 (1967)

    Google Scholar 

  • Borgers, M.: The cytochemical application of new potent inhibitors of alkaline phosphatases. J. Histochem. Cytochem.21, 812–824 (1973)

    Google Scholar 

  • Borgers, M., Thoné, F.: The inhibition of alkaline phosphatase byl-p-bromotetramisole. Histochemistry44, 277–280 (1975)

    Google Scholar 

  • Bossche, H. van den, Janssen, P.A.J.: The biochemical mechanism of action of the anthelmintic drug tetramisole. Life Sci.6, 1781 (1967)

    Google Scholar 

  • Bossche, H. von den, Janssen, P.A.J.: The biochemical mechanism of action of the antinematodal drug tetramisole. Biochem. Pharmacol.18, 35–42 (1969)

    Google Scholar 

  • Brändén, C.-J., Jörnvall, H., Eklund, H., Furugren, B.: Alcohol dehydrogenases. In: The enzymes (Boyer, P.D., ed.), Vol. XI, pp, 103–190. New York-London: Academic Press 1975

    Google Scholar 

  • Brunk, U.T., Ericsson, J.L.E.: The demonstration of acid phosphatase in in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability. In: Fixation in histochemistry (Stoward, P.J., ed.), pp. 121–135. London: Chapman and Hall 1973

    Google Scholar 

  • Burstone, M.S.: The relationship between fixation and techniques for the histochemical localization of hydrolytic enzymes. J. Histochem. Cytochem.6, 322–339 (1958)

    Google Scholar 

  • Cheatum, S.G., Warren, J.C.: Purification and properties of 3β-hydroxysteroid dehydrogenase and Δ5-3-ketosteroid isomerase from bovine corpora lutea. Acta biochim. biophys.122, 1–13 (1966)

    Google Scholar 

  • Deamer, D.W., Utsumi, K., Packer, L.: Oscillatory states of mitochondria. III. Ultrastructure of trapped conformational states. Arch. Biochem. Biophys.121, 641–651 (1967)

    Google Scholar 

  • Ferguson, M.M., Baillie, A.H., Calman, K.C., Hart, D.Mck.: Histochemical distribution of alcohol dehydrogenases in endocrine tissue. Nature (Lond.)210, 1277–1279 (1966)

    Google Scholar 

  • Fishbein, W.N.: Studies on the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay. III. Cryophyllaxis with dimethyl sulfoxide and enzyme localization. Cryobiology8, 293–299 (1971)

    Google Scholar 

  • Goldberg, B., Jones, G.E.S., Borkowf, H.I.: A histochemical study of substrate specificity for the steroid 3β-ol dehydrogenase and isomerase systems in human ovary and testis. J. Histochem. Cytochem.12, 880–889 (1964)

    Google Scholar 

  • Hanker, J.S.: Oxidoreductases. In: Electron microscopy of enzymes (Hayat, M.A., ed.), Vol. 4, pp. 1–139. New York: Van Nostrand Reinhold Company 1975

    Google Scholar 

  • Hanker, J.S., Kusyk, C.J., Bloom, F.E., Pearse, A.G.E.: The demonstration of dehydrogenases and monoamine oxidase by the formation of osmium blacks at the sites of Hatchett's brown. Histochemie33, 105–230 (1973)

    Google Scholar 

  • Hanker, J.S., Kusyk, C.J., Clapp, D.H., Yates, P.E.: Effect of dimethyl sulfoxide (DMSO) on the histochemical demonstration od dehydrogenases. J. Histochem. Cytochem.18, 673 (1970)

    Google Scholar 

  • Hardonk, M.J., Koudstaal, J.: Enzyme histochemistry as a link between biochemistry and morphology. Progr. Histochem. Cytochem.8, 1–68 (1976)

    Google Scholar 

  • Hopwood, D.: The reaction between formaldehyde, Glutaraldehyde and osmium tetroxide and their fixation effects on bovine serum albumin and on tissue blocks. Histochemie24, 56–64 (1970)

    Google Scholar 

  • Høyer, P.E., Andersen, H.: Specificity in steroid histochemistry, with special reference to the use of steroid solvents. Distribution of 11β-hydroxysteroiddehydrogenase in kidney and thymus from the mouse. Histochemie24, 292–306 (1970)

    Google Scholar 

  • Høyer, P.E., Møller, M.: Activity, localisation and specificity of 11β-hydroxysteroid dehydrogenases (11β-HSD) in cat and rat salivary glands. Proc. roy. micr. Soc.11, Suppl., 20–21 (1976)

    Google Scholar 

  • Jörnvall, H.: Rat liver alcohol dehydrogenase. Characterisation of alkylated cysteine residues in the carboxy-methylated protein. FEBS Lett.28, 32–35 (1972)

    Google Scholar 

  • Jörnvall, H.: Functional aspects of structural studies of alcohol dehydrogenases. In: Alcohol and aldehyde metabolizing systems (Thurman, R.G., Yonetani, T., Williamson, J.R., Chance, B., ed.), pp. 23–32. New York-London: Academic Press 1974

    Google Scholar 

  • Jones, G.E.S., Goldberg, B., Woodruff, J.C.: Cell specific steroid inhibitions in histochemical steroid 3β-ol dehydrogenase activities in man. Histochemie14, 131–142 (1968)

    Google Scholar 

  • Kolthoff, I.M., Reddy, T.B.: Acid-base strength in dimethyl sulfoxide. Inorg. Chem.1, 189 (1962)

    Google Scholar 

  • Leeflang-de Pijper, A.M., Hülsmann, W.C.: Pitfalls in histochemical localization studies of NADPH generating enzymes or enzyme systems in rat small intestine. Histochemistry39, 143–153 (1974)

    Google Scholar 

  • Lillie, R.D.: Histopathologic technic and practical histochemistry, 3rd ed. New York: McGraw-Hill Book Co. 1965

    Google Scholar 

  • Makita, T., Sandborn, E.B.: The effect of dimethyl sulfoxide (DMSO) in the incubation medium for the cytochemical localization of succinate dehydrogenase. Histochemie26, 305–310 (1971)

    Google Scholar 

  • Markovič, O., Theorell, H.: Rat-liver alcohol dehydrogenase (RLADH). In: Structure and function of oxidation-reduction enzymes (Åkeson, Å., Ehrenberg, A., ed.), pp. 635–637. Oxford-New York: Pergamon Press 1972

    Google Scholar 

  • Mietkiewski, K. von, Hyla, R., Król, C., Warchol, J.B.: Die alkalische Phosphatase als Exponent der sekretorischen Tätigkeit des Rattenovars. Acta histochem. (Jena)47, 148–152 (1973)

    Google Scholar 

  • Packer, L., Greville, G.D.: Energy-linked oxidation of glutaraldehyde by rat liver mitochondria. FEBS Lett.3, 112–114 (1969)

    Google Scholar 

  • Parker, A.J.: The use of dipolar aprotic solvents in organic chemistry! In: Advances in organic chemistry (Raphael, R.A., Taylor, E.C., Wynberg, H., ed.), Vol. 5, pp. 1–46. New York-London: Interscience Publishers 1965

    Google Scholar 

  • Pearse, A.G.E.: Histochemistry, theoretical and applied, 3rd. ed., Vol. 1. London: Churchill Livingstone 1968

    Google Scholar 

  • Pearse, A.G.E.: Histochemistry, theoretical and applied, 3rd. ed., Vol. 2. London: Churchill Livingstone 1972

    Google Scholar 

  • Pupkin, M., Bratt, H., Weisz, J., Lloyd, C.W., Balogh, K.: Dehydrogenases in the rat ovary. I. A histochemical study of Δ5-3#x03B2; and 20α-hydroxysteroid dehydrogenases and enzymes of carbohydrate oxidation during the estrous cycle. Endocrinol.79, 316–327 (1966)

    Google Scholar 

  • Rubin, B. L., Strecker, H.J.: Further studies on the sex difference in 3β-hydroxysteroid dehydrogenase activity of rat livers. Endocrinol.69, 257–267 (1961)

    Google Scholar 

  • Sandborn, E.B., Makita, T., Lin, K.-N.: The use of dimethyl sulfoxide as an accelerator in the fixation of tissue for ultrastructural and cytochemical studies and in freeze etching of cells. Anat. Rec.163, 255 (1969)

    Google Scholar 

  • Schmidt, W. von, Wendler, D., Gabler, W.: Aktivität und Lokalisation verschiedener Steroiddehydrogenasen (StDH) während der Plazentation der Ratte. Acta histochem. (Jena)38, 318–325 (1970)

    Google Scholar 

  • Shaw, C.R., Koen, A.L.: On the identity of “nothing dehydrogenase”. J. Histochem. Cytochem.13, 431–433 (1965)

    Google Scholar 

  • Sigman, D.S.: Interactions of substrates, inhibitors, and coenzymes at the active site of horse liver alcohol dehydrogenase. J. biol. Chem.242, 3815–3824 (1967)

    Google Scholar 

  • Symoens, J.: The effects of levamisole on host defense mechanisms. A review. Janssen Pharmaceutica Research Laboratories, Beerse, Belgium, 1974

    Google Scholar 

  • Thomas, M., Aldridge, W.N.: The inhibition of enzymes by beryllium. Biochem. J.98, 94–99 (1966)

    Google Scholar 

  • Wartburg, J.P., von, Papenberg, J.: Alcohol dehydrogenase in ethanol metabolism. Psychosom. Med.28, 405 (1966)

    Google Scholar 

  • Wattenberg, L.W.: Microscopic histochemical demonstration of steroid-3β-ol dehydrogenase in tissue sections. J. Histochem. Cytochem.6, 225–232 (1958)

    Google Scholar 

  • Winborn, W.B., Seelig, L.L.: Paraformaldehyde and s-collidine—a fixative for processing large blocks of tissue for electron microscopy. Tex. Rep. Biol. Med.28, 347 (1970)

    Google Scholar 

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  1. Laboratory of Cyto- and Histochemistry, Anatomy Department A, University of Copenhagen, Universitetsparken 1, DK-2100, Copenhagen Ø, Denmark

    Poul Erik Høyer & Helge Andersen

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  1. Poul Erik Høyer
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  2. Helge Andersen
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Høyer, P.E., Andersen, H. Histochemistry of 3β-hydroxysteroid dehydrogenase in rat ovary. Histochemistry 51, 167–193 (1977). https://doi.org/10.1007/BF00567222

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