Cell Tiss. Res. 172, 455476 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Ultrastructural Study of the Shell-repair Membrane in the Snail, Helix pomatia L. * Anna Abolin~-Krogis Institute of Zoophysiology, University of Uppsala, Sweden
Summary. The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretion of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the mineralizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted. Key words: Shell-repair membrane - Initial calcification - Formation of crystalline structures - Amoebocytes - Helix pomatia L.
Introduction The process of shell-repair in molluscs has been extensively studied with the help of light and electron microscopes. The contributing functions of the hepatopancreas and the mantle have been thoroughly examined (Sioli, 1935; Manigault, 1939; Wagge, 1951, 1952; Fretter, 1952; Wagge and Mittler, 1953; Durning, 1957; Abolin~-Krogis, 1958, 1961, 1963a; Wada, 1961, 1972; Wilbur, 1964, 1972; Watabe, 1965; Beedham, 1965; Travis et al., 1967; Tsujii, 1968; Timmermanns, 1969; Bevelander and Nakahara, 1969; Saleuddin and Chan, 1969; Saleuddin, Send offprint requests to: Dr. Anna Abolin~-Krogis, Zoofysiologiska Institutionen Box 560, S-751 22 Uppsala 1, Sweden * This investigation was supported by a grant from the Swedish Natural Science Research Council, which is gratefully acknowledged. I am indebted to Miss Ch. Stensj6 and Mrs. E. Hellm6n for their technical assistance
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1970, 1971 ; K a p u r and G u p t a , 1970; Istin, 1970; Istin and Masoni, 1973; M e e n a k shi et al., 1973). Attention was called to the pigment granules in the mantle epithelial cells by L e v e t z o w (1932). The a u t h o r supposed that these granules m a y have some function in shell g r o w t h and regeneration. M o r e detailed examination o f the pigment granules was undertaken by Abolin~-Krogis (1963 b, 1973). The fluorescence and histochemical studies showed that the pigment granules in the a m o e b o cytes and the mantle epithelial cells are o f lipofuscin type and have characteristics in c o m m o n with lysosomes. It was suggested that the granules were apparently engaged b o t h in the binding o f calcium ions and in the catalysis o f organic substances within the repair membrane. T h e ultrastructure o f thin sectioned shell-repair m e m b r a n e s was, however, n o t satisfactorily investigated. The present study deals with the analysis o f the substructure o f this m e m b r a n e in the snail, Helix pomatia. F r o m the results obtained, it was expected that some information could be provided a b o u t the possible physiological role o f the different cellular constituents o f the calcifying m e m b r a n e in the early stages o f the regeneration. Material and Methods Twenty adult snails, Helix pomatia, weighing about 20 g, were selected for the experiments. The shell-repair membranes, 6-12h after damage, were fixed in ice-cold 2~ glutaraldehyde in 0.1 M phosphate buffer, pH7.3. Subsequent post-fixation was performed in 0.05 M phosphate buffered 1~o osmium tetroxide according to the methods of Sabatini et al. (1963) and Millonig (1962). Some of the membranes were decalcified in 0.5 M EDTA, pH 7.3 and stained en bloc with 0.05~ ruthenium red (Luft, 1971). The specimens were dehydrated in graded ethanol and embedded in Epon 812. Thin sections were double-stained with saturated uranyl acetate followed by lead citrate (Reynolds, 1963). A Siemens Elmiskop 101 at 60 or 80kV was used for the examination of thin sections. The microscope was equipped with an anticontamination device cooled with liquid nitrogen. Some intact shell-repair membranes were fixed in buffered formaldehyde and stained with 0.05~ ruthenium red. After dehydration, the membranes were enclosed in Canada balsam and studied with a light microscope. The examination of the mantle exudate, i.e., the extrapallial fluid, was undertaken with the light microscope. The smear preparations were fixed in 95% alcohol and stained with haematoxylineosin. Results The examination o f the animals with shells d a m a g e d under natural conditions showed always cracks o f the original shell on the surface o f the mantle. U n d e r experimental conditions the a u t h o r was not able to obtain the shell-repair m e m branes w i t h o u t cracks. A r o u n d the cracks and within the repair m e m b r a n e the a m o e b o c y t e s were usually present. Moreover, the liquid droplets exuded f r o m the mantle epithelium, taken up on the cover-glass, contained amoebocytes when examined with the light microscope (Fig. 1). Fig. 1. Amoebocytes within the extrapallial fluid. Haematoxylin-eosin staining of smear preparation. • 800 Fig. 2. Disintegrating amoebocyte within the shell-repair membrane. The cell organelles: Golgi complex, mitochondria and granular endoplasmic reticulum (GER) are largely destroyed. Small osmiophilic vesicles (s) and dense bodies of different opacity are visible within the cell. x 18,000
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The amoebocytes within the shell-repair membrane showed considerable variation in size and content. They were either small, irregularly-shaped, approximately 10~tm in diameter, or large and elongated, approximately 181am in diameter. Despite the observed variations in their size, they are not described as different types. The number of amoebocytes varied notably within each separate membrane. Six h after shell damage, the amoebocytes showed different degrees of disintegration (Fig. 2). Some of them were not significantly transformed and possessed a well-developed nucleus and a great number of mitochondria. The latter frequently contained small dark inclusions, possibly calcium salt (Fig. 3). The majority of the amoebocytes showed signs of more advanced degeneration. Within these ceils the mitochondria appeared dense and their cristae disrupted. Small, round corpuscles were occasionally observed within the mitochondrial matrix (Fig. 4 a, b). The Golgi complex and the granular endoplasmic reticulum (GER) were reduced. The latter consisted of vacuole-like cisternae lined by ribosomes (Fig. 4a). Fine filaments, granules and coarse aggregates were frequently encountered within the cisternae (Figs. 3, 4 b). The amount of glycogen within the amoebocytes was inconspicuous. The more interesting inclusions of the amoebocytes were the cytoplasmic dense bodies. These showed varied structure, density and size. Among the dense bodies, the characteristic lipofuscin-type pigment granules were only seldomly present. The pigment granules possessed homogeneous structure which was somewhat chattered by sectioning (Figs. 2, 6). More frequently, another type of cytoplasmic dense body was observed within the amoebocytes. These bodies were either fibrillar in structure (Fig. 7 a) or they possessed a core-containing inclusion. The core appeared to be a hard mineral formation which bulged out of its surrounding (Fig. 5). The size of these inclusions varied between 0.2 llm and 0.6 ~tm in diameter. Frequently, the above-mentioned dense bodies also possessed a bleb-like protrusion and a vacuole displaced on one side. The blebs sometimes contacted small empty vesicles (Fig. 5). Lipid droplets were usually grouped on the periphery of the dense bodies (Fig. 5). All characteristics indicated that the cytoplasmic dense bodies were comparable with cytosomes, i.e., lysosomelike bodies (Figs. 4b, 5). Small corpuscles and vesicles were often found in the immediate vicinity of the destroyed mitochondria and the dense bodies (Figs. 2, 4 b). In addition, some very large cytosomes were commonly observed within the amoebocytes. They contained variable amounts of polymorphic inclusions, in which different components could be distinguished, such as rounded globules, membranous fragments, and irregular dense material (Fig. 7 b-d). Intact, small calcium spherites, approximately 0.8 ~t in diameter, were rarely observed within Fig. 3. Mitochondria- containing groups of dense granules. Granular material and coarser aggregates within the cisternae of GER. x 30,000 Figs. 4a and b. Mitochondria showing dense matrix and reduced cristae. Round corpuscles within the mitochondria (arrows). a round, empty cisternae of GER lined by ribosomes; b dense bodies of different opacity, two of them contain crystalline inclusions (c), small osmiophilic vesicles (s) among the dense bodies and disintegrated mitochondria. Vacuoles containing fibrillar or dense granular material. • 30,000
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Fig. 5. Disintegrating amoebocyte with different sized and structured dense cytoplasmic bodies. Some of them contain a characteristic inclusion with hard core (c) and a vacuole (v). The others are surrounded by lipid droplets (/). The bleb-like protrusions of the dense bodies are often in contact with small vesicles. At the bottom, the cell membrane of the amoebocyte is disrupted and finely granular, partly filamentous material visible on both sides of the disrupted cell membrane. At some distance from the cell, coarse fibrils, small vesicles, and large aggregates (a) are present within the repair membrane, x 24,000
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Fig. 6. Part of a degenerating amoebocyte. Small vesicles with osmiophilic limiting membrane (s) are present within the cell and among the fibrillar material of the repair membrane. The amoebocyte contains two pigment granules (p). • 24,000
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Fig. 7 a - e . Cytoplasmic dense bodies within the disintegrating amoebocytes: a dense body with fibrillar structure; b dense bodies within a large cytosome showing homogeneous structure; e at the top, a pigment granule of lipofuscin-type; the other dense body contains a less dense inclusion and rounded corpuscles; d right at the top, a large cytosome containing dense bodies and whirled fibers, on the left, a dense body containing crystalline structure; e at the top, calcium spherites within the degenerating amoebocyte, a x 48,000; b - e • 32,000
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Fig. 8. Remnants of a completely destroyed amoebocyte. The nuclear membrane, dense bodies, and large autophagic vacuoles are included in a tight network of fibrils. Among the fibrils small dense vesicles (s) and large aggregates (a). x 28,000 the amoebocytes. T h e calcium spherites were identified by the concentric arrangem e n t o f their fibrillar material (Fig. 7 e). The ultrastructural study o f the shell-repair m e m b r a n e disclosed the m o s t striking observation that the a m o e b o c y t e s were engaged in the p r o d u c t i o n o f a fibrillar protein o f the repair matrix. In some sections it was possible to recognize an incontinuity in the cell m e m b r a n e o f the a m o e b o c y t e and a finely granular,
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partly filamentous material accumulated at this place, i.e., inside and outside the disrupted cell membrane (Fig. 5). At some distance from the cell the fine granular and fibrillar material appeared to be mingled with larger fibrils. The released substance seemed to undergo a fibrillar organization and a self-assembling process outside the cell. In the repair membrane among the fibrillar material granules and small corpuscles or vesicles were interspersed (Fig. 6). The polygonal or ovoid granules, approximately 0.05 gm in diameter (Fig. 9), stained positively with ruthenium red and thus revealed their proteoglycan nature (Luft, 1971; Thyberg et al., 1975b). The small vesicles were approximately 0.1 gm in diameter. They had a thick, electron-dense, osmiophilic limiting membrane (Fig. 6). Occasionally, vesicles of the same size and structure were observed within the disintegrating amoebocytes in the vicinity of destroyed dense bodies and mitochondria (Figs. 2, 4b, 6). The osmiophilic reaction implied a lipid as one of the constituents of the limiting membrane of the vesicles. Apart from the above-mentioned proteoglycan granules and small vesicles, the repair membrane contained dense bodies of different structure and size (approximately 0.2 gm-0.8 gm in diameter). Some of these bodies showed either a vesicular or fibrillar structure and appeared to be formed by aggregates of the osmiophilic vesicles (Figs. 11 a, b). Such aggregation of the vesicles was indeed observed in the repair membranes (Figs. 5, 8). The other dense bodies contained crystalline structures, which were of plate-like or cylindrical construction (Figs. 11 c, d). The bodies containing cylinder-like crystallite were apparently a more advanced form of the cytoplasmic, crystalline core-containing inclusion. Occasionally, a ramification of these crystallites was observed (Fig. 12a). After the decalcification, the organic part of the crystal became visible. It consisted of tightly twisted protein fibrils (Fig. 12b). In other cases the dense material was concentrated on the surface of the filamentous body (Fig. 13). Frequently, large groups of accumulated dense bodies and twisted fibers were observed within the repair membrane (Fig. 14). Some intact lipofuscin pigment granules (Fig. 10) and lysosome-like dense bodies were always present within the repair membrane (Fig. 8). The repair membrane also contained heavily calcified rounded and double fan-shaped organic crystalline structures (Figs. 16, 17). The ultrathin sections of these structures showed an accumulation of the vesicles and dense bodies in the middle and in a concentric ring on the periphery of these structures (Fig. 15). Within a thin section not all of the peripheral bodies were visible due to their location outside the plane of sectioning. The latter were connected to the centrally located bodies by fibrillar projections. The outline of the large crystalline structures was marked by small, dense crystallites, possibly calcium carbonate (Fig. 15). Fig. 9. Thin fibrils and granules within the repair membrane. The granules are positively stained with ruthenium red. x 32,000 Fig. 10. A lipofuscin-type pigment granule within the repair membrane, x 20,000 Fig. 11 a-d. Cytoplasmic dense bodies within the repair membrane: a - b bodies of fibrillar-vesicular type; c - d bodies containing crystalline structures, x 30,000
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Fig. 12. a Ramified crystalline structure within a disrupted dense body. b Crystalline structure after decalcification. The organic substance consists of a twisted fibrillar material, a x 30,000; b x 50,000 Fig. 13. A cross of dense fibrillar material on the surface of a filamentous body. Decalcified preparation. • 30,000 Fig. 14. Aggregate of dense cytoplasmic bodies and fibres in the repair membrane. • 25,000
The amount of proteoglycan granules was greatly reduced in the whole area occupied by the large crystalline structures. The decalcification of the large crystalline structures showed that a row of dense bodies was localized on their surface. The peripherally grouped bodies had a peculiar pattern of organized protein fibrils (Fig. 18). The large organic crystalline structures were subsequently transformed into the needle-clusters of the dendritic spherulites (Abolin~-Krogis, 1958). After decalcification, the radially oriented organic filaments of the needlecluster were visible (Fig. 19). Apparently, the calcium carbonate crystallites in these structures have the same orientation as the filaments, suggesting that the orientation of the inorganic crystals is controlled by the organic compounds.
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Fig. 15. Two large crystalline structures in the repair membrane. Accumulation of vesicles and dense bodies visible in the middle and in a concentric ring in the periphery of these structures. The peripheral bodies are connected with these centrally located by fibrillar projections (c). The outline of the large crystalline structures is marked by small, dense grains, possibly calcium carbonate crystallites, x 25,000 In the case o f the alignment o f the needles, the needle-cluster was converted into the tabular (platelike) calcium c a r b o n a t e crystal. I n the crystals with incomplete leveling o f the needles, some staining with the dyes was observed in their middle and within the concentric rings, where the organic material had n o t totally disappeared (Fig. 16). Twelve h after shell damage, the majority o f a m o e b o c y t e s within the repair m e m b r a n e appeared almost completely disintegrated. The greatest resistance
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Fig. 16. Calcium carbonate crystal of a double fan-shaped type in an intact repair membrane. Stained with r u t h e n i u m red. x 900 Fig.17. A double fan-shaped needle-cluster in an intact repair membrane. Unstained preparation. • 700 Fig. 18. Thin section of a double fan-shaped crystalline structure. After decalcification the arrangement of the dense bodies on the surface of the centrally located organic material is visible. Each of the peripheral bodies shows a peculiar arrangement of the fibrillar material around the central corpuscle. x 16,000 Fig. 19. Part of a thin section of disk-shaped crystalline structure. After decalcification the radial arrangement of the fibrillar material is visible, x 30,000 Fig. 20a and b. The tubular reticulum in the cytoplasm of the amoebocytes. The reticulum consists of: a longitudinal tubules and b rosette-like profiles. Glycogen accumulation visible in the middle of the micrograph, a x 20,000; b x 70,000
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was shown by the homogeneous dense bodies and large autophagic vacuoles. These cellular remnants were surrounded by a tight network of fibrils. Among them, dense vesicles and aggregates of these were always present (Fig. 8). The smooth-surfaced tubular reticulum described previously in the amoebocytes of connective tissue (Abolin~-Krogis, 1972), was only observed in a few cases in the amoebocytes of the repair membrane (Fig. 20a, b). The reticulum was formed by the longitudinal connecting tubules and the rosette-like profiles. The fragile structure of the tubular reticulum was apparently disrupted at the beginning of the disintegration of amoebocytes.
Discussion
The ultrastructural examination of the shell-repair membranes of the snails revealed characteristic details in the structure and composition of these membranes. As was shown with light and fluorescence microscopy (Abolin~-Krogis, 1963b, 1973) the repair membranes contained amoebocytes. The function of these cells, as was shown in this investigation, appeared to be manifold. The large autophagic vacuoles, containing different cell organelles and cracks of the original shell, suggested that the amoebocytes have a phagocytotic capacity. Phagocytosis of the amoebocytes was recently described by Sminia (1972) in the connective tissue of Lymnaea stagnalis and by Wolburg-Buchholz and Nolte (1973) in Cepaea nemoralis. Besides phagocytosis, the amoebocytes enriched the repair membranes with different repair substances and cellular constituents. The fine granular and filamentous material accumulated at the cell membrane was involved in the production of the structural protein of the shell-repair membrane. The ruthenium red positive granules, i.e., the proteoglycan granules, were also a cell product which formed a component of the repair membrane. The small vesicles, the polymorphic cytoplasmic dense bodies and the intact lipofuscin pigment granules were released from the cells. Actually, the number of lipofuscin pigment granules was markedly decreased within the disintegrated amoebocytes, whereas that of the dense bodies of lysosomal-type increased. As both of these structures showed yellow fluorescence, it was not possible to distinguish between them in the fluorescence microscope (Abolin~-Krogis, 1973). The lysosome-like dense bodies appeared to be active in the repair membranes, where their constructive and destructive capacities were needed (de Duve and Wattiaux, 1966). As stated by Dingle (1969), the intact lysosomes appeared to be extruded from the cells already in very early phases of cellular degeneration. Moreover, the dense bodies of lysosome-type were involved in the formation of a special type of granule characterized by a crystalline core. The possibility of the formation of crystalline structures characterizes the intact lipofuscin pigment granules also, as already indicated by Fawcett (1966). Thereby, no marked difference existed in the formation of crystalline structures between the dense bodies of lysosome-type and lipofuscin pigment granules. The present electron microscopical study confirmed the important fact that the vesicles and dense bodies possess the same localization as the fluorescent
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bodies and granules within the large crystalline structures, suggesting that these structures are identical in nature (Abolin~-Krogis, 1973). On the other hand, the hard core-containing bodies, characterized by weak stainability with different dyes and by their size, corresponded to b-granules described previously by Abolin~-Krogis (1961, 1963 b). They apparently represented single crystals within the repair membrane observed by X-ray examination (Abolin~-Krogis, 1968). In a later developmental stage the hard core appeared to be transformed into a cylindrical or ramified crystallite. After decalcification of the ramified crystallite the fibrillar part of it became clearly visible. It was composed of protein fibrils twisted in an unusual manner. The bodies of lower electron density appeared to be composed of fine fibrillar or vesicular material and were regarded as the earlier developmental stages of the previously described structures. The osmiophilic vesicles present within the repair membrane may likewise be attributed to a cell product. Two possibilities of their origin appear to exist. Firstly, they may originate from the disintegrated dense bodies, i.e., lysosomes, showing the common yellow fluorescence with these structures. This statement was obtained from fluorescence and electron microscopic studies. Secondly, the vesicles may originate from the fragmented cristae of the mitochondria. As showed in this investigation, the destruction of mitochondria and disappearance of the mitochondrial cristae was frequently observed. The protein-lipid complex and the enzymes of the membrane may possibly select the necessary substrate for the binding of the mineral salt. Therefore, the small vesicles within the repair membrane of the snail possibly represent the smallest unit of the membrane which was able to localize and concentrate the calcium ions in some organically bound form. The determining factor for the capture of mineral salt appears to be their lipid content, i.e., phospholipids, the chief constituent of lipids in the lipofuscin pigment granules and the membranes. The role of the lipids in the process of mineralization of the mammalian skeleton has been thoroughly examined by Irving (1976), Irving and Wuthier (1968), Wuthier (1971, 1976), and Seimiya and Ohki (1973). Within the repair membrane, besides the crystalline core-containing dense bodies, i.e., b-granules, the aggregation of the previously described vesicles induced formation of a number of additional dense bodies. The latter were also included as the organic components in the large crystalline structures and formed the central and peripheral loci of calcification. Besides the amoebocytes, the greatest part of the lipofuscin-type pigment granules and dense bodies of lysosome-type appears to be released from the mantle epithelial cells (Abolin~-Krogis, 1963a). It was shown that, after shell damage, the whole exposed mantle area appeared dull-white, caused by the loss of the pigment granules. In that way the mantle epithelial cells participated in the production of the dense bodies of the shell-repair membrane. Moreover, the mantle epithelium cells were undoubtedly also engaged in the release of proteinaceous material, polysaccharides, and lipids during the time of shell repair (Abolin~-Krogis, 1963 a). These substances extruded from the cells may aggregate and possibly induce the formation of the vesicles and dense bodies extracellularly. Of striking interest was the involvement of the amoebocytes in the production of the fibrillar protein, possibly collagenous in nature. The involvement of the amoebocytes in the synthesis of collagen was recently shown by Sminia et al.
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(1973). In the process of wound healing in Lymnaea stagnalis, the flattened amoebocytes which appeared 3-5 days after the incision were shown to produce collagen fibers. In the early stages of the development of repair membrane, the mature collagen fibers with periodical structure were not observed. Possibly, the experimental time in this study was too short (6 to 12 h) for the development of mature collagen. However, it can not be excluded that the elaboration of the mature collagen, with banded structure, occurs only rarely within the shell-repair membranes and the original shell. The sparsely distributed mature collagen fibers were described by Travis et al. (1967) in the prismatic and nacreous layers of the shells of some bivalves. In contrast, Stang-Voss (1970) and WalburgBuchholz and Nolte (1973) reasoned that the vesicular cells ("Blasenzellen", i.e., Leydig cells) may have an important role in the collagen production in molluscs. It appears reasonable to believe that in the process of shell repair materials from different sources were utilized. More detailed analyses were performed with the mantle and the hepatopancreas, which contain a large pool of quickly exchangeable calcium ions (Sioli, 1935; Manigault, 1939; Wagge, 1951; Wagge and Mittler, 1953; Fretter, 1952; Durning, 1957; Abolin~-Krogis, 1961, 1963a; Tsujii, 1968; Istin, 1970; Istin and Masoni, 1973). How large the additional part from the intact shell may be is not sufficiently experimentally proven (Sioli, 1935; Wagge, 1951). Generally, very little is known about the demineralization mechanism of the inorganic part of the shell. It is believed that the demineralization is affected by the acids released from the lytic cells (Mueller et al., 1973). The tubular system found in the amoebocytes may possibly represent a specific system for providing acids or hydrogen ions. A structure similar to the tubular reticulum in amoebocytes was described in the acid-secreting cells of the frog stomach by Forte and Forte (1970). Obviously, only after removal of calcium salt may the organic substances be attacked by the lysosomal enzymes. The extracellular release of the lysosomal enzymes within the repair membrane appears very possible. The experimental results obtained by Poole et al. (1974) within the cartilage of the chick confirmed such a possibility. Which of the previously mentioned calcium supplies were utilized, and to what extent, in each separate case, apparently depends on the general metabolism and the physiological condition of the regenerating animal. Chan and Saleuddin (1974) assumed that the general calcium supply in the body of snails appears to be the original shell, which may be used in case of need. Unfortunately, their work contains many confusing points and the results obtained by the authors were hardly comparable with the findings of other investigators. A comparison of the mineralizing processes in the early stages of the calcification of the shell-repair membrane of the snail with the corresponding calcifying processes of the epiphyseal cartilage of mammals (Anderson, 1969; Anderson and Sajdera, 1976; Bonucci, 1969, 1970; Thyberg and Friberg, 1972; Thyberg et al., 1975a, b) showed many common features, in some cases even a striking similarity (Anderson, 1969, Fig. 8). Naturally, the formative cells in the cartilage were of another type, i.e., the chondrocytes, but the released materials of the activated cells were in both cases closely similar if not identical. The repair membrane of the snail and the calcifying cartilage matrix of mammals contain protein-
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aceous nonperiodic filaments, proteoglycan granules, and cytoplasmic dense bodies, i.e., matrix vesicles. Moreover, Anderson (1969), Bonucci (1969), and Thyberg et al. (1975a) suggested that the matrix vesicles were derived from the cells and that they play a role in the initiation of calcification. The authors also considered that the matrix vesicles, at least some of them, were lysosomes and may have the function of hydrolyzing the organic parts of the cartilage. On the other hand, different opinions have been expressed concerning the initial crystal formation in the cartilage matrix. Thyberg et al. (1975 b) supposed that the mineral crystals were first deposited in close vicinity to the membranebounded matrix vesicles, whereas Anderson (1969), Anderson and Sajdera (1976), BonucCi (1970), Bernard (1972), and Eisenmann and Glick (1972) supported the view that the first mineral crystals were laid down within these structures. Evidence concerning the initial crystal formation in the repair membrane of the snail, obtained in this study, appears to be more complicated. Supposing that the protein-phospholipid complex of the small vesicles is engaged in the binding of the mineral ions, the latter would accumulate on the surface of these structures. At a later stage of development, when the vesicles aggregated and formed dense bodies, they were both engaged in the organization of the organic material around the primary loci of mineralization. At this time the captured inorganic material appeared located within a less dense organic substance, as in a compartment. This stage of mineralization was very clearly shown in the fluorescence microscope (Abolin~-Krogis, 1973). The mineral deposition within the large crystalline structures appears somewhat more complicated. In these structures, two or more separate active areas were simultaneously recognizable, the central intracrystalline calcifying area and the peripheral one. The latter is characterized by the accumulation of the calcifying centra on the periphery of the large crystalline structures. In both areas, a forced packing of the fibrillar material around the active centra apparently takes place. The peripheral growth of the crystalline structure may have its own nucleating mechanism. It is only in the later developmental stage that the separate active centra within the large crystalline structures possibly conjugate and form the needle-clusters. The latter finally were transformed into the plate-like calcium carbonate crystals (Abolin~-Krogis, 1958). The lysosomal enzymes appeared firstly to take part in the reduction of the proteoglycan granules (Woessner, 1973), whereas the fibrillar material was concentrated around the calcifying centra. It does not seem improbable that the proteoglycans afforded protection against the accumulation and organization of the protein fibrils around these centra. The loss of the protector suddenly induced the organization of the fibrillar protein and the mineralization could proceed. However, it can not be excluded that the conjugation of the proteoglycan granules with the fibrillar protein occurred before the calcification of the whole complex started. The complete reduction of the amount of organic material within the crystalline structures occured in more advanced stages of mineralization. Thus, the mature calcium carbonate crystal appeared to be located within a compartment formed by the organic sheets. Summarizing the results obtained in this study, the fact appears once more to be verified that different cell structures, small vesicles and dense bodies, were engaged in the formation of the initial calcifying centra. Some of these cellular
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constituents were liberated from the degenerated and disintegrated amoebocytes, w h i c h w e r e also e n g a g e d in t h e p r o d u c t i o n o f t h e f i b r i l l a r p r o t e i n . I n spite o f t h e fact, t h a t t h e w h o l e o r g a n i c m a s s o f t h e d e s t r o y e d a m o e b o c y t e s w a s a d d e d t o the repair membrane, the bulk of the substances needed for the complete restor a t i o n o f t h e d a m a g e d shell a p p a r e n t l y c o m e f r o m o t h e r s o u r c e s o f t h e a n i m a l body.
References Abolin~-Krogis, A.: The morphological and chemical characteristics of organic crystals in the regenerating shell of Helix pomatia L. Acta Zool. 39, 19-38 (1958) Abolin~-Krogis, A.: The histochemistry of the hepatopancreas of Helix pomatia (L.) in relation to the regeneration of the shell. Ark. Zool. 13, 159-201 (1961) Abolin~-Krogis, A.: The histochemistry of the mantle of Helix pomatia (L.) in relation to the repair of the damaged shell. Ark. Zool. 15, 461-474 (1963a) Abolin~-Krogis, A.: On the protein stabilizing substances in the isolated b-granules and in the regenerating membranes of the shell of Helix pomatia (L.). Ark. Zool. 15, 475-484 (1963 b) Abolin~-Krogis, A.: Shell regeneration in Helix pomatia with special reference to the elementary calcifying particles. Symp. Zool. Soc. London No. 22, 75-92 (1968) Abolinw A.: The tubular endoplasmic reticulum in the amoebocytes of the shell-regenerating snail, Helix pomatia L. Z. Zellforsch. 128, 58-68 (1972) Abolin~-Krogis, A.: Fluorescence and histochemical studies of the calcification-initiating lipofuscin type pigment granules in the shell-repair membrane of the snail, Helix pomatia L. Z. Zellforsch. 142, 205-221 (1973) Anderson, H.C.: Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59-72 (1969) Anderson, H.C., Sajdera, S.W.: Calcification of rachitic cartilage to study matrix vesicle function. Fed. Proc. 35, 148-153 (1976) Beedham, G.E.: Repair of the shell in species of Anodonta. Proc. Zool. Soc. London 145, 107 124 (1965) Bernard, G.W.: Ultrastructural observations of initial calcification in dentine and enamel. J. Ultrastruct. Res. 41, 1-17 (1972) Bevelander, G., Nakahara, H.: An electron microscope study of the formation of the nacreous layer in the shell of certain bivalve molluscs. Calcif. Tiss. Res. 3, 84-92 (1969) Bonucci, E.: Further investigation on the organic/inorganic relationship in calcifying cartilage. Calcif. Tiss. Res. 3, 38-54 (1969) Bonucci, E.: Fine structure and histochemistry of "calcifying globules" in epiphyseal cartilage. Z. Zellforsch. 103, 192-217 (1970) Chan, W., Saleuddin, A.S.M.: Evidence that Otala lactea (Miiller) utilizes calcium from the shell. Proc. malac. Soc. London 41, 195-200 (1974) Dingle, J.T.: The extracellular secretion oflysosomal enzymes. In: Lysosomes in biology and pathology, Vol. 2 (J.T. Dingle and H.B. Fell, eds.), pp. 421-436. Amsterdam-London: North-Holland Publ. 1969 Durning, W.C.: Repair of a defect in the shell of the snail Helix aspersa. J. Bone J. Surg. A39, 377-393 (1957) Duve, C. de, Wattiaux, R.: Functions of lysosomes. Ann. Rev. Physiol. 28, 435-492 (1966) Eisenmann, D.R., Glick, P.L.: Ultrastructure of initial crystal formation in dentin. J. Ultrastruct. Res. 41, 18-28 (1972) Fawcett, D.W.: The cell its organelles and inclusions. In: An atlas of fine structure, pp. 281 284. Philadelphia and London: Saunders Comp. 1966 Forte, T.M., Forte, J.G.: Histochemical staining and characterization of glycoproteins in acidsecreting cells of frog stomach. J. Cell Biol. 47, 437-452 (1970) Fretter, V.: Experiments with p32 and 1TM on species of Helix, Arion and Agriolimax. Quart. J. micr. Sci. 93, 133-146 (1952)
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Irving, J.T.: Interrelations of matrix lipids, vesicles and calcification. Fed. Proc. 35, 109-111 (1976) Irving, J.T., Wuthier, R.E.: Histochemistry and biochemistry of calcification with special reference to the role of lipids. Clin. Orthop. Res. 56, 237-260 (1968) Istin, M.: R61e du manteau dans le m6tabolisme du calcium chez les lamellibranches. Bull. d'Inform. Sci. et Techn. du Comm. ~i l'Energie Atomique. No 144, p. 53-80 (1970) Istin, M., Masoni, A.: Absorption et redistribution du calcium dans le manteau des lamellibranches en relation avec la structure. Calcif. Tiss. Res. 11, 151-162 (1973) Kaput, S.P., Gupta, A.S.: The role of amoebocytes in the regeneration of shell in a land pulmonate, Eupleeta indiea (Pfieffer). Biol. Bull. 139, 502-509 (1970) Levetzow, K.G., von: Die Struktur einiger Schneckenschalen und ihre Entstehung durch typisches und atypisches Wachstum. Jena Z. Naturwiss. 66, 41-108 (1932) Luft, J.H.: Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec. 171, 369-416 (1971) Manigault, P.: Recherches sur le calcaire chez les mollusces. Phosphatase et pr6cipitation calcique. Histochimie du calcium. Ann. l'Inst. Oc6anograph., Paris 18, 331-346 (1939) Meenakshi, V.R., Blackwelder, P.L., Wilbur, K.M.! An ultrastructural study of shell regeneration in Mytilus edulis (Mollusca, Bivalvia). J. Zool. London. 171, 475-484 (1973) Millonig, G.: Further observation on a phosphate buffer for osmium solution in fixation. In: Fifth Internat. Congr. for Electron Microscopy in Philadelphia (S.S. Breese, Jr., ed.), vol. 2, P-8. New York and London: Academic Press 1962 Mueller, W.J., Brubaker, R.L., Gay, C.V., Boelkins, J.N.: Mechanisms of bone resorption in laying hens. Fed. Proc. 32, 1951-1954 (1973) Poole, A.R., Hembry, R.M., Dingle, J.T.: Cathepsin D in cartilage. The immunohistochemical demonstration of extracellular enzyme in normal and pathological conditions. J. Cell Sci. 14, 139-161 (1974) Reynolds, E.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Sabatini, D.D., Bensch, K., Barrnett, R.J.: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58 (1963) Saleuddin, A.S.M.: Electron microscopic study of the mantle of normal and regenerating Helix. Canadian J. Zool. 48, 409-416 (1970) Saleuddin, A.S.M.: Fine structure of normal and regenerated shell of Helix. Canadian. J. Zool. 49, 37-41 (1971) Saleuddin, A.S.M., Chan, W.: Shell regeneration in Helix: shell matrix composition and crystal formation. Canadian. J. Zool. 47, 110%1111 (1969) Seimiya, T., Ohki, S.: Ionic structure of phospholipid membranes, and binding of calcium ions. Biochim. biophys. Acta (Amst.) 298, 546-561 (1973) Sioli, H.: I~lber den Chemismus der Reparatur yon Schalendefekten bei Helix pomatia. Zool. Jahrb. 54, 507-534 (1935) Sminia, T.: Structure and function of blood and connective tissue cells of the fresh water pulmonate Lymnaea stagnalis studied by electron microscopy and enzyme histochemistry. Z. Zellforsch. 130, 497-526 (1972) Sminia, T., Pietersma, K., Scheerboom, J.E.M.: Histological and ultrastructnral observations on wound healing in the freshwater pulmonate Lymnaea stagnalis. Z. Zellforsch. 141, 561-573 (1973) Stang-Voss, G.: Zur Ultrastruktur der Blutzellen wirbelloser Tiere. III. Uber die Haemocyten der Schnecke Lymnea stagnalis L. (Pulmonata). Z. Zellforsch. 107, 142-156 (1970) Thyberg, J., Friberg, U.: Electron microscopic enzyme histochemical studies on the cellular genesis of matrix vesicles in the epiphyseal plate. J. Ultrastruct. Res. 41, 43-59 (1972) Thyberg, J., Moskalewski, S., Friberg, U.: Electron microscopic studies on the uptake of colloidal thorium dioxide particles by isolated fetal guinea-pig chondrocytes and the distribution of labeled lysosomes in cartilage formed by transplanted chondrocytes. Cell Tiss. Res. 156, 317-344 (1975b) Thyberg, J., Nilsson, St., Friberg, U.: Electron microscopic and enzyme cytochemical studies on the guinea pig metaphysis with special reference to the lysosomal system of different cell types. Cell Tiss. Res. 156, 273-299 (1975a) Timmermans, L.P.M.: Studies on shell formation in molluscs. Ned. J. Zool. 19, 417-523 (1969) Travis, D.F., Francois, C.J., Bonar, L.C., Glimcher, M.J.: Comparative studies of the organic matrices of invertebrate mineralized tissues. J. Ultrastruct. Res. 18, 519-550 (1967)
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Tsujii, T.: Studies on the mechanism of shell- and pearl-formation. X. The submicroscopic structure of the epithelial cells of the mantle of pearl oyster, Pteria (Pinetada) martensii (Dunker). Rep. Fac. of Fisheries, Univ. of Mie 6, 41 57 (1968) Wada, K.: Crystal growth of molluscan shells. Bull. nat. Pearl Res. Lab. 7, 703-828 (1961) Wada, K.: Nucleation and growth of aragonite crystals in the nacre of some bivalve molluscs. Biomineralization 6, 141-159 (1972) Wagge, L.E.: Amoebocytic activity and alkaline phosphatase during the regeneration of the shell in the snail Helix aspersa. Quart. J. micr. Sci. 92, 307-321 (1951) Wagge, L.E. : Quantitative studies of calcium metabolism in Helix aspersa. J. exp. Zool. 120, 311-342 (1952) Wagge, L.E., Mittler, T.: Shell regeneration in some British molluscs. Nature (Lond.) 171, 528 (1953) Watabe, N.: Studies on shell formation. XI. Crystal-matrix relationships in the inner layers of mollusc shells. J. Ultrastruct. Res. 12, 351-370 (1965) Wilbur, K.M.: Shell formation and regeneration. In: Physiology of Mollusca (K.M. Wilbur and C.W. Yong, eds.), Vol. 1, pp. 243-282. New York: Academic Press 1964 Wilbur, K.M.: Shell formation in molluscs. In: Chemical zoology (M. Florkin and B.T. Sheer, eds.), Vol. 7, pp. 103 146. New York: Academic Press 1972 Woessner, J.F., Jr.: Cartilage cathepsin D and its action on matrix components. Fed. Proc. 32, 14851488 (1973) Wolburg-Buchholz, K., Nolte, A.: Vergleichende Untersuchungen an Amoebozyten und Blasenzellen von Cepaea nemoralis L. (Gastropoda, Stylomatophora). Unterschiedliche Endozytosef~ihigkeit der Zellen. Z. Zellforsch. 137, 281-292 (1973) Wuthier, R.E.: Zonal analysis of phospholipids in the epiphyseal cartilage and bone of normal and rachitic chickens and pigs. Calcif. Tiss. Res. 8, 36-49 (1971) Wuthier, R.E.: Lipids of matrix vesicles. Fed. Proc. 35, 117 121 (1976)
Accepted June 16, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
Ultrastructural Study of the Shell-repair Membrane in the Snail, Helix pomatia L. * Anna Abolin~-Krogis Institute of Zoophysiology, University of Uppsala, Sweden
Summary. The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretion of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the mineralizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted. Key words: Shell-repair membrane - Initial calcification - Formation of crystalline structures - Amoebocytes - Helix pomatia L.
Introduction The process of shell-repair in molluscs has been extensively studied with the help of light and electron microscopes. The contributing functions of the hepatopancreas and the mantle have been thoroughly examined (Sioli, 1935; Manigault, 1939; Wagge, 1951, 1952; Fretter, 1952; Wagge and Mittler, 1953; Durning, 1957; Abolin~-Krogis, 1958, 1961, 1963a; Wada, 1961, 1972; Wilbur, 1964, 1972; Watabe, 1965; Beedham, 1965; Travis et al., 1967; Tsujii, 1968; Timmermanns, 1969; Bevelander and Nakahara, 1969; Saleuddin and Chan, 1969; Saleuddin, Send offprint requests to: Dr. Anna Abolin~-Krogis, Zoofysiologiska Institutionen Box 560, S-751 22 Uppsala 1, Sweden * This investigation was supported by a grant from the Swedish Natural Science Research Council, which is gratefully acknowledged. I am indebted to Miss Ch. Stensj6 and Mrs. E. Hellm6n for their technical assistance
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1970, 1971 ; K a p u r and G u p t a , 1970; Istin, 1970; Istin and Masoni, 1973; M e e n a k shi et al., 1973). Attention was called to the pigment granules in the mantle epithelial cells by L e v e t z o w (1932). The a u t h o r supposed that these granules m a y have some function in shell g r o w t h and regeneration. M o r e detailed examination o f the pigment granules was undertaken by Abolin~-Krogis (1963 b, 1973). The fluorescence and histochemical studies showed that the pigment granules in the a m o e b o cytes and the mantle epithelial cells are o f lipofuscin type and have characteristics in c o m m o n with lysosomes. It was suggested that the granules were apparently engaged b o t h in the binding o f calcium ions and in the catalysis o f organic substances within the repair membrane. T h e ultrastructure o f thin sectioned shell-repair m e m b r a n e s was, however, n o t satisfactorily investigated. The present study deals with the analysis o f the substructure o f this m e m b r a n e in the snail, Helix pomatia. F r o m the results obtained, it was expected that some information could be provided a b o u t the possible physiological role o f the different cellular constituents o f the calcifying m e m b r a n e in the early stages o f the regeneration. Material and Methods Twenty adult snails, Helix pomatia, weighing about 20 g, were selected for the experiments. The shell-repair membranes, 6-12h after damage, were fixed in ice-cold 2~ glutaraldehyde in 0.1 M phosphate buffer, pH7.3. Subsequent post-fixation was performed in 0.05 M phosphate buffered 1~o osmium tetroxide according to the methods of Sabatini et al. (1963) and Millonig (1962). Some of the membranes were decalcified in 0.5 M EDTA, pH 7.3 and stained en bloc with 0.05~ ruthenium red (Luft, 1971). The specimens were dehydrated in graded ethanol and embedded in Epon 812. Thin sections were double-stained with saturated uranyl acetate followed by lead citrate (Reynolds, 1963). A Siemens Elmiskop 101 at 60 or 80kV was used for the examination of thin sections. The microscope was equipped with an anticontamination device cooled with liquid nitrogen. Some intact shell-repair membranes were fixed in buffered formaldehyde and stained with 0.05~ ruthenium red. After dehydration, the membranes were enclosed in Canada balsam and studied with a light microscope. The examination of the mantle exudate, i.e., the extrapallial fluid, was undertaken with the light microscope. The smear preparations were fixed in 95% alcohol and stained with haematoxylineosin. Results The examination o f the animals with shells d a m a g e d under natural conditions showed always cracks o f the original shell on the surface o f the mantle. U n d e r experimental conditions the a u t h o r was not able to obtain the shell-repair m e m branes w i t h o u t cracks. A r o u n d the cracks and within the repair m e m b r a n e the a m o e b o c y t e s were usually present. Moreover, the liquid droplets exuded f r o m the mantle epithelium, taken up on the cover-glass, contained amoebocytes when examined with the light microscope (Fig. 1). Fig. 1. Amoebocytes within the extrapallial fluid. Haematoxylin-eosin staining of smear preparation. • 800 Fig. 2. Disintegrating amoebocyte within the shell-repair membrane. The cell organelles: Golgi complex, mitochondria and granular endoplasmic reticulum (GER) are largely destroyed. Small osmiophilic vesicles (s) and dense bodies of different opacity are visible within the cell. x 18,000
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The amoebocytes within the shell-repair membrane showed considerable variation in size and content. They were either small, irregularly-shaped, approximately 10~tm in diameter, or large and elongated, approximately 181am in diameter. Despite the observed variations in their size, they are not described as different types. The number of amoebocytes varied notably within each separate membrane. Six h after shell damage, the amoebocytes showed different degrees of disintegration (Fig. 2). Some of them were not significantly transformed and possessed a well-developed nucleus and a great number of mitochondria. The latter frequently contained small dark inclusions, possibly calcium salt (Fig. 3). The majority of the amoebocytes showed signs of more advanced degeneration. Within these ceils the mitochondria appeared dense and their cristae disrupted. Small, round corpuscles were occasionally observed within the mitochondrial matrix (Fig. 4 a, b). The Golgi complex and the granular endoplasmic reticulum (GER) were reduced. The latter consisted of vacuole-like cisternae lined by ribosomes (Fig. 4a). Fine filaments, granules and coarse aggregates were frequently encountered within the cisternae (Figs. 3, 4 b). The amount of glycogen within the amoebocytes was inconspicuous. The more interesting inclusions of the amoebocytes were the cytoplasmic dense bodies. These showed varied structure, density and size. Among the dense bodies, the characteristic lipofuscin-type pigment granules were only seldomly present. The pigment granules possessed homogeneous structure which was somewhat chattered by sectioning (Figs. 2, 6). More frequently, another type of cytoplasmic dense body was observed within the amoebocytes. These bodies were either fibrillar in structure (Fig. 7 a) or they possessed a core-containing inclusion. The core appeared to be a hard mineral formation which bulged out of its surrounding (Fig. 5). The size of these inclusions varied between 0.2 llm and 0.6 ~tm in diameter. Frequently, the above-mentioned dense bodies also possessed a bleb-like protrusion and a vacuole displaced on one side. The blebs sometimes contacted small empty vesicles (Fig. 5). Lipid droplets were usually grouped on the periphery of the dense bodies (Fig. 5). All characteristics indicated that the cytoplasmic dense bodies were comparable with cytosomes, i.e., lysosomelike bodies (Figs. 4b, 5). Small corpuscles and vesicles were often found in the immediate vicinity of the destroyed mitochondria and the dense bodies (Figs. 2, 4 b). In addition, some very large cytosomes were commonly observed within the amoebocytes. They contained variable amounts of polymorphic inclusions, in which different components could be distinguished, such as rounded globules, membranous fragments, and irregular dense material (Fig. 7 b-d). Intact, small calcium spherites, approximately 0.8 ~t in diameter, were rarely observed within Fig. 3. Mitochondria- containing groups of dense granules. Granular material and coarser aggregates within the cisternae of GER. x 30,000 Figs. 4a and b. Mitochondria showing dense matrix and reduced cristae. Round corpuscles within the mitochondria (arrows). a round, empty cisternae of GER lined by ribosomes; b dense bodies of different opacity, two of them contain crystalline inclusions (c), small osmiophilic vesicles (s) among the dense bodies and disintegrated mitochondria. Vacuoles containing fibrillar or dense granular material. • 30,000
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Fig. 5. Disintegrating amoebocyte with different sized and structured dense cytoplasmic bodies. Some of them contain a characteristic inclusion with hard core (c) and a vacuole (v). The others are surrounded by lipid droplets (/). The bleb-like protrusions of the dense bodies are often in contact with small vesicles. At the bottom, the cell membrane of the amoebocyte is disrupted and finely granular, partly filamentous material visible on both sides of the disrupted cell membrane. At some distance from the cell, coarse fibrils, small vesicles, and large aggregates (a) are present within the repair membrane, x 24,000
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Fig. 6. Part of a degenerating amoebocyte. Small vesicles with osmiophilic limiting membrane (s) are present within the cell and among the fibrillar material of the repair membrane. The amoebocyte contains two pigment granules (p). • 24,000
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Fig. 7 a - e . Cytoplasmic dense bodies within the disintegrating amoebocytes: a dense body with fibrillar structure; b dense bodies within a large cytosome showing homogeneous structure; e at the top, a pigment granule of lipofuscin-type; the other dense body contains a less dense inclusion and rounded corpuscles; d right at the top, a large cytosome containing dense bodies and whirled fibers, on the left, a dense body containing crystalline structure; e at the top, calcium spherites within the degenerating amoebocyte, a x 48,000; b - e • 32,000
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Fig. 8. Remnants of a completely destroyed amoebocyte. The nuclear membrane, dense bodies, and large autophagic vacuoles are included in a tight network of fibrils. Among the fibrils small dense vesicles (s) and large aggregates (a). x 28,000 the amoebocytes. T h e calcium spherites were identified by the concentric arrangem e n t o f their fibrillar material (Fig. 7 e). The ultrastructural study o f the shell-repair m e m b r a n e disclosed the m o s t striking observation that the a m o e b o c y t e s were engaged in the p r o d u c t i o n o f a fibrillar protein o f the repair matrix. In some sections it was possible to recognize an incontinuity in the cell m e m b r a n e o f the a m o e b o c y t e and a finely granular,
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partly filamentous material accumulated at this place, i.e., inside and outside the disrupted cell membrane (Fig. 5). At some distance from the cell the fine granular and fibrillar material appeared to be mingled with larger fibrils. The released substance seemed to undergo a fibrillar organization and a self-assembling process outside the cell. In the repair membrane among the fibrillar material granules and small corpuscles or vesicles were interspersed (Fig. 6). The polygonal or ovoid granules, approximately 0.05 gm in diameter (Fig. 9), stained positively with ruthenium red and thus revealed their proteoglycan nature (Luft, 1971; Thyberg et al., 1975b). The small vesicles were approximately 0.1 gm in diameter. They had a thick, electron-dense, osmiophilic limiting membrane (Fig. 6). Occasionally, vesicles of the same size and structure were observed within the disintegrating amoebocytes in the vicinity of destroyed dense bodies and mitochondria (Figs. 2, 4b, 6). The osmiophilic reaction implied a lipid as one of the constituents of the limiting membrane of the vesicles. Apart from the above-mentioned proteoglycan granules and small vesicles, the repair membrane contained dense bodies of different structure and size (approximately 0.2 gm-0.8 gm in diameter). Some of these bodies showed either a vesicular or fibrillar structure and appeared to be formed by aggregates of the osmiophilic vesicles (Figs. 11 a, b). Such aggregation of the vesicles was indeed observed in the repair membranes (Figs. 5, 8). The other dense bodies contained crystalline structures, which were of plate-like or cylindrical construction (Figs. 11 c, d). The bodies containing cylinder-like crystallite were apparently a more advanced form of the cytoplasmic, crystalline core-containing inclusion. Occasionally, a ramification of these crystallites was observed (Fig. 12a). After the decalcification, the organic part of the crystal became visible. It consisted of tightly twisted protein fibrils (Fig. 12b). In other cases the dense material was concentrated on the surface of the filamentous body (Fig. 13). Frequently, large groups of accumulated dense bodies and twisted fibers were observed within the repair membrane (Fig. 14). Some intact lipofuscin pigment granules (Fig. 10) and lysosome-like dense bodies were always present within the repair membrane (Fig. 8). The repair membrane also contained heavily calcified rounded and double fan-shaped organic crystalline structures (Figs. 16, 17). The ultrathin sections of these structures showed an accumulation of the vesicles and dense bodies in the middle and in a concentric ring on the periphery of these structures (Fig. 15). Within a thin section not all of the peripheral bodies were visible due to their location outside the plane of sectioning. The latter were connected to the centrally located bodies by fibrillar projections. The outline of the large crystalline structures was marked by small, dense crystallites, possibly calcium carbonate (Fig. 15). Fig. 9. Thin fibrils and granules within the repair membrane. The granules are positively stained with ruthenium red. x 32,000 Fig. 10. A lipofuscin-type pigment granule within the repair membrane, x 20,000 Fig. 11 a-d. Cytoplasmic dense bodies within the repair membrane: a - b bodies of fibrillar-vesicular type; c - d bodies containing crystalline structures, x 30,000
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Fig. 12. a Ramified crystalline structure within a disrupted dense body. b Crystalline structure after decalcification. The organic substance consists of a twisted fibrillar material, a x 30,000; b x 50,000 Fig. 13. A cross of dense fibrillar material on the surface of a filamentous body. Decalcified preparation. • 30,000 Fig. 14. Aggregate of dense cytoplasmic bodies and fibres in the repair membrane. • 25,000
The amount of proteoglycan granules was greatly reduced in the whole area occupied by the large crystalline structures. The decalcification of the large crystalline structures showed that a row of dense bodies was localized on their surface. The peripherally grouped bodies had a peculiar pattern of organized protein fibrils (Fig. 18). The large organic crystalline structures were subsequently transformed into the needle-clusters of the dendritic spherulites (Abolin~-Krogis, 1958). After decalcification, the radially oriented organic filaments of the needlecluster were visible (Fig. 19). Apparently, the calcium carbonate crystallites in these structures have the same orientation as the filaments, suggesting that the orientation of the inorganic crystals is controlled by the organic compounds.
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Fig. 15. Two large crystalline structures in the repair membrane. Accumulation of vesicles and dense bodies visible in the middle and in a concentric ring in the periphery of these structures. The peripheral bodies are connected with these centrally located by fibrillar projections (c). The outline of the large crystalline structures is marked by small, dense grains, possibly calcium carbonate crystallites, x 25,000 In the case o f the alignment o f the needles, the needle-cluster was converted into the tabular (platelike) calcium c a r b o n a t e crystal. I n the crystals with incomplete leveling o f the needles, some staining with the dyes was observed in their middle and within the concentric rings, where the organic material had n o t totally disappeared (Fig. 16). Twelve h after shell damage, the majority o f a m o e b o c y t e s within the repair m e m b r a n e appeared almost completely disintegrated. The greatest resistance
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Fig. 16. Calcium carbonate crystal of a double fan-shaped type in an intact repair membrane. Stained with r u t h e n i u m red. x 900 Fig.17. A double fan-shaped needle-cluster in an intact repair membrane. Unstained preparation. • 700 Fig. 18. Thin section of a double fan-shaped crystalline structure. After decalcification the arrangement of the dense bodies on the surface of the centrally located organic material is visible. Each of the peripheral bodies shows a peculiar arrangement of the fibrillar material around the central corpuscle. x 16,000 Fig. 19. Part of a thin section of disk-shaped crystalline structure. After decalcification the radial arrangement of the fibrillar material is visible, x 30,000 Fig. 20a and b. The tubular reticulum in the cytoplasm of the amoebocytes. The reticulum consists of: a longitudinal tubules and b rosette-like profiles. Glycogen accumulation visible in the middle of the micrograph, a x 20,000; b x 70,000
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was shown by the homogeneous dense bodies and large autophagic vacuoles. These cellular remnants were surrounded by a tight network of fibrils. Among them, dense vesicles and aggregates of these were always present (Fig. 8). The smooth-surfaced tubular reticulum described previously in the amoebocytes of connective tissue (Abolin~-Krogis, 1972), was only observed in a few cases in the amoebocytes of the repair membrane (Fig. 20a, b). The reticulum was formed by the longitudinal connecting tubules and the rosette-like profiles. The fragile structure of the tubular reticulum was apparently disrupted at the beginning of the disintegration of amoebocytes.
Discussion
The ultrastructural examination of the shell-repair membranes of the snails revealed characteristic details in the structure and composition of these membranes. As was shown with light and fluorescence microscopy (Abolin~-Krogis, 1963b, 1973) the repair membranes contained amoebocytes. The function of these cells, as was shown in this investigation, appeared to be manifold. The large autophagic vacuoles, containing different cell organelles and cracks of the original shell, suggested that the amoebocytes have a phagocytotic capacity. Phagocytosis of the amoebocytes was recently described by Sminia (1972) in the connective tissue of Lymnaea stagnalis and by Wolburg-Buchholz and Nolte (1973) in Cepaea nemoralis. Besides phagocytosis, the amoebocytes enriched the repair membranes with different repair substances and cellular constituents. The fine granular and filamentous material accumulated at the cell membrane was involved in the production of the structural protein of the shell-repair membrane. The ruthenium red positive granules, i.e., the proteoglycan granules, were also a cell product which formed a component of the repair membrane. The small vesicles, the polymorphic cytoplasmic dense bodies and the intact lipofuscin pigment granules were released from the cells. Actually, the number of lipofuscin pigment granules was markedly decreased within the disintegrated amoebocytes, whereas that of the dense bodies of lysosomal-type increased. As both of these structures showed yellow fluorescence, it was not possible to distinguish between them in the fluorescence microscope (Abolin~-Krogis, 1973). The lysosome-like dense bodies appeared to be active in the repair membranes, where their constructive and destructive capacities were needed (de Duve and Wattiaux, 1966). As stated by Dingle (1969), the intact lysosomes appeared to be extruded from the cells already in very early phases of cellular degeneration. Moreover, the dense bodies of lysosome-type were involved in the formation of a special type of granule characterized by a crystalline core. The possibility of the formation of crystalline structures characterizes the intact lipofuscin pigment granules also, as already indicated by Fawcett (1966). Thereby, no marked difference existed in the formation of crystalline structures between the dense bodies of lysosome-type and lipofuscin pigment granules. The present electron microscopical study confirmed the important fact that the vesicles and dense bodies possess the same localization as the fluorescent
Shell-repair Membrane in the Snail, Helix pomatia
L.
471
bodies and granules within the large crystalline structures, suggesting that these structures are identical in nature (Abolin~-Krogis, 1973). On the other hand, the hard core-containing bodies, characterized by weak stainability with different dyes and by their size, corresponded to b-granules described previously by Abolin~-Krogis (1961, 1963 b). They apparently represented single crystals within the repair membrane observed by X-ray examination (Abolin~-Krogis, 1968). In a later developmental stage the hard core appeared to be transformed into a cylindrical or ramified crystallite. After decalcification of the ramified crystallite the fibrillar part of it became clearly visible. It was composed of protein fibrils twisted in an unusual manner. The bodies of lower electron density appeared to be composed of fine fibrillar or vesicular material and were regarded as the earlier developmental stages of the previously described structures. The osmiophilic vesicles present within the repair membrane may likewise be attributed to a cell product. Two possibilities of their origin appear to exist. Firstly, they may originate from the disintegrated dense bodies, i.e., lysosomes, showing the common yellow fluorescence with these structures. This statement was obtained from fluorescence and electron microscopic studies. Secondly, the vesicles may originate from the fragmented cristae of the mitochondria. As showed in this investigation, the destruction of mitochondria and disappearance of the mitochondrial cristae was frequently observed. The protein-lipid complex and the enzymes of the membrane may possibly select the necessary substrate for the binding of the mineral salt. Therefore, the small vesicles within the repair membrane of the snail possibly represent the smallest unit of the membrane which was able to localize and concentrate the calcium ions in some organically bound form. The determining factor for the capture of mineral salt appears to be their lipid content, i.e., phospholipids, the chief constituent of lipids in the lipofuscin pigment granules and the membranes. The role of the lipids in the process of mineralization of the mammalian skeleton has been thoroughly examined by Irving (1976), Irving and Wuthier (1968), Wuthier (1971, 1976), and Seimiya and Ohki (1973). Within the repair membrane, besides the crystalline core-containing dense bodies, i.e., b-granules, the aggregation of the previously described vesicles induced formation of a number of additional dense bodies. The latter were also included as the organic components in the large crystalline structures and formed the central and peripheral loci of calcification. Besides the amoebocytes, the greatest part of the lipofuscin-type pigment granules and dense bodies of lysosome-type appears to be released from the mantle epithelial cells (Abolin~-Krogis, 1963a). It was shown that, after shell damage, the whole exposed mantle area appeared dull-white, caused by the loss of the pigment granules. In that way the mantle epithelial cells participated in the production of the dense bodies of the shell-repair membrane. Moreover, the mantle epithelium cells were undoubtedly also engaged in the release of proteinaceous material, polysaccharides, and lipids during the time of shell repair (Abolin~-Krogis, 1963 a). These substances extruded from the cells may aggregate and possibly induce the formation of the vesicles and dense bodies extracellularly. Of striking interest was the involvement of the amoebocytes in the production of the fibrillar protein, possibly collagenous in nature. The involvement of the amoebocytes in the synthesis of collagen was recently shown by Sminia et al.
472
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(1973). In the process of wound healing in Lymnaea stagnalis, the flattened amoebocytes which appeared 3-5 days after the incision were shown to produce collagen fibers. In the early stages of the development of repair membrane, the mature collagen fibers with periodical structure were not observed. Possibly, the experimental time in this study was too short (6 to 12 h) for the development of mature collagen. However, it can not be excluded that the elaboration of the mature collagen, with banded structure, occurs only rarely within the shell-repair membranes and the original shell. The sparsely distributed mature collagen fibers were described by Travis et al. (1967) in the prismatic and nacreous layers of the shells of some bivalves. In contrast, Stang-Voss (1970) and WalburgBuchholz and Nolte (1973) reasoned that the vesicular cells ("Blasenzellen", i.e., Leydig cells) may have an important role in the collagen production in molluscs. It appears reasonable to believe that in the process of shell repair materials from different sources were utilized. More detailed analyses were performed with the mantle and the hepatopancreas, which contain a large pool of quickly exchangeable calcium ions (Sioli, 1935; Manigault, 1939; Wagge, 1951; Wagge and Mittler, 1953; Fretter, 1952; Durning, 1957; Abolin~-Krogis, 1961, 1963a; Tsujii, 1968; Istin, 1970; Istin and Masoni, 1973). How large the additional part from the intact shell may be is not sufficiently experimentally proven (Sioli, 1935; Wagge, 1951). Generally, very little is known about the demineralization mechanism of the inorganic part of the shell. It is believed that the demineralization is affected by the acids released from the lytic cells (Mueller et al., 1973). The tubular system found in the amoebocytes may possibly represent a specific system for providing acids or hydrogen ions. A structure similar to the tubular reticulum in amoebocytes was described in the acid-secreting cells of the frog stomach by Forte and Forte (1970). Obviously, only after removal of calcium salt may the organic substances be attacked by the lysosomal enzymes. The extracellular release of the lysosomal enzymes within the repair membrane appears very possible. The experimental results obtained by Poole et al. (1974) within the cartilage of the chick confirmed such a possibility. Which of the previously mentioned calcium supplies were utilized, and to what extent, in each separate case, apparently depends on the general metabolism and the physiological condition of the regenerating animal. Chan and Saleuddin (1974) assumed that the general calcium supply in the body of snails appears to be the original shell, which may be used in case of need. Unfortunately, their work contains many confusing points and the results obtained by the authors were hardly comparable with the findings of other investigators. A comparison of the mineralizing processes in the early stages of the calcification of the shell-repair membrane of the snail with the corresponding calcifying processes of the epiphyseal cartilage of mammals (Anderson, 1969; Anderson and Sajdera, 1976; Bonucci, 1969, 1970; Thyberg and Friberg, 1972; Thyberg et al., 1975a, b) showed many common features, in some cases even a striking similarity (Anderson, 1969, Fig. 8). Naturally, the formative cells in the cartilage were of another type, i.e., the chondrocytes, but the released materials of the activated cells were in both cases closely similar if not identical. The repair membrane of the snail and the calcifying cartilage matrix of mammals contain protein-
Shell-repair Membrane in the Snail, Helix pomatia L.
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aceous nonperiodic filaments, proteoglycan granules, and cytoplasmic dense bodies, i.e., matrix vesicles. Moreover, Anderson (1969), Bonucci (1969), and Thyberg et al. (1975a) suggested that the matrix vesicles were derived from the cells and that they play a role in the initiation of calcification. The authors also considered that the matrix vesicles, at least some of them, were lysosomes and may have the function of hydrolyzing the organic parts of the cartilage. On the other hand, different opinions have been expressed concerning the initial crystal formation in the cartilage matrix. Thyberg et al. (1975 b) supposed that the mineral crystals were first deposited in close vicinity to the membranebounded matrix vesicles, whereas Anderson (1969), Anderson and Sajdera (1976), BonucCi (1970), Bernard (1972), and Eisenmann and Glick (1972) supported the view that the first mineral crystals were laid down within these structures. Evidence concerning the initial crystal formation in the repair membrane of the snail, obtained in this study, appears to be more complicated. Supposing that the protein-phospholipid complex of the small vesicles is engaged in the binding of the mineral ions, the latter would accumulate on the surface of these structures. At a later stage of development, when the vesicles aggregated and formed dense bodies, they were both engaged in the organization of the organic material around the primary loci of mineralization. At this time the captured inorganic material appeared located within a less dense organic substance, as in a compartment. This stage of mineralization was very clearly shown in the fluorescence microscope (Abolin~-Krogis, 1973). The mineral deposition within the large crystalline structures appears somewhat more complicated. In these structures, two or more separate active areas were simultaneously recognizable, the central intracrystalline calcifying area and the peripheral one. The latter is characterized by the accumulation of the calcifying centra on the periphery of the large crystalline structures. In both areas, a forced packing of the fibrillar material around the active centra apparently takes place. The peripheral growth of the crystalline structure may have its own nucleating mechanism. It is only in the later developmental stage that the separate active centra within the large crystalline structures possibly conjugate and form the needle-clusters. The latter finally were transformed into the plate-like calcium carbonate crystals (Abolin~-Krogis, 1958). The lysosomal enzymes appeared firstly to take part in the reduction of the proteoglycan granules (Woessner, 1973), whereas the fibrillar material was concentrated around the calcifying centra. It does not seem improbable that the proteoglycans afforded protection against the accumulation and organization of the protein fibrils around these centra. The loss of the protector suddenly induced the organization of the fibrillar protein and the mineralization could proceed. However, it can not be excluded that the conjugation of the proteoglycan granules with the fibrillar protein occurred before the calcification of the whole complex started. The complete reduction of the amount of organic material within the crystalline structures occured in more advanced stages of mineralization. Thus, the mature calcium carbonate crystal appeared to be located within a compartment formed by the organic sheets. Summarizing the results obtained in this study, the fact appears once more to be verified that different cell structures, small vesicles and dense bodies, were engaged in the formation of the initial calcifying centra. Some of these cellular
474
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constituents were liberated from the degenerated and disintegrated amoebocytes, w h i c h w e r e also e n g a g e d in t h e p r o d u c t i o n o f t h e f i b r i l l a r p r o t e i n . I n spite o f t h e fact, t h a t t h e w h o l e o r g a n i c m a s s o f t h e d e s t r o y e d a m o e b o c y t e s w a s a d d e d t o the repair membrane, the bulk of the substances needed for the complete restor a t i o n o f t h e d a m a g e d shell a p p a r e n t l y c o m e f r o m o t h e r s o u r c e s o f t h e a n i m a l body.
References Abolin~-Krogis, A.: The morphological and chemical characteristics of organic crystals in the regenerating shell of Helix pomatia L. Acta Zool. 39, 19-38 (1958) Abolin~-Krogis, A.: The histochemistry of the hepatopancreas of Helix pomatia (L.) in relation to the regeneration of the shell. Ark. Zool. 13, 159-201 (1961) Abolin~-Krogis, A.: The histochemistry of the mantle of Helix pomatia (L.) in relation to the repair of the damaged shell. Ark. Zool. 15, 461-474 (1963a) Abolin~-Krogis, A.: On the protein stabilizing substances in the isolated b-granules and in the regenerating membranes of the shell of Helix pomatia (L.). Ark. Zool. 15, 475-484 (1963 b) Abolin~-Krogis, A.: Shell regeneration in Helix pomatia with special reference to the elementary calcifying particles. Symp. Zool. Soc. London No. 22, 75-92 (1968) Abolinw A.: The tubular endoplasmic reticulum in the amoebocytes of the shell-regenerating snail, Helix pomatia L. Z. Zellforsch. 128, 58-68 (1972) Abolin~-Krogis, A.: Fluorescence and histochemical studies of the calcification-initiating lipofuscin type pigment granules in the shell-repair membrane of the snail, Helix pomatia L. Z. Zellforsch. 142, 205-221 (1973) Anderson, H.C.: Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59-72 (1969) Anderson, H.C., Sajdera, S.W.: Calcification of rachitic cartilage to study matrix vesicle function. Fed. Proc. 35, 148-153 (1976) Beedham, G.E.: Repair of the shell in species of Anodonta. Proc. Zool. Soc. London 145, 107 124 (1965) Bernard, G.W.: Ultrastructural observations of initial calcification in dentine and enamel. J. Ultrastruct. Res. 41, 1-17 (1972) Bevelander, G., Nakahara, H.: An electron microscope study of the formation of the nacreous layer in the shell of certain bivalve molluscs. Calcif. Tiss. Res. 3, 84-92 (1969) Bonucci, E.: Further investigation on the organic/inorganic relationship in calcifying cartilage. Calcif. Tiss. Res. 3, 38-54 (1969) Bonucci, E.: Fine structure and histochemistry of "calcifying globules" in epiphyseal cartilage. Z. Zellforsch. 103, 192-217 (1970) Chan, W., Saleuddin, A.S.M.: Evidence that Otala lactea (Miiller) utilizes calcium from the shell. Proc. malac. Soc. London 41, 195-200 (1974) Dingle, J.T.: The extracellular secretion oflysosomal enzymes. In: Lysosomes in biology and pathology, Vol. 2 (J.T. Dingle and H.B. Fell, eds.), pp. 421-436. Amsterdam-London: North-Holland Publ. 1969 Durning, W.C.: Repair of a defect in the shell of the snail Helix aspersa. J. Bone J. Surg. A39, 377-393 (1957) Duve, C. de, Wattiaux, R.: Functions of lysosomes. Ann. Rev. Physiol. 28, 435-492 (1966) Eisenmann, D.R., Glick, P.L.: Ultrastructure of initial crystal formation in dentin. J. Ultrastruct. Res. 41, 18-28 (1972) Fawcett, D.W.: The cell its organelles and inclusions. In: An atlas of fine structure, pp. 281 284. Philadelphia and London: Saunders Comp. 1966 Forte, T.M., Forte, J.G.: Histochemical staining and characterization of glycoproteins in acidsecreting cells of frog stomach. J. Cell Biol. 47, 437-452 (1970) Fretter, V.: Experiments with p32 and 1TM on species of Helix, Arion and Agriolimax. Quart. J. micr. Sci. 93, 133-146 (1952)
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Irving, J.T.: Interrelations of matrix lipids, vesicles and calcification. Fed. Proc. 35, 109-111 (1976) Irving, J.T., Wuthier, R.E.: Histochemistry and biochemistry of calcification with special reference to the role of lipids. Clin. Orthop. Res. 56, 237-260 (1968) Istin, M.: R61e du manteau dans le m6tabolisme du calcium chez les lamellibranches. Bull. d'Inform. Sci. et Techn. du Comm. ~i l'Energie Atomique. No 144, p. 53-80 (1970) Istin, M., Masoni, A.: Absorption et redistribution du calcium dans le manteau des lamellibranches en relation avec la structure. Calcif. Tiss. Res. 11, 151-162 (1973) Kaput, S.P., Gupta, A.S.: The role of amoebocytes in the regeneration of shell in a land pulmonate, Eupleeta indiea (Pfieffer). Biol. Bull. 139, 502-509 (1970) Levetzow, K.G., von: Die Struktur einiger Schneckenschalen und ihre Entstehung durch typisches und atypisches Wachstum. Jena Z. Naturwiss. 66, 41-108 (1932) Luft, J.H.: Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec. 171, 369-416 (1971) Manigault, P.: Recherches sur le calcaire chez les mollusces. Phosphatase et pr6cipitation calcique. Histochimie du calcium. Ann. l'Inst. Oc6anograph., Paris 18, 331-346 (1939) Meenakshi, V.R., Blackwelder, P.L., Wilbur, K.M.! An ultrastructural study of shell regeneration in Mytilus edulis (Mollusca, Bivalvia). J. Zool. London. 171, 475-484 (1973) Millonig, G.: Further observation on a phosphate buffer for osmium solution in fixation. In: Fifth Internat. Congr. for Electron Microscopy in Philadelphia (S.S. Breese, Jr., ed.), vol. 2, P-8. New York and London: Academic Press 1962 Mueller, W.J., Brubaker, R.L., Gay, C.V., Boelkins, J.N.: Mechanisms of bone resorption in laying hens. Fed. Proc. 32, 1951-1954 (1973) Poole, A.R., Hembry, R.M., Dingle, J.T.: Cathepsin D in cartilage. The immunohistochemical demonstration of extracellular enzyme in normal and pathological conditions. J. Cell Sci. 14, 139-161 (1974) Reynolds, E.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Sabatini, D.D., Bensch, K., Barrnett, R.J.: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58 (1963) Saleuddin, A.S.M.: Electron microscopic study of the mantle of normal and regenerating Helix. Canadian J. Zool. 48, 409-416 (1970) Saleuddin, A.S.M.: Fine structure of normal and regenerated shell of Helix. Canadian. J. Zool. 49, 37-41 (1971) Saleuddin, A.S.M., Chan, W.: Shell regeneration in Helix: shell matrix composition and crystal formation. Canadian. J. Zool. 47, 110%1111 (1969) Seimiya, T., Ohki, S.: Ionic structure of phospholipid membranes, and binding of calcium ions. Biochim. biophys. Acta (Amst.) 298, 546-561 (1973) Sioli, H.: I~lber den Chemismus der Reparatur yon Schalendefekten bei Helix pomatia. Zool. Jahrb. 54, 507-534 (1935) Sminia, T.: Structure and function of blood and connective tissue cells of the fresh water pulmonate Lymnaea stagnalis studied by electron microscopy and enzyme histochemistry. Z. Zellforsch. 130, 497-526 (1972) Sminia, T., Pietersma, K., Scheerboom, J.E.M.: Histological and ultrastructnral observations on wound healing in the freshwater pulmonate Lymnaea stagnalis. Z. Zellforsch. 141, 561-573 (1973) Stang-Voss, G.: Zur Ultrastruktur der Blutzellen wirbelloser Tiere. III. Uber die Haemocyten der Schnecke Lymnea stagnalis L. (Pulmonata). Z. Zellforsch. 107, 142-156 (1970) Thyberg, J., Friberg, U.: Electron microscopic enzyme histochemical studies on the cellular genesis of matrix vesicles in the epiphyseal plate. J. Ultrastruct. Res. 41, 43-59 (1972) Thyberg, J., Moskalewski, S., Friberg, U.: Electron microscopic studies on the uptake of colloidal thorium dioxide particles by isolated fetal guinea-pig chondrocytes and the distribution of labeled lysosomes in cartilage formed by transplanted chondrocytes. Cell Tiss. Res. 156, 317-344 (1975b) Thyberg, J., Nilsson, St., Friberg, U.: Electron microscopic and enzyme cytochemical studies on the guinea pig metaphysis with special reference to the lysosomal system of different cell types. Cell Tiss. Res. 156, 273-299 (1975a) Timmermans, L.P.M.: Studies on shell formation in molluscs. Ned. J. Zool. 19, 417-523 (1969) Travis, D.F., Francois, C.J., Bonar, L.C., Glimcher, M.J.: Comparative studies of the organic matrices of invertebrate mineralized tissues. J. Ultrastruct. Res. 18, 519-550 (1967)
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Tsujii, T.: Studies on the mechanism of shell- and pearl-formation. X. The submicroscopic structure of the epithelial cells of the mantle of pearl oyster, Pteria (Pinetada) martensii (Dunker). Rep. Fac. of Fisheries, Univ. of Mie 6, 41 57 (1968) Wada, K.: Crystal growth of molluscan shells. Bull. nat. Pearl Res. Lab. 7, 703-828 (1961) Wada, K.: Nucleation and growth of aragonite crystals in the nacre of some bivalve molluscs. Biomineralization 6, 141-159 (1972) Wagge, L.E.: Amoebocytic activity and alkaline phosphatase during the regeneration of the shell in the snail Helix aspersa. Quart. J. micr. Sci. 92, 307-321 (1951) Wagge, L.E. : Quantitative studies of calcium metabolism in Helix aspersa. J. exp. Zool. 120, 311-342 (1952) Wagge, L.E., Mittler, T.: Shell regeneration in some British molluscs. Nature (Lond.) 171, 528 (1953) Watabe, N.: Studies on shell formation. XI. Crystal-matrix relationships in the inner layers of mollusc shells. J. Ultrastruct. Res. 12, 351-370 (1965) Wilbur, K.M.: Shell formation and regeneration. In: Physiology of Mollusca (K.M. Wilbur and C.W. Yong, eds.), Vol. 1, pp. 243-282. New York: Academic Press 1964 Wilbur, K.M.: Shell formation in molluscs. In: Chemical zoology (M. Florkin and B.T. Sheer, eds.), Vol. 7, pp. 103 146. New York: Academic Press 1972 Woessner, J.F., Jr.: Cartilage cathepsin D and its action on matrix components. Fed. Proc. 32, 14851488 (1973) Wolburg-Buchholz, K., Nolte, A.: Vergleichende Untersuchungen an Amoebozyten und Blasenzellen von Cepaea nemoralis L. (Gastropoda, Stylomatophora). Unterschiedliche Endozytosef~ihigkeit der Zellen. Z. Zellforsch. 137, 281-292 (1973) Wuthier, R.E.: Zonal analysis of phospholipids in the epiphyseal cartilage and bone of normal and rachitic chickens and pigs. Calcif. Tiss. Res. 8, 36-49 (1971) Wuthier, R.E.: Lipids of matrix vesicles. Fed. Proc. 35, 117 121 (1976)
Accepted June 16, 1976
