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Cysts of the outer root sheath of wool follicles: A study of abnormal keratinization
© 1970 by Academic Press, Inc.
116
J. ULTRASTRUCTtJRERESEARCH31, 116--124 (1970)
Cysts of the Outer Root Sheath of Wool Follicles: A Study of Abnormal Keratinization R. C. HENRIKSON1 and R. E. CHAPMAN Division of Animal Physiology, C.S.I.R.O., Prospect, N.S.W., Australia Received September 4, 1969
Cystic enlargements of the outer root sheath in the suprabulbar region of wool follicles have been studied by electron microscopy. The first indication of cyst formation is a perinuclear condensation of filaments in cells that otherwise resemble normal cells of the outer root sheath. Keratohyalin and membranecoating granules are rarely seen. A mass of keratinizecl cells progressively differentiates in the cyst. In cells close to the center of the cyst, organelles are centripetally located in the cytoplasm. A gradation of glycogen exists radially in the cyst wall, the higher concentrations being found in peripheral cells. The cyst may eventually reach a diameter of about 150 l~. Cysts developing from the epidermis and hair follicles, and containing keratinized epithelial cells have been described in humans by Epstein and Kligman (6). Similar cellular proliferations have been studied by McGavran and Binnington (7), who classified the cysts as "epidermal" or "pilar" depending upon their origin and upon the presence or absence of keratohyalin. During the loss of pelage by hairless mice, dermal cysts are formed which undergo sebaceous transformation and eventually keratinize (8). Cysts also have been found in the skin of the sheep. Carne et al. (2) have described a relatively large cyst (3 mm-3 cm in diameter) which differentiates from interfollicular epidermis and is thought to be a hereditary condition. A squamous cell carcinoma may develop from the wall of the cyst, and the ultrastructure of these tumors has been reported by Borland and Webber (1). Another type of cutaneous cyst found in the sheep is derived from the outer root sheath (3, 9). This type of cyst is commonly found in apparently healthy animals and is not breed-specific. The outer root sheath cysts are relatively small and appear at the suprabulbar level of the follicle. The cysts are formed peripherally by outer root sheath cells and centrally by lamellae of keraz Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York.
ABNORMAL KERATINIZATION
117
Fro. 1. Light micrograph of a longitudinal section of a follicle with a well-developed cyst (arrow) of the outer root sheath. Periodic acid-Schiff, x 100. FIG. 2. Light micrograph of a 2 # transverse section of cystic follicles. Early signs of cyst formation are present at (1) and a small cyst is seen at (2). A more fully developed cyst (3) which contains a lenticular mass of cornified cells, is attached to an adjacent follicle. Azure B. × 120.
tinized cells. There is evidence suggesting t h a t the cysts are related to the g r o w t h of straight, instead of n o r m a l l y crimped, w o o l fibers (so-called " d o g g y " wool) (3). The present s t u d y is an e x a m i n a t i o n , p r i m a r i l y with the electron microscope, of o u t e r r o o t sheath cysts, giving p a r t i c u l a r a t t e n t i o n to the d e v e l o p m e n t of the cysts a n d to the p a r t i c i p a t i o n of various organelles in this e x a m p l e of keratinization.
MATERIALS AND METHODS Skin samples, obtained from 4 adult sheep (Merino, English Leicester, Border Leicester) were fixed in osmium tetroxide buffered with Veronal acetate, collidine, or phosphate. Tissue blocks were dehydrated in ethanol and embedded in Araldite. Thick sections (2 #), stained with 1% aqueous Azure B, were used to identify cystic follicles. Thin sections of 22 cysts were examined with an Hitachi HU-11C electron microscope.
118
ttENRIKSON AND CHAPMAN
FIG. 3. Electron micrograph of normal outer root sheath cells of a wool follicle. Glycogen (G) occupies much of the cytoplasm. Perifollicular connective tissue (CT) and a keratinized cell of the inner root sheath (IRS) are indicated, x 11,000.
OBSERVATIONS Keratinized cysts develop f r o m hyperplastic enlargements or bulges of the outer r o o t sheath in the suprabulbar region of the follicle (Fig. 1). By light microscopy the cyst is first seen as a basophilic mass surrounded by cells which resemble normal outer r o o t sheath cells and are continuous with the root sheath of the follicle (Figs. 1 and 2). A t a later stage the basophilic keratinized portion of the cyst is lenticular in shape and encloses an apparently empty space (Figs. 1 and 2). Cysts eventually m a y reach a diameter of approximately 150 #. The first indication of cyst development seen with the electron microscope is a juxtanuclear condensation of cytoplasmic filaments (Figs. 4 and 5; cf. Fig. 3). Glycogen is c o m m o n l y f o u n d in these cells, as in other cells of the outer r o o t sheath. As keratinization proceeds in the cyst, the a m o u n t of glycogen is reduced, organelles become less distinct, and the cell becomes increasingly electron opaque (Fig. 6). Projections f r o m keratinized cells are sometimes surrounded by less differentiated FIG. 4. Edge of an early cyst. Connective tissue surrounding the cyst is seen in the upper left area of the field. Note the condensation of filaments around a nucleus (arrows); this is the first indication of cyst formation. Large amounts of glycogen (G) are found in the cells, which at this stage still resemble outer root sheath cells. The glycogen is frequently localized in protuberances of the cell surface. × 9000.
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120
HENRIKSON AND CHAPMAN
FIG. 5. A field near the center of a developing cyst. The nucleus (N) is surrounded by filaments which enmesh a few keratohyalin granules in addition to mitochondria and vesicles. The junction between this cell and more peripheral cells is extensivelyinfolded (arrows). x 22,500.
cells (Fig. 7). The number of dense keratinized cells forming the center of the cyst increases as a result of keratinization, but the cornified cells are not arranged in lamellae at this stage (Fig. 8). Organelles appear to be stratified in cells close to the keratinized mass. In general, nuclei, filaments, and mitochondria are found centripetally in the cells (Fig. 8). This intracellular distribution is less pronounced in peripheral cells of the cyst where larger amounts of glycogen occur. Small keratohyalin granules are found only infrequently in either early or more developed cysts (Fig. 5). Structures resembling membrane-coating granules are sometimes present but are not common in the epithelial cells forming the cyst wall. The junction between the noncornified and cornified layers of the cyst wall in many instances is deeply infolded and shows a complex array of interdigitations (Fig. 5). This type of cell surface is not seen in other keratinizing tissues, such as epidermis and hair cortex. Cells forming the lenticular mass of the larger cysts (Figs. 9 and 10) are similar
ABNORMAL KERATIN1ZATION
121
FIG. 6. A keratinizing cell at the edge of a cyst. The nucleus may still be identified (N). In general, keratinized cells are not commonly found at the surface of a cyst. x 9000. FIO. 7. A tangential section through the periphery of a cornified cell which is surrounded by noncornified cells, x 30,000. to cornified cells of the epidermis. Few o r g a n d i e s m a y be identified; the ceils are n o t parakeratotic; the extracellular space is quite n a r r o w ; a n d the plasma m e m b r a n e of the cornified cell is thickened (Fig. 10). T o w a r d the center of the cyst the c o m p a c t nature of the cornified layer is lost, a n d at the j u n c t i o n of this layer with the hollow interior of the cyst the cornified cells are disrupted (Fig. 10).
DISCUSSION The morphological changes d u r i n g keratinization in outer root sheath cysts resemble those seen in the wool fiber a n d epidermis. As differentiation of the cyst proceeds, nuclei become less distinct a n d a dense mass of keratinized cells is formed. FIG. 8. Approximately a quarter of a large cyst. A mass of keratinized cells is surrounded by noncornified cells in which organelles are centripetally arranged (*). Glycogen (G) is found in the cyst wall and is more concentrated toward the periphery of the cyst. x 6500. FIG. 9. Part of the wall of a large, fully differentiated cyst. One end of the lens-shaped mass of cornified cells (cf. Figs. 1 and 2) is seen in the upper right area of the field; an apparently empty space is enclosed by the cornified cells, x 5500. Fro. 10. Layers of cornified cells from the interior of a large cyst. The cells have thickened plasma membranes, and the intercellular spaces are not dilated. Cells closer to the interior of the cyst, toward the top of the field, are less well organized and appear disrupted (*). x 37,500.
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124
HENRIKSON AND CHAPMAN
Some apparently unique features, however, are present. The perinuclear condensation of filaments, which is the first indication of cyst formation in the hyperplastic cell mass, is not characteristically seen in other types of keratinizing cells. In large cysts the eornified cells are usually arranged in lamellae which form a lenticular mass. This part of the cyst wall is similar to the stratum corneum of epidermis with the exception that the keratin pattern, commonly found in proximal cornified cells of the epidermis, has not been observed in cornified cells of cysts. Membrane-coating granules and keratohyalin granules are sparse in cells of the cyst wall, and it seems unlikely that these organelles play an important role in keratinization of the cyst. The centripetal location of organelles in epithelial cells of the cyst and also the complex junction betweeen cornified and noncornified cells are unusual and are of unknown significance. The keratinizing cysts derived from the outer root sheaths of wool follicles resemble milia (6) and pilar cysts (7), and are another example of the multipotentiality of epidermis and its derivatives. The development of cysts from the outer root sheath of wool follicles appears to be an aging process. Cysts are generally not found in the skin of lambs, and in young sheep about 12 months old less than 1% of the follicles are cystic. In some sheep 3 years and older, 20 % or more of the follicles in the wool-growing regions bear cysts (4). In contrast, cysts have not been observed on follicles in the hairy regions of sheep. Although histologically the bulbar portions of both wool and hair follicles are very similar, their synthetic activities differ. Wool follicles are engaged in continuous protein synthesis and formation of wool fibers for several years in those breeds which do not have an annual cycle of shedding. Hair follicles, however, pass through periods of activity and inactivity [anagen and telogen (5)]. The possibility may be considered that the commonly observed hyperplasia of the outer root sheath of wool follicles is related to the continuous high level of protein synthesis maintained by these follicles. REFERENCES 1. BORLAND, R. and WEBBER, A. J., Cancer Res. 26, 172 (1966). 2. CARNE, ~--LR., LLOYD,L. C. and CARTER,H. B., J. Pathol. Bacteriol. 86, 305 (1963). 3. CHAPMAN,R. E., SHORT,B. F. and HYLAND,P. G., Nature 187, 960 (1960). 4. CHAPMAN,R. E. and SHORT,B. F., Australian dr. Agr. Res. 16, 211 (1965). 5. DRY, F. W., J. Genet. 16, 287 (1926). 6. EPSTEIN,W. and KLIGMAN,A. M., J. Invest. Dermatol. 26, 1 (1956). 7. McGAVRAN,M. H. and BINNINGTON,B., Arch. Dermatol. 14, 499 (1966). 8. MONTAGNA,W., CHASE,H. B. and MELARAGNO,H. P., J. Invest. Dermatol. 19, 83 (1952). 9. RYDER,M. L. and AUBER,L., Nature 174, 743 (1954).
116
J. ULTRASTRUCTtJRERESEARCH31, 116--124 (1970)
Cysts of the Outer Root Sheath of Wool Follicles: A Study of Abnormal Keratinization R. C. HENRIKSON1 and R. E. CHAPMAN Division of Animal Physiology, C.S.I.R.O., Prospect, N.S.W., Australia Received September 4, 1969
Cystic enlargements of the outer root sheath in the suprabulbar region of wool follicles have been studied by electron microscopy. The first indication of cyst formation is a perinuclear condensation of filaments in cells that otherwise resemble normal cells of the outer root sheath. Keratohyalin and membranecoating granules are rarely seen. A mass of keratinizecl cells progressively differentiates in the cyst. In cells close to the center of the cyst, organelles are centripetally located in the cytoplasm. A gradation of glycogen exists radially in the cyst wall, the higher concentrations being found in peripheral cells. The cyst may eventually reach a diameter of about 150 l~. Cysts developing from the epidermis and hair follicles, and containing keratinized epithelial cells have been described in humans by Epstein and Kligman (6). Similar cellular proliferations have been studied by McGavran and Binnington (7), who classified the cysts as "epidermal" or "pilar" depending upon their origin and upon the presence or absence of keratohyalin. During the loss of pelage by hairless mice, dermal cysts are formed which undergo sebaceous transformation and eventually keratinize (8). Cysts also have been found in the skin of the sheep. Carne et al. (2) have described a relatively large cyst (3 mm-3 cm in diameter) which differentiates from interfollicular epidermis and is thought to be a hereditary condition. A squamous cell carcinoma may develop from the wall of the cyst, and the ultrastructure of these tumors has been reported by Borland and Webber (1). Another type of cutaneous cyst found in the sheep is derived from the outer root sheath (3, 9). This type of cyst is commonly found in apparently healthy animals and is not breed-specific. The outer root sheath cysts are relatively small and appear at the suprabulbar level of the follicle. The cysts are formed peripherally by outer root sheath cells and centrally by lamellae of keraz Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York.
ABNORMAL KERATINIZATION
117
Fro. 1. Light micrograph of a longitudinal section of a follicle with a well-developed cyst (arrow) of the outer root sheath. Periodic acid-Schiff, x 100. FIG. 2. Light micrograph of a 2 # transverse section of cystic follicles. Early signs of cyst formation are present at (1) and a small cyst is seen at (2). A more fully developed cyst (3) which contains a lenticular mass of cornified cells, is attached to an adjacent follicle. Azure B. × 120.
tinized cells. There is evidence suggesting t h a t the cysts are related to the g r o w t h of straight, instead of n o r m a l l y crimped, w o o l fibers (so-called " d o g g y " wool) (3). The present s t u d y is an e x a m i n a t i o n , p r i m a r i l y with the electron microscope, of o u t e r r o o t sheath cysts, giving p a r t i c u l a r a t t e n t i o n to the d e v e l o p m e n t of the cysts a n d to the p a r t i c i p a t i o n of various organelles in this e x a m p l e of keratinization.
MATERIALS AND METHODS Skin samples, obtained from 4 adult sheep (Merino, English Leicester, Border Leicester) were fixed in osmium tetroxide buffered with Veronal acetate, collidine, or phosphate. Tissue blocks were dehydrated in ethanol and embedded in Araldite. Thick sections (2 #), stained with 1% aqueous Azure B, were used to identify cystic follicles. Thin sections of 22 cysts were examined with an Hitachi HU-11C electron microscope.
118
ttENRIKSON AND CHAPMAN
FIG. 3. Electron micrograph of normal outer root sheath cells of a wool follicle. Glycogen (G) occupies much of the cytoplasm. Perifollicular connective tissue (CT) and a keratinized cell of the inner root sheath (IRS) are indicated, x 11,000.
OBSERVATIONS Keratinized cysts develop f r o m hyperplastic enlargements or bulges of the outer r o o t sheath in the suprabulbar region of the follicle (Fig. 1). By light microscopy the cyst is first seen as a basophilic mass surrounded by cells which resemble normal outer r o o t sheath cells and are continuous with the root sheath of the follicle (Figs. 1 and 2). A t a later stage the basophilic keratinized portion of the cyst is lenticular in shape and encloses an apparently empty space (Figs. 1 and 2). Cysts eventually m a y reach a diameter of approximately 150 #. The first indication of cyst development seen with the electron microscope is a juxtanuclear condensation of cytoplasmic filaments (Figs. 4 and 5; cf. Fig. 3). Glycogen is c o m m o n l y f o u n d in these cells, as in other cells of the outer r o o t sheath. As keratinization proceeds in the cyst, the a m o u n t of glycogen is reduced, organelles become less distinct, and the cell becomes increasingly electron opaque (Fig. 6). Projections f r o m keratinized cells are sometimes surrounded by less differentiated FIG. 4. Edge of an early cyst. Connective tissue surrounding the cyst is seen in the upper left area of the field. Note the condensation of filaments around a nucleus (arrows); this is the first indication of cyst formation. Large amounts of glycogen (G) are found in the cells, which at this stage still resemble outer root sheath cells. The glycogen is frequently localized in protuberances of the cell surface. × 9000.
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120
HENRIKSON AND CHAPMAN
FIG. 5. A field near the center of a developing cyst. The nucleus (N) is surrounded by filaments which enmesh a few keratohyalin granules in addition to mitochondria and vesicles. The junction between this cell and more peripheral cells is extensivelyinfolded (arrows). x 22,500.
cells (Fig. 7). The number of dense keratinized cells forming the center of the cyst increases as a result of keratinization, but the cornified cells are not arranged in lamellae at this stage (Fig. 8). Organelles appear to be stratified in cells close to the keratinized mass. In general, nuclei, filaments, and mitochondria are found centripetally in the cells (Fig. 8). This intracellular distribution is less pronounced in peripheral cells of the cyst where larger amounts of glycogen occur. Small keratohyalin granules are found only infrequently in either early or more developed cysts (Fig. 5). Structures resembling membrane-coating granules are sometimes present but are not common in the epithelial cells forming the cyst wall. The junction between the noncornified and cornified layers of the cyst wall in many instances is deeply infolded and shows a complex array of interdigitations (Fig. 5). This type of cell surface is not seen in other keratinizing tissues, such as epidermis and hair cortex. Cells forming the lenticular mass of the larger cysts (Figs. 9 and 10) are similar
ABNORMAL KERATIN1ZATION
121
FIG. 6. A keratinizing cell at the edge of a cyst. The nucleus may still be identified (N). In general, keratinized cells are not commonly found at the surface of a cyst. x 9000. FIO. 7. A tangential section through the periphery of a cornified cell which is surrounded by noncornified cells, x 30,000. to cornified cells of the epidermis. Few o r g a n d i e s m a y be identified; the ceils are n o t parakeratotic; the extracellular space is quite n a r r o w ; a n d the plasma m e m b r a n e of the cornified cell is thickened (Fig. 10). T o w a r d the center of the cyst the c o m p a c t nature of the cornified layer is lost, a n d at the j u n c t i o n of this layer with the hollow interior of the cyst the cornified cells are disrupted (Fig. 10).
DISCUSSION The morphological changes d u r i n g keratinization in outer root sheath cysts resemble those seen in the wool fiber a n d epidermis. As differentiation of the cyst proceeds, nuclei become less distinct a n d a dense mass of keratinized cells is formed. FIG. 8. Approximately a quarter of a large cyst. A mass of keratinized cells is surrounded by noncornified cells in which organelles are centripetally arranged (*). Glycogen (G) is found in the cyst wall and is more concentrated toward the periphery of the cyst. x 6500. FIG. 9. Part of the wall of a large, fully differentiated cyst. One end of the lens-shaped mass of cornified cells (cf. Figs. 1 and 2) is seen in the upper right area of the field; an apparently empty space is enclosed by the cornified cells, x 5500. Fro. 10. Layers of cornified cells from the interior of a large cyst. The cells have thickened plasma membranes, and the intercellular spaces are not dilated. Cells closer to the interior of the cyst, toward the top of the field, are less well organized and appear disrupted (*). x 37,500.
i
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ii~iiii~iiiii,,~ii~,
124
HENRIKSON AND CHAPMAN
Some apparently unique features, however, are present. The perinuclear condensation of filaments, which is the first indication of cyst formation in the hyperplastic cell mass, is not characteristically seen in other types of keratinizing cells. In large cysts the eornified cells are usually arranged in lamellae which form a lenticular mass. This part of the cyst wall is similar to the stratum corneum of epidermis with the exception that the keratin pattern, commonly found in proximal cornified cells of the epidermis, has not been observed in cornified cells of cysts. Membrane-coating granules and keratohyalin granules are sparse in cells of the cyst wall, and it seems unlikely that these organelles play an important role in keratinization of the cyst. The centripetal location of organelles in epithelial cells of the cyst and also the complex junction betweeen cornified and noncornified cells are unusual and are of unknown significance. The keratinizing cysts derived from the outer root sheaths of wool follicles resemble milia (6) and pilar cysts (7), and are another example of the multipotentiality of epidermis and its derivatives. The development of cysts from the outer root sheath of wool follicles appears to be an aging process. Cysts are generally not found in the skin of lambs, and in young sheep about 12 months old less than 1% of the follicles are cystic. In some sheep 3 years and older, 20 % or more of the follicles in the wool-growing regions bear cysts (4). In contrast, cysts have not been observed on follicles in the hairy regions of sheep. Although histologically the bulbar portions of both wool and hair follicles are very similar, their synthetic activities differ. Wool follicles are engaged in continuous protein synthesis and formation of wool fibers for several years in those breeds which do not have an annual cycle of shedding. Hair follicles, however, pass through periods of activity and inactivity [anagen and telogen (5)]. The possibility may be considered that the commonly observed hyperplasia of the outer root sheath of wool follicles is related to the continuous high level of protein synthesis maintained by these follicles. REFERENCES 1. BORLAND, R. and WEBBER, A. J., Cancer Res. 26, 172 (1966). 2. CARNE, ~--LR., LLOYD,L. C. and CARTER,H. B., J. Pathol. Bacteriol. 86, 305 (1963). 3. CHAPMAN,R. E., SHORT,B. F. and HYLAND,P. G., Nature 187, 960 (1960). 4. CHAPMAN,R. E. and SHORT,B. F., Australian dr. Agr. Res. 16, 211 (1965). 5. DRY, F. W., J. Genet. 16, 287 (1926). 6. EPSTEIN,W. and KLIGMAN,A. M., J. Invest. Dermatol. 26, 1 (1956). 7. McGAVRAN,M. H. and BINNINGTON,B., Arch. Dermatol. 14, 499 (1966). 8. MONTAGNA,W., CHASE,H. B. and MELARAGNO,H. P., J. Invest. Dermatol. 19, 83 (1952). 9. RYDER,M. L. and AUBER,L., Nature 174, 743 (1954).