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Incorporation of strontium into the calcium carbonate crystals of the endolymphatic sac in the tree frog (Hyla arborea japonica).

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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.
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