Cell Tiss. Res. 167, 37-47 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
The Ultrastructure and Calcification of the Scales of Tilapia mossambica (Peters) W.J.R. Lanzing and R.G. Wright School of Biological Sciences and Electronmicroscope Unit, University of Sydney, Australia
Summary. The scales of Tilapia are surrounded by an envelope of scleroblasts responsible for the production of layers of collagen that constitute the bulk of the scale. The scleroblasts adjoining the lateral face of the oldest scale region gradually atrophy. New collagen layers are deposited against the inner face of the scale, the adjoining scleroblasts showing evidence of high metabolic activity. Calcification occurs by inotropic deposition of crystals alongside the fibres. There is no sharp demarcation between calcified and non-calcified scale regions, a calcification front gradually moving towards newly formed collagen layers. It is felt that fish scales should be considered as calcified derivatives of dermal collagen layers.
Key words: Scale - Tilapia - Calcification - Ultrastructure. Introduction
The fine structure of the skin incorporating the scales of Tilapia rnossambica was recently described by Lanzing and Wright (1974). Earlier, ultramicroscopic evidence of the presence of collagen bundles was found in scales of the sole Hippoglossoides (Brown and Wellings, 1969). At the same time, teleost scales were reported to consist of calcified and non-calcified parts (van Oosten, 1957; Maekawa and Yamada, 1970). The present paper attempts to present a detailed investigation of the fine structure of the scale of Tilapia. Scanningmicroscopic observations on the morphology of the scale were presented elsewhere (Lanzing and Higginbottom, 1974). Materials and Methods The Tilapia mossambica culture procedures as well as the fixation and staining methods have been described previously (Lanzing and Wright, 1974). Send offprint requests to: Dr. W.J.R. Lanzing, School of Biological Sciences, University of Sydney, Carslaw Building, Sydney, N.S.W. 2006, Australia.
38
W.J.R. Lanzing and R.G. Wright
Results
Light microscopic observations reveal that the scales are embedded in dermal connective tissue. Each scale consists of a number of differently oriented collagen layers, staining alternatively with Orange G and Light Green SF Yellowish. The scales, - even the posterior edges that appear to protrude from the s k i n - are at all times covered by dermal cells, in turn protected from the environment by a thin layer of epidermal cells. In between the scales as well as in the other parts of the skin occur considerable masses of collagen. Fig. 1 presents a generalized view of various dermal and epidermal components. Electron microscopic observations show that the epidermis overlying the scale is composed primarily of type B cells, separated from the epidermis by the continuous basal lamina. The fine structure of the outer or lateral part of the mature scale is heavily calcified. Fig. 2A shows that the circuli adjacent to the collagen layers incorporate a considerable quantity of calcium salts. The older, more developed parts of the scale are covered by regressed scleroblasts, showing few ribosome-lined cisternae and other cytoplasmic organelles. It is clear from decalcified preparations that the circuli are formed by bundles of collagen fibres that course across the scale surface (Fig. 1 C) to form a loop, the ends of which are joined by the tubercula in the rostral field of the scale. In the young, developing scale region calcification sites or nuclei are visible in the collagenous mass forming the bulk of the rather thin scale (Fig. 2B). The inner or medial lateral face of the scale is joined by large scleroblasts showing an exceptionally well developed rough endoplasmic reticulum (Fig. 3 A). The cisternae often course parallel to one another with a fairly uniform diameter of 80 nm. Clearly visible are the desmosomes (Fig. 3A, B) that join the scleroblasts together. In so doing those scleroblasts that are involved in scale formation form a continuous and uninterrupted envelope, separating the scale from the surrounding tissue. New collagen fibres are being deposited against the inner or medial surface of the scale. These fibres are deposited in layers which measure between 1,200-1,800 nm in thickness. Newer (younger) fibre layers course in a direction almost at right angles to the adjacent older layers (Fig. 3 A), resembling the structure of plywood. In cross section the collagen fibres are round and measure between 61 and 190 nm. It appears (Fig. 6A) that the diameter of the newly-formed fibres is sometimes less than that of older fibres, although even in the older region of the scale the diameter of the fibres is not constant. There are three possible explanations for this: a) that b) that c) that collagen is
the fibres are relatively short and acicular, t.i. tapering at the ends, the individual fibre is of variable thickness when formed and some fibres retain their newly-formed appearance because little extra added to the original fibre.
Irrespective of their diameter, the collagen fibres show a periodicity of about 63 nm, which is typical for collagen. Bundles of microfilaments are often found in the scleroblasts, but no evidence was found to indicate that the collagen fibres are actually preformed in the scleroblasts before appearing in the matrix around the scale.
Fig. 1 A-C. Diagram of a scale showing its relation to other skin components and its ultrastructural aspects. (A) Median section through a scale showing several collagen layers. Near the scale are a melanophore, a dermal fibroblast, dermal collagen and epidermal tissue. The thickness of the dermal collagen layers is not necessarily less than that of scalar collagen. (B) Enlarged section of a scale. Note the changing position of the different collagen layers and the difference in cytoplasmic development between distal (SBd) and median (SB,,) scleroblasts. A calcification front is gradually proceeding towards the median side of the scale. The calcified part of the scale is represented by a narrow black strip drawn across the older region of the scale (arrows). (C) Hypothetical block diagram of part of the surface of a scale showing the relationship between a circulus and the other collagen formations. BL basal lamina; C circulus; COL collagen; D desmosome; E eoidermis; ESP epidermal surface pattern; ER ergastoplasm; FB fibroblast; F' F " calcification front; M melanophore; M I T mitochondrion; N nucleus; S scale; SBa scleroblast (distal); SB., scleroblast (median); SD scalar denticle
40
W.J.R. Lanzing and R.G. Wright
Fig. 2 A and B. (A) Cross section through a fully calcified circulus. Note the paramedian section through a scalar denticle (SD) The epidermis is separated from the dermis by a basal lamina. • 30,000. (B) Cross section through a developing region of a scale. Note the calcifying part of the circulus and the scale, as well as the well-developed distal scleroblast, x 13,500
Fig. 3A and B. Median aspect of a growing scale. Note the parallel arrangement of the ribosomelinked cisternae. (A) Desmosome-linked scleroblasts and several layers of collagen are shown. x 21,000. (B) Highly magnified scleroblasts showing pericisternal vesicles and microfilaments; the collagen fibres are characterized by their typical periodicity. • 80,000
42
W.J.R. Lanzing and R.G. Wright
Scales not only increase in thickness but also in width. The growing region of the rostral or anterior field of the scale is completely surrounded by scleroblasts characterized by an extensively developed rough endoplasmic reticulum. Fig. 4 shows the very edge of the rostral field of a scale. The scleroblasts are held together by a number of desmosomes measuring about 200 nm in width. At all times the developing edge of the scale is separated from the
Fig. 4. Growing edge of the anterior (rostral) aspect of a scale. Scleroblasts form a tight envelope around the developing scale edge. Numerous developing collagen fibres appear in the scalar matrix. Note desmosomes and well-developed ergastoplasm, x 13,500
Scales of Tilapia mossambica (Peters)
43
surrounding dermal tissue by scleroblasts. The apparently empty space around the scale shown in Fig. 4 is artificial, yet underlines the existence of the scale pocket as an entity separate from the remainder of the dermis. Although the scleroblasts are thought to produce collagen fibres, the central or earliest-formed part of the scale appears to consist of homogeneous moderately electron-dense material in which the collagen fibres become embedded (Fig. 4). This is the region that shows the first signs of calcification in the form of small aggregations of electron-dense crystals radiating outwards from the site of origin. At the other, posterior, edge of the scale the scleroblasts again form a tight envelope around the scale (Fig. 5). Although otherwise little dermal tissue is present, the protruding tip of the scale is always completely protected from the environment by epidermal tissue consisting of at least three layers of cells. Calcification of the scale begins in the oldest, earliest-formed region of the scale, showing up in the form of small aggregations of electron dense crystals, as discussed above. However, soon after the new hydroxy-apatite crystals are formed simultaneously along a broad front over the whole width of the scale.
Fig. 5. Growing edge of the posterior (caudal) aspect of a scale. Scleroblasts again forming a tight envelope around the scalar edge. Note how the epidermis is wrapped around the scale, separating it from the external environment, x 5,250
Fig. 6A and B. (A) Showing the calcification front of a scale. Cross sections of the collagen fibres display different diameters (arrows). The apatite crystals gradually fill up the extracellular fibrillar space, emphasizing the circular outline of the fibres. • 66,750. (B) Showing the calcification front of a scale. The collagen fibres are sectioned longitudinally or at oblique angles. Note the plate-like apatite crystals that appear to form an electron dense mantle around the fibres. • 88,000
Scales of Tilapia mossambica (Peters)
45
There is, in other words, a calcification front which steadily moves towards the younger, median part of the scale (Fig. 6A and B). Fig. 6A shows how the newly-formed needle shaped crystals, measuring between 40450 nm in length, are grouped around each collagen fibre. Both transverse and longitudinal sections (Fig. 6B) illustrate that the crystals are arranged with their long axis parallel to the axis of the collagen fibre. It is not certain whether some of these crystals actually penetrate the collagen fibre or simply grow close to but separate from the adjacent fibre. The fact that in older calcified regions collagen fibres become masked by heavy apatite deposits (Fig. 6A) suggests fibre penetration. There is no doubt that much of the interfibrillar space is filled gradually with the apatite crystals. The scales of Tilapia mossambica do not show a distinct separation between calcified and non-calcified regions. Fig. 1A illustrates that calcification of the scale is a gradual process, with the calcified region steadily moving inwards, thereby increasing the volume of the hard part of the scale.
Discussion
A conspicuous feature of the skin of Tilapia is the complex system formed by layers of collagen fibres located in between such dermal components as chromatophores and scales. These scales also display a similar complex fibre system. However, at all times the scales are completely surrounded by a layer of so-called scleroblasts. Several terms have been used in the literature to describe scale-forming cells, but the term scleroblasts mentioned by van Oosten (1957) has been used here to separate them from fibroblasts, odontoblasts and osteoblasts. Odontoblasts (Sicker and Bhaskar, 1972), osteoblasts (Decker, 1966), fibroblasts (Lupulescu, 1974) as well as scleroblasts secrete collagen and are involved in calcification, but only odontoblasts, tendon fibroblasts and scleroblasts appear to develop desmosomes. They can therefore form a tight envelope around such organs as teeth and scales. The piscine dermal fibroblasts share with the scleroblasts the property of producing complex collagen fibre systems, but these systems are not of discrete or finite size and show neither circulus formation nor calcification. Scleroblasts, like odontoblasts (Reith, 1968), are polarized cells in that collagen is released to only one particular side of the cell. With scleroblasts the collagen is released only on the side of the growing scale. Collagen precursor molecules are thought to be produced in the ribosome-lined cisternae of the endoplasmic reticulum (Ross, 1968; Kobayashi, 1971), the precursors being released in vesicles, a form of reverse pinocytosis. Such vesicles could be identical with those found in fibroblasts of the Tilapia skin. Once released into the extracellular space, tropocollagen molecules aggregate to form thin collagen molecules. The increase, with time, of the diameter of the collagen fibre in Tilapia skin and scale occurs also in mammalian tissue (Reith, 1967; Sicher and Bhaskar, 1972). According to Jackson and Bentley (1968) the thickening of the fibres would be caused by a mucopolysaccharide-induced accretion of additional collagen fibres. However, in scales the collagen fibres soon reach a maximum diameter which is rarely exceeded.
46
W.J.R. Lanzing and R.G. Wright
The tightly-packed collagen of the earliest-formed part of the scale probably corresponds to the osteoid region described in the scale of the rainbow trout by Maekawa and Yamada (1970). There does not seem to be a fundamental difference between the origin and the later additions to the scale, or between the calcified and the non-calcified parts of the scale of Tilapia. It is felt that the distinction between "bony plate" and "fibrillary plate" (Maekawa and Yamada, 1970; Kobayashi, 1971 ; Brown and Wellings, 1969) is over-emphasized. Initial mineralization in rodent cartilage appears to take place in special nucleation sites consisting of extracellular vesicles containing crystallites (Anderson, 1969; Simon et al., 1973). Similarly, Maekawa and Yamada (1970) noted dense bodies in the calcifying region of the trout scale. Such mineralization sites were not clearly visible in Tilapia scales. However, in the oldest part of the Tilapia scale the calcification pattern appeared less regular than in the younger, newly formed collagen layers. In some scales the initial pattern could be described as globular or spheritic. Following Orvig's classification (Sicher and Bhaskar, 1971) calcification in all scales appeared to gradually assume an inotropic pattern, the axes of the crystals lying in alignment with those of the adjacent collagen fibres. Whether these crystals are composed of calcium hydroxyapatite remains to be confirmed; Bocciarelli (1973) suggested that octocalcium phosphate-which actually produces needle or plate like crystals-and hydroxyapatite can be found concurrently in bony tissues. A comparison of the mode of calcification, the existence of a calcification front and the nature of the crystals between the scale of a fish and the tooth of a mammal (Reith, 1968; Sicher and Bhaskar, 1971) reveals a distinct similarity between these organs. It is not yet certain what agents initiate calcification. A succession of different acid mucopolysaccharides may be involved (Kobayashi, 1971). In higher vertebrates parathyroid hormone and calcitonin are known to influence calcium and phosphate cellular exchange in bone (Talmage, 1972), with calcitonin also promoting collagen production (Lupulescu, 1969). Calcitonin, at least, occurs also in fish (Copp, 1969).
References Anderson, H.C. : Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59-72 (1969) BocciareUi, S.: Apatite microcrystals in bone and dentine. J. de Microscopie 16, 21-34 (1973) Brown, G.A., Wellings, S.R. : Collagen formation and calcification in teleost scales. Z. Zellforsch. 93, 571-582 (1969) Copp, D.H.: The ultimobranchial glands and calcium regulation. In (Hoar, W.S., Randall, D.J., eds.), Fish physiology, voL 2. New York: Academic Press Decker, J. : An electronmicroscopic investigation of osteogenesis in the embryonic chick. Amer. J. Anat. 118, 591q514 (1966) Greenlee, T.K., Ross, R.: The development of the rat flexor digital tendon. J. Ultrastruct. Res. 18, 354-376 (1967) Jackson, D.S., Bentley, J.P. : CoUagen-glycosaminoglycan interactions. In (Gould, B.S., ed.), Treatise on collagen, vol. 2. New York: Academic Press 1968 Kobayashi, S. : Acid mucopolysaccharides in calcified tissue. In (Bourne, G.H., Danielli, J.F., eds.), International review of cytology, vol. 30. New York: Academic Press 1971
Scales of Tilapia mossambiea (Peters)
47
Lanzing, W.J.R., Higginbotham, D.R. : Scanning microscopy of surface structures of Tilapia mossamniea scales. J. Fish. Biol. 6, 30%310 (1974) Lanzing, W.J.R., Wright, R.G. : The ultrastructure of the skin of Tilapia mossambica. Cell Tiss. Res. 154, 251-264 (1974) Lupulescu, A. : Effects of calcitonin on fibroblasts and collagen formation in rabbits. J. Morph. 142, 447M66 (1974) Maekawa, K., Yamada, J. : Some histochemical and fine structural aspects of growing scales of the rainbow trout. Bull. Fac. Fish. Hokkaido Univ. 21, 70-78 (1970) Reith, E.J. : Collagen formation in developing molar teeth of rats. J. Ultrastruct. Res. 21, 383-414 (1968) Ross, R.: The connective tissue fibre forming cell. In (Gould, B.S., ed.), Treatise on collagen, vol. 2. New York: Academic Press 1968 Sicher, H., Bhaskar, S.N.: Orban's oral histology and embryology. St. Louis: C.V. Mosby 1972 Simon, D.R., Berman, I., Howell, D.S. : Relationship of extracellular matrix vesicles to calcification in normal and healing rhachitis cartilage. Anat. Rec. 176, 167-180 (1973) Talmage, R.V. : Further studies on the control of calcium homeostasis by parathyroidal hormone. In (Talmage, R.V., Muson, P., eds.), Calcium, parathyroid and the calcitonins. Amsterdam: Excerpta Medica 1972
Received July 3, 1975
Cell and Tissue Research 9 by Springer-Verlag 1976
The Ultrastructure and Calcification of the Scales of Tilapia mossambica (Peters) W.J.R. Lanzing and R.G. Wright School of Biological Sciences and Electronmicroscope Unit, University of Sydney, Australia
Summary. The scales of Tilapia are surrounded by an envelope of scleroblasts responsible for the production of layers of collagen that constitute the bulk of the scale. The scleroblasts adjoining the lateral face of the oldest scale region gradually atrophy. New collagen layers are deposited against the inner face of the scale, the adjoining scleroblasts showing evidence of high metabolic activity. Calcification occurs by inotropic deposition of crystals alongside the fibres. There is no sharp demarcation between calcified and non-calcified scale regions, a calcification front gradually moving towards newly formed collagen layers. It is felt that fish scales should be considered as calcified derivatives of dermal collagen layers.
Key words: Scale - Tilapia - Calcification - Ultrastructure. Introduction
The fine structure of the skin incorporating the scales of Tilapia rnossambica was recently described by Lanzing and Wright (1974). Earlier, ultramicroscopic evidence of the presence of collagen bundles was found in scales of the sole Hippoglossoides (Brown and Wellings, 1969). At the same time, teleost scales were reported to consist of calcified and non-calcified parts (van Oosten, 1957; Maekawa and Yamada, 1970). The present paper attempts to present a detailed investigation of the fine structure of the scale of Tilapia. Scanningmicroscopic observations on the morphology of the scale were presented elsewhere (Lanzing and Higginbottom, 1974). Materials and Methods The Tilapia mossambica culture procedures as well as the fixation and staining methods have been described previously (Lanzing and Wright, 1974). Send offprint requests to: Dr. W.J.R. Lanzing, School of Biological Sciences, University of Sydney, Carslaw Building, Sydney, N.S.W. 2006, Australia.
38
W.J.R. Lanzing and R.G. Wright
Results
Light microscopic observations reveal that the scales are embedded in dermal connective tissue. Each scale consists of a number of differently oriented collagen layers, staining alternatively with Orange G and Light Green SF Yellowish. The scales, - even the posterior edges that appear to protrude from the s k i n - are at all times covered by dermal cells, in turn protected from the environment by a thin layer of epidermal cells. In between the scales as well as in the other parts of the skin occur considerable masses of collagen. Fig. 1 presents a generalized view of various dermal and epidermal components. Electron microscopic observations show that the epidermis overlying the scale is composed primarily of type B cells, separated from the epidermis by the continuous basal lamina. The fine structure of the outer or lateral part of the mature scale is heavily calcified. Fig. 2A shows that the circuli adjacent to the collagen layers incorporate a considerable quantity of calcium salts. The older, more developed parts of the scale are covered by regressed scleroblasts, showing few ribosome-lined cisternae and other cytoplasmic organelles. It is clear from decalcified preparations that the circuli are formed by bundles of collagen fibres that course across the scale surface (Fig. 1 C) to form a loop, the ends of which are joined by the tubercula in the rostral field of the scale. In the young, developing scale region calcification sites or nuclei are visible in the collagenous mass forming the bulk of the rather thin scale (Fig. 2B). The inner or medial lateral face of the scale is joined by large scleroblasts showing an exceptionally well developed rough endoplasmic reticulum (Fig. 3 A). The cisternae often course parallel to one another with a fairly uniform diameter of 80 nm. Clearly visible are the desmosomes (Fig. 3A, B) that join the scleroblasts together. In so doing those scleroblasts that are involved in scale formation form a continuous and uninterrupted envelope, separating the scale from the surrounding tissue. New collagen fibres are being deposited against the inner or medial surface of the scale. These fibres are deposited in layers which measure between 1,200-1,800 nm in thickness. Newer (younger) fibre layers course in a direction almost at right angles to the adjacent older layers (Fig. 3 A), resembling the structure of plywood. In cross section the collagen fibres are round and measure between 61 and 190 nm. It appears (Fig. 6A) that the diameter of the newly-formed fibres is sometimes less than that of older fibres, although even in the older region of the scale the diameter of the fibres is not constant. There are three possible explanations for this: a) that b) that c) that collagen is
the fibres are relatively short and acicular, t.i. tapering at the ends, the individual fibre is of variable thickness when formed and some fibres retain their newly-formed appearance because little extra added to the original fibre.
Irrespective of their diameter, the collagen fibres show a periodicity of about 63 nm, which is typical for collagen. Bundles of microfilaments are often found in the scleroblasts, but no evidence was found to indicate that the collagen fibres are actually preformed in the scleroblasts before appearing in the matrix around the scale.
Fig. 1 A-C. Diagram of a scale showing its relation to other skin components and its ultrastructural aspects. (A) Median section through a scale showing several collagen layers. Near the scale are a melanophore, a dermal fibroblast, dermal collagen and epidermal tissue. The thickness of the dermal collagen layers is not necessarily less than that of scalar collagen. (B) Enlarged section of a scale. Note the changing position of the different collagen layers and the difference in cytoplasmic development between distal (SBd) and median (SB,,) scleroblasts. A calcification front is gradually proceeding towards the median side of the scale. The calcified part of the scale is represented by a narrow black strip drawn across the older region of the scale (arrows). (C) Hypothetical block diagram of part of the surface of a scale showing the relationship between a circulus and the other collagen formations. BL basal lamina; C circulus; COL collagen; D desmosome; E eoidermis; ESP epidermal surface pattern; ER ergastoplasm; FB fibroblast; F' F " calcification front; M melanophore; M I T mitochondrion; N nucleus; S scale; SBa scleroblast (distal); SB., scleroblast (median); SD scalar denticle
40
W.J.R. Lanzing and R.G. Wright
Fig. 2 A and B. (A) Cross section through a fully calcified circulus. Note the paramedian section through a scalar denticle (SD) The epidermis is separated from the dermis by a basal lamina. • 30,000. (B) Cross section through a developing region of a scale. Note the calcifying part of the circulus and the scale, as well as the well-developed distal scleroblast, x 13,500
Fig. 3A and B. Median aspect of a growing scale. Note the parallel arrangement of the ribosomelinked cisternae. (A) Desmosome-linked scleroblasts and several layers of collagen are shown. x 21,000. (B) Highly magnified scleroblasts showing pericisternal vesicles and microfilaments; the collagen fibres are characterized by their typical periodicity. • 80,000
42
W.J.R. Lanzing and R.G. Wright
Scales not only increase in thickness but also in width. The growing region of the rostral or anterior field of the scale is completely surrounded by scleroblasts characterized by an extensively developed rough endoplasmic reticulum. Fig. 4 shows the very edge of the rostral field of a scale. The scleroblasts are held together by a number of desmosomes measuring about 200 nm in width. At all times the developing edge of the scale is separated from the
Fig. 4. Growing edge of the anterior (rostral) aspect of a scale. Scleroblasts form a tight envelope around the developing scale edge. Numerous developing collagen fibres appear in the scalar matrix. Note desmosomes and well-developed ergastoplasm, x 13,500
Scales of Tilapia mossambica (Peters)
43
surrounding dermal tissue by scleroblasts. The apparently empty space around the scale shown in Fig. 4 is artificial, yet underlines the existence of the scale pocket as an entity separate from the remainder of the dermis. Although the scleroblasts are thought to produce collagen fibres, the central or earliest-formed part of the scale appears to consist of homogeneous moderately electron-dense material in which the collagen fibres become embedded (Fig. 4). This is the region that shows the first signs of calcification in the form of small aggregations of electron-dense crystals radiating outwards from the site of origin. At the other, posterior, edge of the scale the scleroblasts again form a tight envelope around the scale (Fig. 5). Although otherwise little dermal tissue is present, the protruding tip of the scale is always completely protected from the environment by epidermal tissue consisting of at least three layers of cells. Calcification of the scale begins in the oldest, earliest-formed region of the scale, showing up in the form of small aggregations of electron dense crystals, as discussed above. However, soon after the new hydroxy-apatite crystals are formed simultaneously along a broad front over the whole width of the scale.
Fig. 5. Growing edge of the posterior (caudal) aspect of a scale. Scleroblasts again forming a tight envelope around the scalar edge. Note how the epidermis is wrapped around the scale, separating it from the external environment, x 5,250
Fig. 6A and B. (A) Showing the calcification front of a scale. Cross sections of the collagen fibres display different diameters (arrows). The apatite crystals gradually fill up the extracellular fibrillar space, emphasizing the circular outline of the fibres. • 66,750. (B) Showing the calcification front of a scale. The collagen fibres are sectioned longitudinally or at oblique angles. Note the plate-like apatite crystals that appear to form an electron dense mantle around the fibres. • 88,000
Scales of Tilapia mossambica (Peters)
45
There is, in other words, a calcification front which steadily moves towards the younger, median part of the scale (Fig. 6A and B). Fig. 6A shows how the newly-formed needle shaped crystals, measuring between 40450 nm in length, are grouped around each collagen fibre. Both transverse and longitudinal sections (Fig. 6B) illustrate that the crystals are arranged with their long axis parallel to the axis of the collagen fibre. It is not certain whether some of these crystals actually penetrate the collagen fibre or simply grow close to but separate from the adjacent fibre. The fact that in older calcified regions collagen fibres become masked by heavy apatite deposits (Fig. 6A) suggests fibre penetration. There is no doubt that much of the interfibrillar space is filled gradually with the apatite crystals. The scales of Tilapia mossambica do not show a distinct separation between calcified and non-calcified regions. Fig. 1A illustrates that calcification of the scale is a gradual process, with the calcified region steadily moving inwards, thereby increasing the volume of the hard part of the scale.
Discussion
A conspicuous feature of the skin of Tilapia is the complex system formed by layers of collagen fibres located in between such dermal components as chromatophores and scales. These scales also display a similar complex fibre system. However, at all times the scales are completely surrounded by a layer of so-called scleroblasts. Several terms have been used in the literature to describe scale-forming cells, but the term scleroblasts mentioned by van Oosten (1957) has been used here to separate them from fibroblasts, odontoblasts and osteoblasts. Odontoblasts (Sicker and Bhaskar, 1972), osteoblasts (Decker, 1966), fibroblasts (Lupulescu, 1974) as well as scleroblasts secrete collagen and are involved in calcification, but only odontoblasts, tendon fibroblasts and scleroblasts appear to develop desmosomes. They can therefore form a tight envelope around such organs as teeth and scales. The piscine dermal fibroblasts share with the scleroblasts the property of producing complex collagen fibre systems, but these systems are not of discrete or finite size and show neither circulus formation nor calcification. Scleroblasts, like odontoblasts (Reith, 1968), are polarized cells in that collagen is released to only one particular side of the cell. With scleroblasts the collagen is released only on the side of the growing scale. Collagen precursor molecules are thought to be produced in the ribosome-lined cisternae of the endoplasmic reticulum (Ross, 1968; Kobayashi, 1971), the precursors being released in vesicles, a form of reverse pinocytosis. Such vesicles could be identical with those found in fibroblasts of the Tilapia skin. Once released into the extracellular space, tropocollagen molecules aggregate to form thin collagen molecules. The increase, with time, of the diameter of the collagen fibre in Tilapia skin and scale occurs also in mammalian tissue (Reith, 1967; Sicher and Bhaskar, 1972). According to Jackson and Bentley (1968) the thickening of the fibres would be caused by a mucopolysaccharide-induced accretion of additional collagen fibres. However, in scales the collagen fibres soon reach a maximum diameter which is rarely exceeded.
46
W.J.R. Lanzing and R.G. Wright
The tightly-packed collagen of the earliest-formed part of the scale probably corresponds to the osteoid region described in the scale of the rainbow trout by Maekawa and Yamada (1970). There does not seem to be a fundamental difference between the origin and the later additions to the scale, or between the calcified and the non-calcified parts of the scale of Tilapia. It is felt that the distinction between "bony plate" and "fibrillary plate" (Maekawa and Yamada, 1970; Kobayashi, 1971 ; Brown and Wellings, 1969) is over-emphasized. Initial mineralization in rodent cartilage appears to take place in special nucleation sites consisting of extracellular vesicles containing crystallites (Anderson, 1969; Simon et al., 1973). Similarly, Maekawa and Yamada (1970) noted dense bodies in the calcifying region of the trout scale. Such mineralization sites were not clearly visible in Tilapia scales. However, in the oldest part of the Tilapia scale the calcification pattern appeared less regular than in the younger, newly formed collagen layers. In some scales the initial pattern could be described as globular or spheritic. Following Orvig's classification (Sicher and Bhaskar, 1971) calcification in all scales appeared to gradually assume an inotropic pattern, the axes of the crystals lying in alignment with those of the adjacent collagen fibres. Whether these crystals are composed of calcium hydroxyapatite remains to be confirmed; Bocciarelli (1973) suggested that octocalcium phosphate-which actually produces needle or plate like crystals-and hydroxyapatite can be found concurrently in bony tissues. A comparison of the mode of calcification, the existence of a calcification front and the nature of the crystals between the scale of a fish and the tooth of a mammal (Reith, 1968; Sicher and Bhaskar, 1971) reveals a distinct similarity between these organs. It is not yet certain what agents initiate calcification. A succession of different acid mucopolysaccharides may be involved (Kobayashi, 1971). In higher vertebrates parathyroid hormone and calcitonin are known to influence calcium and phosphate cellular exchange in bone (Talmage, 1972), with calcitonin also promoting collagen production (Lupulescu, 1969). Calcitonin, at least, occurs also in fish (Copp, 1969).
References Anderson, H.C. : Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59-72 (1969) BocciareUi, S.: Apatite microcrystals in bone and dentine. J. de Microscopie 16, 21-34 (1973) Brown, G.A., Wellings, S.R. : Collagen formation and calcification in teleost scales. Z. Zellforsch. 93, 571-582 (1969) Copp, D.H.: The ultimobranchial glands and calcium regulation. In (Hoar, W.S., Randall, D.J., eds.), Fish physiology, voL 2. New York: Academic Press Decker, J. : An electronmicroscopic investigation of osteogenesis in the embryonic chick. Amer. J. Anat. 118, 591q514 (1966) Greenlee, T.K., Ross, R.: The development of the rat flexor digital tendon. J. Ultrastruct. Res. 18, 354-376 (1967) Jackson, D.S., Bentley, J.P. : CoUagen-glycosaminoglycan interactions. In (Gould, B.S., ed.), Treatise on collagen, vol. 2. New York: Academic Press 1968 Kobayashi, S. : Acid mucopolysaccharides in calcified tissue. In (Bourne, G.H., Danielli, J.F., eds.), International review of cytology, vol. 30. New York: Academic Press 1971
Scales of Tilapia mossambiea (Peters)
47
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Received July 3, 1975
