JOURNAL OF U L T RA ST RUCTURE R E S E A R C H ~ ,
359-373 \
(1976)
The Ultrastructure of the Hair Follicles in Early and Late Catagen, with Special Reference to the Alteration of the Junctional Structure between the Dermal Papilla and Epithelial Component SADAO SUGIYAMA, MAKOTO TAKAHASHI, AND MITSUO KAMIMURA Department of Dermatology, Sapporo Medical College, Sapporo, Japan Received May 14, 1975, and in revised form, September 15, 1975 The morphology of mouse h a i r follicles in the catagen stage was examined u n d e r a n electron microscope. In the early and late catagen stage, compact fine fibrils (120-130 A in diameter) with a hollow profile and a beaded p a t t e r n along t h e i r long axis were found throughout the dilated intercellular spaces in dermal papilla. These fibrils were not only contiguous to papilla cell m e m b r a n e s b u t were also attached, and prepared to be inserted into the basal lamina-like structure t h a t protruded from the basal l a m i n a into the dilated intercellular spaces at the upper portion of the papilla. In the late catagen stage, hemi-desmosomes were found along the keratinocyte m e m b r a n e s adjacent to t h e basal l a m i n a at the papilla-epithelial junction. Both the fine fibrils and hemidesmosomes, which were characteristic structures in hair follicles in the catagen stage, seem to act mechanically, in cooperation with the basal lamina, to form a firm adhesion structure between the papilla and the epithelial column during this stage.
The cyclic change in the metabolism of hair production is accompanied by predictable anatomical changes of both epithelial and mesodermal components in the hair follicle (4). During the catagen stage, the transitional phase between active growth (anagen) and resting stage (telogen), complicated and dramatic anatomical changes take place in the lower part of the hair follicle, leading to the formation of resting hair follicle. This process has been described in detail in light microscopic studies (5, 12, 17, 27). Parakkal (20-22) reported various findings on the morphogenesis of hair follicles of the catagen stage at the ultrastructural level. A characteristic morphological relation between the epithelial component and dermal papilla in the catagen stage is the adhesion of both components, which is invariably maintained when the involution of the hair bulb enclosing the papilla and extensive shortening of the lower hair follicle occur. When these changes of the epithelial component alone are considered, it may be said that they provide a rather 359 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
unfavorable condition for maintenance of adhesion of the epithelial component to subjacent dermal papilla. Thus, it may be surmised that some reinforced attachment structure may be formed at the papillaepithelial junction during this phase. However, the ultrastructure responsible for the mechanical function of maintenance of adhesion between the papilla and the epithelial component during the catagen remains unclarified, especially in late catagen when the lower follicle undergoes extensive shortening. Thus, the present study was undertaken to clarify or otherwise elucidate the fine structural organization of both the papilla and the papilla-epithelial junction of hair follicles in the early and late catagen stage. MATERIALS AND METHODS Small pieces of skin were excised from the backs of 19- to 22-day-old C57 black mice. These specimens were fixed for 2 h r in 1% osmium tetroxide buffered with cacodylate, or in 4% glutaraldehyde buffered with cacodylate for 2 hr and t h e n in 1% osmium tetroxide buffered with cacodylate for 2 hr. Subse-
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SUGIYAMA, TAKAHASHI AND KAMIMURA
quently, they were dehydrated in an ascending series of ethanol and embedded in epoxy resin (16). Thin sections were cut with a Porter-Blum microtome equipped with a diamond knife. These sections were stained with uranyl acetate and lead citrate (23), and then examined with a Hitachi HS-8 electron microscope. RESULTS I. HAIR FOLLICLE IN EARLY
CATAGEN
A. Epithelial Component The following is presented to show the actual site and phase studied. The club, surrounded by several layers of keratinocytes (epithelial sac), is seen formed in the suprabulbar region (Fig. la). Some keratinocytes of the epithelial sac show a limited number of trichohyalin granules in their cytoplasm (Fig. lb). The hair bulb is seen transformed into a short column of keratinocytes (epithelial column) between the epithelial sac and the dermal papilla, and shows a degenerative process. The column is composed of a centrally located large mass of degenerated cell materials with outer layers of undifferentiated keratinocytes containing numerous cytoplasmic vacuoles and the outer root sheath (Figs. la, c). In the epithelial column at the level from the top of the papilla to a higher level, the outer root sheath cells exhibit characteristic features. One of the striking features is that the contour of the cells on the dermal side shows pronounced irregular cytoplasmic indentations and illdefined cell membranes. The basal lamina measuring about 900 A in thickness follows the contour of the cytoplamic indentations, lacking an electron-lucent zone facing the indentations (Fig. lc).
At the base of the epithelial column, two or three layers of undifferentiated keratinocytes cover only the upper third portion of the spherical dermal papilla, and these keratinocytes are characterized by the presence of large phagosomes in the cytoplasm (Fig. 2). No hemi-desmosomes are found on the plasma membranes of the cells abutting upon the basal lamina between the epithelial column and the dermal papilla.
B. Dermal Papilla Papilla cells. The papilla cells in the early catagen stage are moderately irregular in shape, and show oval nuclei with one or two small nucleolei. The cells are adjacent to each other with very narrow intercellular spaces. However, numerous dilated intercellular spaces are also encountered in the dermal papilla (Fig. 2). The papilla cell shows a few dilated cisterns of rough-surfaced endoplasmic reticulum which contain fine filamentous materials. The Golgi complex, located.in the juxtanuclear region, is moderately well developed (Fig. 5), i.e., a communication of Golgi vacuoles and flattened cisternae and small bleb-like protrusions from the cisternae are frequently encountered. Golgi cisternae and vesicles contain moderately dense amorphous materials. Numerous smooth vesicles, presumably of pinocytotic character, and coated vesicles are seen mainly in the periphery of the cells, and moderate numbers of lysosomes and residual bodies are seen scattered in the cytoplasm (Figs. 3, 4). Other usual organelles and sparse cytoplasmic filaments are seen
FIG. la. The club (CL) surrounded by the epithelial sac (ES) is seen in suprabulbar region. A large mass of degenerated cell materials (DC) and its outer layers of keratinocytes (K) with numerous cytoplasmic vacuoles are seen between the dermal papilla (DP) and the epithelial sac (ES). Arrow indicates the direction of the hair follicle. Specimen fixed with 1% OsO4; section stained with uranyl and lead. x 3300. Fia. lb. Enlarged view of the enclosed area shown in Fig. la. Note t h a t the cell in the epithelial sac has trichohyalin granules (tg). cc, club cell with pyknotic nucleus. × 20 000. Fro. lc. The outer root sheath cells (ORS) in the epithelial column exhibit irregular cytoplasmic indentations on the dermal side. A set of cytoplasmic indentations is surrounded directly by the basal lamina (bl). x 5900.
FIGS. la-c. Portions of bulb and suprabulbar region in the early catagen stage. 361
FIG. 2. At the base of the epithelial column in the early catagen stage, two layers of undifferentiated keratinocytes (K) with large phagosomes (ps) cover only the upper t h i r d portion of spherical dermal papilla (DP). Dilated intercellular spaces (IS) are seen in several places of the dermal papilla (DP), especially in the upper portion. Arrow indicates the direction of the h a i r follicle. Specimen fixed and stained as in Fig. 1. x 6200. 362
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
363
in the r e l a t i v e l y electron-lucent cytoplasm. Basal lamina and intercellular substances. At the junction between the papilla and the epithelial column in the early catagen stage, a distinct basal lamina measuring about 450 A in thickness is present, and is seen separated from the keratinocyte membranes by a relatively electron-lucent zone measuring about 450 A in width. The basal lamina-like strncture, displaying a fine filamentous texture embedded in relatively dense amorphous material, protrudes from some parts of the basal lamina into the dilated intercellular spaces at the upper portion of the dermal papilla, forming its characteristic network (Figs. 3, 4). On the other hand, compact fine fibrils are found throughout the dilated intercellular spaces of the papilla (Figs. 3, 4, 6a, b). These fibrils are approximately from 120 to 130 A in diameter. In cross sections, the fibrils have a hollow appearance with a light core surrounded by a dense circumferential outer layer. In the longitudinal section of the fibrils, two parallel strata with medium density are separated by a central light interval. When observed as long strands, the fibrils exhibit a beaded pattern along their long axis (Figs. 6a, b). The periodicity of the beaded pattern appears to exhibit a slight irregularity. These intercellular fine fibrils are revealed to be embedded into the network of the basal lamina-like structure at the exterior of the network (Fig. 4). In addition, the fibrils are not only closely contiguous to the papilla cell membranes but also attached to them (Figs. 3, 4, 6a, b). A few light small oval areas are interspersed among the fibrils.
umn, the basal lamina of the glassy merebrane is only slightly thickened, and the undifferentiated keratinocytes in the column have irregularly shaped nuclei, a moderate number of lysosomes and large residual bodies in their cytoplasm (Figs. 7a, b). A special feature of the ultrastructure of the epithelial column in this period is that hemi-desmosomes are seen along the kerationcyte membranes adjacent to the basal lamina between the column and the dermal papilla (Figs. 8, 9).
II. HAIR FOLLICLE IN LATE CATAGEN
DISCUSSION
A. Epithelial Column In the late catagen stage, the epithelial column is shortened due to the degenerative process occurring in the keratinocytes. In this period of the shortening of the col-
B. Dermal Papilla The ultrastructural organization of the dermal papilla in the late catagen stage is generally similar to that in the early catagen stage. The basal lamina-like structure, which no longer shows a mesh-like pattern such as seen in the early catagen stage, is present underneath the basal lamina and in some dilated intercellular spaces in the upper portion of the papilla (Figs. 8, 9). A structural continuity between the basal lamina and the underlaying basal lamina-like structure is clearly observed (Fig. 9). On the other hand, fine fibrils (120-130 A in diameter) are seen to fill compactly throughout the dilated intercellular spaces of the papilla (Figs. 8, 9). Some of the fibrils are attached to the plasma membranes of papilla cells, and are also seen embedded into the basal lamina-like structure (Figs. 9, 10). These intercellular fine fibrils exhibit a hollow appearance in cross sections and an irregular beaded pattern along their long axis (Fig. 10). The papilla cell in the late catagen stage predominantly shows a small Golgi complex, smooth-surfaced endoplasmic reticulum, lysosomes, residual bodies, and smooth or coated vesicles (Fig. 11).
It was described in electron microscopic studies on mouse hair follicles in the anagen stage (19, 26) that the extracellular substances in the dermal papillae consist of abundant ground substance and a few
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collagen fibers. The present study shows that the intercellular substance in dermal papillae in the early and late catagen stage is comprised of the fine fibrils embedded in relatively light amorphous material. With respect to ultrastructural features, these fibrils clearly correspond to the "microfibrils" reported as a morphological entity of extracellular connective tissue components (10, 14, 15). The relatively small fibrils corresponding to the microfibrils were found to be both free and in association with the connective tissue structures of various organs, including elastic fibers (2, 6, 8, 10, 11, 15, 24), basal laminae (2, 7, 14), and collagen fibers (10,
11, 15). The intercellular fine fibrils observed in the present study are characterized by small diameters, a hollow appearance in cross sections, and a beaded pattern along the long axis, which appears to have irregular periodicity. Fine structure similar to that of these fibrils were reported in the microfibrils of elastic fibers (6, 10, 24), in the filaments bordering the basal lamina of muscle capillaries (2), in the anchoring filaments of lymphatic vessels (13), and in the microfibrils connected with the skeletal muscles (9). The relatively small fibrils are assumed to have a mechanical function to anchor the organs to surrounding connective tissue structures, and have been so reported; Leak and Burke (13) found lymphatic anchoring filaments t h a t were connected with t h e endothelial cell m e m b r a n e s , which extended into the adjoining connective tissue area. Their interpretation is that the filaments have a role to bind the lymphatics to the surrounding connective
tissue components. H a n a k and B6ck (9) found the microfibrils that were in contact with skeletal muscle cell membranes, passing the basal lamina of the muscle, and extending into a cementing substance among the collagen fibers. They considered that the microfibrils were a connecting link between the muscle and the tendon. A unique aspect of the fine fibrils in the dermal papilla in the catagen stage is that the dilated intercellular spaces are filled with the fibrils that are attached to the papilla cell membranes and are embedded in the basal lamina-like structure t h a t protrudes downward from the basal lamina into the dilated intercellular spaces. On the basis of this relationship of the fibrils, as well as an ultrastructural similarity of the fibrils to the lymphatic anchoring filaments and the microfibrils connected with the skeletal muscle, it is suggested that the fibrils may have a mechanical function, connecting the papilla cells with the basal l a m i n a - l i k e s t r u c t u r e , which may bind the papilla cells. The basal lamina at the dermal-squamous epithelial junction is generally considered to act mechanically as a framework to support the epithelial cells and anchor these cells to the subjacent dermis. This f u n c t i o n of t h e b a s a l l a m i n a is strongly suggested by the structural relation of the basal lamina to basal cell membranes with hemi-desmosomes, and to subjacent fibrillar connective tissue elements of special type (1, 28, 29), called anchoring fibrils by Palade and Farquhar (18). However, the specialized a t t a c h m e n t structures such as the hemi-desmosomes and the anchoring fibrils have not been found at the junction between the dermal papilla
FIG. 3. At the papilla-epithelial junction in the early catagen stage, the basal l a m i n a (bl) follows the plasma m e m b r a n e s of undifferentiated keratinocytes (K) adjacent to the dermal papilla, and is separated from these by a relatively electron-lucent, zone. The network of the basal lamina-like structure (bls) is present in the dilated intercellular spaces in the upper portion of the papilla, and joins with the basal l a m i n a (bl) in places (*). Compact fine fibrils (ff) in the dilated intercellular spaces are closely contiguous to plasma m e m b r a n e s of papilla cells (PC) and to the network of the basal lamina-like structure (bls). Many small vesicles (v), coated vesicles (cv), and several lysosomes (ly) are seen in the cytoplasm of papilla cells (PC). Specimen fixed and stained as in Fig. 1. x 23 000.
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and the hair bulb in ihe anagen Stage (19, 26). Therefore, the attachment at this junction may not be so strong in the anagen stage. The present study strongly suggests that the attachment between the dermal, papilia and the epithelial column may be supported by .the characteristic structures in hair follicles in the catagen Stage, as follows. (1) The attachment Of the dermal papilla to the basal lamina is considered to be reinforced in the early and late catagen stage by intercellular fine fi-
"
brilsl in association wi~h the basal lamina: like structure with both a structural continuity and an ultrastructural similarity to the basal lamina. (2) It is apparent that the attachment of the epithelial column to the basal lamina is strengthened by the hemidesmos0mes of the keratinocytes of the column in the late catagen stage. Thus, the structural complex of the hemi-desmosomes, the basal lamina and basal laminalike structure, and the intercellular fine fibrils may be responsible for the firm
FIG. 4. Portion of the papilla-epithelial junction in the early catagen stage. A structural continuity (arrow) between the basal l a m i n a (bl) and the basal lamina-like structure (bls) and a fine-structural identity of both structures as fine filamentous texture are demonstrable. The compact intercellular fine fibrils (t"f) are not only closely continguous to papilla cell m e m b r a n e s but also embedded in the basal lamina-like structure (bls) at the peripheral part of its network (*). Small vesicles (v), coated vesicles (cv), and lysosomes (ly) are seen in the cytoplasm of the papilla cell (PC). Specimen fixed and stained as in Fig. 1. x 36 000. FIG. 5. Papilla cell in the hair follicle in the early catagen stage. A communication of Golgi vacuoles and flattened cisternae (arrow) and small bleb-like protrusions from the cisternae (double-headed arrow) are seen in relatively well-developed Golgi complex (G). Inset shows a residual body t h a t is bounded by a singleunit membrane, and has a n internal structure consisting of fine dense granules, several curved dense bands, and peripherally located vacuoles. Specimen fixed and stained as in Fig. 1. × 29 000. Inset, × 34 000. FIGS. 6a, b. Fine fibrils in the dilated intercellular spaces of dermal papilla in the early catagen stage. A hollow appearance (thick arrow) and a n i r r e g u l a r beaded p a t t e r n are clearly seen, respectively, in cross and longitudinal sections of the intercellular fine fibrils. Both t h i n and white arrows indicate distinct contact of the fibrils with papilla cell membranes. U, small oval area without fine fibrils. Specimen fixed and stained as in Fig. 1. Figure 6a, × 95 000. Figure 6b, × 130 000. FIG. 7a. Electron micrograph of longitudinal section though the dermal papilla and the epithelial column in the late catagen stage. Structural contact between hemispherical dermal papilla (DP) and shortened epithelial column (EC) is demonstrable. Note the irregular shape of nuclei in the cells of the epithelial column (EC). Inset shows slightly thickened basal l a m i n a (bl) and layers of collagen fibers (Co) around the epithelial column (EC). Specimen prefixed in 4% glutaraldehyde with cacodylate buffer, followed by 1% OsO4 with cacodylate buffer. Sections stained with uranyl and lead stain. × 2200. Inset, × 20 000. FIG. 7b. Enlarged view of the area indicated by arrow in Fig. 7a. Various sized residual bodies (rsb) are located in the vicinity of the nucleus of the cell in the epithelial column. × 20 000. FIG. 8. Upper portion of dermal papilla and base of epithelial column in the late catagen stage. Hemidesmosomes (hd) are Seen along the plasma m e m b r a n e s of undifferentiated keratinocytes (K) adjacent to the basal l a m i n a (bl) at the papilla-epithelial junction. The basal lamina-like structure (bls) and fine fibrils (if) are seen to fill the dilated intercellular spaces in the dermal papilla (DP). Specimen fixed and stained as in Fig. 7. × 25 000. FIG. 9. Junctional area between dermal papilla and epithelial column in the late catagen stage. Tonofilaments embedded in the a t t a c h m e n t plates of hemi-desmosomes (hd) are sparse. A structural continuity between the basal l a m i n a (bl) and the underlaying basal lamina-like structure (bls) is seen. A contact of intercellular fine fibrils (fl) with the plasma m e m b r a n e s of papilla cells (PC) is demonstrable (arrow). The fibrils are also embedded in the basal lamina-like structure (*). Specimen fixed and stained as in Fig. 7. × 87 000. FIG. 10. Intercellular fine fibrils and basal lamina-like structure in the dilated intercellular space of dermal papilla in the late catagen stage. In cross sections, the fine fibrils exhibit a hollow appearance with a light central core surrounded by a dense outer layer (arrow). In longitudinal sections, the fibrils exhibit a beaded pattern along their long axis (parenthesized passage). (*) indicates an insertion of the fibrils into the basal lamina-like structure (bls). Specimen fixed and stained as in Fig. 7. × 120 000. FIG. 11. Papilla cells in h a i r follicle in the late catagen stage. Note the presence oflysosomes (ly), smoothsurfaced endoplasmic reticulum (se), small vesicles (v), and coated vesicles (cv) in the cytoplasm of papilla cells. Inset shows the residual body (rsb). Specimen fixed and stained as in Fig. 7. × 34 000. Inset, × 34 000.
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
FIGURES 4-5
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FIGURE 6
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
FIGURS 7
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SUGIYAMA, TAKAHASHI AND KAMIMURA
FIaURES 8-9
'
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
FmuRss 10-11
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SUGIYAMA, TAKAHASHI AND KAMIMURA
adhesion between the epithelial column and the dermal papilla in the follicles in the late catagen stage, which are in the process of shortening the column. It has been reported that the dermal papilla is very important for the growth of hair in the anagen stage, since only a fragile hair is produced by the depapillated follicle (3). Thus, it may be said that the contact between the epithelial component and the dermal papilla during the catagen stage may be an important event in the preparation of one of the necessary conditions for subsequent hair growth. The cellular component in mouse dermal papillae in the anagen stage has been shown to consist of fibroblasts with welldeveloped rough-surfaced endoplasmic reticulum and Golgi complexes (19). Although the papilla cells in the catagen stage are characterized by the predominance in the Golgi complex, lysosomes, residual bodies, and small vesicles presumably of pinocytotic character, they may be derived from the fibroblasts in the preceding dermal papilla in the anagen stage, and may undergo changes in their ultrastructural appearance. This ultrastructural change of the cells seems to be a reflection of the increased endocytotic activity rather than the active synthesis and secretion of a proteinaceous substance. With regard to the synthesis of the intercellular fine fibrils in dermal papilla in the catagen stage, the intimate relation of the fibrils to the papilla cell membranes suggests the possibility that the precursors of the fibrils may be synthesized by the papilla cells before the completion of the ultrastructural change in the cells, namely, presumably in the earliest phase of catagen. In mouse hair follicles in the telogen stage, Roth (25) found that the hemi-desmosomes of the germ cells are attached to the basal lamina between the hair germ and the dermal papilla, and that the fine filaments (70-80/k in diameter) are joined to the basal lamina and extend into the intercellular spaces of the
papilla. This junctional structure in the resting hair follicle is approximately similar to that in the hair follicle in the catagen stage. The present study suggests that both the synthesis of the intercellular fine fibrils by the papilla cells, presumably in the earliest phase of catagen, and the formation of the hemi-desomosomes by the keratinocytes in the epithelial column in the late catagen stage are essential events for the alteration of the junctional structure between the dermal papilla and the epithelial component in the follicle during the catagen stage. REFERENCES 1. BRODY,I., J. Ultrastruct. Res. 4, 246 (1960). 2. BRUNS, R. R., AND PALADE, G. E., J, Cell Biol. 37, 244 (1968). 3. BUTCHER, E. O., Anat. Rec. 151, 231 (1965). 4. CHASE, H. B., Physiol. Rev. 34, 113 (1954). 5. ELLIS, R. A., AND MORETTI, G., Ann. N . Y . Acad. Sci. 83, 448 (1959). 6. FAHRENBACH, W. H., AND SANDBERG, J. L., Anat. Rec. 155, 563 (1966). 7. FARQUHAR, M. G., WISSIG, S. L., AND PALADE, G. E., J. Exp. Med. 113, 47 (1961). 8. GREENLEE, T. K., Ross, R., AND HARTMAN, J. L., J. Cell Biol. 30, 59 (1966). 9. HANAK, H., AND B0CK, P., J. Ultrastruct. Res. 36, 68 (1968). 10. HAUST, M. D., Amer. J. Pathol. 47, 1113 (1965). 11. KARRER, H. E., J. Ultrastruct. Res. 2, 96 (1958). 12. KLIGMAN, A. M., J. Invest. Dermatol. 33, 370 (1959). 13. LEAK, L. V., AND BURKE, J. F., J. Cell Biol. 36, 129 (1968). 14. Low, F. N., Anat. Rec. 139, 105 (1961). 15. Low, F. N., Anat. Rec. 142, 131 (1962). 16. LuFr, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 17. MONTAGNA, W., The Structure and Function of Skin, first ed., p. 173. Academic Press, New York, 1956. 18. PALADE, G., AND FARQUHAR, M. G., J. CellBiol. 37, 215 (1965). 19. PARAKKAL, P. F., in MONTAGNA, W., AND DOBSON, R. (Eds.), Hair Growth: Advances in B i ology of Skin, Vol. 9, p. 441. P e r g a m o n Press Oxford, 1969. 20. PARAKKAL, P. F., J. Cell Biol. 40, 561 (1969). 21. PARAKKAL, P. F., J. Ultrastruct. Res. 29, 21~ (1969). 22. PAaAKKAL, P. F., Z. Zellforsch. 107, 174 (1970). 23. REYNOLDS, E. S., J. Cell Biol. 46, 366 (1963).
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN 24. Ross, R., AND BORNSTEIN, P., J. CellBiol. 46, 366 (1969). 25. ROTH, S. I., in LYNE, A. G., AND SHORT, B. F. (Eds.), Biology of the Skin and Hair Growth. p. 233. Angus and Robertson, Sydney, 1965. 26. ROTH, S. I., ANn HELWIG, E. B., J. Ultrastruct. Res. 11, 33 (1964).
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27. STRAILE, W. E., CHASE, H. B., AND ARSENAULT, C., J. Exp. Zool. 148, 205 (1961). 28. SusI, F. R., BELT, W. D., AND KELLY, D. W., J. Cell Biol. 34, 686 (1967). 29. YOUNES, M. S., STEELE, H. D., ROBERTSON,E. M.~ ANn BENCOSME, S. A., Amer. J. Obstet. Gyneeol. 92, 163 (1965).
359-373 \
(1976)
The Ultrastructure of the Hair Follicles in Early and Late Catagen, with Special Reference to the Alteration of the Junctional Structure between the Dermal Papilla and Epithelial Component SADAO SUGIYAMA, MAKOTO TAKAHASHI, AND MITSUO KAMIMURA Department of Dermatology, Sapporo Medical College, Sapporo, Japan Received May 14, 1975, and in revised form, September 15, 1975 The morphology of mouse h a i r follicles in the catagen stage was examined u n d e r a n electron microscope. In the early and late catagen stage, compact fine fibrils (120-130 A in diameter) with a hollow profile and a beaded p a t t e r n along t h e i r long axis were found throughout the dilated intercellular spaces in dermal papilla. These fibrils were not only contiguous to papilla cell m e m b r a n e s b u t were also attached, and prepared to be inserted into the basal lamina-like structure t h a t protruded from the basal l a m i n a into the dilated intercellular spaces at the upper portion of the papilla. In the late catagen stage, hemi-desmosomes were found along the keratinocyte m e m b r a n e s adjacent to t h e basal l a m i n a at the papilla-epithelial junction. Both the fine fibrils and hemidesmosomes, which were characteristic structures in hair follicles in the catagen stage, seem to act mechanically, in cooperation with the basal lamina, to form a firm adhesion structure between the papilla and the epithelial column during this stage.
The cyclic change in the metabolism of hair production is accompanied by predictable anatomical changes of both epithelial and mesodermal components in the hair follicle (4). During the catagen stage, the transitional phase between active growth (anagen) and resting stage (telogen), complicated and dramatic anatomical changes take place in the lower part of the hair follicle, leading to the formation of resting hair follicle. This process has been described in detail in light microscopic studies (5, 12, 17, 27). Parakkal (20-22) reported various findings on the morphogenesis of hair follicles of the catagen stage at the ultrastructural level. A characteristic morphological relation between the epithelial component and dermal papilla in the catagen stage is the adhesion of both components, which is invariably maintained when the involution of the hair bulb enclosing the papilla and extensive shortening of the lower hair follicle occur. When these changes of the epithelial component alone are considered, it may be said that they provide a rather 359 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
unfavorable condition for maintenance of adhesion of the epithelial component to subjacent dermal papilla. Thus, it may be surmised that some reinforced attachment structure may be formed at the papillaepithelial junction during this phase. However, the ultrastructure responsible for the mechanical function of maintenance of adhesion between the papilla and the epithelial component during the catagen remains unclarified, especially in late catagen when the lower follicle undergoes extensive shortening. Thus, the present study was undertaken to clarify or otherwise elucidate the fine structural organization of both the papilla and the papilla-epithelial junction of hair follicles in the early and late catagen stage. MATERIALS AND METHODS Small pieces of skin were excised from the backs of 19- to 22-day-old C57 black mice. These specimens were fixed for 2 h r in 1% osmium tetroxide buffered with cacodylate, or in 4% glutaraldehyde buffered with cacodylate for 2 hr and t h e n in 1% osmium tetroxide buffered with cacodylate for 2 hr. Subse-
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SUGIYAMA, TAKAHASHI AND KAMIMURA
quently, they were dehydrated in an ascending series of ethanol and embedded in epoxy resin (16). Thin sections were cut with a Porter-Blum microtome equipped with a diamond knife. These sections were stained with uranyl acetate and lead citrate (23), and then examined with a Hitachi HS-8 electron microscope. RESULTS I. HAIR FOLLICLE IN EARLY
CATAGEN
A. Epithelial Component The following is presented to show the actual site and phase studied. The club, surrounded by several layers of keratinocytes (epithelial sac), is seen formed in the suprabulbar region (Fig. la). Some keratinocytes of the epithelial sac show a limited number of trichohyalin granules in their cytoplasm (Fig. lb). The hair bulb is seen transformed into a short column of keratinocytes (epithelial column) between the epithelial sac and the dermal papilla, and shows a degenerative process. The column is composed of a centrally located large mass of degenerated cell materials with outer layers of undifferentiated keratinocytes containing numerous cytoplasmic vacuoles and the outer root sheath (Figs. la, c). In the epithelial column at the level from the top of the papilla to a higher level, the outer root sheath cells exhibit characteristic features. One of the striking features is that the contour of the cells on the dermal side shows pronounced irregular cytoplasmic indentations and illdefined cell membranes. The basal lamina measuring about 900 A in thickness follows the contour of the cytoplamic indentations, lacking an electron-lucent zone facing the indentations (Fig. lc).
At the base of the epithelial column, two or three layers of undifferentiated keratinocytes cover only the upper third portion of the spherical dermal papilla, and these keratinocytes are characterized by the presence of large phagosomes in the cytoplasm (Fig. 2). No hemi-desmosomes are found on the plasma membranes of the cells abutting upon the basal lamina between the epithelial column and the dermal papilla.
B. Dermal Papilla Papilla cells. The papilla cells in the early catagen stage are moderately irregular in shape, and show oval nuclei with one or two small nucleolei. The cells are adjacent to each other with very narrow intercellular spaces. However, numerous dilated intercellular spaces are also encountered in the dermal papilla (Fig. 2). The papilla cell shows a few dilated cisterns of rough-surfaced endoplasmic reticulum which contain fine filamentous materials. The Golgi complex, located.in the juxtanuclear region, is moderately well developed (Fig. 5), i.e., a communication of Golgi vacuoles and flattened cisternae and small bleb-like protrusions from the cisternae are frequently encountered. Golgi cisternae and vesicles contain moderately dense amorphous materials. Numerous smooth vesicles, presumably of pinocytotic character, and coated vesicles are seen mainly in the periphery of the cells, and moderate numbers of lysosomes and residual bodies are seen scattered in the cytoplasm (Figs. 3, 4). Other usual organelles and sparse cytoplasmic filaments are seen
FIG. la. The club (CL) surrounded by the epithelial sac (ES) is seen in suprabulbar region. A large mass of degenerated cell materials (DC) and its outer layers of keratinocytes (K) with numerous cytoplasmic vacuoles are seen between the dermal papilla (DP) and the epithelial sac (ES). Arrow indicates the direction of the hair follicle. Specimen fixed with 1% OsO4; section stained with uranyl and lead. x 3300. Fia. lb. Enlarged view of the enclosed area shown in Fig. la. Note t h a t the cell in the epithelial sac has trichohyalin granules (tg). cc, club cell with pyknotic nucleus. × 20 000. Fro. lc. The outer root sheath cells (ORS) in the epithelial column exhibit irregular cytoplasmic indentations on the dermal side. A set of cytoplasmic indentations is surrounded directly by the basal lamina (bl). x 5900.
FIGS. la-c. Portions of bulb and suprabulbar region in the early catagen stage. 361
FIG. 2. At the base of the epithelial column in the early catagen stage, two layers of undifferentiated keratinocytes (K) with large phagosomes (ps) cover only the upper t h i r d portion of spherical dermal papilla (DP). Dilated intercellular spaces (IS) are seen in several places of the dermal papilla (DP), especially in the upper portion. Arrow indicates the direction of the h a i r follicle. Specimen fixed and stained as in Fig. 1. x 6200. 362
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in the r e l a t i v e l y electron-lucent cytoplasm. Basal lamina and intercellular substances. At the junction between the papilla and the epithelial column in the early catagen stage, a distinct basal lamina measuring about 450 A in thickness is present, and is seen separated from the keratinocyte membranes by a relatively electron-lucent zone measuring about 450 A in width. The basal lamina-like strncture, displaying a fine filamentous texture embedded in relatively dense amorphous material, protrudes from some parts of the basal lamina into the dilated intercellular spaces at the upper portion of the dermal papilla, forming its characteristic network (Figs. 3, 4). On the other hand, compact fine fibrils are found throughout the dilated intercellular spaces of the papilla (Figs. 3, 4, 6a, b). These fibrils are approximately from 120 to 130 A in diameter. In cross sections, the fibrils have a hollow appearance with a light core surrounded by a dense circumferential outer layer. In the longitudinal section of the fibrils, two parallel strata with medium density are separated by a central light interval. When observed as long strands, the fibrils exhibit a beaded pattern along their long axis (Figs. 6a, b). The periodicity of the beaded pattern appears to exhibit a slight irregularity. These intercellular fine fibrils are revealed to be embedded into the network of the basal lamina-like structure at the exterior of the network (Fig. 4). In addition, the fibrils are not only closely contiguous to the papilla cell membranes but also attached to them (Figs. 3, 4, 6a, b). A few light small oval areas are interspersed among the fibrils.
umn, the basal lamina of the glassy merebrane is only slightly thickened, and the undifferentiated keratinocytes in the column have irregularly shaped nuclei, a moderate number of lysosomes and large residual bodies in their cytoplasm (Figs. 7a, b). A special feature of the ultrastructure of the epithelial column in this period is that hemi-desmosomes are seen along the kerationcyte membranes adjacent to the basal lamina between the column and the dermal papilla (Figs. 8, 9).
II. HAIR FOLLICLE IN LATE CATAGEN
DISCUSSION
A. Epithelial Column In the late catagen stage, the epithelial column is shortened due to the degenerative process occurring in the keratinocytes. In this period of the shortening of the col-
B. Dermal Papilla The ultrastructural organization of the dermal papilla in the late catagen stage is generally similar to that in the early catagen stage. The basal lamina-like structure, which no longer shows a mesh-like pattern such as seen in the early catagen stage, is present underneath the basal lamina and in some dilated intercellular spaces in the upper portion of the papilla (Figs. 8, 9). A structural continuity between the basal lamina and the underlaying basal lamina-like structure is clearly observed (Fig. 9). On the other hand, fine fibrils (120-130 A in diameter) are seen to fill compactly throughout the dilated intercellular spaces of the papilla (Figs. 8, 9). Some of the fibrils are attached to the plasma membranes of papilla cells, and are also seen embedded into the basal lamina-like structure (Figs. 9, 10). These intercellular fine fibrils exhibit a hollow appearance in cross sections and an irregular beaded pattern along their long axis (Fig. 10). The papilla cell in the late catagen stage predominantly shows a small Golgi complex, smooth-surfaced endoplasmic reticulum, lysosomes, residual bodies, and smooth or coated vesicles (Fig. 11).
It was described in electron microscopic studies on mouse hair follicles in the anagen stage (19, 26) that the extracellular substances in the dermal papillae consist of abundant ground substance and a few
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collagen fibers. The present study shows that the intercellular substance in dermal papillae in the early and late catagen stage is comprised of the fine fibrils embedded in relatively light amorphous material. With respect to ultrastructural features, these fibrils clearly correspond to the "microfibrils" reported as a morphological entity of extracellular connective tissue components (10, 14, 15). The relatively small fibrils corresponding to the microfibrils were found to be both free and in association with the connective tissue structures of various organs, including elastic fibers (2, 6, 8, 10, 11, 15, 24), basal laminae (2, 7, 14), and collagen fibers (10,
11, 15). The intercellular fine fibrils observed in the present study are characterized by small diameters, a hollow appearance in cross sections, and a beaded pattern along the long axis, which appears to have irregular periodicity. Fine structure similar to that of these fibrils were reported in the microfibrils of elastic fibers (6, 10, 24), in the filaments bordering the basal lamina of muscle capillaries (2), in the anchoring filaments of lymphatic vessels (13), and in the microfibrils connected with the skeletal muscles (9). The relatively small fibrils are assumed to have a mechanical function to anchor the organs to surrounding connective tissue structures, and have been so reported; Leak and Burke (13) found lymphatic anchoring filaments t h a t were connected with t h e endothelial cell m e m b r a n e s , which extended into the adjoining connective tissue area. Their interpretation is that the filaments have a role to bind the lymphatics to the surrounding connective
tissue components. H a n a k and B6ck (9) found the microfibrils that were in contact with skeletal muscle cell membranes, passing the basal lamina of the muscle, and extending into a cementing substance among the collagen fibers. They considered that the microfibrils were a connecting link between the muscle and the tendon. A unique aspect of the fine fibrils in the dermal papilla in the catagen stage is that the dilated intercellular spaces are filled with the fibrils that are attached to the papilla cell membranes and are embedded in the basal lamina-like structure t h a t protrudes downward from the basal lamina into the dilated intercellular spaces. On the basis of this relationship of the fibrils, as well as an ultrastructural similarity of the fibrils to the lymphatic anchoring filaments and the microfibrils connected with the skeletal muscle, it is suggested that the fibrils may have a mechanical function, connecting the papilla cells with the basal l a m i n a - l i k e s t r u c t u r e , which may bind the papilla cells. The basal lamina at the dermal-squamous epithelial junction is generally considered to act mechanically as a framework to support the epithelial cells and anchor these cells to the subjacent dermis. This f u n c t i o n of t h e b a s a l l a m i n a is strongly suggested by the structural relation of the basal lamina to basal cell membranes with hemi-desmosomes, and to subjacent fibrillar connective tissue elements of special type (1, 28, 29), called anchoring fibrils by Palade and Farquhar (18). However, the specialized a t t a c h m e n t structures such as the hemi-desmosomes and the anchoring fibrils have not been found at the junction between the dermal papilla
FIG. 3. At the papilla-epithelial junction in the early catagen stage, the basal l a m i n a (bl) follows the plasma m e m b r a n e s of undifferentiated keratinocytes (K) adjacent to the dermal papilla, and is separated from these by a relatively electron-lucent, zone. The network of the basal lamina-like structure (bls) is present in the dilated intercellular spaces in the upper portion of the papilla, and joins with the basal l a m i n a (bl) in places (*). Compact fine fibrils (ff) in the dilated intercellular spaces are closely contiguous to plasma m e m b r a n e s of papilla cells (PC) and to the network of the basal lamina-like structure (bls). Many small vesicles (v), coated vesicles (cv), and several lysosomes (ly) are seen in the cytoplasm of papilla cells (PC). Specimen fixed and stained as in Fig. 1. x 23 000.
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and the hair bulb in ihe anagen Stage (19, 26). Therefore, the attachment at this junction may not be so strong in the anagen stage. The present study strongly suggests that the attachment between the dermal, papilia and the epithelial column may be supported by .the characteristic structures in hair follicles in the catagen Stage, as follows. (1) The attachment Of the dermal papilla to the basal lamina is considered to be reinforced in the early and late catagen stage by intercellular fine fi-
"
brilsl in association wi~h the basal lamina: like structure with both a structural continuity and an ultrastructural similarity to the basal lamina. (2) It is apparent that the attachment of the epithelial column to the basal lamina is strengthened by the hemidesmos0mes of the keratinocytes of the column in the late catagen stage. Thus, the structural complex of the hemi-desmosomes, the basal lamina and basal laminalike structure, and the intercellular fine fibrils may be responsible for the firm
FIG. 4. Portion of the papilla-epithelial junction in the early catagen stage. A structural continuity (arrow) between the basal l a m i n a (bl) and the basal lamina-like structure (bls) and a fine-structural identity of both structures as fine filamentous texture are demonstrable. The compact intercellular fine fibrils (t"f) are not only closely continguous to papilla cell m e m b r a n e s but also embedded in the basal lamina-like structure (bls) at the peripheral part of its network (*). Small vesicles (v), coated vesicles (cv), and lysosomes (ly) are seen in the cytoplasm of the papilla cell (PC). Specimen fixed and stained as in Fig. 1. x 36 000. FIG. 5. Papilla cell in the hair follicle in the early catagen stage. A communication of Golgi vacuoles and flattened cisternae (arrow) and small bleb-like protrusions from the cisternae (double-headed arrow) are seen in relatively well-developed Golgi complex (G). Inset shows a residual body t h a t is bounded by a singleunit membrane, and has a n internal structure consisting of fine dense granules, several curved dense bands, and peripherally located vacuoles. Specimen fixed and stained as in Fig. 1. × 29 000. Inset, × 34 000. FIGS. 6a, b. Fine fibrils in the dilated intercellular spaces of dermal papilla in the early catagen stage. A hollow appearance (thick arrow) and a n i r r e g u l a r beaded p a t t e r n are clearly seen, respectively, in cross and longitudinal sections of the intercellular fine fibrils. Both t h i n and white arrows indicate distinct contact of the fibrils with papilla cell membranes. U, small oval area without fine fibrils. Specimen fixed and stained as in Fig. 1. Figure 6a, × 95 000. Figure 6b, × 130 000. FIG. 7a. Electron micrograph of longitudinal section though the dermal papilla and the epithelial column in the late catagen stage. Structural contact between hemispherical dermal papilla (DP) and shortened epithelial column (EC) is demonstrable. Note the irregular shape of nuclei in the cells of the epithelial column (EC). Inset shows slightly thickened basal l a m i n a (bl) and layers of collagen fibers (Co) around the epithelial column (EC). Specimen prefixed in 4% glutaraldehyde with cacodylate buffer, followed by 1% OsO4 with cacodylate buffer. Sections stained with uranyl and lead stain. × 2200. Inset, × 20 000. FIG. 7b. Enlarged view of the area indicated by arrow in Fig. 7a. Various sized residual bodies (rsb) are located in the vicinity of the nucleus of the cell in the epithelial column. × 20 000. FIG. 8. Upper portion of dermal papilla and base of epithelial column in the late catagen stage. Hemidesmosomes (hd) are Seen along the plasma m e m b r a n e s of undifferentiated keratinocytes (K) adjacent to the basal l a m i n a (bl) at the papilla-epithelial junction. The basal lamina-like structure (bls) and fine fibrils (if) are seen to fill the dilated intercellular spaces in the dermal papilla (DP). Specimen fixed and stained as in Fig. 7. × 25 000. FIG. 9. Junctional area between dermal papilla and epithelial column in the late catagen stage. Tonofilaments embedded in the a t t a c h m e n t plates of hemi-desmosomes (hd) are sparse. A structural continuity between the basal l a m i n a (bl) and the underlaying basal lamina-like structure (bls) is seen. A contact of intercellular fine fibrils (fl) with the plasma m e m b r a n e s of papilla cells (PC) is demonstrable (arrow). The fibrils are also embedded in the basal lamina-like structure (*). Specimen fixed and stained as in Fig. 7. × 87 000. FIG. 10. Intercellular fine fibrils and basal lamina-like structure in the dilated intercellular space of dermal papilla in the late catagen stage. In cross sections, the fine fibrils exhibit a hollow appearance with a light central core surrounded by a dense outer layer (arrow). In longitudinal sections, the fibrils exhibit a beaded pattern along their long axis (parenthesized passage). (*) indicates an insertion of the fibrils into the basal lamina-like structure (bls). Specimen fixed and stained as in Fig. 7. × 120 000. FIG. 11. Papilla cells in h a i r follicle in the late catagen stage. Note the presence oflysosomes (ly), smoothsurfaced endoplasmic reticulum (se), small vesicles (v), and coated vesicles (cv) in the cytoplasm of papilla cells. Inset shows the residual body (rsb). Specimen fixed and stained as in Fig. 7. × 34 000. Inset, × 34 000.
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
FIGURES 4-5
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FIGURE 6
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
FIGURS 7
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SUGIYAMA, TAKAHASHI AND KAMIMURA
FIaURES 8-9
'
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN
FmuRss 10-11
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SUGIYAMA, TAKAHASHI AND KAMIMURA
adhesion between the epithelial column and the dermal papilla in the follicles in the late catagen stage, which are in the process of shortening the column. It has been reported that the dermal papilla is very important for the growth of hair in the anagen stage, since only a fragile hair is produced by the depapillated follicle (3). Thus, it may be said that the contact between the epithelial component and the dermal papilla during the catagen stage may be an important event in the preparation of one of the necessary conditions for subsequent hair growth. The cellular component in mouse dermal papillae in the anagen stage has been shown to consist of fibroblasts with welldeveloped rough-surfaced endoplasmic reticulum and Golgi complexes (19). Although the papilla cells in the catagen stage are characterized by the predominance in the Golgi complex, lysosomes, residual bodies, and small vesicles presumably of pinocytotic character, they may be derived from the fibroblasts in the preceding dermal papilla in the anagen stage, and may undergo changes in their ultrastructural appearance. This ultrastructural change of the cells seems to be a reflection of the increased endocytotic activity rather than the active synthesis and secretion of a proteinaceous substance. With regard to the synthesis of the intercellular fine fibrils in dermal papilla in the catagen stage, the intimate relation of the fibrils to the papilla cell membranes suggests the possibility that the precursors of the fibrils may be synthesized by the papilla cells before the completion of the ultrastructural change in the cells, namely, presumably in the earliest phase of catagen. In mouse hair follicles in the telogen stage, Roth (25) found that the hemi-desmosomes of the germ cells are attached to the basal lamina between the hair germ and the dermal papilla, and that the fine filaments (70-80/k in diameter) are joined to the basal lamina and extend into the intercellular spaces of the
papilla. This junctional structure in the resting hair follicle is approximately similar to that in the hair follicle in the catagen stage. The present study suggests that both the synthesis of the intercellular fine fibrils by the papilla cells, presumably in the earliest phase of catagen, and the formation of the hemi-desomosomes by the keratinocytes in the epithelial column in the late catagen stage are essential events for the alteration of the junctional structure between the dermal papilla and the epithelial component in the follicle during the catagen stage. REFERENCES 1. BRODY,I., J. Ultrastruct. Res. 4, 246 (1960). 2. BRUNS, R. R., AND PALADE, G. E., J, Cell Biol. 37, 244 (1968). 3. BUTCHER, E. O., Anat. Rec. 151, 231 (1965). 4. CHASE, H. B., Physiol. Rev. 34, 113 (1954). 5. ELLIS, R. A., AND MORETTI, G., Ann. N . Y . Acad. Sci. 83, 448 (1959). 6. FAHRENBACH, W. H., AND SANDBERG, J. L., Anat. Rec. 155, 563 (1966). 7. FARQUHAR, M. G., WISSIG, S. L., AND PALADE, G. E., J. Exp. Med. 113, 47 (1961). 8. GREENLEE, T. K., Ross, R., AND HARTMAN, J. L., J. Cell Biol. 30, 59 (1966). 9. HANAK, H., AND B0CK, P., J. Ultrastruct. Res. 36, 68 (1968). 10. HAUST, M. D., Amer. J. Pathol. 47, 1113 (1965). 11. KARRER, H. E., J. Ultrastruct. Res. 2, 96 (1958). 12. KLIGMAN, A. M., J. Invest. Dermatol. 33, 370 (1959). 13. LEAK, L. V., AND BURKE, J. F., J. Cell Biol. 36, 129 (1968). 14. Low, F. N., Anat. Rec. 139, 105 (1961). 15. Low, F. N., Anat. Rec. 142, 131 (1962). 16. LuFr, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). 17. MONTAGNA, W., The Structure and Function of Skin, first ed., p. 173. Academic Press, New York, 1956. 18. PALADE, G., AND FARQUHAR, M. G., J. CellBiol. 37, 215 (1965). 19. PARAKKAL, P. F., in MONTAGNA, W., AND DOBSON, R. (Eds.), Hair Growth: Advances in B i ology of Skin, Vol. 9, p. 441. P e r g a m o n Press Oxford, 1969. 20. PARAKKAL, P. F., J. Cell Biol. 40, 561 (1969). 21. PARAKKAL, P. F., J. Ultrastruct. Res. 29, 21~ (1969). 22. PAaAKKAL, P. F., Z. Zellforsch. 107, 174 (1970). 23. REYNOLDS, E. S., J. Cell Biol. 46, 366 (1963).
ULTRASTRUCTURE OF HAIR FOLLICLE IN CATAGEN 24. Ross, R., AND BORNSTEIN, P., J. CellBiol. 46, 366 (1969). 25. ROTH, S. I., in LYNE, A. G., AND SHORT, B. F. (Eds.), Biology of the Skin and Hair Growth. p. 233. Angus and Robertson, Sydney, 1965. 26. ROTH, S. I., ANn HELWIG, E. B., J. Ultrastruct. Res. 11, 33 (1964).
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27. STRAILE, W. E., CHASE, H. B., AND ARSENAULT, C., J. Exp. Zool. 148, 205 (1961). 28. SusI, F. R., BELT, W. D., AND KELLY, D. W., J. Cell Biol. 34, 686 (1967). 29. YOUNES, M. S., STEELE, H. D., ROBERTSON,E. M.~ ANn BENCOSME, S. A., Amer. J. Obstet. Gyneeol. 92, 163 (1965).
