Arch. Histol. Cytol., Vol. 53, No. 4 (1990) p. 351-369

Lectin the

Cytochemistry

Papillary

Hiroaki Department

NAKAMURA

Layer and

Hidehiro

on the in the

Stratum

Rat

Incisor

Intermedium Enamel

and

Organ

OZAWA

of Oral Anatomy, Niigata University School of Dentistry, Niigata, Japan

Received January 16, 1990

Summary. Lectin cytochemistry was carried out to elucidate the role of stratum intermedium cells and papillary layer cells in amelogenesis, especially in the process of sugar incorporation and on membrane characteristics according to their cytodifferentiation. Regarding the lectin-reaction on the plasma membrane, little or at best a weak reaction of Con A, UEA-I, PNA, MPA and WGA was seen in stratum intermedium cells from the late differentiation stage to the early secretory stage of ameloblasts. Lectinstainability in the stratum intermedium increased in accordance with the cytodifferentiation of ameloblasts. At the active secretory stage of ameloblasts, lectins intensely stained the plasma membranes of stratum intermedium cells. The plasma membranes of papillary layer cells at both stages of ruffle-ended and smoothended ameloblasts were stained by same lectins as well. The results therefore suggest that: 1) stratum intermedium cells bring about changes in the glycolipids and glycoproteins of their plasma membranes in accordance with the cytodifferentiation of ameloblasts; 2) they regulate the transport of mineral and/or organic materials between ameloblasts and extracellular fluid via highly charged plasma membranes generated by glycocalyx; 3) the cell-cell interaction of stratum intermedium cells with ameloblasts, in which carbohydrateprotein (endogenous lectin) interaction plays a significant role, is important for the cytodifferentiation of these cells. Regarding the papillary layer cells, the results suggest that they also regulate the transport of minerals by their charged plasma membranes and participate in the removal of the enamel matrix.

tance to mechanical forces and the maintenance of the shape of the developing enamel organ by their well-developed desmosomes (MATTHIESSENand M(LLGARD,1973). Stratum intermedium cells also play a role in the transport of mineral and organic materials for the formation of the enamel matrix and/or act as a barrier between the extracellular fluid and ameloblasts (WILLIAMS, 1896; MARSLAND, 1951). Additionally, they have been reported to have several characteristics, e.g., being rich in mitochondria and microvilli, which are commonly found in cells participating in the transport function (KALLENBACH, 1978; MATTHIESSEN and R0MERT, 1980). Papillary layer cells have been thought to partici-

Stratum intermedium cells have been reported as cells differentiating into preameloblasts (WALDEYER, 1871; TEN CATS, 1961) or participating in the formation of stellate reticulum cells (THOMAS, 1925; HUNT and PAYNTER, 1963). They are involved in the resis351

pate in the removal of organic components and water from the enamel matrix (MARSLAND,1952). They are also characterized by a well-developed mitochondria and microvilli, suggesting the active intracellular transport of ions, water or other substances (KALLENBACH, 1966; ELWOODand BERNSTEIN, 1968; GARANT and NALBANDIAN,1968; REITH, 1970). Stratum intermedium cells and papillary layer cells have been histocytochemically characterized by their membrane-bound enzymes, e.g., ALPase (TEN LATE, 1962; AOYAMA,1970; KURAHASHI and YOSHIKI,1972; DEPORTERand TEN CATE,1976; TAKANO et al., 1986), Ca-ATPase (TAKANO et al., 1986; SASAKI and GARANT, 1986) and Na-K-ATPase (GARANT et al., 1987). These membrane-bound enzymes are thought to participate in the active transport of ions and organic materials for amelogenesis, suggesting that both stratum intermedium cells and papillary layer cells play a role in the transcellular transport mechanism. Glycocalyx, originating in glycolipids and membrane-bound glycoproteins, is known to change according to the differentiation of cells (BRABEC et

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al., 1980; ZIESKE and BERNSTEIN, 1982). Recently the interactions between carbohydrates and their specific endogenous lectins have been suggested to play a major role in cell-cell recognition (GABIUS et al., 1987). Thus, glycocalyx could also be an important structure for cell-cell recognition, triggering subsequent cell-cell interaction and cell differentiation. Lectin histocytochemistry is a method commonly used for the investigation of carbohydrates and/or glycocalyx (DAMJANOV,1987). Lectin has sometimes been used in the enamel organ (NAKAI et al., 1985; AKITA et al., 1988; NANCI et al., 1989), but the available reports fail to discuss in detail both the process of sugar incorporation as well as the role of glycocalyx. This study was designed to elucidate the localization of carbohydrate residues-mainly that on the plasma membranes of stratum intermedium cells and papillary layer cells-at the electron microscopic level using HRP-conjugated lectins, and thereby clarify the roles of these cells in the amelogenesis of the rat incisor.

MATERIALS

AND METHODS

Mandibles of male Wistar rats (100 g) were used for cytochemical observation. The rats were anaesthetized with nembutal and perfused through the left ventricle, first with Ringer's solution, and then with 2% paraformaldehyde and 2.5% glutaraldehyde in

Table

1.

Lectins

used

in the present

Man: mannose, Glc: glucose, G1cNAc: N-acetylglucosamine,

0.05 M sodium cacodylate buffer (pH 7.3) for 10 min. The mandibles were dissected, immersed in the same fixative for 2 h and decalcified in 4.13% EDTA for 2 weeks at 4C. Following this, Microslicer-and Cryostat-sections about 50 pm in thickness were obtained. The sections were rinsed overnight with PBS containing 0.02% saponin in order to allow the lectins to thoroughly penetrate into the cell organelle, and then incubated in the following HRP-conjugated lectin solution for 24-48 h at 4'C: 10 jig/ml Con A (Concanavalin A); 10 jig/ml UEA-I (Ulex europeus agglutinin I); 20,ug/ml PNA (Peanut agglutinin); 5 ug/ml WGA (Wheat germ agglutinin) (Seikagaku Kogyo Co., Ltd.); and 10ug/ml MPA (Maclura pomifera agglutinin) (E. Y. Laboratories, Inc.) (Table 1). The sections were washed with PBS and then refixed with 2.5% glutaraldehyde. Once rinsed with PBS and 0.05 M Tris-HC1 buffer (pH 7.6), they were immersed in DAB solution (0.05% Diaminobenzidine in 0.05 M Tris-HC1 buffer, pH 7.6) first, and then in DAB-H202 solution for 10 min at room temperature. They were postfixed with 1 % 0s04 in 0.1 M phosphate buffer (pH 7.3) for 1h at 4C. After dehydration in a graded acetone series, the sections were embedded in Epok 812. Ultrathin sections were obtained using a Porter-Blum MT-1 and stained with lead citrate. The samples thus obtained were observed under a Hitachi H-500 electron microscope at an accelerating voltage of 75 kV. Controls. Some sections were immersed in DAB and DAB-H202 solution without incubation in HRPconjugated lectin. Non-specific HRP-binding was

study

Fuc: fucose, Gal: galactose, NANA: N-acetylneuraminic

Ga1NAC: acid.

N-acetylgalactosamine,

Lectin

examined by incubating the sections with 1 purpurogallin/ml of native HRP. Specificity of the binding was tested by incubating the sections with inhibitory sugars: 0.1 M a-methyl-D-mannoside for Con A; 0.1 M L-fucose for UEA-I; 0.1 M galactose for PNA; 0.1 or 0.5 M a-D-melibiose for MPA; 0.1 or 0.5 M Nacetyl-D-glucosamine for WGA (Table 1).

RESULTS Light microscopic

observations

From the late differentiation stage to the early secretory stage of ameloblasts, stratum intermedium cells were visually distinguishable from ameloblasts, stel-

Cytochemistry

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Organ

353

late reticulum or outer enamel epithelium. Ameloblasts were columnar in shape and their nuclei situated in the proximal pole of the cytoplasm. At the distal end of the ameloblasts, Tomes' processes were developing in accordance with their cytodifferentiation into secretory ameloblasts, and stratum intermedium cells were seen on the proximal end. In these stages, no lectin-reaction was found in the stratum intermedium cells although a reaction was detectable in ameloblasts, stellate reticulum cells and outer enamel epithelium (Fig. 1a). However, with the progress of the secretory stage of ameloblasts, the lectin-reaction gradually appeared in the stratum intermedium cells. Ameloblasts at their active secretory stage were tall and possessed well-developed Tomes' processes. Their nuclei were found in the proximal part and the

a

b

Fig. 1. Light micrographs of HRP-Con A-stained sections. a. From the late differentiation stage to the early secretory stage of ameloblasts (Am), the reaction in stratum intermedium cells (SI) is merely visible. X 370. b. Reaction observed in stratum intermedium cells (SI) at the active secretory stage of ameloblasts (Am). x 370

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area of mitochondria accumulation positioned proximal to the nucleus. Cuboidal stratum intermedium cells lined up on the proximal end of secretory ameloblasts. At this stage, a stronger lectin-reaction was detected in the stratum intermedium cells rather than the other cells in the enamel organ (Fig. ib). The binding patterns of lectins used in this study (Con A, UEA-I, PNA, MPA and WGA) were similar with one another. Electron

microscopic

observations

1. Lectin-reaction on the plasma membrane From the late differentiation stage to the early secretory stage of ameloblasts, stratum intermedium cells, roughly polyhedral in shape, were located at the proximal end of ameloblasts and formed two or three lines. They were connected with each other, ameloblasts and stellate reticulum cells by desmosomes and gap junctions. Though a few mitochondria were seen in their cytoplasm, other cell organelles were not well-developed. In these stages, a lectin-reaction was found on the plasma membrane of the outer enamel epithelium and stellate reticulum. However, the lectin-reaction on the plasma membrane of stratum intermedium cells was either slight or nonexistent (Figs. 2a, 3a, 4a, 5a, 6a). As their cytodifferentiation advanced, the lectin-reaction first appeared on the plasma membrane facing the stellate reticulum cells and gradually spread along their plasma membrane. At the active secretory stage of the ameloblasts, stratum intermedium cells lined up along their proximal ends. These were polyhedral, and interconnected with each other, ameloblasts and stellate reticulum cells by desmosomes and gap junctions. They developed numerous microvilli and possessed mitochondria, rough endoplasmic reticulum and a well-developed Golgi apparatus in their cytoplasm. At this stage, a strong lectin-reaction was detected on the plasma membrane of the stratum intermedium cells (Figs. 2b, 3b, 4b, 5b, 6b). Stainability tended to be most intense on the plasma membrane of microvilli and less intense on the region facing ameloblasts. The lectinreaction was also positive in coated pits and coated vesicles of stratum intermedium cells (Fig. 5c). No difference was found in any binding patterns among the various lectins. At the maturation stage of ameloblasts, stratum intermedium cells, stellate reticulum cells and outer enamel epithelium turned into irregularly shaped polygonal cells, called papillary layer cells. These three types of cells were no longer discernible as separate layers. Papillary layer cells were linked

with each other and ameloblasts by well-developed desmosomes and gap junctions. Mitochondria accumulated mainly in the proximal part facing the blood capillary. Rough endoplasmic reticulum and a well-developed Golgi apparatus were found in the supranuclear region. Lectin-reaction was detected on the plasma membrane of papillary layer cells at the stage of ruffle-ended ameloblasts as well as smoothended ameloblasts (Figs. 7a, 8a, 9a, 10a, ha). No difference was found in the stainability of each lectin. The reaction on the plasma membrane tended to be more intense on microvilli than on the region in contact with ameloblasts. The reaction was also positive in coated pits and coated vesicles of papillary layer cells (Figs. 7c, lOc, lic). 2. Lectin-reaction in the cell organelle In the cell organelle of stratum intermedium cells at the stage of secretory ameloblasts, Con A, which binds to glucose and mannose residues, reacted in the nuclear envelope, rough endoplasmic reticulum and the Golgi apparatus (Fig. 2c). PNA binding to galactose residue and WGA binding to N-acetylglucosamine and N-acetylneuraminic acid residues reacted in the Golgi apparatus (Figs. 4c, 6c). WGA reaction was also positive in the nuclear pores (Fig. 6c). However, no UEA-I or MPA reaction was recognized in the cell organelle of stratum intermedium cells. In papillary layer cells at the maturation stage of ameloblasts, a Con A reaction was detected in the nuclear envelope, rough endoplasmic reticulum and the Golgi apparatus (Fig. 7b). UEA-I binding to fucose residue also reacted in the nuclear envelope (Fig.8b), but its binding pattern was different from Con A. Although a Con A reaction was found continuously along the nuclear envelope, the detection of UEA-I reaction was irregular. WGA reacted in the Golgi apparatus and nuclear pores (Fig. lib). No PNA or MPA reaction was seen in the cell organelle of papillary layer cells. 3. Reaction in the extracellular space From the late differentiation stage to the early secretory stage of ameloblasts, the space between stratum intermedium cells narrowed. The extracellular space between stratum intermedium cells widened slightly at the secretory stage of ameloblasts. In these stages, no reaction was detected in the extracellular space. At the maturation stage of ameloblasts, the extracellular space between papillary layer cells was occupied by interdigitating microvilli arising from the lateral cell membranes. At this stage, matrix-like

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Cytochemistry

in Rat Enamel

Organ

355

a

b

c

Fig. 2. Electron micrographs of HRP-Con A-stained sections. a. Late differentiation stage of ameloblasts (Am). No Con A reaction is seen in stratum intermedium cells (SI) x6,100 b. At the secretory stage of ameloblasts (Am), Con A reactions are observed in the nuclear envelopes and the plasma membranes of stratum intermedium cells (SI). x 6,300. c. Highly magnified stratum intermedium cell at the secretory stage of ameloblasts Con A reactions detected in the nuclear envelope (arrowheads), rough endoplasmic reticuli (rER), the Golgi apparatus (Go) and the plasma membrane. N nucleus. x 18,000

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a

b Fig. 3. Electron micrographs of HRP-UEA-I-stained sections a. Few UEA-I reaction seen in stratum intermedium cells (SI) at the late differentiation stage of ameloblasts (Am) x 6,200 b. UEA-I reaction was observed on the plasma membranes of stratum intermedium cells (SI) at the secretory stage of ameloblasts (Am) x 5,500

Lectin

Cytochemistry

in Rat

Enamel

Organ

357

a

b

C

Fig. 4. Electron micrographs of HRP-PNA-stained sections. a. Late differentiation stage (Am) PNA reaction is weak in stratum intermedium cells (SI) x6,300 b. Secretory stage (Am) PNA reaction is intense on the plasma membranes of stratum intermedium cells (SI) x magnified stratum intermedium cell at the secretory stage of ameloblasts PNA reaction is Golgi apparatus (Go) and the plasma membrane N nucleus. X22,000

of ameloblasts of ameloblast 6,300 c. Highly observed in the

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a

b

c

Fig. 5. Electron micrographs of HRP-MPA-stained sections a. Late differentiation stage of ameloblasts (Am) A weak MPA reaction in stratum intermedium cells (SI) is seen x 6,100 b. Secretory stage of ameloblasts (Am), MPA reaction was intense on the plasma membranes of stratum intermedium cells (SI) x 5,900 c. Highly magnified stratum intermedium cell (SI) at the secretory stage of ameloblasts (Am) MPA reaction observed on the coated pits and vesicle (arrowheads) x 21,000

Lectin

Cytochemistry

in Rat Enamel

Organ

359

a

b

C

Fig. 6. Electron micrographs of HRP WGA stained sections. a. Late differentiation stage of ameloblasts (Am) WGA reaction is weak in stratum mtermedium cells (SI) X5,900 b. Secretory stage of ameloblasts (Am) Note the WGA intense reaction on the plasma membranes of stratum itermedium cells (SI) x 5,900 c. Highly magnified stratum mtermedium cell at the secretory stage of ameloblasts. WGA reactions can be seen in the nuclear pores (arrowheads) and the Golgi apparatus (Go) N nucleus. x 15,000

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a

b

c

Fig. 7. Electron micrographs of HRP-Con A-stained sections. a. At the maturation stage of ameloblasts (Am), Con A reactions are seen in the nuclear envelopes and the plasma membranes of papillary layer cells (PL). BC blood capillary. X4,300. b. Highly magnified papillary layer cell at the maturation stage. Con A reactions observed in the nuclear envelope (arrowheads), rough endoplasmic reticuli (rER), the Golgi apparatus (Go) and the plasma membrane. N nucleus. X 18,000. c. Matrix-like substance (asterisk) detected between the papillary layer cells (PL). A WGA reaction also observed on the coated pit and vesicle (arrowheads). X 16,000

Lectin

Cytochemistry

in Rat Enamel

Organ

361

a

b

c

Fig. 8. Electron micrographs of HRP-UEA-I-stained sections. a. At the maturation stage of ameloblasts (Am), an UEA-I reaction is observable on the plasma membranes of papillary layer cells (PL). BC blood capillary. x6,100. b. Highly magnified papillary layer cell at the maturation stage. UEA-I reaction is sporadically observed in the nuclear envelope (arrowheads). N nucleus. X21,000. c. Matrix-like substance (asterisk) detected between the papillary layer cells (PL). x 21,000

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a

b Fig. 9. Electron micrographs of HRP-PNA-stained sections. a. At the maturation stage of ameloblasts (Am), a PNA reaction is observable on the plasma membranes of papillary layer cells (PL). BC blood capillary. x 4,900. b. Matrix-like substances (asterisks) detected between the papillary layer cells (PL). X 22,000

Lectin

Cytochemistry

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a

b Fig. 10. Electron micrographs of HRP MPA-stained sections a. MPA reaction seen on the plasma membranes of papillary layer cells (PL) at the maturation stage of ameloblasts (Am) BC blood capillary x 6,100 b. Matrix-like substances (asterisks) detected between the papillary layer cells (PL) MPA reaction also observed on the coated pits and vesicles (arrowheads) x 13,000

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a

b

C

Fig. 11. Electron micrographs of HRP-WGA-stained sections. a. WGA reaction on the plasma membranes of papillary layer cells (PL) at the maturation stage of ameloblasts (Am). BC blood capillary. X4,400. b. Highly magnified papillary layer cell at the maturation stage. WGA reactions can be observed in the nuclear pores (arrowheads) and the Golgi apparatus (Go). N nucleus. X 24,000. c. Matrix-like substance (asterisk) detected between the papillary layer cells (PL). WGA reaction also observed on the coated pits and vesicle (arrowheads). X 21,000

Lectin

substances which resembled detected by Con A, UEA-I, (Figs. 7c, 8c, 9b, lob, llc) in The stainability by PNA and compared to other lectins.

stippled-materials were PNA, MPA and WGA the extracellular space. MPA was more intense

Controls No reactions were detected in those sections that were incubated with native HRP or without lectin. In the sections incubated with inhibitory sugar, the lectin-reaction was either eliminated or greatly diminished.

Cytochemistry

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DISCUSSIONS 1. Lectin-reaction in the cell organelle of stratum intermedium cells and papillary layer cells The lectin-staining pattern in stratum intermedium cells and papillary layer cells resulting from this study suggests that mannose and glucose are incorporated in the nuclear envelope, rough endoplasmic reticulum and the Golgi apparatus. N-acetylglucosamine and/or N-acetylneuraminic acid are, in their turn, transferred in the Golgi apparatus. In addition, galactose is transferred in the Golgi apparatus of stratum intermedium cells and fucose is incorporated in the nuclear envelope of papillary layer cells.

a b c Fig. 12. A schematic diagram of a lectin-reaction at differentiation (a), secretory (b) and maturation (c) stages of ameloblasts. a. Few reaction in stratum intermedium cells (SI) at the differentiation stage of ameloblasts (Am). b. Intense reaction in stratum intermedium cells (SI) at the active secretory stage of ameloblasts (Am). Note that the stainability is more intense on the plasma membrane except for the region facing to ameloblasts. c. Relatively intense reaction in papillary layer cells (PL) at the maturation stage of ameloblasts (Am). Note the weak reaction on the plasma membrane in contact with ameloblasts. BC blood capillary.

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The lectin-reaction in the cell organelle of stratum intermedium cells and papillary layer cells suggests that the two types of cells are engaged in an active sugar metabolism. This concept has also been supported by a Tunicamycin experiment (CHARDIN et al., 1989): large glycogen accumulation in the cytosol of stratum intermedium cells caused by Tunicamycin administration, which inhibits N-linked glycosylation, would likely result from a dysfunction of the sugar metabolism. Incorporated sugars would be for the most part transported to the plasma membranes as a carbohydrate moiety of glycolipids and/or membranebound glycoproteins rather than that of secretory glycoproteins, due to the absence of secretory granules in stratum intermedium cells and papillary layer cells of the rat incisor enamel organ. Carbohydrates are generally structured by enzymatic transfer; developmental alteration of the structure of cell surface carbohydrates is usually due to the developmental alteration of glycosyltransferase activity. In our study, UEA-I reaction was detected in the nuclear envelope in papillary layer cells but not in that of stratum intermedium cells. This finding suggests that fucosyltransferase activity increases in accordance with the cytodiff erentiation of stratum intermedium cells into papillary layer cells. Regarding the WGA reaction on nuclear pores, nuclear pore glycoprotein has been reported to contain 0-linked N-acetylglucosamine (HOLT et al., 1987). Therefore, WGA reaction on nuclear pores of stratum intermedium cells and papillary layer cells would reflect a N-acetylglucosamine residue of nuclear pore glycoprotein. 2. Transport function of stratum cells and papillary layer cells

intermedium

In this study and the observations using the HIDTCH-SP method (KOGAYAand FURUHASHI,1988) and lectin histochemistry (NAKAI et al., 1985; AKITA et al., 1988), the plasma membranes of stratum intermedium cells were revealed to be highly covered with glycocalyx. The glycocalyx of stratum intermedium cells would bear a net negative charge on their plasma membranes trapping cations (FARACHCARSONet al., 1989) and/or positively charged organic materials. Indeed, the findings of the potassium pyroantimonate technique and other calcium detecting methods (EISENMANNet al., 1982; OZAWA et al., 1984) revealed cations on the plasma membranes of stratum intermedium cells. Additionally, the autoradiography of 45Ca (BAWDEN and WENNBERG, 1977;

KAWAMOTOand SHIMIZU, 1987), 125I-labeled albumin (KINOSHITA, 1979) and 125I-labeled various proteins (MCKEE et al., 1986) suggests the presence of a barrier system between the enamel matrix and the capillary. The stratum intermedium cells would therefore play an important role in the barrier system by their charged plasma membranes. Stratum intermedium cells are interconnected each other and with ameloblasts and stellate reticulum cells by gap junctions, which are responsible for the transport of ions and small molecules between linked cells (GARANT and NALBANDIAN,1968; MATTHIESSEN and MLLGARD, 1973). Moreover, stratum intermedium cells show characteristic fine structures, e.g., rich in mitochondria and microvilli, commonly found in cells participating in transport (KALLENBACH, 1978; MATTHIESSEN and ROMERT, 1980). At the secretory stage of ameloblasts, the mineralization of enamel is restricted: the transport of calcium from the extracellular fluids to the mineralizing matrix at this stage is controlled by the enamel organ (BAWDENand WENNBERG, 1977; BAWDENet al., 1982). The above reports and the results of this study suggest the following transport mechanism: Firstly, Ca ions are trapped on the plasma membranes of stratum intermedium cells by the negative charge generated by glycocalyx. They are then transported into the cytoplasm through a Ca-channel supposedly present in the plasma membrane of the stratum intermedium cells, and then into the ameloblasts through the gap junctions between ameloblasts and stratum intermedium cells. Finally, Ca ions are carried into the enamel matrix by Ca-ATPase on the plasma membranes of secretory ameloblasts (INAGE and WEINSTOCK, 1979; TAKANO et al., 1986). Therefore, stratum intermedium cells and ameloblasts could be regarded as a single functional unit and stratum intermedium cells would play a role in the transport of ions from the capillary to the secretory ameloblasts, especially Ca ion, and possibly organic materials for enamel formation. Papillary layer cells have been known to possess Na-K-ATPase (GARANT et al., 1987) on their plasma membranes. The lectin-reaction in the papillary layer cells would therefore reflect such enzymatic membrane-bound glycoprotein to some extent. Papillary layer cells also have appearances such as are commonly found in cells involved in the transport of ions, water or other substances (KALLENBACH,1966, 1967; ELWOOD and BERNSTEIN, 1968; GARANT and NALBANDIAN,1968; REITH,1970). Furthermore, papillary layer cells are interconnected with each other and ameloblasts at the maturation stage by gap

Lectin

junctions (GARANT and NALBANDIAN,1968; GARANT, 1972). Therefore, some Ca ions required for enamel maturation would be trapped on the plasma membranes of papillary layer cells, transported into ruffleended ameloblasts via gap junctions, then carried into the enamel matrix by Ca-ATPase on the plasma membranes of ruffle-ended ameloblasts (SALAMA et al., 1987; TAKANO and AKAI, 1987). In addition, experiments using HRP (SKOBE and GARANT, 1974; SASAKI and HIGASHI, 1983) suggest that papillary layer cells are closely related with the removal of materials from the enamel matrix. Matrix-like substances detected by lectins between papillary layer cells may be in the process of the removal of the enamel matrix during enamel maturation. These matrix-like substances would contain much galactose and/or galactosamine residues, as they were stained more intensely by PNA and MPA than other lectins. Therefore, papillary layer cells would play a role in the removal of the enamel matrix as well as in the transport of minerals from the capillary to the enamel. 3. Cytodifferentiation cells and ameloblasts

of stratum

intermedium

Lectin-stainability in stratum intermedium cells increased according to the cytodiff erentiation of ameloblasts. The tendency resembles the localization of ALPase activity. Therefore, the lectin-reaction reflects the localization of ALPase to some extent because ALPase is a membrane-bound glycoprotein. However, lectin-reaction was also detected on the plasma membranes of stellate reticulum cells and the outer enamel epithelium,although these cells did not show ALPase activity clearly; the phenomena suggest that the lectin-reaction and the localization of ALPase activity are not completely equivalent. As to the relationship between the cytodifferentiation of stratum intermedium cells and that of ameloblasts, interesting evidence has been reported for the enamel-free area; in mouse molars, the inner enamel epithelium differentiates into preameloblastlike cells which secrete a small amount of enamellike matrix and are accompanied by irregularly shaped stratum intermedium cells at the proximal end. Preameloblast-like cells cannot differentiate into secretory ameloblasts with Tomes' processes; stratum intermedium cells change their appearance and gradually lose their detectability (SAKAKURAet al., 1989). These observations suggest that stratum intermedium cells play an important role in the cytodiff erentiation of the inner enamel epithelium

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into secretory ameloblasts and/or maintaining the secretory function of secretory ameloblasts. Selective carbohydrate-protein interaction is nowadays thought to participate in the regulation of the developmental process. Endogenous sugar-binding proteins such as lectins have been detected in various tissue (DODDand JESSELL, 1986; GABIUS et al., 1987). The developmental changes of glycocalyx and endogenous sugar receptors suggest that specific carbohydrate-protein interaction is critical for the development of organs. In this respect, the glycocalyx of the plasma membrane would function as a structure for cell-cell and/or cell-matrix recognition, both of which contribute toward determining the sociological behavior of the cell and responses to regulatory factors during development (HARRISON and CHESTERTON, 1980; DODD and JESSELL, 1986; GABIUS et al., 1987). In our lectin cytochemistry, the glycocalyx of stratum intermedium cells changed in accordance with the cytodiff erentiation of ameloblasts, suggesting that stratum intermedium cells and ameloblasts regulate their mutual cytodiff erentiation by cell-cell interaction, for which carbohydrateprotein interaction constitutes an important cell-cell recognition process. Acknowledgments. We thank Associate Prof. S. EJIRI, Department of Oral Anatomy, Niigata University School of Dentistry, for his technical advice. We are also grateful to the staff of the Department of Oral Anatomy for much assistance.

REFERENCES AKITA, H., Y. KOBAYASHI and M. KAGAYAMA: A histochemical study on lectin binding in the immature enamel and secretory ameloblasts of rat incisors. Tohoku J. Exp. Med. 155: 139-149 (1988). AOYAMA,I.: Studies on the enamel forming cells by means of enzyme histochemistry (In Japanese). Jap. J. Oral Biol. 12: 346-372 (1970). BAWDEN,J. W. and A. WENNBERG:In vitro study of cellular influence on 45Ca uptake in developing rat enamel. J. Dent. Res. 56: 313-319 (1977). BAWDEN,J. W., M. A. CRENSHAW,Y. TAKANOand L. HAMMARSTROM: Session III-Enamel organ morphology, function and transport. Ion transport through the enamel organ-An update. J. Dent. Res. 61 (spec. iss.): 1552-1554 (1982). BRABEC,R. K., B. P. PETERS, I. A. BERNSTEIN,R. H. GRAYand I. J. GOLDSTEIN:Differential lectin binding to cellular membranes in the epidermis of the newborn rat. Proc. Nat. Acad. Sci. USA 77: 477-479 (1980).

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Dr. Hiroaki NAKAMURA Department of Oral Anatomy Niigata University School of Dentistry Gakkocho-dori-2, Niigata 951 Japan 中 村 浩 彰 951新 潟市 学校 町通2番 町 新 潟大 学歯 学部 口腔解 剖学 第一 教 室

Lectin cytochemistry on the stratum intermedium and the papillary layer in the rat incisor enamel organ.

Lectin cytochemistry was carried out to elucidate the role of stratum intermedium cells and papillary layer cells in amelogenesis, especially in the p...
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