Incorporation of strontium into the calcium carbonate crystals of the endolymphatic sac in the tree frog (Hyla arborea japonica).код для вставкиСкачать
THE ANATOMICAL RECORD 218223-228 (1987) Incorporation of Strontium Into the Calcium Carbonate Crystals of the Endolymphatic Sac in the Tree Frog (Hyla arborea japonica) SEIICHI KAWAMATA Department of Anatomy, Faculty of Medicine, Toyama Medical and Pharmaceutical Uniuersity, 2630 Sugitani, Toyama, 930-01, Japan ABSTRACT Tree frogs were loaded with strontium chloride (SrC12).The incorporation of strontium metal into the calcium carbonate (CaC03) crystals located both in the inner ear and in the endolymphatic sac was studied by x-ray microanalysis (XMA) and scanning electron microscopy (SEM). In the inner ear, strontium was not recognized except for traces in a few crystals. When observed by SEM, these crystals had a faceted body and two pointed ends with rather smooth surfaces. However, in the endolymphatic sac, which greatly expands into the spinal canal, strontium was clearly present a t every surface of all crystals. Careful examinations by paint and line XMA revealed that strontium x-ray counts were highest at the pointed ends and decreased sharply and then gradually toward the equator of the crystals. SEM observations revealed that the crystals in the endolymphatic sac always had rough and irregular surfaces regardless of their shapes and sizes. Calcium was always found in crystals of both organs. Except for calcium and stronitium, other elements including sodium and heavier elements were negligible in XMA. These findings suggest that strontium is incorporated into the crystals only in the endolymphatic sac, and the rough-surfaced covering of these crystals reflects newly deposited strontium salt. It seems to indicate that these crystals grow predominantly by accretion. Otoconia (statoconia) are essential elements in the inner ear for receiving stimuli such as the forces of gravity and linear acceleration. They are well known to be composed of CaC03 (Funaoka and Toyota, 1928; Carlstrom et al., 1953; Carlstrom and Engstrom, 1955; Carlstrom, 1963; Anniko et al., 1984).Numerous papers have accumulated dealing wiith their morphology (Ross et al., 1976), development (Veenhof, 1969; Salamat et al., 1980; Ballarino and Howland, 1982; Ballarino et al., 1985), and metabolism (Lim, 1973; Harada and Tagashira, 1981; Imoto et al., 1983) in various species. In the tree frog, CaC03 crystals are produced, not only in the inner ear, but also in the endolymphatic sac. Though the fine structure of the endolymphatic sac has been described (Whiteside, 1922; Dlempster, 1930; Kawamata et al., 1987, the mechanism by which crystals are formed is not well understood. Radioactive calcium has been employed to clarify the metabolism of otoconia by light microscopy (Belanger, 1960; Veenhof, 1969) or scintillation counting (Prestlon et al., 1975; Mechigian et al., 1979; Ross, 1979). Unfortunately, CaC03 crystals are so hard to thin section ithat autoradiographic study a t the electron microscopic level has not been reported. On the other hand, strontiurn, which belongs to the same group as calcium in the periodic table of elements, behaves similarly to calcium in metabolism (Olsen and Jonsen, 1979).A study employing this metal in order to monitor the metabolism of CaC03 spherites (Morgan, 1981) has been published. In the tree frog, it has been found that 0 1987 ALAN R. LISS, INC a SrCl2 solution promotes the crystal formation of the paravertebral lime sacs (bulges of the endolymphatic sac) and enhances x-ray photo density (Krause, 1935; Sulze, 1942; Schlumberger and Burk, 1953). However, incorporation of this metal into the crystals has not been confirmed directly and its distribution pattern in each crystal is unknown. I loaded the tree frog with this metal and observed the CaC03 crystals by XMA and SEM to determine their formation mechanism. MATERIALS AND METHODS Seven tree frogs weighing approximately 1 gm each were used in this study. They were captured at Toyama City. Five of them were maintained in a 0.8% SrClz solution for 7 days. Feeding was omitted. They were then decapitated and the inner ears and spinal canal were removed in a fixative. The fixative contained 1% paraformaldehyde and 1.25% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4) with CaCl2 at 250 mgAiter. After fixation, CaC03 crystals of both the inner ear and the endolymphatic sac were smeared with a small amount of distilled water on a carbon plate, allowed to dry, and rinsed with distilled water. In some tree frogs, crystals of both the inner ear and the endolymphatic sac ~ Received October 9, 1986; accepted December 9, 1986. Address reprint requests to Dr. Seiichi Kawamata, Department of Anatomy, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-01, Japan. 224 S. KAWAMATA Fig. 1. Crystals in the inner ear of the strontium-loaded tree frog. The letters “a-c” indicate the spots a t which point XMA was carried out. The results are shown in Figure 2. Carbon coating. ~ 3 , 0 0 0 . Fig. 2. Spectra of point XMA of the crystals seen in Figure 1.Spectra of spots a-c correspond to panels a-c, respectively. Strontium (indi- cated by a vertical bar), is not detected. Ca, calcium; Sr, strontium. Fig. 3. Crystals in the inner ear of the strontium-loaded tree frog. They have rather smooth surfaces. Gold coating. ~ 6 , 0 0 0 . 225 INCORPORATION OF Sr INTO CaC03 CRYSTALS were smeared on the same carbon plates to equalize the effects of following processes. After they were dried, specimens for XMA and SEM were coated with carbon and gold, respectively. SEM observations, point XMA (xray counting from all1 elements at a spot), and line XMA (x-ray counting in the strontium specific energy range along a scanning line) were carried out using a Hitachi X-650 type electron imicroscope equipped with a n energy dispersive x-ray microanalysis system Kevex 7000 operated a t 20 kV. For study of incorporation of strontium in vitro, two tree frogs were decaipitated without strontium loading. Unfixed endolympbatic sacs were immersed in a 0.8% SrC12 solution for seven days at room temperature. Then these specimens were fixed and prepared as described above, and XMA was performed. RESULTS Crystals in the Inner Ear X-ray microanalysis Only calcium was detected (Figs. 1, 2). Strontium was not detected (Fig. 2 ) except for slight amounts in a few crystals. SEM observations Crystals had a faceted body and two pointed ends. Their size varied considerably and their surfaces were generally smooth (Fig. 3). Crystals in the Endolymphatic Sac X-ray microanalysis XMA revealed that these crystals contained both calcium and strontium (Figs. 4, 5). The instrument I used can detect sodium or heavier elements, but x-ray counts of other elements were few. Strontium was detected at every surface of all crystals in the endolymphatic sac (Fig. 5). Careful point XMA revealed that the counts of strontium were highest at the tips of the pointed ends and lowest at the equator of the crystal. This finding was also confirmed by line XMA (Fig. 7). Line XMA along the long axes of crystals traced a U-shaped curve (Fig. 7, line B), whereas line XMA a t right angles to the long axes did not have such a profile. Furthermore, in the latter case, higher x-ray counts were measured near the pointed ends of crystals than crossing the equator. No crystal was found to contain only strontium or only calcium (Figs. 5, 8). These observations were usually verified in numerous crystals, but absolute strontium xray counts of comparable points varied somewhat from one crystal to another. In rare cases, “strontium-rich” crystals were found. In these crystals, the strontium xray counts at the equator were nearly as high as at the pointed ends (Fig. 7). Every point on these crystals gave much higher strontium x-ray counts than did the prevalent crystals (Figs. 7,8). SEM observations Crystals in the endolymphatic sac were similar in shape and size to those in the inner ear. However, all these crystals had rough and irregular surfaces regardless of their shapes and sizes (Fig. 6). Incorporation of Strontium In Vitro No crystal in the endolymphatic sac was found to contain strontium by XMA following immersion in a 0.8% SrClz solution. DISCUSSION The mechanism by which CaC03 crystals are formed is not well understood. Some experiments to elucidate calcium turnover in the crystals were attempted with radioactive calcium (Belanger, 1960; Veenhof, 1969; Preston et al., 1975; Ross, 1979; Mechigian et al., 1979). Incorporation of a calcium radioisotope into the otoconia was confirmed, although it was much lower than for bone (Belanger, 1960; Preston et al., 1975; Veenhof, 1969; Ross, 1979). Using autoradiography at the light microscopic level, Veenhof (1969) observed incorporation of radioactive calcium into the otoconia of mice but only during the late fetal or neonatal period. No incorporation was seen in adult mice. Tetracycline was also used to survey the growing fronts of otoconia by fluorescent microscopy in chick embryos with positive results (Balsamo et al., 1969) and in adult mice with negative results Weenhof, 1969). In this study, strontium was employed as a tracer, since this metal behaves similarly to calcium in metabolism (Olsen and Jonsen, 1979). Strontium was always detected in the crystals in the endolymphatic sac, but not in those in the inner ear. It is known that the amount of crystals in the endolymphatic sac increases rapidly when the frog is loaded with calcium or strontium salts (Krause, 1935; Sulze, 1942; Schlumberger and Burk, 1953) and vitamin D (Schlumberger and Burk, 1953). Furthermore, this crystalline calcium is easily mobilized for the growth of the skeleton (Guardabassi, 1960). On the other hand, the metabolism of the crystals in the inner ear is very slow or arrested (Belanger, 1960; Veenhof, 1969; Ross, 1979). Even when frogs are loaded with CaClz and vitamin D, no demonstrable alteration in the total size or density of the otoconia is observed (Schlumberger and Burk, 1953). In this study also, no remarkable change i n the amount of crystals in the inner ear was recognized. Based on these facts, the difference in strontium incorporation may be attributed to the different turnover rates of crystals in the endolymphatic sac and the inner ear, although other possibilities cannot be excluded. With respect to the distribution pattern of strontium in each crystal in the endolymphatic sac, strontium salt probably covered all surfaces of these crystals. Similarly, Balsam0 et al. (1969) found that the fluorescence of tetracycline was emitted from a superficial layer covering otoconia of the tetracycline-loaded chick embryo. The covering of strontium salt seemed to make the surface of the crystals rough and irregular when observed by SEM. On the other hand, in the inner ear, the surfaces of crystals that contained no strontium were generally smooth. This difference in the surfaces cannot be explained by different conditions of coating or other preparation processes, since crystals from the inner ear and the endolymphatic sac were treated on the same carbon plate. The advantage of employing strontium as a tracer rather than radioactive calcium or tetracycline is that 226 S. KAWAMATA Fig. 4. Crystals in the endolymphatic sac of the strontium-loaded Fig. 6. Crystals in the endolymphatic sac of the strontium-loaded tree frog. The letters “a-c” indicate the spots at which point XMA was tree frog. They have rough and irregular surfaces. Gold coating. carried out. The results are shown in Figure 5. Carbon coating. x 7,000. ~ 6 , 0 0 0 . Fig. 5. Spectra of point XMA of the crystals seen in Figure 4.Spectra of spots a-c, correspond to panels a-c, respectively. Strontium is clearly observable. INCORPORATION OF Sr INTO CaCOS CRYSTALS 227 Fig. 7. A micrograph showing a strontium-rich crystal (upper) and one of the prevalent crystals (lower) in the endolymphatic sac. The data for line XMA along scanning lines are also shown. Lines A,B indicate x-ray counts specific to strontium along the upper and lower scanning lines, respectively. Line B is a typical U-shaped curve. The letters “a,b” indicate the equatorial spots at which XMA were carried out. The results are shown in Figure 8. Carbon coating. ~ 9 , 0 0 0 . XMA and SEM make it possible to examine the distribution of strontium at the electron microscopic level. This experiment cllearly demonstrated that all CaC03 crystals in the endolymphatic sac can grow mainly by accretion of strontium salt. The strontium x-ray counts varied from one crystal to another, and from one point to another even in the same crystal. The x-ray counts are not directly proportionate to the content of strontium; however, growth seems most active at the pointed ends and rather inhibited at the equator of the crystals. These findings are in close agreement with the speculation reported by Ross and Peacor (1975) and Ross et al. (1976).However, strontium-rich crystals may result from other mechanisms such as fusion of smaller crystals (Belanger, 1960; Campos et al., 1984) or simultaneous crystallization around multiple nucleation sites (Nakahara and Bevelander, 1979, 1980). Presumably such mechanisms apply to the formation of CaC03 crystals under natural conditions, at least in the endolymphatic sac. This hypothesis may be extended to the crystals in the inner ear of the tree frog, because their chemical and crystallographic natures are the same as for crystals in the endolymphatic sac (Schlumberger and Burk, 1953). Crystals in the inner ear are formed during a short period when the animal is young, and after this period it is assumed that their growth is very slow or arrested (Veenhof, 1969) except under pathologic conditions (Harada and Tagashira, 1981).Consequently strontium may no longer be incorporated. Fig. 8. Spectra of point XMA of the crystals seen in Figure 7. Spectra of spots a,b correspond to panels a and b, respectively. Extremely high x-ray counts of strontium at spot a are obvious. ACKNOWLEDGMENTS The author is grateful to Mr. M. Kawahara for his excellent technical assistance and to Ms. Y. Yasukawa and Ms. T. Kawamata for their secretarial help. The 228 S. KAWAMATA author also thanks Prof. K. Takaya for critical reading of the manuscript. LITERATURE CITED Anniko, M., J. Ylikoski, and R. Wroblewski (1984)Microprobe analysis of human otoconia. Acta Otolaryngol. (Stockh.), 97:283-289. Ballarino, J., and H.C. Howland (1982) Otoconial morphology of the developing chick. Anat. Rec., 204t83-87. Ballarino, J., H.C. Howland, H. Catherine, W. Skinner, E.B. Brothers, and W. Bassett (1985) Studies of otoconia in the developing chick by polarized light microscopy. Am. J . Anat., 174:131-144. Balsamo, G., M. De Vincentiis, and F. Marmo (1969) The effect of tetracyclin on the processes of calcification of the otoliths in the developing chick embryo. 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