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Glycoconjugates in the epidermis of Pleurodeles waltlii
JOURNAL OF ULTRASTRUCTURE RESEARCH
77, 354-359 (1981)
Glycoconjugates in the Epidermis of Pleurodeles waltlii CH. BUENO, P. NAVAS, J. AUoy, AYD J. L. LOPEZ-CAMPOS Department of Cytology and Histology, Faculty of Biology, University of Seville, Spain Received March 31, 1981, and in revised form July 28, 1981 A cytochemical study on the epidermis of Pleurodeles waltlii has been carried out. We describe the existence of glycoconjugates associated with plasma membranes of stratum corneum and outermost cells of stratum granulosum or replacement cell layer. Glycoconjugates are also demonstrated in small granules localized in the stratum granulosum. The cytological and cytochemical characteristics of these granules lead us to consider them as membrane-coating granules. We propose that these granules may produce the surface coat of stratum corneum and replacement cell layer and may function in the permeability of these cells.
Ultrastructure of amphibian epidermis has been described by several authors (Vofite, 1963; Farquhar and Palade, 1965; Lavker, 1972; Budtz and Larsen, 1975; Warburg and Lewinson, 1977). The stratum corneum (SC) is formed by flattened cells filled with filaments and electron-dense cytoplasm. Below these is the outermost cells of the stratum granulosum (SG) or replacement cell layer (RCL) (Vofite and Ussing, 1968; Brown and Ilic, 1979). Two sets of tight junctions appear in the frog skin (Farquhar and Palade, 1965; Martinez-Palomo et al., 1971). The first is localized between the cells of SC and the second joins the outer membranes of RCL. As a consequence, these two sets limit a compartment which corresponds to the intercellular space between the SC and the outermost cells of SG (Farquhar and Palade, 1965; Martinez-Palomo et al., 1971; Budtz and Larsen, 1975; Navas et al., 1980). The outer membrane of RCL is considered to be the first selective barrier for the movement of substances through the amphibian epidermis (Vofite and Ussing, 1968; Vofite et al., 1975), and an additional barrier is formed by the cells of SC (Mart[nezPalomo et al., 1971). The object of this study is the localization of glycoconjugates in the ceils of SC and SG of amphibian epidermis. Their origin and their possible role in the permeability of amphibian skin will be discussed.
MATERIALS AND METHODS Small fragments of dorsal and ventral skin of Pleurodeles waltlii (Amphibia, Urodela) were removed and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.0, then postfixed in 1% osmium tetroxide in the same buffer and embedded in resin (Spurt, 1969). Thin sections were contrasted with uranyl acetate and lead citrate. Cytochemical methods. (1) Thin sections of skin fixed in glutaraldehyde and embedded in Epon were mounted on gold grids and stained with the P A - T C H Ag proteinate method of Thiery (1967). Oxidized control sections were treated either with silver proteinate without previous TCH treatment or with TCH alone. (2) Thin sections of skin fixed in glutaraldehyde and embedded in Epon were floated on 1% phosphotungstic acid in 1 N HC1 during 40 to 60 rain according to Flechon (1970), and were then washed with 1.25 N HCI.
RESULTS
The intercellular space between the SC and SG in the epidermis of Pleurodeles waltlii is full of a material of branched, fibrous appearance (Fig. 1) with some accumulations among them. This fibrous material comes from the plasma membrane of both cells, forming a glycocalyx. This material's appearance is the same as that which appears in small granules, surrounded by a membrane unit, with a dense center (Fig. 2). They appear in the SG and are mainly distributed on the cellular borders (Fig. 4). They release their content into the intercellular space of the SG, although the majority of them do this on the cellular sur-
354 0022-5320/81/120354-06502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
G L Y C O C O N J U G A T E S IN A M P H I B I A N S K I N
355
FIG. 1. Intercellular space b e t w e e n s t r a t u m c o r n e u m (SC) and stratum g r a n u l o s u m (SG) in urodelan skin. Note the c o n t e n t of this c o m p a r t m e n t , x 70 000. FIG. 2. Cells of the stratum g r a n u l o s u m in the skin of Pleurodeles waltlii. A r r o w s sign MCGs. Inset: M C G in the urodelan epidermis. Fig. 2, x 22 500; inset, x 60 000.
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GLYCOCONJUGATES IN AMPHIBIAN SKIN
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FIG. 7. Intercellular space between stratum corneum (SC) and stratum granulosum (SG) stained with PTA at low pH. x 45 000. FIG. 8. MCGs in the skin of P. waltlii stained with phosphotungstic acid. x 60 000.
face of the outermost cells of this layer (Fig. 3). The P A - T C H - s i l v e r proteinate technique reveals the existence of glycoconjugates in the intercellular space between the SC and SG (Fig. 3) and on the most external surface of the SC (Fig. 6). This technique reveals the same positivity in the abovementioned granules (Figs. 4 and 5), in both the center and the internal portion of the membrane surrounding it, as well as fillform fragments that join the center and the membrane (Fig. 5). The PTA at low pH technique produces similar results; precipitates of phosphotungstic acid appear in the intercellular
space between SC and SG (Fig. 7) and in the aforementioned granules (Fig. 8). No reaction appears on the free surface of SC. DISCUSSION
In the compartment of intercellular space formed between the SC and the outermost cells of SG a substance appears, which corresponds to the carbohydrate moieties of glycoconjugates associated with the plasma membranes of cells that limit it. This is demonstrated by PATAg and PTA at low pH. The internal structure of the granules described under Results, within the SG of the P. waltlii epidermis, remind one of the
FIG. 3. Intercellular space between stratum corneum (SC) and stratum granulosum (SG) stained with P A TCH-Ag proteinate method. Note the fusion point of MCG and plasma membrane (arrow). x 60 000. FIG. 4. MCGs in the urodelan skin nearby the intercellular space of SG. They are stained with P A - T C H Ag proteinate method, x 18 000. FIG. 5. MCGs in the skin of P. waltlii stained with Thiery method. Note the peripheral zone, center and fibrillar connections between them. x 60 000. FIG. 6. Outermost surface of SC of urodelan skin stained with PA-TCH-Ag proteinat¢ method, x 60 000.
358
BUENO ET AL.
membrane-coating granules (MCGs) described in human buccal mucosa (Hayward and Hackeman, 1973; Hayward, 1979) and in the hyperkeratinized epidermis in the Kyrle-Flegel disease (Squier et al., 1978), which do not usually possess a lamellated internal structure. This internal structure, its distribution on the surface of SG cells, the release of their content into the intercellular space (Hayward, 1979), and its glycoconjugate content (Hayward and Hackeman, 1973) are sufficient criteria to consider these as MCGs. However, MCGs were not observed by Matoltsy and Parakkal (1965) in frog skin and do not correspond to "crystal-containing granules" described in the epidermis of R a n a pipiens (Lavker, 1973), but they may correspond to PAS-positive granules described by Parakkal and Matoltsy (1964) in frog epidermis. These small granules have similar cytological behavior to those described in the epidermis of P. waltlii. In view of these results, we suppose that the glycoconjugates associated with the plasma membranes of SC and RCL proceed from MCGs, and those which appear on the free surface of SC are remains of those which had been in the RCL, after the moulting cycle (Budtz and Larsen, 1975). Some authors have indicated the possible origin of the cell surface coat from MCGs in other epithelia (Matoltsy and Parakkal, 1965; Hayward, 1979). Recently, Brown and Ilic (1979) described the presence of intramembrane particles in the most external plasma membranes of SC and RCL. These particles are associated with the permeability which occurs in the outermost cell layer of anuran epidermis, as transport of water and solutes occurs through hydrophilic canals which are considered to be proteins of the membrane and are visible as intramembrane particles (Solomon, 1968; Yu and Branton, 1976). It is also thought that the intramembrane particles correspond to large protein complexes (Segrest et al., 1974) which ap-
pear as a specialization of the glycocalyx (Sandoz et al., 1979). We think that the intramembrane particles described in the external membranes of SC and RCL (Brown and Ilic, 1979) will correspond to glycoconjugates, possibly glycoproteins, which appear associated with these membranes. The function of these glycoconjugates would be related to the characteristic permeability of these membranes to water and solutes as possible components of intramembrane particles and glycoconjugate moieties may have some effect upon ion transport; the external surface of SC being less selective due to deterioration of the carbohydrate moieties which can not be renewed in this layer. This assertion is comprehensible, as it has been indicated several times that the MCGs provide a permeable barrier when their content is released into the intercellular space (Wolff and H6nigsmann, 1971; Elias and Friend, 1975). REFERENCES BROWN, D., AND ILIC, V. (1979) J. Ultrastruct. Res. 67, 55-64. BUDTZ, P. E., AND LARSEN, L. O. (1975) Cell Tissue Res. 159, 459483. ELIAS, P. M., AND FRIEND, D. S. (1975) J. Cell Biol. 65, 180-191. FARQUHAR, M. G., AND PALADE, G. E. (1965) J. Cell Biol. 26, 263-290. FLECHON, J. E. (1970) J. Microsc. 9, 221-242. HAYWARD, A. F. (1979) Int. Rev. Cytol. 59, 97-127. HAYWARD, A. F., AND HACKEMANN, M. (1973) J. Ultrastruct. Res. 43, 205-219. LAVKER, R. M. (1972) Tissue Cell 4, 663-675. LAVKER, R. M. (1973) J. Ultrastruct. Res. 45, 223230. MARTiNEZ-PALOMO, A., ERLIJ, D., AND BRACHO, H. (1971) J. Cell Biol. 50, 277-287. MATOLTSY, A. G., AND PARAKKAL, P. F. (1965) J. Cell Biol. 24, 297-307. NAVAS, P., LOPEZ-CAMPOS,J. L., AND DIAZ-FLORES, L. (1980) Morf. Norm. Patol. 4, 313-321. PARAKKAL, P. F., AND MATOLTSY, A. G. (1964) J. Cell Biol. 20, 85-94. SANDOZ, D., BOISVIEUX-ULRICH,E., AND CHAILLEY, B. (1979) Biol. Cell. 36, 267-280.
SEGREST, J. P., GULtK-IcRzYWIC~I, T., ANO SARDET, C. (1974) Proc. Nat. Acad. Sci. USA 71, 3294-3298.
GLYCOCONJUGATES IN AMPHIBIAN SKIN SOLOMON, A. K. (1968) J. Gen. Physiol. 51, 33553645. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31-37. SQUIER, C. A., EADY, R. A. J., AND HOPPS, R. M. (1978) J. Invest. Dermatol. 70, 361-364. THIERY, J. P. (1967) J. Mierosc. 6, 987-1018. VOt3TE, C. L. (1963) J. Ultrastruct. Res. 9, 497-510. VOUTE, C. L., MOLLGARD, K., AND USSING, H. H. (1975) J. Membr. Biol. 21,273-289.
359
VOt3TE, C. L., AND USSING, H. H. (1968) J. Cell Biol. 36, 625-638. WARBURG, M. R., AND LEWINSON, D. (1977) Cell Tissue Res. 181, 369-393. WOLFF, K., AND HONIGSMANN, H. (1971) J. Ultrastruct. Res. 36, 176-190. Yu, J., AND BRANTON, D. (1976) Proc. Nat. Aead. Sci. U S A 73, 3891-3895.
77, 354-359 (1981)
Glycoconjugates in the Epidermis of Pleurodeles waltlii CH. BUENO, P. NAVAS, J. AUoy, AYD J. L. LOPEZ-CAMPOS Department of Cytology and Histology, Faculty of Biology, University of Seville, Spain Received March 31, 1981, and in revised form July 28, 1981 A cytochemical study on the epidermis of Pleurodeles waltlii has been carried out. We describe the existence of glycoconjugates associated with plasma membranes of stratum corneum and outermost cells of stratum granulosum or replacement cell layer. Glycoconjugates are also demonstrated in small granules localized in the stratum granulosum. The cytological and cytochemical characteristics of these granules lead us to consider them as membrane-coating granules. We propose that these granules may produce the surface coat of stratum corneum and replacement cell layer and may function in the permeability of these cells.
Ultrastructure of amphibian epidermis has been described by several authors (Vofite, 1963; Farquhar and Palade, 1965; Lavker, 1972; Budtz and Larsen, 1975; Warburg and Lewinson, 1977). The stratum corneum (SC) is formed by flattened cells filled with filaments and electron-dense cytoplasm. Below these is the outermost cells of the stratum granulosum (SG) or replacement cell layer (RCL) (Vofite and Ussing, 1968; Brown and Ilic, 1979). Two sets of tight junctions appear in the frog skin (Farquhar and Palade, 1965; Martinez-Palomo et al., 1971). The first is localized between the cells of SC and the second joins the outer membranes of RCL. As a consequence, these two sets limit a compartment which corresponds to the intercellular space between the SC and the outermost cells of SG (Farquhar and Palade, 1965; Martinez-Palomo et al., 1971; Budtz and Larsen, 1975; Navas et al., 1980). The outer membrane of RCL is considered to be the first selective barrier for the movement of substances through the amphibian epidermis (Vofite and Ussing, 1968; Vofite et al., 1975), and an additional barrier is formed by the cells of SC (Mart[nezPalomo et al., 1971). The object of this study is the localization of glycoconjugates in the ceils of SC and SG of amphibian epidermis. Their origin and their possible role in the permeability of amphibian skin will be discussed.
MATERIALS AND METHODS Small fragments of dorsal and ventral skin of Pleurodeles waltlii (Amphibia, Urodela) were removed and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.0, then postfixed in 1% osmium tetroxide in the same buffer and embedded in resin (Spurt, 1969). Thin sections were contrasted with uranyl acetate and lead citrate. Cytochemical methods. (1) Thin sections of skin fixed in glutaraldehyde and embedded in Epon were mounted on gold grids and stained with the P A - T C H Ag proteinate method of Thiery (1967). Oxidized control sections were treated either with silver proteinate without previous TCH treatment or with TCH alone. (2) Thin sections of skin fixed in glutaraldehyde and embedded in Epon were floated on 1% phosphotungstic acid in 1 N HC1 during 40 to 60 rain according to Flechon (1970), and were then washed with 1.25 N HCI.
RESULTS
The intercellular space between the SC and SG in the epidermis of Pleurodeles waltlii is full of a material of branched, fibrous appearance (Fig. 1) with some accumulations among them. This fibrous material comes from the plasma membrane of both cells, forming a glycocalyx. This material's appearance is the same as that which appears in small granules, surrounded by a membrane unit, with a dense center (Fig. 2). They appear in the SG and are mainly distributed on the cellular borders (Fig. 4). They release their content into the intercellular space of the SG, although the majority of them do this on the cellular sur-
354 0022-5320/81/120354-06502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
G L Y C O C O N J U G A T E S IN A M P H I B I A N S K I N
355
FIG. 1. Intercellular space b e t w e e n s t r a t u m c o r n e u m (SC) and stratum g r a n u l o s u m (SG) in urodelan skin. Note the c o n t e n t of this c o m p a r t m e n t , x 70 000. FIG. 2. Cells of the stratum g r a n u l o s u m in the skin of Pleurodeles waltlii. A r r o w s sign MCGs. Inset: M C G in the urodelan epidermis. Fig. 2, x 22 500; inset, x 60 000.
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®
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~'
i ,~-
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©
GLYCOCONJUGATES IN AMPHIBIAN SKIN
357
z. ,%
~
¢
- .
. .
¢
,
g
SG
FIG. 7. Intercellular space between stratum corneum (SC) and stratum granulosum (SG) stained with PTA at low pH. x 45 000. FIG. 8. MCGs in the skin of P. waltlii stained with phosphotungstic acid. x 60 000.
face of the outermost cells of this layer (Fig. 3). The P A - T C H - s i l v e r proteinate technique reveals the existence of glycoconjugates in the intercellular space between the SC and SG (Fig. 3) and on the most external surface of the SC (Fig. 6). This technique reveals the same positivity in the abovementioned granules (Figs. 4 and 5), in both the center and the internal portion of the membrane surrounding it, as well as fillform fragments that join the center and the membrane (Fig. 5). The PTA at low pH technique produces similar results; precipitates of phosphotungstic acid appear in the intercellular
space between SC and SG (Fig. 7) and in the aforementioned granules (Fig. 8). No reaction appears on the free surface of SC. DISCUSSION
In the compartment of intercellular space formed between the SC and the outermost cells of SG a substance appears, which corresponds to the carbohydrate moieties of glycoconjugates associated with the plasma membranes of cells that limit it. This is demonstrated by PATAg and PTA at low pH. The internal structure of the granules described under Results, within the SG of the P. waltlii epidermis, remind one of the
FIG. 3. Intercellular space between stratum corneum (SC) and stratum granulosum (SG) stained with P A TCH-Ag proteinate method. Note the fusion point of MCG and plasma membrane (arrow). x 60 000. FIG. 4. MCGs in the urodelan skin nearby the intercellular space of SG. They are stained with P A - T C H Ag proteinate method, x 18 000. FIG. 5. MCGs in the skin of P. waltlii stained with Thiery method. Note the peripheral zone, center and fibrillar connections between them. x 60 000. FIG. 6. Outermost surface of SC of urodelan skin stained with PA-TCH-Ag proteinat¢ method, x 60 000.
358
BUENO ET AL.
membrane-coating granules (MCGs) described in human buccal mucosa (Hayward and Hackeman, 1973; Hayward, 1979) and in the hyperkeratinized epidermis in the Kyrle-Flegel disease (Squier et al., 1978), which do not usually possess a lamellated internal structure. This internal structure, its distribution on the surface of SG cells, the release of their content into the intercellular space (Hayward, 1979), and its glycoconjugate content (Hayward and Hackeman, 1973) are sufficient criteria to consider these as MCGs. However, MCGs were not observed by Matoltsy and Parakkal (1965) in frog skin and do not correspond to "crystal-containing granules" described in the epidermis of R a n a pipiens (Lavker, 1973), but they may correspond to PAS-positive granules described by Parakkal and Matoltsy (1964) in frog epidermis. These small granules have similar cytological behavior to those described in the epidermis of P. waltlii. In view of these results, we suppose that the glycoconjugates associated with the plasma membranes of SC and RCL proceed from MCGs, and those which appear on the free surface of SC are remains of those which had been in the RCL, after the moulting cycle (Budtz and Larsen, 1975). Some authors have indicated the possible origin of the cell surface coat from MCGs in other epithelia (Matoltsy and Parakkal, 1965; Hayward, 1979). Recently, Brown and Ilic (1979) described the presence of intramembrane particles in the most external plasma membranes of SC and RCL. These particles are associated with the permeability which occurs in the outermost cell layer of anuran epidermis, as transport of water and solutes occurs through hydrophilic canals which are considered to be proteins of the membrane and are visible as intramembrane particles (Solomon, 1968; Yu and Branton, 1976). It is also thought that the intramembrane particles correspond to large protein complexes (Segrest et al., 1974) which ap-
pear as a specialization of the glycocalyx (Sandoz et al., 1979). We think that the intramembrane particles described in the external membranes of SC and RCL (Brown and Ilic, 1979) will correspond to glycoconjugates, possibly glycoproteins, which appear associated with these membranes. The function of these glycoconjugates would be related to the characteristic permeability of these membranes to water and solutes as possible components of intramembrane particles and glycoconjugate moieties may have some effect upon ion transport; the external surface of SC being less selective due to deterioration of the carbohydrate moieties which can not be renewed in this layer. This assertion is comprehensible, as it has been indicated several times that the MCGs provide a permeable barrier when their content is released into the intercellular space (Wolff and H6nigsmann, 1971; Elias and Friend, 1975). REFERENCES BROWN, D., AND ILIC, V. (1979) J. Ultrastruct. Res. 67, 55-64. BUDTZ, P. E., AND LARSEN, L. O. (1975) Cell Tissue Res. 159, 459483. ELIAS, P. M., AND FRIEND, D. S. (1975) J. Cell Biol. 65, 180-191. FARQUHAR, M. G., AND PALADE, G. E. (1965) J. Cell Biol. 26, 263-290. FLECHON, J. E. (1970) J. Microsc. 9, 221-242. HAYWARD, A. F. (1979) Int. Rev. Cytol. 59, 97-127. HAYWARD, A. F., AND HACKEMANN, M. (1973) J. Ultrastruct. Res. 43, 205-219. LAVKER, R. M. (1972) Tissue Cell 4, 663-675. LAVKER, R. M. (1973) J. Ultrastruct. Res. 45, 223230. MARTiNEZ-PALOMO, A., ERLIJ, D., AND BRACHO, H. (1971) J. Cell Biol. 50, 277-287. MATOLTSY, A. G., AND PARAKKAL, P. F. (1965) J. Cell Biol. 24, 297-307. NAVAS, P., LOPEZ-CAMPOS,J. L., AND DIAZ-FLORES, L. (1980) Morf. Norm. Patol. 4, 313-321. PARAKKAL, P. F., AND MATOLTSY, A. G. (1964) J. Cell Biol. 20, 85-94. SANDOZ, D., BOISVIEUX-ULRICH,E., AND CHAILLEY, B. (1979) Biol. Cell. 36, 267-280.
SEGREST, J. P., GULtK-IcRzYWIC~I, T., ANO SARDET, C. (1974) Proc. Nat. Acad. Sci. USA 71, 3294-3298.
GLYCOCONJUGATES IN AMPHIBIAN SKIN SOLOMON, A. K. (1968) J. Gen. Physiol. 51, 33553645. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31-37. SQUIER, C. A., EADY, R. A. J., AND HOPPS, R. M. (1978) J. Invest. Dermatol. 70, 361-364. THIERY, J. P. (1967) J. Mierosc. 6, 987-1018. VOt3TE, C. L. (1963) J. Ultrastruct. Res. 9, 497-510. VOUTE, C. L., MOLLGARD, K., AND USSING, H. H. (1975) J. Membr. Biol. 21,273-289.
359
VOt3TE, C. L., AND USSING, H. H. (1968) J. Cell Biol. 36, 625-638. WARBURG, M. R., AND LEWINSON, D. (1977) Cell Tissue Res. 181, 369-393. WOLFF, K., AND HONIGSMANN, H. (1971) J. Ultrastruct. Res. 36, 176-190. Yu, J., AND BRANTON, D. (1976) Proc. Nat. Aead. Sci. U S A 73, 3891-3895.