Interaction of rabbit sperm and egg

Summary The localization of proteoglycans in the predentin of the rat incisor was investigated by ultrastructural histochemistry. Ruthenium red stained the cell coat of the odontoblasts as well as intracellular vesicles. There was also a staining of the extracellular matrix, but not of collagen fibers in the predentin. Treatment with the enzyme hyaluronidase prior to staining with ruthenium red abolished the staining of the vesicles and the extracellular matrix but not that of the cell coat. Bismuth nitrate and phosphotungstic acid gave similar staining of odontoblast vesicles and extracellular matrix. It is likely that the stained structures contain proteoglycans. The importance of these proteoglycans and their ultrastructural localization are discussed in relation to intracellular transport and the calcification process.     

Temporo-spatial distribution of matrix and microfilament components during odontoblast and ameloblast differentiation

Summary Several extracellular matrix components (procollagen type III, fibronectin, collagen type IV, laminin and nidogen) and microfilament constituents (actin, α-actinin and vinculin) were localized by indirect immunofluorescence microscopy in frozen sections of embryonic mouse molars. Nidogen was present at the epithelio-mesenchymal junction during polarization and initial steps of functional differentiation of odontoblasts. Nidogen disappeared at a stage where direct contacts between preameloblasts and predentin were required to allow the initiation of ameloblast polarization. Our observations concerning the distribution of procollagen type III and fibronectin during odontoblast differentiation add to current knowledge. Procollagen type III and fibronectin surrounding preodontoblasts accumulated at the apical part of polarizing and functional odontoblasts secreting “initial” predentin. Procollagen type III, but not fibronectin, disappeared in front of functional odontoblasts synthesizing “late” predentin and dentin. Fibronectin, present in “initial” predentin, was no longer detected in “late” predentin and dentin but was found between odontoblasts secreting “late” predentin and dentin. Actin, α-actinin and vinculin were concentrated in the peripheral cytoplasm of preameloblasts and accumulated at the apical and basal poles of functional ameloblasts. During differentiation of odontoblasts, the three proteins accumulated at the apical pole of these cells. Time and space correlations between matrix and microfilament modifications during odontoblast and ameloblast differentiation are documented. The possibility is discussed that there is transmembranous control of the cytoskeletal activities of odontoblasts and ameloblasts by the extracellular matrix.     

Differential expression of decorin and biglycan genes during mouse tooth development.

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.     

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.     

TM14 is a new member of the fibulin family (fibulin-7) that interacts with extracellular matrix molecules and is active for cell binding

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin beta1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.     

A simple,disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars

Summary Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and ligual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.     

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.     

Expression pattern, subcellular localization, and functional implications of ODAM in ameloblasts, odontoblasts, osteoblasts, and various cancer cells

During tooth development and tumorigenesis, the odontogenic ameloblast-associated protein (ODAM) is involved in cellular differentiation and matrix protein production. However, the precise function of ODAM remains largely unknown. To suggest new functional roles of ODAM, we investigated the cellular expression and subcellular localization of ODAM in tooth and cancer cells. ODAM was expressed in ameloblasts, odontoblasts, and osteoblasts in vivo and in vitro. Furthermore, ODAM was localized in both the nucleus and cytoplasm of MMP-20 expressing ameloblasts and odontoblasts, but only in the cytoplasm of non-MMP-20 expressing osteoblasts. The extracellular secretion of ODAM was not observed in odontoblasts and osteoblasts, but was seen in ameloblasts. In addition, ODAM was discovered in the nucleus, cytoplasm, and extracellular matrix of various cancer cells. These results suggest that the expression pattern and subcellular localization of ODAM is highly variable and dependent on cell types and their differentiation states, and that functional correlations exist between ODAM and MMP-20. This study provides the first evidence for ODAM in multiple cellular compartments of differentiating odontogenic and cancer cell lines with important functional implications.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

Effects of suramin,a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro

Summary— Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [³H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.     

MEPE Localization in the Craniofacial Complex and Function in Tooth Dentin Formation

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein found in dental and skeletal tissues. Although information regarding the role of MEPE in bone and disorders of phosphate metabolism is emerging, the role of MEPE in dental tissues remains unclear. We performed RNA in situ hybridization and immunohistochemistry analyses to delineate the expression pattern of MEPE during embryonic and postnatal development in craniofacial mineralizing tissues. Mepe RNA expression was seen within teeth from cap through root formation in association with odontoblasts and cellular cementoblasts. More intense expression was seen in the alveolar bone within the osteoblasts and osteocytes. MEPE immunohistochemistry showed biphasic dentin staining in incisors and more intense staining in alveolar bone matrix and in forming cartilage. Analysis of Mepe null mouse molars showed overall mineralized tooth volume and density of enamel and dentin comparable with that of wild-type samples. However, Mepe^-/- molars exhibited increased thickness of predentin, dentin, and enamel over controls and decreased gene expression of Enam, Bsp, Dmp1, Dspp, and Opn by RT-PCR. In vitro Mepe overexpression in odontoblasts led to significant reductions in Dspp reporter activity. These data suggest MEPE may be instrumental in craniofacial and dental matrix maturation, potentially functioning in the maintenance of non-mineralized matrix.     

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.     

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Distribution of group II phospholipase A2 protein and mRNA in rat tissues.

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.     

Immunocytochemical and biochemical detection of EMMPRIN in the rat tooth germ: differentiation-dependent co-expression with MMPs and co-localization with caveolin-1 in membrane rafts of dental epithelial cells

In tooth development matrix metalloproteinases (MMPs) are under the control of several regulatory mechanisms including the upregulation of expression by inducers and downregulation by inhibitors. The aim of the present study was to monitor the occurrence and distribution pattern of the extracellular matrix metalloproteinase inducer (EMMPRIN), the metalloproteinases MMP-2 and MT1-MMP and caveolin-1 during the cap and bell stage of rat molar tooth germs by means of immunocytochemistry. Strong EMMPRIN immunoreactivity was detected on the cell membranes of ameloblasts and cells of the stratum intermedium in the bell stage of the enamel organ. Differentiating odontoblasts exhibited intense EMMPRIN immunoreactivity, especially at their distal ends. Caveolin-1 immunoreactivity was evident in cells of the internal enamel epithelium and in ameloblasts. Double immunofluorescence studies revealed a focal co-localization between caveolin-1 and EMMPRIN in ameloblastic cells. Finally, western blotting experiments demonstrated the expression of EMMPRIN and caveolin-1 in dental epithelial cells (HAT-7 cells). A substantial part of EMMPRIN was detected in the detergent-insoluble caveolin-1-containing low-density raft membrane fraction of HAT-7 cells suggesting a partial localization within lipid rafts. The differentiation-dependent co-expression of MMPs with EMMPRIN in the enamel organ and in odontoblasts indicates that EMMPRIN takes part in the induction of proteolytic enzymes in the rat tooth germ. The localization of EMMPRIN in membrane rafts provides a basis for further investigations on the role of caveolin-1 in EMMPRIN-mediated signal transduction cascades in ameloblasts.     

Localization of 28 kDa calbindin in human odontoblasts

Summary The presence of 28 kDa calbindin in human odontoblasts was studied by use of specific antibodies raised against chick duodenal 28 kDa calbindin, in immunofluorescence, immuno-peroxidase, and electron-microscopic labelling experiments.The calbindin-like protein was detected mainly in the cytoplasm of odontoblast cell bodies, in their processes and occasionally in their nuclei. Correspondingly, at the ultrastructural level, immunoreactive material was associated with the cytosol, microfilaments and cilia. These findings suggest that human odontoblasts express a 28 kDa vitamin D-dependent calcium-binding protein, unlike those of rats and mice in which ameloblasts are the only cells immunoreactive for the protein.