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Membrane-coating granules of the epidermis
510
J. ULTRASTRUCTURERESEARCH15, 510-515 (1966)
Membrane-Coating Granules of the Epidermis ~ A. GEDEON MATOLTSY
Department of Dermatology, Boston University School of Medicine, and Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, Boston, Massachusetts. Received September 15, 1965 The epidermis of young kangaroos was found to be a suitable tissue to study the formation and fate of cytoplasmic granules, called membrane-coating granules (MCG). In this tissue MCG are formed during an early stage of cell differentiation, and as the cells ascend the MCG migrate toward the cell periphery. During the granular stage of differentiation the MCG are discharged into the intercellular space. While the cells change into horny cells, their plasma membrane thickens and the intercellular space becomes filled with an amorphous substance; remnants of MCG can no longer be seen. These findings are essentially identical to those previously noted in the keratinizing oral epithelium of the mouse (7) and further the view that MCG are important differentiation products of keratinizing epithelia and participate in the formation of an intercellular cement and a protective envelope around the horny cells.
Small cytoplasmic granules have been observed in keratinizing epithelia of mammals by several investigators and have been called by different names and assigned various functions (1, 3-5, 8, 9, 11, 13). They were thought to be small keratohyalin granules (11), attenuated mitochondria (8), secretory granules (3), vesicles (13), virus particles (12), or some unknown cell constituents (9). In a previous study we surveyed keratinizing epithelia of different vertebrates in the electron microscope and noted these granules, with a membranous inner structure, in the differentiating cells. Our studies indicated that these cell constituents represent differentiation products which are involved in formation of an intercellular cement and a resistant thick envelope around the horny cells. We studied in great detail the formation and fate of these granules in the keratinizing oral epithelium of the mouse and made the following observations. The small granules develop in the Golgi region of differentiating cells located above the basal layer. While these cells ascend, the granules move toward the cell periphery and during an advanced stage of differentiation fuse with the plasma membrane and 1 This investigation was supported by research grant AM 05779, National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.
MEMBRANE-COATING GRANULES
511
empty their contents into the intercellular space. The contents of the granules spread over the cell surface, and a coated and thickened plasma membrane is formed as the cells enter their final horny stage. The thick envelopes of horny cells were found to resist the action of keratinolytic agents indicating that they protect the cell contents. To distinguish these granules from other granular elements it was proposed to call them "membrane-coating granules" (MCG) (7). Although small granules, identical to M C G , have been seen in the epidermis by m a n y investigators (3, 5, 7, 8, 11, 13), clear evidence for the discharge of M C G into the intercellular spaces has not yet been presented for this tissue. The epidermis of the young kangaroo was found to be particularly suitable for studying the fate of M C G because their migration toward the cell periphery can be readily recognized by comparing cells of the different strata and their remnants remain well preserved in the intercellular spaces for a relatively long time. This tissue was therefore chosen for detailed studies and the results are presented in this paper.
MATERIAL AMD METHOD Small pieces of the scalp and back skin of red kangaroos 1-3 weeks old were fixed for 2 hours at 4°C in 1% osmium tetroxide buffered with Veronal-acetate to pH 7.5. After fixation the tissues were dehydrated in alcohol and embedded in epoxy resin according to the method of Luft (6). Thin sections were cut with a LKB or Porter-Blum microtome and stained with lead hydroxide. The specimens were examined in a Siemens Elmiscope and an RCA EMU-3 electron microscope. RESULTS In the epidermis of young kangaroos M C G are not present in basal cells; they are first seen in the central portion of cells located above the basal layer. In the lower portion of the epidermis the cells are separated by wide gaps and the M C G are preferentially located in the apical part of the cells (Fig. 1). At higher levels the intercellular spaces appear narrower and the M C G are close to the plasma membrane. At the level where the keratohyalin granules start to develop the intercellular spaces are narrow, and the M C G are aligned in multiple rows next to the plasma membrane (Fig. 2). Above this layer the cells begin to flatten and discharge their M C G into the intercellular space (Fig. 2). Remnants of M C G are seen as parallel or disoriented lamellar structures in the intercellular space. Above this layer the keratinizing cells are encased by a thickened plasma membrane; the plasma membrane does not seem to thicken at desmosomal contact points. Remnants of M C G are not recognizable between the fully formed horny cells, the intercellular space being filled with some amorphous material.
512
A. G E D E O N MATOLTSY
DISCUSSION The observations made in this study indicate that M C G are formed during an early stage of differentiation in the epidermis and that they migrate toward the cell periphery as the cells mature and are finally extruded into the intercellular space. The remnants of the M C G spread between the cells and contribute to the formation of an intercellular cement and thickened plasma membranes. These sequential events are identical to those found in the oral epithelium of the mouse (7) and indicate that the formation and fate of M C G are similar in these tissues. Recently Rupec and Braun-Falco studied the human epidermis and noted M C G either attached to or in the vicinity of the plasma membrane of granular cells. They consider these granules to be formed by the rounding and tying-off of the invaginations of the plasma membrane. According to their observations these granules migrate toward the center of the cell and finally dissolve. They state that the granules are formed to reduce the surface of the plasma membrane and that the change of the folded plasma membrane into a smooth one during keratinization of epidermal cells is facilitated by this mechanism (10). Isolated observations made by other workers, however, favor the view that M C G are discharged from m a t u r e epidermal cells and are involved in the development of an intercellular cement and thick envelope around the horny cells. Frei and Sheldon (3) noted M C G close to the plasma membrane of the granular cells in the normal and hyperplastic epidermis of the mouse. In some specimens they saw M C G within invaginations of the "cell surface membrane." Furthermore, they found that some of the uppermost granular cells do not contain M C G and between such cells a dense extracellular material is present. Brody (2) described a similar extracellular material in this region of the epidermis and also found that the envelope of the horny cells is much thicker, than that of the granular cells. In the light of these observations and our own results it is difficult to accept Rupec and Braun-Falco's interpretation. This work was started during the tenure of a Visiting Fellowship at the John Curtin School of Medicine, The Australian National University, in Canberra. The author wishes to thank Dr. Edgar H. Mercer, Head of the Electron Microscope Department, and the Council of The Australian National University for the kind invitation and the excellent facilities provided for the work.
FIG. 1. Mid-portion of the epidermis of a 3-week-old kangaroo. Membrane-coating granules (MCG) can be seen migrating toward the plasma membrane. Note that the intercellular spaces are wider in the lower layers than the upper ones. × 17,000.
i~ii~¸~i ,~ii
~i~ ~i~,,,~i!~
514
A. GEDEON MATOLTSY
FIG. 2. Upper part of the epidermis of a 3-week-old kangaroo. Membrane-coating granules (MCG) appear close to the folded plasma membrane of the lower granular cell; the intercellular space is narrow. M C G were discharged from the upper granular cell; their remnants appear as lamellar structures (arrows) in the intercellular space. The keratinizing cells are enveloped by a thickened and smoothed out plasma membrane (PM) and the intercellular space is filled with an amorphous substance, x 28,000.
MEMBRANE-COATINGGRANULES
515
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
ALBRIGHT,J. T. and LISTGARTEN,M. A., Arch. Oral. Biol. 7, 613 (1962). BRODY, I., J. Ultrastruct. Res. 2, 482 (1959). FREt, J. V. and SHELDON, H., J. Biophys. Biochem. Cytol. 11, 719 (1961). FRITmOF, L. and WEgS~LL, J., J. Ultrastruct. Res. 12, 371 (1965). HORSTMANN,E. and I(NOOV, A., Z. ZellJbrsch. Mikroskop. Anat. 47, 348 (1958). LusT, J. M., J. Biophys. Bioehem. Cytol. 9, 409 (1961). MATOLTSY,A. G. and PARAKKAL,P. F., J. Cell Biol. 24, 297 (1965). ODLAND, G. F., J. Invest. Dermatol. 34, 11 (1960). RHODIN, J. A. G. and REITH, E. J., in BUTCHER, E. O. and SOGNNAES,R. F. (Eds.), Fundamentals of Keratinization, p. 61. Am. Assoc. Advancem. Sci. Publ. 70, Washington, D. C., 1962. Ruv~c, M. and BRAuN-FALCO, O., Arch. Kiln. Exptl. Dermatol. 221, 184 (1965). SELBY, C. C., J. Invest. Dermatol. 29, 131 (1957). WETTSTEIN,B., LAGERHOLM,B., and ZECH, H., A cta Dermato-Venereol. 41, 115 (1961). ZELICKSON,A. S. and HARTMANN,J. F., J. Invest. Dermatol. 38, 99 (1962).
J. ULTRASTRUCTURERESEARCH15, 510-515 (1966)
Membrane-Coating Granules of the Epidermis ~ A. GEDEON MATOLTSY
Department of Dermatology, Boston University School of Medicine, and Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, Boston, Massachusetts. Received September 15, 1965 The epidermis of young kangaroos was found to be a suitable tissue to study the formation and fate of cytoplasmic granules, called membrane-coating granules (MCG). In this tissue MCG are formed during an early stage of cell differentiation, and as the cells ascend the MCG migrate toward the cell periphery. During the granular stage of differentiation the MCG are discharged into the intercellular space. While the cells change into horny cells, their plasma membrane thickens and the intercellular space becomes filled with an amorphous substance; remnants of MCG can no longer be seen. These findings are essentially identical to those previously noted in the keratinizing oral epithelium of the mouse (7) and further the view that MCG are important differentiation products of keratinizing epithelia and participate in the formation of an intercellular cement and a protective envelope around the horny cells.
Small cytoplasmic granules have been observed in keratinizing epithelia of mammals by several investigators and have been called by different names and assigned various functions (1, 3-5, 8, 9, 11, 13). They were thought to be small keratohyalin granules (11), attenuated mitochondria (8), secretory granules (3), vesicles (13), virus particles (12), or some unknown cell constituents (9). In a previous study we surveyed keratinizing epithelia of different vertebrates in the electron microscope and noted these granules, with a membranous inner structure, in the differentiating cells. Our studies indicated that these cell constituents represent differentiation products which are involved in formation of an intercellular cement and a resistant thick envelope around the horny cells. We studied in great detail the formation and fate of these granules in the keratinizing oral epithelium of the mouse and made the following observations. The small granules develop in the Golgi region of differentiating cells located above the basal layer. While these cells ascend, the granules move toward the cell periphery and during an advanced stage of differentiation fuse with the plasma membrane and 1 This investigation was supported by research grant AM 05779, National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.
MEMBRANE-COATING GRANULES
511
empty their contents into the intercellular space. The contents of the granules spread over the cell surface, and a coated and thickened plasma membrane is formed as the cells enter their final horny stage. The thick envelopes of horny cells were found to resist the action of keratinolytic agents indicating that they protect the cell contents. To distinguish these granules from other granular elements it was proposed to call them "membrane-coating granules" (MCG) (7). Although small granules, identical to M C G , have been seen in the epidermis by m a n y investigators (3, 5, 7, 8, 11, 13), clear evidence for the discharge of M C G into the intercellular spaces has not yet been presented for this tissue. The epidermis of the young kangaroo was found to be particularly suitable for studying the fate of M C G because their migration toward the cell periphery can be readily recognized by comparing cells of the different strata and their remnants remain well preserved in the intercellular spaces for a relatively long time. This tissue was therefore chosen for detailed studies and the results are presented in this paper.
MATERIAL AMD METHOD Small pieces of the scalp and back skin of red kangaroos 1-3 weeks old were fixed for 2 hours at 4°C in 1% osmium tetroxide buffered with Veronal-acetate to pH 7.5. After fixation the tissues were dehydrated in alcohol and embedded in epoxy resin according to the method of Luft (6). Thin sections were cut with a LKB or Porter-Blum microtome and stained with lead hydroxide. The specimens were examined in a Siemens Elmiscope and an RCA EMU-3 electron microscope. RESULTS In the epidermis of young kangaroos M C G are not present in basal cells; they are first seen in the central portion of cells located above the basal layer. In the lower portion of the epidermis the cells are separated by wide gaps and the M C G are preferentially located in the apical part of the cells (Fig. 1). At higher levels the intercellular spaces appear narrower and the M C G are close to the plasma membrane. At the level where the keratohyalin granules start to develop the intercellular spaces are narrow, and the M C G are aligned in multiple rows next to the plasma membrane (Fig. 2). Above this layer the cells begin to flatten and discharge their M C G into the intercellular space (Fig. 2). Remnants of M C G are seen as parallel or disoriented lamellar structures in the intercellular space. Above this layer the keratinizing cells are encased by a thickened plasma membrane; the plasma membrane does not seem to thicken at desmosomal contact points. Remnants of M C G are not recognizable between the fully formed horny cells, the intercellular space being filled with some amorphous material.
512
A. G E D E O N MATOLTSY
DISCUSSION The observations made in this study indicate that M C G are formed during an early stage of differentiation in the epidermis and that they migrate toward the cell periphery as the cells mature and are finally extruded into the intercellular space. The remnants of the M C G spread between the cells and contribute to the formation of an intercellular cement and thickened plasma membranes. These sequential events are identical to those found in the oral epithelium of the mouse (7) and indicate that the formation and fate of M C G are similar in these tissues. Recently Rupec and Braun-Falco studied the human epidermis and noted M C G either attached to or in the vicinity of the plasma membrane of granular cells. They consider these granules to be formed by the rounding and tying-off of the invaginations of the plasma membrane. According to their observations these granules migrate toward the center of the cell and finally dissolve. They state that the granules are formed to reduce the surface of the plasma membrane and that the change of the folded plasma membrane into a smooth one during keratinization of epidermal cells is facilitated by this mechanism (10). Isolated observations made by other workers, however, favor the view that M C G are discharged from m a t u r e epidermal cells and are involved in the development of an intercellular cement and thick envelope around the horny cells. Frei and Sheldon (3) noted M C G close to the plasma membrane of the granular cells in the normal and hyperplastic epidermis of the mouse. In some specimens they saw M C G within invaginations of the "cell surface membrane." Furthermore, they found that some of the uppermost granular cells do not contain M C G and between such cells a dense extracellular material is present. Brody (2) described a similar extracellular material in this region of the epidermis and also found that the envelope of the horny cells is much thicker, than that of the granular cells. In the light of these observations and our own results it is difficult to accept Rupec and Braun-Falco's interpretation. This work was started during the tenure of a Visiting Fellowship at the John Curtin School of Medicine, The Australian National University, in Canberra. The author wishes to thank Dr. Edgar H. Mercer, Head of the Electron Microscope Department, and the Council of The Australian National University for the kind invitation and the excellent facilities provided for the work.
FIG. 1. Mid-portion of the epidermis of a 3-week-old kangaroo. Membrane-coating granules (MCG) can be seen migrating toward the plasma membrane. Note that the intercellular spaces are wider in the lower layers than the upper ones. × 17,000.
i~ii~¸~i ,~ii
~i~ ~i~,,,~i!~
514
A. GEDEON MATOLTSY
FIG. 2. Upper part of the epidermis of a 3-week-old kangaroo. Membrane-coating granules (MCG) appear close to the folded plasma membrane of the lower granular cell; the intercellular space is narrow. M C G were discharged from the upper granular cell; their remnants appear as lamellar structures (arrows) in the intercellular space. The keratinizing cells are enveloped by a thickened and smoothed out plasma membrane (PM) and the intercellular space is filled with an amorphous substance, x 28,000.
MEMBRANE-COATINGGRANULES
515
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
ALBRIGHT,J. T. and LISTGARTEN,M. A., Arch. Oral. Biol. 7, 613 (1962). BRODY, I., J. Ultrastruct. Res. 2, 482 (1959). FREt, J. V. and SHELDON, H., J. Biophys. Biochem. Cytol. 11, 719 (1961). FRITmOF, L. and WEgS~LL, J., J. Ultrastruct. Res. 12, 371 (1965). HORSTMANN,E. and I(NOOV, A., Z. ZellJbrsch. Mikroskop. Anat. 47, 348 (1958). LusT, J. M., J. Biophys. Bioehem. Cytol. 9, 409 (1961). MATOLTSY,A. G. and PARAKKAL,P. F., J. Cell Biol. 24, 297 (1965). ODLAND, G. F., J. Invest. Dermatol. 34, 11 (1960). RHODIN, J. A. G. and REITH, E. J., in BUTCHER, E. O. and SOGNNAES,R. F. (Eds.), Fundamentals of Keratinization, p. 61. Am. Assoc. Advancem. Sci. Publ. 70, Washington, D. C., 1962. Ruv~c, M. and BRAuN-FALCO, O., Arch. Kiln. Exptl. Dermatol. 221, 184 (1965). SELBY, C. C., J. Invest. Dermatol. 29, 131 (1957). WETTSTEIN,B., LAGERHOLM,B., and ZECH, H., A cta Dermato-Venereol. 41, 115 (1961). ZELICKSON,A. S. and HARTMANN,J. F., J. Invest. Dermatol. 38, 99 (1962).