An Electron Microscope Study of Crystal Calcium Carbonate Formation in the Mouse Otolith HIROSHI NAKAHARA ' AND GERRIT BEVELANDER ' Josai Dental College, Sakado, Saitama-Ken, Japan
ABSTRACT The ultrathin sections of the developing otolith of mouse fetuses 15.5 days to 20 days after birth were observed with the aid of the electron microscope. The first step of otolith formation is an aggregation of organic clusters observed in the sacculus of the 15.5-day fetus. These organic structures are modified and assume a hexagonal shape in the 17.5-day fetus. The unmineralized stages of the otolith referred to as thepreotolzth, serve as the template for future development. One day after birth, a t either end of the preotolith, minute tube-like structures develop in which needle-shaped crystallites are initiated. Crystallites continue to develop throughout the hexagonal template which give rise to a structure containing many regularly arranged needle-shaped crystallites. Based on the present observations, the mouse otolith is considered a multi-iso-oriented crystal. Despite the several studies utilizing diverse disciplines, our knowledge concerning the formation and structure of the mammalian otolith is incomplete and fragmentary. Lyon ('551, Sher ('711, de Vincentis and Marmo ('661, reported that the formation of an organic matrix or complex precedes mineralization of the otolith in the mouse and chick, respectively. Other investigators, Wislocki and Ladman ('551, Belanger ('601, Mugiya ('681, and Degens et al. ('69), have noted the presence of protein and protein-polysaccharides in the mature otolith. Following the administration of trace doses of 'Ta (Belanger, '60; de Vincentis and Marmo, '661, the establishment of a mineral-polysaccharide relationship was demonstrated. Carlstrom et al. ('53) have shown that the mineral composition of the mammalian otolith is calcite. Subsequent studies (Carlstrom and Engstrom, '55) suggest that otoliths behave like single crystals. These latter observations were confirmed and amplified by Iurato and DePetris ('67). Previous studies (Lyon, '55; Belanger, '60) have shown that the vestibular epithelium contributes to the precursor of the otolith. This study is concerned only with the ultrastructural sequence of extracellular events ANAT.
REC. (1979)193: 233-242.
that lead to the formation and mineralization of the otolith. MATERIALS AND METHODS
Mouse fetuses (15.5, 17.5 days) and young specimens ranging in age from newborn to 20 days of the ICR strain obtained from a commercial source were used in this study. Portions of the utricle and saccule in which the otoliths are located were carefully removed and fixed in collidine buffered 5% glutaraldehyde for six hours. After fixation they were washed twice in buffer solution, routinely dehydrated and then embedded in Araldite 502. Ultra-thin sections were cut with a glass or a diamond knife, stained in uranyl acetate (2%in H20) and lead citrate (2% in N/10 NaOH). Unstained sections were also prepared for study of the presence of mineralized material. OBSERVATIONS
Examination of a section of the macula sacculus of a 15.5-day fetus (fig. 11,shows an aggregation of organic material arranged in clusters situated in close proximity to the surface of the epithelium of the macula. The peReceived June 23, '78. Accepted Sept. 5, "78. Present address: Box 2656. Camel. California 93921.
233
234
HIROSHI NAKAHARA AND GERRIT BEVELANDER
ripheral portion of t h e aggregations exhibits a dense accumulation of fine thread-like fibrils and granules; t h e central region contains only a few of these structures: this and subsequent stages of t h e otolith t h a t are unmineralized will be referred to as t h e preotolith. The epithelial cells of t h e 15.5- and 17.5-day specimens exhibit blebs on t h e surface of t h e cells. Although comparable in size and cytological constituents to t h e non-hexagonal early preotoliths, these blebs do not appear to become transformed into preotoliths since t h e latter do not have a cell membrane (limiting membrane) or its remnants within or around it. I t would appear more likely t h a t t h e preotoliths are t h e result of a reorganization of materials secreted in t h e extracellular space. At 17.5 days, t h e preotoliths still located in the same position relative to t h e surface epithelium have undergone a striking change when compared with those of t h e 15.5-day specimens. As shown in figures 2 and 3 these structures have enlarged and assumed a hexagonal shape. In addition, t h e organic material consisting of t h e fibrils and granules has become distributed not only on the periphery but also in t h e central region of t h e hexagon. This rearrangement (shown in fig. 3) gives rise to a relatively clear diamond-shaped area a t both ends of the hexagon. At birth t h e preotolith appears as a fairly regularly shaped hexagon exhibiting a meshwork of fibrillar and granular material t h a t is distributed quite uniformly throughout t h e entire structure. Two modifications relative to the meshwork components have occurred at this stage of development: (1) they are now radially arranged in reference to t h e center of t h e hexagon; (2) a t either end of t h e hexagon they acquire a n intense electron density (figs. 4, 5). Examination of a section of thesacculus of a 1-day animal shows t h a t some of t h e electron dense granules referred to above have aligned themselves in such a manner t h a t they appear as minute tube-like structures. Further, t h e initiation and formation of needle-shaped crystallites seems to have occurred in t h e space enclosed by t h e aligned granules (figs. 6, 7). The subsequent development of t h e otolith consists of a repetition of t h e events just described, t h a t is, formation of tubules followed by t h e initiation of crystallites in them. This occurs first in both ends of the hexagon (figs.
8,9), and continues until the entire hexagonal template is occupied by crystallites (fig. 10). Specimens examined from t h e fifth through t h e twentieth day show t h a t crystallites are present throughout t h e entire extent of the otolith in both t h e utricle and saccule. In the peripheral part of t h e otolith the crystallites are more densely packed than in t h e central portion. Apparently, this uneven distribution is characteristic of t h e mature otolith. The crystallites in t h e older specimens retain their needle- or thread-like appearance and are oriented from t h e surface to t h e center in a radial direction (fig. 10). The events occurring in t h e utricle are similar in all respects except t h a t development, especially t h e crystal formation after birth, occurs approximately one day later than development in t h e saccule. DISCUSSION
As previously stated several investigators reported t h a t a n organic matrix precedes mineralization. Lyon ('55) described t h e presence of a n organic precipitate and granules which she designated otolith precursors. Our study confirms Lyon's observations to t h e extent t h a t organic aggregations serve as the forer u n n e r s of otoliths. We have, however, amplified t h e above observations considerably. The irregularly shaped aggregations assume t h e size and shape of the future otolith. The preotolith undergoes ultrastructural changes during development that give rise to t h e formation of minute tubules or envelopes in which crystallite initiation occurs, these crystallites eventually occupy t h e entire hexagonal template. Although details concerning crystal initiation and growth in otoliths have not been described until this juncture, we wish to point out t h a t crystallite formation in minute tubelike envelopes is similar to t h e formation of calcium carbonate crystals in mollusc nacre (Bevelander and Nakahara, '69), in the calciferous glands of earthworms, t h e prismatic layer of molluscs and in t h e green alga, Hulimedu (Nakahara and Bevelander, '69, '71, '78). The consensus in regard to the crystallographic properties of t h e mammalian otolith is as follows: they a r e calcite (Carlstrom e t al. ('531, consist of well aligned subunits and behave like a single crystal with the crystallographic c axis parallel to t h e major axis (Carl-
CA-CARBONATE FORMATION IN THE OTOLITH
strom and Engstrom, ' 5 5 ) .Iurato and DePetris ('67) also maintain that the mammalian otolith behaves as a single crystal of calcite. These latter authors remark, however, that it is difficult to ascertain whether the otoliths are true single crystals or regular assemblies of many small iso-oriented crystallites. They further state that this point may have a bearing on the manner in which the crystals are formed. Our studies have shown that the crystal (otolith) does indeed consist of many crystallites. Also, the fact that the otolith has a t least two and probably more nucleation sites precludes the possibility that the otolith is a single crystal. On the basis of our investigations we submit that the mouse otolith is an iso-oriented multiple crystal. SUMMARY
1. The first observable extracellular precursor of the otolith consists of organic aggregations. 2. These aggregations are modified and assume a hexagonal shape which serves as the template for future development. 3. At either end of the template minute tubules develop in which needle-shaped crystallites are initiated; this occurs in the 1-day juvenile specimens. 4. Crystallites continue to develop throughout the hexagonal template which gives rise to a structure containing many regularly arranged needle-shaped crystallites. 5 . On the basis of the above considerations we maintain that the otolith is a multi-iso-oriented crystal.
235
LITERATURE CITED Belanger, L. F. 1960 Development, structure and composition of the otolithic organs of the rat. In: Calcification in Biological Systems. R. F. Sognnaes, ed. AAAS, Washington, D.C., pp. 151-162. Bevelander, G., and H. Nakahara 1969 An electron microscope study of the formation of thenacreous layer in the shell of certain bivalve molluscs. Calc. Tiss. Res., 3: 84-92. Carlstrom, D., and E. Engstrom 1955 The ultrastructure of Statoconia. Acta Otolaryng., 45: 14-18. Carlstrom, D., E. Engstrom and S. Hjorth 1953 Electron microscopic and X-ray diffraction studies of statoconia. Laryngoscope, 63: 1052-1057. Degens, E. T., W. G. Duser and R. L. Haedrich 1969 Molecular structure and composition of fish otoliths. Marine Biol., 2: 105-113. Iurato, S., and S. DePetris 1967 Otolithic membranes. In: Submicroscopic Structure of the Inner Ear. S.Iurato, ed. Pergamon Press, Oxford, pp. 210-216. Lyon, M. F. 1955 The development of the otoliths of the mouse. J. Embryol. exp. Morph., 3: 213-229. Mugiya, Y. 1968 Calcification in fish and shell-fish VII. Histochemical similarities between the otolith and the macula region of sacculus in juvenile rainbow trout, with special reference to the otolith formation of fish. Bull. Sci. Fish., 34: 1096-1106. Japan. SOC. Nakahara, H., and G. Bevelander 1969 An electron microscope and autoradiographic study of the calciferous glands of the earthworm Lumbricus terrestris. Calc. Tiss. Res., 4: 193-201. 1971 The formation and growth of the prismatic layer of Pinctada radiata. Calc. Tiss. Res., 7: 31-45. 1978 The formation of calcium carbonate crystals in Halimeda incrassata with special reference to the role of t h e organic matrix. Japan. SOC.Phycol., 26: 9-12 Sher, A. E. 1971 The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryng., Suppl., 285: 1-77. de Vincentis, M., and F. Marmo 1966 The "Ca turnover in the membranous labyrinth of the chick embryo duringdevelopment. J. Embryol. exp. Morph., 3: 349-354. Wislocki, G. B., and A. J. Ladman 1955 Selective and histochemical staining of the otolithic membranes, capulae and tectorial membrane of t h e inner ear. J. Anat., 89: 3-12.
All sections with the exception of those shown in figures 8 and 10 are stained in uranyl acetate-lead citrate (double stain).
PLATE 1 EXPLANATION OF FIGURES
1 Part of macula sacculus from 15.5-day mouse fetus. E, epithelium; PO, early stage of preotolith. X 6,000.
236
2
Part of macula utriculi. Seventeen and one-half-day fetus. E, epithelium; PO, preotolith. x 5,000.
3
Longitudinally sectioned preotolith from utriculus. Seventeen and one-half-day fetus, note distribution of granules. X 27,000.
CA-CARBONATE FORMATION IN THE OTOLITH Hiroshi Nakahara and Gerrit Brvelander
237
PLATE 2 EXPLANATION OF FIGURES
238
4
Mature preotolith from utriculus of newborn mouse. Cluster of electron dense organic granules form tubular structures near both ends of preotolith. X 30.000.
5
Enlarged from figure 4. Note t h e tube-like structure of electron dense material. x 75,000.
6
Showing otolith from utricule of 1-day-oldmouse; stage of t h e earliest Ca-Carbonate formation. Section decalcified during staining process. Spaces formerly occupied by small Ca-Carbonate crystals (enclosed by electron dense material) a r e shown near both ends of otolith. X 25,000.
7
Enlarged from a part of figure 5 . CR, spaces formerly occupied by Ca-Carbonate crystals. x 70,000.
CA CAKBONATE FORMATION IN THE OTOLITH Hiroshl N a k a h a r s a n d tierrit Bevrlander
PLATE 2
239
PLATE 3
X
Otolith from utriculr of 1 day-old mouse Note the groups of small Ca-Carbonate crystals a t either end of otolith (dark1 X 30,000.
Y
Otolith froin utricule of' 3-day-old mouse Crystals occupy two large areas of otolith (light artW. X 15.000
10
Nearl? m a t u r e otolith from ulricule of 7 ~ d a y - o l dmouse Unstained. rod^ 01' needlrshaprd Ca-Carbonate crystals a r e c l ~ s e l ypacked in LTcater part of otolith except I'ur the central area where only a few crystals are present x 50.000.
CA CARBONATE FOKMATIVN IN THE OTOLITH Hiroshi Ndkahnra and G e r n t Bevelander
PLAl’t
i
24 1
ABSTRACT The ultrathin sections of the developing otolith of mouse fetuses 15.5 days to 20 days after birth were observed with the aid of the electron microscope. The first step of otolith formation is an aggregation of organic clusters observed in the sacculus of the 15.5-day fetus. These organic structures are modified and assume a hexagonal shape in the 17.5-day fetus. The unmineralized stages of the otolith referred to as thepreotolzth, serve as the template for future development. One day after birth, a t either end of the preotolith, minute tube-like structures develop in which needle-shaped crystallites are initiated. Crystallites continue to develop throughout the hexagonal template which give rise to a structure containing many regularly arranged needle-shaped crystallites. Based on the present observations, the mouse otolith is considered a multi-iso-oriented crystal. Despite the several studies utilizing diverse disciplines, our knowledge concerning the formation and structure of the mammalian otolith is incomplete and fragmentary. Lyon ('551, Sher ('711, de Vincentis and Marmo ('661, reported that the formation of an organic matrix or complex precedes mineralization of the otolith in the mouse and chick, respectively. Other investigators, Wislocki and Ladman ('551, Belanger ('601, Mugiya ('681, and Degens et al. ('69), have noted the presence of protein and protein-polysaccharides in the mature otolith. Following the administration of trace doses of 'Ta (Belanger, '60; de Vincentis and Marmo, '661, the establishment of a mineral-polysaccharide relationship was demonstrated. Carlstrom et al. ('53) have shown that the mineral composition of the mammalian otolith is calcite. Subsequent studies (Carlstrom and Engstrom, '55) suggest that otoliths behave like single crystals. These latter observations were confirmed and amplified by Iurato and DePetris ('67). Previous studies (Lyon, '55; Belanger, '60) have shown that the vestibular epithelium contributes to the precursor of the otolith. This study is concerned only with the ultrastructural sequence of extracellular events ANAT.
REC. (1979)193: 233-242.
that lead to the formation and mineralization of the otolith. MATERIALS AND METHODS
Mouse fetuses (15.5, 17.5 days) and young specimens ranging in age from newborn to 20 days of the ICR strain obtained from a commercial source were used in this study. Portions of the utricle and saccule in which the otoliths are located were carefully removed and fixed in collidine buffered 5% glutaraldehyde for six hours. After fixation they were washed twice in buffer solution, routinely dehydrated and then embedded in Araldite 502. Ultra-thin sections were cut with a glass or a diamond knife, stained in uranyl acetate (2%in H20) and lead citrate (2% in N/10 NaOH). Unstained sections were also prepared for study of the presence of mineralized material. OBSERVATIONS
Examination of a section of the macula sacculus of a 15.5-day fetus (fig. 11,shows an aggregation of organic material arranged in clusters situated in close proximity to the surface of the epithelium of the macula. The peReceived June 23, '78. Accepted Sept. 5, "78. Present address: Box 2656. Camel. California 93921.
233
234
HIROSHI NAKAHARA AND GERRIT BEVELANDER
ripheral portion of t h e aggregations exhibits a dense accumulation of fine thread-like fibrils and granules; t h e central region contains only a few of these structures: this and subsequent stages of t h e otolith t h a t are unmineralized will be referred to as t h e preotolith. The epithelial cells of t h e 15.5- and 17.5-day specimens exhibit blebs on t h e surface of t h e cells. Although comparable in size and cytological constituents to t h e non-hexagonal early preotoliths, these blebs do not appear to become transformed into preotoliths since t h e latter do not have a cell membrane (limiting membrane) or its remnants within or around it. I t would appear more likely t h a t t h e preotoliths are t h e result of a reorganization of materials secreted in t h e extracellular space. At 17.5 days, t h e preotoliths still located in the same position relative to t h e surface epithelium have undergone a striking change when compared with those of t h e 15.5-day specimens. As shown in figures 2 and 3 these structures have enlarged and assumed a hexagonal shape. In addition, t h e organic material consisting of t h e fibrils and granules has become distributed not only on the periphery but also in t h e central region of t h e hexagon. This rearrangement (shown in fig. 3) gives rise to a relatively clear diamond-shaped area a t both ends of the hexagon. At birth t h e preotolith appears as a fairly regularly shaped hexagon exhibiting a meshwork of fibrillar and granular material t h a t is distributed quite uniformly throughout t h e entire structure. Two modifications relative to the meshwork components have occurred at this stage of development: (1) they are now radially arranged in reference to t h e center of t h e hexagon; (2) a t either end of t h e hexagon they acquire a n intense electron density (figs. 4, 5). Examination of a section of thesacculus of a 1-day animal shows t h a t some of t h e electron dense granules referred to above have aligned themselves in such a manner t h a t they appear as minute tube-like structures. Further, t h e initiation and formation of needle-shaped crystallites seems to have occurred in t h e space enclosed by t h e aligned granules (figs. 6, 7). The subsequent development of t h e otolith consists of a repetition of t h e events just described, t h a t is, formation of tubules followed by t h e initiation of crystallites in them. This occurs first in both ends of the hexagon (figs.
8,9), and continues until the entire hexagonal template is occupied by crystallites (fig. 10). Specimens examined from t h e fifth through t h e twentieth day show t h a t crystallites are present throughout t h e entire extent of the otolith in both t h e utricle and saccule. In the peripheral part of t h e otolith the crystallites are more densely packed than in t h e central portion. Apparently, this uneven distribution is characteristic of t h e mature otolith. The crystallites in t h e older specimens retain their needle- or thread-like appearance and are oriented from t h e surface to t h e center in a radial direction (fig. 10). The events occurring in t h e utricle are similar in all respects except t h a t development, especially t h e crystal formation after birth, occurs approximately one day later than development in t h e saccule. DISCUSSION
As previously stated several investigators reported t h a t a n organic matrix precedes mineralization. Lyon ('55) described t h e presence of a n organic precipitate and granules which she designated otolith precursors. Our study confirms Lyon's observations to t h e extent t h a t organic aggregations serve as the forer u n n e r s of otoliths. We have, however, amplified t h e above observations considerably. The irregularly shaped aggregations assume t h e size and shape of the future otolith. The preotolith undergoes ultrastructural changes during development that give rise to t h e formation of minute tubules or envelopes in which crystallite initiation occurs, these crystallites eventually occupy t h e entire hexagonal template. Although details concerning crystal initiation and growth in otoliths have not been described until this juncture, we wish to point out t h a t crystallite formation in minute tubelike envelopes is similar to t h e formation of calcium carbonate crystals in mollusc nacre (Bevelander and Nakahara, '69), in the calciferous glands of earthworms, t h e prismatic layer of molluscs and in t h e green alga, Hulimedu (Nakahara and Bevelander, '69, '71, '78). The consensus in regard to the crystallographic properties of t h e mammalian otolith is as follows: they a r e calcite (Carlstrom e t al. ('531, consist of well aligned subunits and behave like a single crystal with the crystallographic c axis parallel to t h e major axis (Carl-
CA-CARBONATE FORMATION IN THE OTOLITH
strom and Engstrom, ' 5 5 ) .Iurato and DePetris ('67) also maintain that the mammalian otolith behaves as a single crystal of calcite. These latter authors remark, however, that it is difficult to ascertain whether the otoliths are true single crystals or regular assemblies of many small iso-oriented crystallites. They further state that this point may have a bearing on the manner in which the crystals are formed. Our studies have shown that the crystal (otolith) does indeed consist of many crystallites. Also, the fact that the otolith has a t least two and probably more nucleation sites precludes the possibility that the otolith is a single crystal. On the basis of our investigations we submit that the mouse otolith is an iso-oriented multiple crystal. SUMMARY
1. The first observable extracellular precursor of the otolith consists of organic aggregations. 2. These aggregations are modified and assume a hexagonal shape which serves as the template for future development. 3. At either end of the template minute tubules develop in which needle-shaped crystallites are initiated; this occurs in the 1-day juvenile specimens. 4. Crystallites continue to develop throughout the hexagonal template which gives rise to a structure containing many regularly arranged needle-shaped crystallites. 5 . On the basis of the above considerations we maintain that the otolith is a multi-iso-oriented crystal.
235
LITERATURE CITED Belanger, L. F. 1960 Development, structure and composition of the otolithic organs of the rat. In: Calcification in Biological Systems. R. F. Sognnaes, ed. AAAS, Washington, D.C., pp. 151-162. Bevelander, G., and H. Nakahara 1969 An electron microscope study of the formation of thenacreous layer in the shell of certain bivalve molluscs. Calc. Tiss. Res., 3: 84-92. Carlstrom, D., and E. Engstrom 1955 The ultrastructure of Statoconia. Acta Otolaryng., 45: 14-18. Carlstrom, D., E. Engstrom and S. Hjorth 1953 Electron microscopic and X-ray diffraction studies of statoconia. Laryngoscope, 63: 1052-1057. Degens, E. T., W. G. Duser and R. L. Haedrich 1969 Molecular structure and composition of fish otoliths. Marine Biol., 2: 105-113. Iurato, S., and S. DePetris 1967 Otolithic membranes. In: Submicroscopic Structure of the Inner Ear. S.Iurato, ed. Pergamon Press, Oxford, pp. 210-216. Lyon, M. F. 1955 The development of the otoliths of the mouse. J. Embryol. exp. Morph., 3: 213-229. Mugiya, Y. 1968 Calcification in fish and shell-fish VII. Histochemical similarities between the otolith and the macula region of sacculus in juvenile rainbow trout, with special reference to the otolith formation of fish. Bull. Sci. Fish., 34: 1096-1106. Japan. SOC. Nakahara, H., and G. Bevelander 1969 An electron microscope and autoradiographic study of the calciferous glands of the earthworm Lumbricus terrestris. Calc. Tiss. Res., 4: 193-201. 1971 The formation and growth of the prismatic layer of Pinctada radiata. Calc. Tiss. Res., 7: 31-45. 1978 The formation of calcium carbonate crystals in Halimeda incrassata with special reference to the role of t h e organic matrix. Japan. SOC.Phycol., 26: 9-12 Sher, A. E. 1971 The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryng., Suppl., 285: 1-77. de Vincentis, M., and F. Marmo 1966 The "Ca turnover in the membranous labyrinth of the chick embryo duringdevelopment. J. Embryol. exp. Morph., 3: 349-354. Wislocki, G. B., and A. J. Ladman 1955 Selective and histochemical staining of the otolithic membranes, capulae and tectorial membrane of t h e inner ear. J. Anat., 89: 3-12.
All sections with the exception of those shown in figures 8 and 10 are stained in uranyl acetate-lead citrate (double stain).
PLATE 1 EXPLANATION OF FIGURES
1 Part of macula sacculus from 15.5-day mouse fetus. E, epithelium; PO, early stage of preotolith. X 6,000.
236
2
Part of macula utriculi. Seventeen and one-half-day fetus. E, epithelium; PO, preotolith. x 5,000.
3
Longitudinally sectioned preotolith from utriculus. Seventeen and one-half-day fetus, note distribution of granules. X 27,000.
CA-CARBONATE FORMATION IN THE OTOLITH Hiroshi Nakahara and Gerrit Brvelander
237
PLATE 2 EXPLANATION OF FIGURES
238
4
Mature preotolith from utriculus of newborn mouse. Cluster of electron dense organic granules form tubular structures near both ends of preotolith. X 30.000.
5
Enlarged from figure 4. Note t h e tube-like structure of electron dense material. x 75,000.
6
Showing otolith from utricule of 1-day-oldmouse; stage of t h e earliest Ca-Carbonate formation. Section decalcified during staining process. Spaces formerly occupied by small Ca-Carbonate crystals (enclosed by electron dense material) a r e shown near both ends of otolith. X 25,000.
7
Enlarged from a part of figure 5 . CR, spaces formerly occupied by Ca-Carbonate crystals. x 70,000.
CA CAKBONATE FORMATION IN THE OTOLITH Hiroshl N a k a h a r s a n d tierrit Bevrlander
PLATE 2
239
PLATE 3
X
Otolith from utriculr of 1 day-old mouse Note the groups of small Ca-Carbonate crystals a t either end of otolith (dark1 X 30,000.
Y
Otolith froin utricule of' 3-day-old mouse Crystals occupy two large areas of otolith (light artW. X 15.000
10
Nearl? m a t u r e otolith from ulricule of 7 ~ d a y - o l dmouse Unstained. rod^ 01' needlrshaprd Ca-Carbonate crystals a r e c l ~ s e l ypacked in LTcater part of otolith except I'ur the central area where only a few crystals are present x 50.000.
CA CARBONATE FOKMATIVN IN THE OTOLITH Hiroshi Ndkahnra and G e r n t Bevelander
PLAl’t
i
24 1
