ACBTE Vol. 3:19-28, 1993

Iron accumulation in the dental epithelial cells of Tilapia nilotica, a teleost.



Ichiro SASAGAWA



Department of Anatomy,
School of Dentistry at Niigata, The Nippon Dental University,
1-8 Hamaura-cho, Niigata 951, JAPAN


Introduction
The deposition of iron in the enameloid of teleosts has been noted by several researchers1-3). It has been postulated that outer dental epithelial (ODE) cells transport iron into inner dental epithelial (IDE) cells4), and IDE cells concentrate the iron as a form of ferritin and then release it to the enameloid during enameloid maturation5-8). The mechanism is thought to be similar to that in rat incisors9). Motta10) assumed that feeding ecology causes the iron deposition in teleost enameloid. Suga and colleagues11-14), conversely, suggested that the deposition of iron in enameloid is related to the phylogeny of teleosts rather than to their feeding habits and/or environment. If so, a phylogenic approach will be needed to explain the meaning of selective deposition of iron in teleost enameloid. However, the details of iron deposition in the enameloid are unclear and a comparison with higher vertebrates is inadequate. The time of appearance of pigment in the dental epithelial cells and the outline of fine structure concerning the IDE and ODE cells during enameloid maturation have recently been presented by Sasagawa15). The aims of this study were to reveal further details of the fine structure of the dental epithelial cells during pigmentation, to detect iron from the cells by energy-dispersive X-ray microanalysis and to detect the presence of acid phosphatase (ACPase) activity in the cells.

Materials and Methods
Four adult tilapias, Tilapia nilotica (total length of each, 35-37cm), were used in this study.
The animals were decapitated, and samples of pharyngeal teeth and tooth germs were removed and stored in 10% buffered formalin (cacodylate buffer, pH 7.4) for 3 days. The specimens were then demineralized in 5% formic acid for 2 weeks, dehydrated and embedded in paraffin. Sections were stained with hematoxylin-eosin (H-E), with azan and by the Prussian blue method for the detection of ferric iron, and examined under a light microscope.
For transmission electron microscopy, the portions containing pharyngeal tooth germs were placed in Karnovsky fixative (cacodylate buffer, pH 7.4) overnight at room temperature. The majority of the specimens were demineralized in a 2.5% solution of EDTA-2Na after initial fixation and then placed in a 1% solution of osmium tetroxide. After postfixation and dehydration, these specimens were embedded in Araldite-Epon resin. Ultrathin sections were cut with glass or diamond knives, stained with uranyl acetate and lead citrate or lead citrate alone, and examined under a transmission electron microscope (TEM, Hitachi H-500 or JEOL JEM-1200EX). To confirm the presence of iron and other elements, energy-dispersive X-ray microanalysis (EDX) of the ultrathin sections was also carried out using a JEOL JEM-200CX electron microscope equipped with an EDS system.
Acid phosphatase activity: The portions containing pharyngeal tooth germs were fixed with 2% glutar-paraformaldehyde mixture (0.1M cacodylate buffer, pH7.2) for 2 h at 4 ¡C, and then demineralized in a 2.5% solution of EDTA-2Na for 12 days. For light microscopy, the specimens were dehydrated and embedded in Technovit 7100 (KULZER) at 4 ¡C. Sections approximately 4(I5(Jm in thickness were cut with glass knives, and they were incubated in a modified Barka-Anderson's medium containing disodium naphthol AS-BI phosphate (SIGMA) as substrate at pH5.0 for 60 min at 37 ¡C. After the incubation, the sections were stained with methyl green and examined under a light microscope. Medium free of substrate was used as the control. For transmission electron microscopy, the specimens were embedded in OCT compound (MILES), and frozen sections were obtained from a cryostat. Cryostat sections approximately 40(I5(Jm in thickness were rinsed with cacodylate buffer and incubated in Gomori's medium containing sodium b-glycerophosphate (WAKO) as substrate, at pH5.0 for 30 min at 37 ¡C. After the incubation was completed, specimens were rinsed with cacodylate buffer, postfixed in a 1% solution of osmium tetroxide, dehydrated through the graded alcohol, and embedded in Araldite-Epon resin. Ultrathin sections were stained with uranyle acetate and lead citrate, and examined under a TEM (JEOL JEM-1200EX).

Results and Discussion
A. Light microscopy
Early stage of enameloid maturation
Maturation of enameloid began at the time when the mineralization that had started at the boundary between the enameloid and the dentine, reached the surface of the enameloid. In the enameloid maturation stage, the dental epithelial cells, which consisted of just two cell layers, that is, the IDE and the ODE cells, markedly changed their form from that in the former stages. Many capillaries penetrated into the dental epithelial cells, so that the heights of the IDE cells that were tall and columnar in shape, became irregular at this stage. The elliptical nuclei were situated at the proximal portion of the IDE cells. The ODE cells were clear cells with round nuclei at the center, and formed a simple cuboidal cell layer which penetrated into the IDE cell layer with the capillaries. There were no cells between the IDE and the ODE cells.
Dental epithelial cells that were hardly stained by the Prussian blue method were often observed at the cervical portion of a tooth germ in longitudinal sections, although the shape of the cells showed the features of the enameloid maturation stage. In cross sections, a reaction for ferric iron was sometimes negative in part of the dental epithelial cell layer. At the regions that exhibited the negative reaction, the capillaries penetrated superficially and the differences in height of the IDE cells were unobtrusive. It is assumed that the dental epithelial cells showing negative reaction for ferric iron at the cervical portion display the early stage of enameloid maturation, not differences among the portions in a tooth germ, because iron deposition was visible on the matured cervical enameloid and the dental epithelial cells situated at the cervical portion were often stained by the Prussian blue method. However, no tooth germs in which most of the dental epithelial cells exhibited a negative reaction at the stage of enameloid maturation were found in this study. It is likely that the period in which little iron accumulates in the dental epithelial cells, that is, the early stage, is quite short. The reaction for ferric iron was entirely negative on tooth germs before the stage of enameloid maturation.
Middle stage of enameloid maturation
The capillaries deeply penetrated into the dental epithelial cells which caused marked differences in the heights of IDE cells. The tall IDE cells seemed to be three or four times taller than the short ones. The nuclei of the IDE cells were situated at the proximal portion. The ODE cells having nearly round nuclei were clear cuboidal cells that formed a simple layer surrounding the capillaries. A positive reaction for ferric iron appeared in the IDE cells except for the distal ends. The density of the reaction was almost equivalent in the cells. The reaction was initially evident in regions distal to nuclei, that is, probably in the Golgi area in paraffin sections which were about 10 $B&L(Jm in thickness. However, in Technovit sections which were about 4$B&L(Jm in thickness, the reaction gradually appeared in nearly the whole of the cytoplasm. The ODE cells showed little positive reaction for ferric iron.
Late stage of enameloid maturation
Capillaries penetrating deeply into the dental epithelial cells were no longer visible, so the heights of all IDE cells were similar at this stage. Oval nuclei occupied the proximal portion of the IDE cells. Two or three layers of clear and flat ODE cells were situated beyond the tall IDE cells. A large number of yellow or yellowish-brown granules were observed in the IDE cells in the sections stained with H-E and azan. The reaction for ferric iron in the IDE cells was strongest at this stage. The cytoplasm, particularly the large granules at the central portion of the cells, was stained dark blue. Dotlike or bacilliform substances stained with Prussian blue were visible in the ODE cells.

B. Transmission electron microscopy
The early stage of enameloid maturation
Few ferritin particles were found in the cytoplasm of the IDE cells (Fig. 1). A ruffled border was present at the distal end of the IDE cells (Fig. 2). The proximal end of the ruffled border often expanded like a cisterna and contained electron-dense flocculent materials. Pinocytotic vesicles were sometimes associated with the expanded cisternae. Golgi apparatus that consisted of lamellae arranged in stacks of 4-5, many vesicles and vacuoles were seen around the nucleus. Several electron-dense granules which were probably primary lysosomes were exhibited in the cytoplasm between the ruffled border and nucleus. Granules that seemed to be aggregations of ferritin particles, approximately 200nm in diameter, were sometimes observed in the cytoplasm (Fig. 3). There were small numbers of rough endoplasmic reticula (rER) in the IDE cells at this stage. Several mitochondria, many free ribosomes and small vesicles were scattered throughout the cytoplasm. Interdigitations between adjacent IDE cells were developed, and there were usually desmosomes and gaplike junctions at the interdigitation. Intercellular spaces tended to open in the latter half of the early stage. Most of the cytoplasm in the ODE cells was occupied by labyrinthine canalicular spaces. A small number of mitochondria, filaments, short rER, granules containing electron-dense materials, ribosomes, vacuoles and vesicles were seen in the narrow spaces between the canaliculi. The interface between the IDE and the ODE cells wase relatively straight, and intercellular spaces were scarcely found there. There were many desmosomes and gaplike junctions at the interface. At the opposite side, the ODE cells faced the capillaries through a narrow space and thin basal lamina.
Middle stage of enameloid maturation
Presence of a well-developed ruffled border at the distal end was a prominent feature of the IDE cells at the middle stage. Cisternae containing electron-dense flocculent materials were visible at the proximal end of the ruffled border. Coated vesicles and pinocytotic vesicles were often present around the cisternae. Many mitochondria, primary lysosomelike bodies and multivesicular bodies usually gathered in the distal cytoplasm near the ruffled border. Widely distributed Golgi apparatus that consisted of lamellae in stacks of 4-5, vesicles and vacuoles mainly occupied the central cytoplasm. There were a few mitochondria, short rER, smooth endoplasmic reticula (sER), electron-dense bodies and ribosomes around the Golgi apparatus. A large nucleus was situated at the proximal portion. Short rER, mitochondria and parts of Golgi apparatus were found around the nucleus. Desmosomes and gaplike junctions were the usual means of adhesion between the IDE cells, and intercellular spaces were often seen between the sites of attachment. However, distal portions with ruffled borders tightly touched each other, and cell-adhesions like junctional complex were visible there. This suggests that the lateral cell membranes of the IDE cells are closed by a junctional complex at the distal portion. On the other hand, well-developed interdigitations between the IDE cells were seen at the proximal portion. Enlarged views of the cytoplasm of the IDE cells exhibited large numbers of ferritin particles scattered in it. There were also many ferritin particles at the distal portion, although their density was low in comparison with the other portions. No ferritin particles were observed in nuclei and the inside of membrane-bound organelles, that is, Golgi apparatus, rER and sER. Ferritin particles were electron-dense, round and approximately 5.5nm in diameter. The particles often seemed to have an electron-lucent core. There were many large granules that contained electron-dense fine particles, but the contents of the granules varied in density and texture. The state of electron-dense particles similar to ferritin, but slightly smaller in diameter, in the granules seemed to cause the variation (Figs.4-6), suggesting that ferritin particles gather at the primary lysosomes. Few large crystalloid aggregations of ferritinlike particles were found at the middle stage. Well-developed labyrinthine canalicular spaces occupied most of the cytoplasm in the ODE cells. A number of mitochondria, coated vesicles, electron-dense small granules, ribosomes and primary lysosomes were seen in the narrow spaces between the canaliculi. A few rER and limited Golgi apparatus were situated near the nucleus. There was no evidence that the canaliculi opened to the outside of the ODE cells at the side that faced the capillary. However, the canaliculi were usually close to the outside, separated from it only by the thin cytoplasm. At the side facing the IDE cells, the canaliculi sometimes seemed to open to the intercellular space. There were several desmosomes, but few expanded spaces and gaplike junctions between adjacent ODE cells. Serial arrangement of desmosomes and gaplike junctions were visible between the IDE and the ODE cells. No expanded intercellular spaces were present there (Fig. 7). There were few ferritin particles in the ODE cells. However, some small aggregations of ferritinlike particles were occasionally seen in the ODE cells, although it is possible that the aggregations were part of the IDE cell processes.
Late stage of enameloid maturation
Many electron-dense large granules were found in the central portion between the proximal nucleus and distal fuffled border (Fig. 8). Thelarge granules packed with electron-dense fine particles were divided into two categories in terms of texture: round granules limited by a unit membrane, which usually exhibited amorphous contents, and granules that mostly contained crystalloids and had an irregular outline without a unit membrane. Small electron-dense bodies often attached themselves to the round large granules. The majority of such electron-dense large granules were probably ferritin granules6 16 17). There were still many ferritin particles in the cytoplasm of the IDE cells, although the density of the particles was low. Many mitochondria were found near the large granules. A ruffled border was present at the distal end of the IDE cells, and many vacuoles and vesicles were seen at the proximal portion of the ruffled border. The other organelles in the IDE cells were under-developed, while limited Golgi apparatus and short rER were situated around the nucleus. In particular, organelles were scarcely seen at the proximal quarter of the IDE cells. The IDE cells tightly touched each other and there were no expanded intercellular spaces between them. The lateral cell membranes were almost straight except for the proximal portions that had interdigitation. At the distal portions, tight junctions and desmosomes were visible, implying the presence of a junctional complex. The ODE cells were now flat, clear cells with mitochondria, rER, Golgi apparatus, small granules and abundant filaments around the nuclei. There were a few large electron-dense granules that resembled the ferritin granules in the cytoplasm, supporting the observation of a positive reaction for ferric iron in the ODE cells at this stage by light microscopy. No expanded intercellular spaces were seen among the stratified ODE cells, and between the IDE and the ODE cells. The cell membrane that faced adjacent ODE cells was mostly straight except for small-scale jigsaw interlockings, and there were several desmosomes and gaplike junctions between the ODE cells. From current observations, the shape of the IDE and the ODE cells of the tilapia, a teleost, during enameloid maturation seems to be similar to that of ruffle-ended ameloblasts and papillary cells during enamel maturation in mammals18 19), although there are several differences; e.g., the capillaries penetrating into the IDE cell layer, the labyrinthine canaliculi in the ODE cells, and the absence of cells corresponding to the smooth-ended ameloblasts. It is, therefore, possible that the role of the IDE and the ODE cells during enameloid maturation in tilapias resembles that of the ruffle-ended ameloblasts and papillary cells during enamel maturation. Consequently, these cells in tilapias might be involved in the selective removal of the organic matrix including collagen from the enameloid and in the active transport of inorganic ions, such as calcium, to the enameloid20-22). According to the shape of the dental epithelial cells and the distribution of ferritin particles and granules, the middle stage of the enameloid maturation should correspond to the early and middle pigmentation stages in rat incisors19), and the late stage of enameloid maturation should correspond to the late pigmentation stage 9 19). It is likely that iron is taken into the ODE cells from the capillaries in tilapias as in the rat incisor23), because of the morphological features of the ODE cells and the positive reaction for ferric iron at the late stage. It is definite that the accumulation of iron in the IDE and the ODE cells of tilapias occurs in two stages: the middle enameloid-maturation stage, in which iron accumulates in the IDE cells as ferritin particles through the ODE cells, and the late enameloid-maturation stage, in which iron in the IDE cells aggregates and forms the large granules, that is, ferritin granules. The mechanism that transports iron to the enameloid might differ from that of calcium, because most of the iron moves to the enameloid after the late enameloid-maturation stage. It is rare to find tooth germs after the late stage of enameloid maturation and before eruption, so further studies will be needed in order to reveal the fine structure of the dental epithelial cells at the pigment release stage. How iron becomes part of the matured enameloid of teleosts is also a subject for future study. In any case, it is quite interesting that the mechanism of iron accumulation is similar between enamel and enameloid, mammal and teleost.

C. Energy-dispersive X-ray microanalysis (EDX).
Iron was always detected in the regions that contained many electron-dense ferritin particles, but few organelles in the IDE cells at the middle stage of enameloid maturation, and that included several electron-dense large granules at the late stage of enameloid maturation.

D. Acid phosphatase (ACPase) activity
Middle stage of enameloid maturation
Light micrographs of tooth germs showed feeble scattered ACPase activity in the IDE cells.
By means of transmission electron microscopy, strong ACPase activity was detected in the granules that were situated at the central portion of the IDE cells, suggesting that the granules were lysosomes. Crystalloids were often found inside the heavily stained granules, although the crystalloids were not stained (Figs. 9,10).
Late stage of enameloid maturation
By light microscopy, the IDE cells were found to contain many large granules that exhibited relatively obvious ACPase activity, in the central portion. The sites showing ACPase activity seemed to be slightly different from the positions of pale-yellow pigment.
ACPase activity was found in large granules and vacuoles in transmission electron micrographs. However, there were a few large granules that contained an intense reaction. The fine reaction for ACPase tended to scatter in many granules. It seemed that crystalloids having no unit membrane exhibited scarcely any ACPase activity (Figs. 11,12).
Takano & Ozawa24) postulated that ferritin is digested in the ferritin-containing vesicles for the release of the iron pigment to the enamel in the rat incisor. In this study, it is suggested that lysosomes are strongly involved in the aggregation of ferritinlike particles, that is, electron-dense large granules. It is assumed that lysosome enzymes remove superficial proteins of ferritin, and then hemosiderin, which is slightly smaller than ferritin25-26), is produced in the IDE cells. In Kupffer cells of mammalian liver, ferritin goes on to form hemosiderin permanently retained in the cell within solid membranous particles, representing mainly a response to iron overload25). In general, such hemosiderin rarely returns to the cardiovascular system, and tends to stay in the tissues27). It is, therefore, possible that ferritin is changed to hemosiderin by lysosomal digestion in the autophagosomes of the IDE cells in tilapia. If so, it is probable that iron deposition to teleost enameloid means a response to iron overload and actually an excretion of superfluous elements to enameloid as suggested by Suga13).

Acknowledgement
The author thanks the staff members at Hokuetsu Mizugiken Inc. for their kindness in offering their facilities for the collection of specimens, and Dr. J. Akai of Department of Geology and Mineralogy, Niigata University, for his advice and assistance.

References

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Legends


Plate 1
Fig. 1. Inner dental epithelial (IDE) and outer dental epithelial (ODE) cells at the early stage of enameloid maturation. A capillary (B) is close to the ODE cells. Undemineralized, stained with uranyl acetate and lead citrate (U-Pb), bar=5(I5(Jm.

Fig. 2. Enlarged view of fig.1. Mineralized enameloid (E) and the distal portion of the IDE cells that exhibits infoldings of the distal cell membrane. Few ferritin particles are visible in the cytoplasm. Bar=1(I5(Jm.

Fig. 3. Granule that is similar to the ferritin aggregation in the IDE cells at the early stage of enameloid maturation. A few ferritin particles (arrow heads) are also visible. Demineralized by EDTA-2Na, stained with lead citrate alone, bar=100nm.

Fig. 4. Electron-dense large granule that seems a lysosome containing fine ferritinlike particles in the IDE cell at the middle stage of enameloid maturation. Demineralized, U-Pb, bar=100nm.

Fig. 5. Granule limited by a unit membrane in the IDE cell at the middle stage. Particles contained in the granule are slightly smaller than ferritin particles surrounding them. Undemineralized, U-Pb, bar=100nm.

Fig. 6. Granules in the IDE cell at the middle stage. Undemineralized, Pb alone, bar=200nm.

Fig. 7. Junction between the IDE and ODE cells at the middle stage. Demineralized, U-Pb, bar=1(I5(Jm.


Plate 2
Fig. 8. IDE cells at the late stage of enameloid maturation. E: enameloid space, demineralized, U-Pb, bar=3(I5(Jm.

Fig. 9. ACPase activity in the IDE cells at the middle stage of enameloid maturation. The strong reaction seems to be detected in the large granules. Demineralized, U-Pb, bar=500nm.

Fig.10. Granule exhibits strong reaction for ACPase in the IDE cells at middle stage. Demineralized, U-Pb, bar=500nm.

Fig.11. Central portion of IDE cells showing ACPase activity at the late stage of enameloid maturation. Demineralized, U-Pb, bar=1(I5(Jm.

Fig.12. Enlarged view of the central portion of the IDE cells at late stage. ACPase activity is hardly found at the crystalloids. Demineralized, U-Pb, bar=500nm.