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Light- and electron-microscope study of acid phosphatase in mouse oral mucosa
Light- and electron-microscopestudy of acid phosphatasein mouseoral mucosa Prank
R. Xusi, D.M.D., Ph.D., Boston, Mass.
DEPARTMENTS TUFTS
OF ANATOMY
UNIVERSITY
AND
SCHOOLS
ORAL
OF MEDICINE
PATHOLOGY, AND
DENTAL
MEDICINE
A
cid phosphatase activity has been studied in the oral mucous membranes of many species, including mouse1 and manZ+ It has been observed that the stratum granulosum of these epithelia contains the strongest acid phosphatase activity but no typical lysosomal structures. The present investigation, therefore, was undertaken in an effort to examine and correlate the sites of acid phosphatase activity in mouse labial mucosa by both light- and electron-microscope methods. MATERIALS AND METHODS The keratinized labial mucosae of the lower lips of 6-week-old male Swiss albino mice of the Webster strain were used in this study. For light-microscope and calcium-formol-fixed determination of acid phosphatase, fresh-frozen cryostat sections were incubated for 10 to 60 minutes according to the metal-salt method of Gomoris and an azo dye method.7 Adequate controls were carried out for both procedures. For the electron-microscope localization of acid phosphatase activity, slices of mouse labial mucosa were fixed in cold, buffered, 4 per cent glutaraldehyde8 for 2 hours, washed overnight in buffer, and then rapidly frozen. Forty-micron sections were cut with the cryostat and incubated for 5 to 15 minutes according to the metal-salt method,6 except for omission of the ammonium sulfide step, since lead phosphate is visible in the electron microscope. The 40micron sections were subsequently postfixed in 1 per cent 0~0, for 3 hours and embedded in Epon 812.9 Unstained, thin (500 to 900 A) sections were examined in the Philips EM 200. This study wa8 163 and GM 1451.
824
supported
in
part
by
United
States
Public
Health
Service
Grants
DE
Volume Number
26 6
Acid phosphatase in mouse oral mucosa
825
Fig. I. Light micrograph demonstrating acid phosphatase activity of mouse labial mucosa. Most intense reaction is rroted throughout stratum granulosum, as well as in deeper layers of stratum corneum. (Hexazonium pararosaniline stain. Magnification, x180.)
Fig. 8’. Electron mierograph demonstrating acid phosphatase activity of stratum granulosum of mouse labial mucosa. Reaction product is noted as discrete dense particles in flat, membrane-bound structure (N). Less intense reaction is seen scatterd throughout cytopIasm, at cell membrane (C), and at desmosomes (arrows). (Unstained section. Magnification, ~25,750.)
RESULTS
The distribution of acid phosphatase activity as demonstrated by the metalsalt method and the azo dye method was essentially the same. The greatest enzymatic activity was observed as diffuse staining in the cytoplasm of cells of the stratum granulosum (Fig. 1). Slight reactivity was noted in the other epithelial layers. Electron micrographs revealed deposition of reaction product primarily in cells of the stratum granulosum. Fine particles of lead precipitate were scattered throughout the cytoplasm of these cells, while larger, more numerous particles were observed in flattened, membrane-bound structures, which appeared to be
OS., ox & O.P. December, 1968
Fig. 8’. Electron micrograph demonstrating acid phosphatase activity of stratum eorneum of mouse labial mucosa. Discrete particles of reaction product are scattered throughout cytoplasm of cornified cells. (Unstained section. Magnification, x31,250.)
degenerating nuclei (Fig. 2). Reaction product was also observed at the cell membrane, especially at sites presumed to represent desmosomes. Keratohyalin granules were devoid of staining, except for their inherent electron density. Cells of the stratum corneum contained discrete particles of reaction product throughout their cytoplasm (Fig. 3). DISCUSSION The distribution of acid phosphatase activity in mouse labial mucosa is similar to that in human gingiva2-* and skin.“’ Although evidence for the existence of lysosomes in human and monkey gingival epithelium is based solely on acid phosphatase determinations,llI2 structures resembling lysosomes have recently been described in posterior human buccal mucosa,13 human skin,14 and mouse skin and esophagus. l5 An extensive search for such structures in mouse l.abial mucosa, however, has failed to reveal their presence. It would be premature, at this time, to speculate whether lysosomes are not demonstrable in this epithelium because of their fragility and subsequent loss during tissue prepara-
Volume Number
Acid phosphatase in mouse ord mucosa 827
26 6
tion or because of the fact that acid phosphatase in this tissue is not associated with lysosomal structures. Although the metabolic role of acid phosphatase is not as yet completely clear, its hydrolytic activity in osteoclasts, macrophages, and giant cells has been well documented.le* I7 In addition, a relationship between this enzyme and protein synthesis in regenerating liver has been suggested.ls The intense acid phosphatase activity in the stratum granulosum, therefore, may be associated not only with the dissolution of cellular components but also with protein synthesis, since the stratum granulosum is the region of keratinizing epithelia in which cellular organelles disappear and keratinous proteins are further differentiated. Acid phosphatase located at the cell membranes and desmosomesof the superficial layers of this epithelium may be associated with changes in cellular adhesion and subsequent desquamation of keratinized cells. Further intensive study of this and other related enzymes in normal and pathologic oral mucous membranes with both light and electron microscopes should be of great value. SUMMARY
Acid phosphatase activity was studied in mouse labial mucosa at the lightand electron-microscope levels. Activity of this enzyme was greatest in the stratum granulosum, where it appeared to be scattered throughout the cytoplasm of the cells, at cell membranes, and in structuresaconsidered to be degenerating nuclei. Cells of the stratum corneum also exhibited cytoplasmic acid phosphatase activity. The possible role of this enzyme in the process of keratinization has been discussed. REFERENCES
1. Susi, F. R.: A Histochemical Comparison of Oral J. D. Res. 43: 816. 1964. 2. Cabrini, R. L., and- Carranza, F. A.: Histoehemical Human Gingiva, J. Periodont. 29: 34-37, 1958. 3. Lisanti, V. F.: Hydrolytic Enzymes in Periodontal 86: -.. 461-466. -_-
Mucosa
and Skin
Distribution Tissues,
in the Mouse Lip,
of Acid Phosphatase Ann. New York
Acad.
in Sci.
1960.
4. Quintare%: GI:?Iistochemistry of Mucous Membranes, Ann. Histochem. 7: 47-50, 1962. and Electronmicroscopical Aspects of Human Buccal 5. Van Bulow, F.: Histochemical Mucosa, Acta path. et microbial. scandinav. 66: 409-425, 1966. 6. Gomori, G.: An Improved Histochemical Technique for Acid Phosphatase, Stain Technol. 25: 81-85, 1950. 7. Bark?,. T., and Anderson, P.: Acid Phosphatase Techniques IJsing Hexazonium Pararosanilme as Coupler, J. Histochem. 10: 741-753, 1962. 8. Sabatini, D. D., Miller, F., and Barmett, R. J.: Aldehycle Fixation for Morphological and Enzyme Histochemical Studies With the Electron Microscope, J. Histochem. 12: 57, 1964. 9. Luft, J. H.: Improvements in Epoxy Resin Embedding Methods, 5. Biophys. & Bioehem. Cytol. 9: 409-414, 1961. 10. Eisen, A. Z., Am&, K. A., and Clark, W. H., Jr.: The Ultrastructural Localization of Acid Phosphatase in Human Epidermis, J. Invest. Dermat. 43: 319-326, 1964. 11. Ten Cate, A. R.: The Distribution of Acid Phosphatase, Nonspecific Esterase and Lipid in Oral Epithelia in Man and the Macaque Monkey, Arch. Oral Biol. 8: 747.753, 1963. 12. Smith, C. J., and Cimasoni, G.: Stability of Oral Epithelial Lysosomee During Local Anesthesia, Helvet. odont. acta 9: 95-100, 1965. 13. Hashimoto, K., DiBella, R. J., and Shklar, G.: Electron Microscopic Studies of the Normal Human Buccal Mucosa, J. Invest. Dermat. 47: 512-525, 1966.
a28
hi
O.S., 0.x & O.P. December, 1968
14. Olson, R. L., and Nordyuist, R. E.: Ultramicroscopic Localization of Acid Phosphatase in Human Eoidermis. J. Invest. Dermat. 46: 431-435. lRG6. 15. Rowden, G.: Ultrastructural Studies of ReratinizedEpithelia of the Mouse. I. Combined Electron Microscope and Cyt’ochemical Study of Lysosomes in Mouse Epidermis and Esophageal Epithelium, J. Invest. Dermat. 49: 181-197, 1967. 16. De Duve, C.: Lysosomes, a New Group of Cytoplasmic Particles. In Hayashi, ‘I., (editor) : Subcellular Particles, New York. 1959. Ronald Press. 17. S&i, F. R., Goldhaber, PI, and Jennings, 3.: A Histochemical and Biochemical Study of Acid Ph,osphatase in Resorbing Bone in Culture, Am. J. Physiol. 211: 959-962, 1966. 18. Nor-berg, B.: On Proteins in Regenerating Liver; Phosphomonoesterase, Acta physiol. scandinav. 19: 246-259, 1950.
R. Xusi, D.M.D., Ph.D., Boston, Mass.
DEPARTMENTS TUFTS
OF ANATOMY
UNIVERSITY
AND
SCHOOLS
ORAL
OF MEDICINE
PATHOLOGY, AND
DENTAL
MEDICINE
A
cid phosphatase activity has been studied in the oral mucous membranes of many species, including mouse1 and manZ+ It has been observed that the stratum granulosum of these epithelia contains the strongest acid phosphatase activity but no typical lysosomal structures. The present investigation, therefore, was undertaken in an effort to examine and correlate the sites of acid phosphatase activity in mouse labial mucosa by both light- and electron-microscope methods. MATERIALS AND METHODS The keratinized labial mucosae of the lower lips of 6-week-old male Swiss albino mice of the Webster strain were used in this study. For light-microscope and calcium-formol-fixed determination of acid phosphatase, fresh-frozen cryostat sections were incubated for 10 to 60 minutes according to the metal-salt method of Gomoris and an azo dye method.7 Adequate controls were carried out for both procedures. For the electron-microscope localization of acid phosphatase activity, slices of mouse labial mucosa were fixed in cold, buffered, 4 per cent glutaraldehyde8 for 2 hours, washed overnight in buffer, and then rapidly frozen. Forty-micron sections were cut with the cryostat and incubated for 5 to 15 minutes according to the metal-salt method,6 except for omission of the ammonium sulfide step, since lead phosphate is visible in the electron microscope. The 40micron sections were subsequently postfixed in 1 per cent 0~0, for 3 hours and embedded in Epon 812.9 Unstained, thin (500 to 900 A) sections were examined in the Philips EM 200. This study wa8 163 and GM 1451.
824
supported
in
part
by
United
States
Public
Health
Service
Grants
DE
Volume Number
26 6
Acid phosphatase in mouse oral mucosa
825
Fig. I. Light micrograph demonstrating acid phosphatase activity of mouse labial mucosa. Most intense reaction is rroted throughout stratum granulosum, as well as in deeper layers of stratum corneum. (Hexazonium pararosaniline stain. Magnification, x180.)
Fig. 8’. Electron mierograph demonstrating acid phosphatase activity of stratum granulosum of mouse labial mucosa. Reaction product is noted as discrete dense particles in flat, membrane-bound structure (N). Less intense reaction is seen scatterd throughout cytopIasm, at cell membrane (C), and at desmosomes (arrows). (Unstained section. Magnification, ~25,750.)
RESULTS
The distribution of acid phosphatase activity as demonstrated by the metalsalt method and the azo dye method was essentially the same. The greatest enzymatic activity was observed as diffuse staining in the cytoplasm of cells of the stratum granulosum (Fig. 1). Slight reactivity was noted in the other epithelial layers. Electron micrographs revealed deposition of reaction product primarily in cells of the stratum granulosum. Fine particles of lead precipitate were scattered throughout the cytoplasm of these cells, while larger, more numerous particles were observed in flattened, membrane-bound structures, which appeared to be
OS., ox & O.P. December, 1968
Fig. 8’. Electron micrograph demonstrating acid phosphatase activity of stratum eorneum of mouse labial mucosa. Discrete particles of reaction product are scattered throughout cytoplasm of cornified cells. (Unstained section. Magnification, x31,250.)
degenerating nuclei (Fig. 2). Reaction product was also observed at the cell membrane, especially at sites presumed to represent desmosomes. Keratohyalin granules were devoid of staining, except for their inherent electron density. Cells of the stratum corneum contained discrete particles of reaction product throughout their cytoplasm (Fig. 3). DISCUSSION The distribution of acid phosphatase activity in mouse labial mucosa is similar to that in human gingiva2-* and skin.“’ Although evidence for the existence of lysosomes in human and monkey gingival epithelium is based solely on acid phosphatase determinations,llI2 structures resembling lysosomes have recently been described in posterior human buccal mucosa,13 human skin,14 and mouse skin and esophagus. l5 An extensive search for such structures in mouse l.abial mucosa, however, has failed to reveal their presence. It would be premature, at this time, to speculate whether lysosomes are not demonstrable in this epithelium because of their fragility and subsequent loss during tissue prepara-
Volume Number
Acid phosphatase in mouse ord mucosa 827
26 6
tion or because of the fact that acid phosphatase in this tissue is not associated with lysosomal structures. Although the metabolic role of acid phosphatase is not as yet completely clear, its hydrolytic activity in osteoclasts, macrophages, and giant cells has been well documented.le* I7 In addition, a relationship between this enzyme and protein synthesis in regenerating liver has been suggested.ls The intense acid phosphatase activity in the stratum granulosum, therefore, may be associated not only with the dissolution of cellular components but also with protein synthesis, since the stratum granulosum is the region of keratinizing epithelia in which cellular organelles disappear and keratinous proteins are further differentiated. Acid phosphatase located at the cell membranes and desmosomesof the superficial layers of this epithelium may be associated with changes in cellular adhesion and subsequent desquamation of keratinized cells. Further intensive study of this and other related enzymes in normal and pathologic oral mucous membranes with both light and electron microscopes should be of great value. SUMMARY
Acid phosphatase activity was studied in mouse labial mucosa at the lightand electron-microscope levels. Activity of this enzyme was greatest in the stratum granulosum, where it appeared to be scattered throughout the cytoplasm of the cells, at cell membranes, and in structuresaconsidered to be degenerating nuclei. Cells of the stratum corneum also exhibited cytoplasmic acid phosphatase activity. The possible role of this enzyme in the process of keratinization has been discussed. REFERENCES
1. Susi, F. R.: A Histochemical Comparison of Oral J. D. Res. 43: 816. 1964. 2. Cabrini, R. L., and- Carranza, F. A.: Histoehemical Human Gingiva, J. Periodont. 29: 34-37, 1958. 3. Lisanti, V. F.: Hydrolytic Enzymes in Periodontal 86: -.. 461-466. -_-
Mucosa
and Skin
Distribution Tissues,
in the Mouse Lip,
of Acid Phosphatase Ann. New York
Acad.
in Sci.
1960.
4. Quintare%: GI:?Iistochemistry of Mucous Membranes, Ann. Histochem. 7: 47-50, 1962. and Electronmicroscopical Aspects of Human Buccal 5. Van Bulow, F.: Histochemical Mucosa, Acta path. et microbial. scandinav. 66: 409-425, 1966. 6. Gomori, G.: An Improved Histochemical Technique for Acid Phosphatase, Stain Technol. 25: 81-85, 1950. 7. Bark?,. T., and Anderson, P.: Acid Phosphatase Techniques IJsing Hexazonium Pararosanilme as Coupler, J. Histochem. 10: 741-753, 1962. 8. Sabatini, D. D., Miller, F., and Barmett, R. J.: Aldehycle Fixation for Morphological and Enzyme Histochemical Studies With the Electron Microscope, J. Histochem. 12: 57, 1964. 9. Luft, J. H.: Improvements in Epoxy Resin Embedding Methods, 5. Biophys. & Bioehem. Cytol. 9: 409-414, 1961. 10. Eisen, A. Z., Am&, K. A., and Clark, W. H., Jr.: The Ultrastructural Localization of Acid Phosphatase in Human Epidermis, J. Invest. Dermat. 43: 319-326, 1964. 11. Ten Cate, A. R.: The Distribution of Acid Phosphatase, Nonspecific Esterase and Lipid in Oral Epithelia in Man and the Macaque Monkey, Arch. Oral Biol. 8: 747.753, 1963. 12. Smith, C. J., and Cimasoni, G.: Stability of Oral Epithelial Lysosomee During Local Anesthesia, Helvet. odont. acta 9: 95-100, 1965. 13. Hashimoto, K., DiBella, R. J., and Shklar, G.: Electron Microscopic Studies of the Normal Human Buccal Mucosa, J. Invest. Dermat. 47: 512-525, 1966.
a28
hi
O.S., 0.x & O.P. December, 1968
14. Olson, R. L., and Nordyuist, R. E.: Ultramicroscopic Localization of Acid Phosphatase in Human Eoidermis. J. Invest. Dermat. 46: 431-435. lRG6. 15. Rowden, G.: Ultrastructural Studies of ReratinizedEpithelia of the Mouse. I. Combined Electron Microscope and Cyt’ochemical Study of Lysosomes in Mouse Epidermis and Esophageal Epithelium, J. Invest. Dermat. 49: 181-197, 1967. 16. De Duve, C.: Lysosomes, a New Group of Cytoplasmic Particles. In Hayashi, ‘I., (editor) : Subcellular Particles, New York. 1959. Ronald Press. 17. S&i, F. R., Goldhaber, PI, and Jennings, 3.: A Histochemical and Biochemical Study of Acid Ph,osphatase in Resorbing Bone in Culture, Am. J. Physiol. 211: 959-962, 1966. 18. Nor-berg, B.: On Proteins in Regenerating Liver; Phosphomonoesterase, Acta physiol. scandinav. 19: 246-259, 1950.