Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs.код для вставкиСкачать
THE ANATOMICAL RECORD 229:259-270 (1991) Immunohistochemical Demonstration of Amelogenin Penetration Toward the Dental Pulp in the Early Stages of Ameloblast Development in Rat Molar Tooth Germs TETSUICHIRO INAI, TOSHIO KUKITA, YASUYOSHI OHSAKI, KENGO NAGATA, AKIKO KUKITA, AND KOJIRO KURISU Second Department of Conservative Dentistry (T.I.),and Second Department of Anatomy (T.K., Y.O., K.N., A.K., K.K.), Faculty of Dentistry, Kyushu University, Fukuoka, Japan ABSTRACT In order to examine the synthesis and secretion of enamel protein by ameloblasts in their early stages of development, immunohistochemical localization was carried out a t light and electron microscopic levels using a monoclonal antibody produced in a preliminary experiment. Materials used were tooth germs of mandibular first molars of rats a t 0-5 days after birth. Immunoblot analysis after two-dimensional electrophoresis revealed that antigen molecules recognized by the monoclonal antibody were amelogenins of 26-28 kDa (PI, 6.6-7.0). An immunohistochemical examination using this monoclonal antibody demonstrated that the presecretory ameloblasts in their early stages of differentiation both synthesized amelogenin and secreted through a classical merocrine secretory pathway. In some presecretory ameloblasts as well as ameloblasts we observed the distended cisternae of rough endoplasmic reticulum (rER) which demonstrated heterogenous immunolabelling. The immunolabellings were also detected in the predentin a s well a s the intercellular spaces of odontoblasts and dental pulp cells which indicated penetration of amelogenin from the presecretory ameloblast layer to the dental pulp. The presence of coated pits a t the plasma membrane of odontoblasts in close proximity to enamel protein along with the immunolabelling of lysosomes of the odontoblasts suggests the phagocytosis of the enamel protein into the odontoblasts. These observations suggest the possibility that the penetration of enamel protein toward the dental pulp and odontoblasts plays a role in the interaction between ameloblasts and odontoblasts. The developing enamel matrix contains a complex mixture of proteins. It is, therefore, essential to clearly elucidate the nature of these proteins in order to reach a better understanding of amelogenesis. The enamel proteins secreted by ameloblasts can be divided into two classes, amelogenins and enamelins, based on their solubility properties, amino acid composition, molecular weight, and isoelectric point (Termine et al., 1980; Belcourt e t al., 1983; Rosenbloom et al., 1986; Ogata et al., 1988). Amelogenins are hydrophobic and prolinerich proteins with a relatively low molecular weight ranging from 6 to 27 kDa, whereas enamelins are hydrophilic glycoproteins with a relatively high molecular weight ranging from 8 to 72 kDa. Newly secreted enamel proteins are deposited into the enamel matrix and are rapidly reduced to less than 2% of the original amount during the process of enamel maturation. The immunohistochemical localization of enamel proteins in tooth germs has been previously described a t the light microscopic (Graver et al., 1978; Slavkin et al., 1981,1982)and electron microscopic level (Nanci et al., 1984, 1985,1987,1989;Herold et al., 1987; Slavkin et al., 1988; Inage e t al., 1989).Recently some investic 1991 WILEY-LISS. INC gators (Slavkin et al., 1988; Nanci et al., 1989; Inage et al., 1989) have reported that presecretory ameloblasts synthesize and secrete enamel proteins into predentin prior to or after the breakdown of the basement membrane. However, the exact role of these “ectopic” enamel proteins is still unclear. In the present study, we describe the detailed immunohistochemical localization of amelogenin in the early stages of ameloblast differentiation using the monoclonal antibody against amelogenin obtained in a preliminary study. Immunolabelling was detected in the presecretory ameloblasts both intra- and intercellularly as well as in the predentin, intercellular spaces of the odontoblasts and the dental pulp cells. These results suggest the penetration of amelogenin from the presecretory ameloblast layer toward the dental pulp. Received May 7, 1990; accepted J u l y 24, 1990. Address reprint requests to K. Kurisu, Second Department of‘ Anatomy, Faculty of Dentistry, Kyushu University 63, Maidashi. Higashiku, Fukuoka 812, Japan. 260 T.INAI ET A L MATERIALS AND METHODS preparation of Antibodies lmmunoblot Analysis Protein extraction One group of samples was prepared according to the Preparation of irnmunogen method of Zeichner-David et al. (1988) with some modIncisor tooth germs with some surrounding tissues ifications. Mandibular incisors were dissected from 7 were dissected from 3-6 week-old rats in 0.1 M phos- day-old rats and washed with ice-cold PBS. They were phate-buffered saline (PBS). Apical ends of the tooth homogenized with Polytron (Kinematica, Luzern, Switgerms containing differentiating, presecretory, and zerland) in ice-cold 0.5 M acetic acid and then extracted secretory ameloblasts were cut away and homogenized with 0.5 M acetic acid using a dialysis bag (3,500 Da with tef lon-glass homogenizer in ice-cold Iscove’s mod- cut-off) for 7 days a t 4°C. The extract was centrifuged ified Dulbecco’s medium (IMDM; Gibco, Grand Island, a t 13,000g for 10 minutes a t 4°C and the supernatant NY) containing 0.5% sodium dodecylsulfate. was lyophilized. Other groups of samples were prepared according to the method of Termine e t al. (1980). In vitro immunization and production of hybridornas Unerupted fetal bovine molars were dissected and the The in vitro immunization was performed a s de- enamel surfaces were wiped off to remove any tissue scribed by Reading (1982) and Boss (1984) with some covering their surfaces. The scratchable enamel (cheesy modifications. Briefly, a single cell suspension was pre- enamel) was serially extracted with 4 M guanidine HCl pared from spleens which were removed aseptically (“G’ extract) and then with 4 M guanidine HC1 confrom 6-8 week-old unimmunized female Balbic mice. taining 0.5 M ethylenediamine tetraacetic acid (EDTA) After washing and centrifugation, the spleen cells in (“E” extract). All procedures were performed at 4°C in IMDM (supplemented with 5 x M 2-mercapto- the presence of protease inhibitors. The extracts were ethanol, 100 unitdm1 penicillin, and 100 pgiml strep- concentrated using dialysis bags which were aspirated tomycin) containing 20% fetal bovine serum (FBS; in flasks. They were then dialyzed against distilled Whittaker Co., Walkersville, MD) were incubated for 4 water a t 4”C, and then freeze-dried. days in a CO, incubator in the presence of 1-100 pl Electrophoresis and imrnunodetection tooth germ homogenate and 20 pgiml N-acetylmuSingle-dimensional sodium dodecylsulfate polyacrylramyl-L-alanyl-D-isoglutamine (Peptide Institute Inc., Minoo, Japan) as adjuvant. The immune spleen cells amide gel electrophoresis (SDS-PAGE) was carried out and mouse myeloma cells (P3-X63-Ag8-U1) were fused according to the Laemmli procedure (1970) using a t a ratio of 1O:l in 0.5 ml solution containing 50% 12.5% acrylamide gel under both reducing and nonrepolyethylene glycol 4000 and 5% dimethyl sulfoxide in ducing conditions. The stacking gel contained 4% polydouble distilled water. The fused cells were adjusted to acrylamide while the separating gel contained 12.5% 1 x lo6 spleen cellsiml in HAT medium (IMDM sup- polyacrylamide. SDS-PAGE Standards (Low range; Bio plemented with hypoxanthine, aminopterin, and thy- Rad Lab., Richmond, CA) were used for molecular midine) containing 20% FBS and plated into 24-well weight standards. After electrophoresis, electroblotplates (Falcon, Lincoln Park, NJ). The fused cells were ting of proteins onto nitrocellulose membrane (Transfed in HAT medium for 10-14 days by exchanging 0.5 Blot; Bio Rad Lab.) was done using TEFCO blot equipment (TEF Co., Machida, Japan) as described by ml of the medium on every third day. Towbin et al. (1979). The gel was transferred a t 180 mA for 1 hour in a buffer containing 25 mM Tris, 192 Antibody screening and hybridorna cloning mM glycine, and 20% methanol. The culture media from the wells containing macroTwo-dimensional SDS-PAGE was conducted by the scopically visible colonies were screened by indirect im- method described by O’Farrell (1975). The first dimenmunofluorescence using paraffin sections of rat jaws sion employed 2% Ampholine (pH 3.5-10; Pharmacia containing tooth germs. The details are described in LKB Biotechnology, Piscataway, NJ) in a 2.5 x 130 the section on immunofluorescence procedure. The hy- mm tube and was focused for 1 hour a t 200 V, 15 hours bridomas producing the antibodies which react with at 300 V, and then 1 hour a t 500 V. SDS-PAGE Stanspecific cells or extracellular components in tissue sec- dards were used for the molecular Standard. The septions were cloned four times by limiting dilution. arated proteins were transferred to nitrocellulose In the present study the monoclonal antibody En3 membranes as previously described (Towbin et al., (Inai, unpublished data), which reacted with amelo- 1979). blasts and enamel matrix, is used. Both in single- and two-dimensional immunoblot analyses, some of the nitrocellulose membranes were Production of polyclonal anti-laminin antibody stained with 0.1% amido black 1OB to detect the transPolyclonal anti-laminin antibody was prepared a s ferred proteins. Others were incubated first with monofollows: laminin was extracted from EHS sarcoma tis- clonal antibody En3 and second with horseradish persue and purified by chromatography on DEAE-cellu- oxidase-conjugated goat anti-mouse p chain antibody lose and Agarose A15 and A5 column, a s described by (Zymed Lab. Inc., South San Francisco, CA) according Timpl et al. (1979). The mouse tissue of EHS sarcoma to the methods of Slavkin et al. (1988) to detect the was generously donated by K. Kimata, Faculty of Sci- antigen. ence, Nagoya University, Japan. The antibody to lamiImmunofluorescence Procedure nin was raised in rabbits and then affinity-purified The 0-5 day-old rats were perfused with Periodatewith Sepharose-4B column, a s described previously Lysine-paraformaldehyde fixative (McLean and Na(Linsenmayer, 1981; Ruoslahti et al., 1981). 26 1 ENAMEL PROTEIN IN DENTAL PULP kDa 9 76 64 3- 3 1- 2 2- Fig. 1. Immunoblot analysis of acetic acid extract of rat incisor tooth germs separated by one-dimensional SDS-PAGE. The samples were fractionated by gel electrophoresis under reducing conditions, transferred to nitrocellulose paper, and then incubated with En3. The antigen-antibody complex was identified by an enzymatic colorimetric reaction using horseradish peroxidase. Lane 1 was stained with amido black. Lane 2 was a n immunoblot. A single band of approximately 23 kDa is detected with En3 in lane 2. 14- 8 kDa Fig. 2. Immunoblot analysis of " G extract of bovine enamel protein separated by two-dimensional SDS-PAGE. The samples were run in tube isoelectric focusing gels, separated by SDS-PAGE under reducing conditions, and then transferred to nitrocellulose paper. (a)Amido black stain; (b)immunoblot using En3. A single spot with a molecular mass of 26-28 kDa and PI of 6.6-7.0 is detected with En3 in b. 2 1 I 7 I 6 I 5 I 4 pH I 8 I 7 I 6 I 5 I 4pH I 9 76 64 3- 3 1- 2 214kane, 1974) through the left ventricle and the dissected mandibles were immersed in the same fixative for 3-5 hours at 4°C. After decalcification by 10% EDTA for 1-3 weeks at 4"C, the specimens were embedded in paraffin. Sections mounted on glass slides were incu- bated with either the hybridoma medium under examination or with monoclonal antibody En3 for 1 hour a t room temperature. Control sections were treated with either normal mouse serum (Zymed Lab. Inc.) or PBA (PBS containing 1%chick albumin) instead of mono- 262 T. INAI ET AI, Figs 3-5 ENAMEL PROTEIN I N DENTAL I ’ U I 2 clonal antibody. After repeated rinsing with PBS, the tissue sections were incubated with fluorescein isothiocyanate (FITC) labelled goat anti-mouse immunoglobulin (Ig) or FITC labelled goat anti-mouse p. chain antibody (Zymed Lab. Inc.) for 30 minutes a t room temperature. The entire procedure was carried out in a moist chamber to prevent the samples from drying. Finally the sections were washed extensively with PBS and sealed with PBS-buffered glycerol to which p-phenylenediamine had been added to decrease fluorescent fading a s described by Johnson and Araujo (1981). The specimens were then examined by a Zeiss fluorescence microscope. 26: body. Colloidal gold labelling of goat anti-mouse p. chain antibody (Zymed Lab. Inc.) was carried out as described by Slot and Geuze (1984). The sections were then washed three times for 5 minutes each with PBS and finally again in distilled water. Throughout the entire procedure, care was taken to prevent the sections from drying by keeping them in a moist chamber. The sections were finally stained with uranyl acetate and lead citrate and then were examined with a JEOL 2OOCX electron microscope. RESULTS Characterization of Antigen of Monoclonal Antibody En3 Figure 1 shows the SDS-PAGE pattern and immunoblot analysis of acetic acid extract of the homogenate The 0-5 day-old rats were perfused through the left of rat incisor tooth germs. Since the preliminary imventricle with a mixture of 1%glutaraldehyde and 3% munohistochemical study demonstrated that En3 paraformaldehyde in 0.1 M cacodylate buffer contain- cross-reacted with the bovine enamel matrix, the saming 0.05% CaCl,, pH 7.4. The mandibles were dissected ples from the bovine enamel matrix, which were easy and immersed in the same fixative for 3-5 hours a t to get in large amounts, were also examined. Using 4°C. After decalcification in 10% EDTA for 1-3 weeks En3 a positive band was detected a t approximately 23 at 4”C, the mandibular first molars were cut with a kDa in the acetic acid extract under reducing condition Microslicer into thick sections of 200-300 p. which (Fig. 1).Similar result was obtained under nonreducwere postfixed in osmium tetroxide reduced with po- ing conditions (data not shown). Two-dimensional immunoblot analysis on bovine tassium ferrocyanide (Karnovsky, 1971) for 2 hours a t room temperature. The thick sections were washed in a “G’ extract revealed protein with a molecular weight of sodium cacodylate buffer, dehydrated in a graded se- 26-28 kDa (PI, 6.6-7.0) using the En3 (Fig. 2). No ries of ethanol, and embedded in Epon 812. Ultrathin reaction was detected when two-dimensional immunosections were mounted on nickel grids and floated on blot analysis on bovine “E” extract using En3 was condrops of a saturated aqueous solution of sodium ducted (data not shown). These results definitely sugmetaperiodate for 1hour and on PBL (0.1 M PBS con- gest that En3 specifically binds with amelogenin but taining 1 % bovine serum albumin (BSA) and 0.1 M not with other proteins including enamelin. FITC lalysine-HC1) for 30 minutes. They were then treated belled goat anti-mouse p. chain antibody reacted with En3 antibody for 1 hour a t room temperature. strongly with tissue sections of tooth germs binding Control sections were treated with either normal En3, indicating the Ig class of En3 being IgM (data not mouse serum or PBL instead of En3. After repeated shown). rinsing with PBS, the sections were incubated with lrnrnunofluorescentLocalization of the colloidal gold labelled goat anti-mouse IJ- chain antiEnamel Protein Antigen lrnrnunoelectron Microscopy The first appearance of positive staining for monoclonal antibody En3 was detected at the distal end of the early stage presecretory ameloblast (Fig. 3a). The intensity of staining in the presecretory ameloblast layer increased with the development of the presecreFig. 3. Micrographs of early stage presecretory amelohlasts ( P A ) tory ameloblast. In order to examine the relation beand odontoblasts (Od) stained with En3 (a) and H-E (h).To examine tween the staining pattern with En3 and the basement the basement membrane, an adjacent section was stained with anti- membrane, the adjacent section was stained with antilaminin antibody ( e ) .The first appearance of positive staining for En3 laminin antibody (Fig. 3c). The results demonstrated (large arrow) is detected in the presecretory ameloblast layer along that the first appearance of staining for En3 was obthe basement membrane prior to its breakdown (arrowheads),and the intensity of staining in the presecretory ameloblast layer increases served prior to the breakdown of the basement memwith the development of the presecretory ameloblasts. Note the presbrane and that there was positive staining in the ence of positive staining in the odontoblast layer (small arrows) prior odontoblast layer prior to and after the breakdown of to or after the breakdown of the basement membrane, although the the basement membrane, although the intensity of the intensity of staining increases after the breakdown o f t h e basement membrane. a x : x 290. staining increased after its breakdown. Along with the development of the presecretory ameloblasts, heavy Fig. 4. Micrographs of the late stage of presecretory ameloblasts staining was also detected in the predentin and odon(PA) stained with En3 (a) and H-E (b). Heavy staining for En3 is observed in the predentin (large arrow) and odontoblast layer (Od) toblast layer (Fig. 4). Retention of immunoreactive ma(small arrow). The staining in the odontoblast layer markedly de- terials in the subodontoblastic zone was detected at the creases after the initiation of enamel matrix deposition (large arrow- pulp horn (Fig. 5). Accompanying the enamel matrix head in b). Spot-like staining detected in the predentin seems to cordeposition, the staining in the odontoblast layer was respond to hematoxylin positive material (small arrowheads in a and also found to decrease markedly, and spot-like staining h). a,b: x340. was detected in the predentin (Fig. 4). Fig. 5. Micrographs of the late stage of presecretory ameloblasts at Control sections stained using normal mouse serum the crest of a cusp stained with En3 (a)and H-E (h).Note the positive or PBA in place of En3 showed either negative or very staining a t the subodontoblastic zone of the dental pulp (arrow).a.b: faint background staining (data not shown). x 290. Figs. 3-5. Sections of mandibular first molars of 2 day-old rats. After recording of the immunofluorescent reaction using En3 the same sections were stained with hematoxylin and eosin (H-E) for histological observations. 264 rr. I N A I Err XI, ENAMEL PROTEIN IN DENTAL PULP 265 Lysosomes with labelling were often seen in the odontoblasts (Fig. 12). The presence of labelled SM in the After applying En3 to the ultrathin sections, a spe- dental pulp continued until initial enamel formation. At the late stage of presecretory ameloblasts, correcific labelling was first detected in the early stage of sponding to Differentiation V in which the basement the presecretory ameloblasts corresponding to the Differentiation I1 of Kellenbach (1971),in which the base- membrane cannot be recognized, the labelled SM were ment membrane between presecretory ameloblasts and observed in the intercellular spaces of the stratum inodontoblasts is uninterrupted and smooth. Gold parti- termedium and the proximal end of the presecretory cles were present particularly over the Golgi appara- ameloblasts (Fig. 14). The distal surface of the pretus, rough-surfaced endoplasmic reticulum (rER),pale secretory ameloblasts a t this stage was rough and the or dark staining lysosome-like granules, and the mul- basement membrane could not be recognized (Fig. 15). tivesicular bodies distributed throughout the supranu- Numerous patches of labelled SM were observed (Fig. clear compartment (Figs. 6, 7). However, the intensity 15). At the very late stage of presecretory ameloblasts, of labelling over these large granules was variable. Lacorresponding to Differentiation VI in which the first belling was also observed over an irregular mass of the granular material in the intercellular space (Fig. 6). In mineral deposits in the predentin occur, heavy deposits this paper we call the granular material “stippled ma- of SM, of so called fine-textured materials with labelterial-like substance” (SM). In this stage, the distal end ling, were detected a t the distal end of the cells (Fig. of the presecretory ameloblast was smooth and the 16). At the early stage of secretory ameloblasts, secretory basement membrane was intact (Fig. 8 ) . Labelled SM vesicles in Tomes’ processes and enamel matrix were was also observed in the intercellular space between the presecretory ameloblasts and odontoblasts (Fig. 8). labelled (Fig. 17). In the dentin matrix a t this stage, In the middle stage of the presecretory ameloblasts labelled patch-like materials of various sizes were obcorresponding to Differentiation 111-IV of Kallenbach served (inset of Fig. 17). Control sections incubated with PBL in the place of (Differentiation 111, the basement membrane is smooth and uninterrupted except for penetrating filaments. A En3 showed a very small amount of background labelfew small droplets of coarse-textured material are ling (Fig. 18). present in the predentin; Differentiation IV, the preDISCUSSION secretory ameloblasts send processes of varying size The present a s well a s previous results (Nanci et al., and shape through the basement membrane into the 1985, 1989; Inage et al., 1989) suggest that the secrepredentin), distended rERs containing moderately dense material with labelling were often observed, tion products of both presecretory ameloblasts and while no labelling was detected in those containing ameloblasts follow a classical merocrine secretory pale material (Fig. 9). These structures were observed pathway through the rER, the Golgi apparatus, the either in the supranuclear compartment or in the in- secretory granules, and release at the cell surface. Imfranuclear compartment. At this stage numerous munolabelling with anti-amelogenin antibody En3 remasses of SM with labelling were detected in the pre- veals antigenicity in all these compartments. The fact dentin (Fig. lo), and in the intercellular spaces of both that the rER is immunoreactive indicates that the anthe odontoblasts (Figs. 11, 12) and the dental pulp su- tigenic domain of amelogenin is already defined a s the bodontoblastic zone (Fig. 13). Coated pits were ob- proteins are translated (Nanci et al., 1984, 1985). The labelling for amelogenin was first detected a t served on the lateral plasma membrane of the odontoblasts in contact with the labelled SM (Fig. 11). the presecretory ameloblasts intracellularly a s well a s extracellularly in the early stages of presecretory ameloblasts corresponding to Kallenbach’s Differentiation I1 (Kallenbach, 1971) in which the basement membrane was intact. This finding is consistent with those of Nanci et al. (1989) and Inage et al. (1989). Figs. 6-1 8. Immunoelectron microgaphs of mandibular first molars The burst of amelogenin penetration toward the denof 1-2 day-old rats. Ultrathin sections were incubated wtih En3 or PBL for control, and then incubated with colloidal gold labelled goat tal pulp was prominently detected after a breakdown of anti-mouse p chain antibody. the basement membrane (Differentiation IV). This penetration decreased accompanying the dentin minFig. 6. Supranuclear zone of the early stage presecretory ameloeralization and almost disappeared when the initial blasts. Golgi apparatus (GI, dark type lysosomes (dLy) and stippled enamel was formed. Although immunohistochemical material-like substance (SM) are labelled. x 31,700. localizations of enamel proteins in tooth germs have Fig. 7 . Supranuclear zone of the early presecretory ameloblasts. Dark type lysosomes tdLy) a s well a s pale type lysosomes (inset,pLy1 been reported by many researchers a t the light microscopic level (Graver et al., 1978; Slavkin et al., 1981, are labelled. x 24,900. Inset, x 18,000. 1982) as well as a t the electron microscopic level Fig. 8. Distal end of the early stage presecretory ameloblasts (PA). (Nanci et al., 1984, 1985, 1987, 1989; Herold e t al., Dark type lysosomes (large arrow) and stippled material-like sub1987; Slavkin e t al., 1988; Inage et al., 1989), the posstance (small arrow) in intercellular space (ICS)between presecretory itive immunolabelling for the enamel proteins has not ameloblasts and odontoblasts are labelled. Note the presence of a n yet been described in the dental pulp. One of the reaintact basement membrane. x 39,600. sons why previous authors failed to investigate this lies Flg. 9. Infranuclear compartment of the middle stage presecretory in the difference of the objectives of our respective studameloblast ( P A ) .Distended rERs (dER) containing moderately dense ies. Another reason may be the difference of antibodies materials show labelling, while no labelling is observed in that conused. The previous researchers, except for Herold et al. taining pale material (PER,. x 37,900. lmmunoelectron Microscopic Localization of the Enamel Protein Antigen 266: T.I N A I ET AI, ENAMEL, I’KOTEIN I N I)ENTAI, I’LJLP (1987), used polyclonal antibodies, while we used a monoclonal antibody in the present study. Polyclonal antibodies against a molecule are a mixture of monoclonal antibodies against several antigenic determinants of the molecule. Therefore, it is believed that, in immunohistochemistry, polyclonal antibodies generally show more intensive staining than monoclonal antibodies. However, the reverse is true when the antigenic determinant of a molecule for the monoclonal antibody is dominant in a tissue although polyclonal antibodies contain very few antibodies against the determinant (Yasuda e t al., 1988).The antigenicity of the enamel protein may be reduced by the modification or degradation of antigenic determinants during the process of penetration into the dental pulp, which induces a decrease in immunoreactivity of the polyclonal antibodies against the protein. However, the antigenic determinant of the monoclonal antibody En3 is conserved during the process of penetration into the dental pulp. This may be one of the reasons why the previous investigators using polyclonal antibodies have not detected the penetration of enamel proteins into the dental pulp. Reith (1967) reported that the enamel precursor-like material referred to as stippled material was observed in predentin, on the surface of the enamel matrix, and between ameloblasts in rat molar tooth germs. On the other hand, Kallenbach (1971) found several types of extracellular dense material differing from each other by texture and time of appearance in rat incisors. Coarse-textured material appeared first between presecretory ameloblasts, then within predentin when the basement membrane was perfectly intact. Fine-textured material appeared, after .the disappearance of the basement membrane, in the same position within the predentin formerly occupied by coarse-textured material. These materials, however, have often been detected also in calcified dentin (Suga, 1960; Watson, 1960; Reith, 1967) or deep within predentin (Kallenbach, 1971) in both r a t incisors and molars. Yamamoto et al. (1980) have also observed the SM in dentin and deep in predentin a s well as between odontoblasts. In the present paper, we call the SM present in the area Fig. 10. Predentin (PDI, distal end of odontoblast (Odl, and cytoplasmic process of odontoblast (PI facing the middle stage presecretory ameloblasts in which the basement membrane is penetrated by fine filaments or cytoplasmic processes of presecretory ameloblasts. Stippled material-like substance (SMI deep in predentin is labelled. x 51,000. Fig. 11. Odontoblasts tOd1 and the intercellular space a t the middle stage of presecretory ameloblasts. Stippled material-like substance in the intercellular space is labelled. Note the presence of coated pits (arrows) on the lateral plasma membrane of the odontoblasts. x 41,300. Fig. 12. Odontoblasts (Odl and the intercellular space a t the middle stage of presecretory ameloblasts. Note the presence of labelling on stippled material-like substance tSM) in the intercellular space and the lysosome (Lyl. x 42,900. Fig. 13. The intercellular space at the proximal end of odontoblasts and cells of the dental pulp ( P u ) a t the middle stage of presecretory ameloblasts. Stippled material-like substance tSM) in the space is labelled. x 32,400. 267 other than the enamel matrix the “ectopic” stippled material (ESM). The origin of ESM is controversial. Reith (1967) and Yamamoto et al. (1980) regarded ESM a s products of ameloblasts because of the structural resemblance of it to the enamel matrix before calcification. Kallenbach (1971), however, doubts that ESM is of ameloblast-origin, because 1) ESM were detected in the predentin layer while the basement membrane was still perfectly intact, 2) they were never in contact with the basement membrane but at varying distances away from it, and 3) many ESM were in close proximity to, or even in contact with, the odontoblast processes. Our immunohistochemical examination a s well a s previous studies (Slavkin e t al., 1988; Nanci et al., 1989; Inage et al., 1989) revealed that ESM contain enamel protein. And Snead et al. (1988)demonstrated that only ameloblasts showed amelogenin gene expression while odontoblasts did not. The basement membrane cannot be a perfect barrier even for large molecules. These data support the view that ESM is of ameloblast-origin. The role of ESM is still obscure. ESM may play a role in the initial mineralization of dentin (Slavkin e t al., 1988; Inage et al., 1989). In our decalcified sections, the concomitant absence of “crystal ghosts” (Bonucci, 1969) or “electron lucent clefts” (Warshawsky and Vugman, 1977) in ESM, which would have suggested a trace of crystal formation in the decalcified tissue, suggests that crystals had not yet appeared in ESM. We observed coated pits on the lateral plasma membrane of the odontoblasts in close proximity to ESM with labelling and labelled lysosomes in them. These observations suggest that the odontoblasts endocytosed the ESM and processed it in lysosomes. The penetration of ESM into the dental pulp occurred at a restricted developmental period, namely that it started before dentin mineralization, even before the breakdown of the basement membrane and then disappeared a t initial enamel formation. Inage et al. (1989) supposed that ESM might have a n effect on the differentiation of odontoblasts since these seemed to start the secretion of phosphoproteins a t this stage. These observations suggest the possibility that the penetration of ESM into the dental pulp plays a role in the interaction between presecretory ameloblasts and odontoblasts. However, there is a possibility that this penetration might be the result of physical diffusion without having a specific biological role and might be blocked by the presence of the layer of mineralized dentin. Further investigation is needed to clarify the role and the fate of ESM. Nanci and Warshawsky (1984), and Uchida (1988) reported that the stippled material appearing in the secretory stage of ameloblasts was a n artifact depending on the fixation method. Whatever the true structure of the material is, it is considered to be a secretory product of either the presecretory ameloblasts or ameloblasts and to contain amelogenin. Moe (1971)described the distended cisternae of rER in some presecretory ameloblasts and ameloblasts. Kallenbach ( 1972) detected spherical granules of electron dense materials within the distended cisternae of rER and supposed that these granules were caused by the accumulation of enamel proteins. The present immunohistochemical examination revealed immunola 268 T. I N A I KT AIL Figs. 14-17. 269 ENAMEL PROTEIN IN DENTAL PU1,I’ nor differences between them. Although there were clear circular gaps between the highly electron dense granules and walls of cisternae in the figures of Kallenbach (1972), we observed homogenous and moderately electron dense material in the distended cisternae without clear gaps. This discrepancy of the structure may come from differences of tissue preparation. In the present study, the immunolabelling of distended cisternae was heterogenous, namely some distended rERs were positive but other ones were negative. In our previous study on the immunocytochemical localization of type I collagen in cultured mesenchymal cells from palatal shelves of mouse fetuses, immunolabelling of the distended cisternae was similarly heterogenous (unpublished data). Referring to the experiments of Weinstock (19701, Kallenbach (1972) supposed that the formation of intracisternal granules was caused by a n interruption in the transport of newly synthesized protein from the rER to the Golgi apparatus. If his hypothesis is true, the heterogenous distribution of immunolabelling detected in both our previous as well a s present study may suggest the presence of a sorting system in rER before the transport of newly synthesized proteins from the rER to the Golgi apparatus. Therefore, the relationship between the heterogenous immunolabelling and the phenomena occurring in the cell needs to be investigated further. ACKNOWLEDGMENTS Fig. 18. Control section, incubated with PBL instead of En3, of the odontoblast (Od) at the middle stage of presecretory ameloblasts and stippled material-like substance (SM)in the predentin (PD). No specific labelling and very little background labelling are observed. x 27.000. belling for amelogenin over some distended cisternae, supporting Kallenbach’s suggestion. The morphology of distended cisternae with labelling are very similar to that shown by Kallenbach (1972), but there are mi- Fig. 14. Cells of stratum intermedium (SI)and proximal end of the late stage presecretory ameloblasts (PA).Stippled material-like substance (SM) in the intercellular space is labelled. x 26,800. Fig. 15. Distal end of the late stage presecretory amelohlasts (PA). Stippled material-like substance or fine-textured material (arrows)in contact with presecretory amelohlasts or in predentin (PD) are labelled. Note the indentation of the distal surface of the presecretory ameloblasts and the breakdown of the basement membrane. Some stippled material-like substance is in contact with the cytoplasmic process (Pi of the odontoblast. x 20,000 Fig. 16 The distal end of the late stage presecretory amelohlasts (PA).Stippled material-like substance (large arrow) in the invaginations of the distal cell membrane and fine-textured material (small arrow), which appears more finely granular than stippled materiallike substance, in contact with the distal surface membrane of presecretory ameloblasts and in the dentin (D) are labelled. Secretory granules (arrowheads) in the presecretory ameloblasts are also labelled. x 26,400. Fig. 17. Distal end of the early secretory ameloblasts. Secretory granules (arrows) in Tomes’ processes (TP) and enamel matrix (EM) are labelled. x 17,700. Inset shows stippled material-like substance in the dentin matrix a t this stage which reveals labelling. x 15,700. 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