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Otoconial morphology of the developing chick.

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THE ANATOMICAL RECORD 204:83-87 (1982)
Otoconial Morphology of the Developing Chick
JOANNE BALLARINO m
i ) HOWARD C. HOWLAND
Sections of Physiology and Neurobiology and Behauior, W-201 Seeley Mudd
Building, Cornell University, Ithaca, NY 14853
ABSTRACT
We examined with scanning electron microscopy the otolithic
organs of chick embryos aged 6 to 21 days. Otoconial forms not previously reported in adult birds or mammals were found. On the basis of the size and
prevalence of various types of otoconia at different embryonic stages we organized
the otoconial forms into a possible growth sequence starting with an early “doublefluted” or skeletal form and terminating with the normal mature form found in
adults.
The utricular otolithic organ of birds and
mammals is a gravity receptor located in the
inner ear in the plane of the horizontal semicircular canal. It is composed of a sensory epithelium of hair cells and supporting cells over
which lies the otolithic membrane, consisting
of a mass of calcium carbonate crystals embedded in a mucopolysaccharide gel. These
crystals, or otonconia, have the crystalline
structure of calcite (as determined by x-ray
crystallography, Carlstrom, 1963). Each otoconium contains significant amounts of organic material in addition to calcium carbonate
(Lim, 1973; Ross and Peacor, 1975; Salamat et
al., 1980).
The otoconial membrane functions as a highdensity weight which, upon tilting of the head,
shears the underlying hair cells causing a
change in frequency of firing of neurons in the
vestibular nerve. Although the mass of the otoconial membrane remains approximately constant in the adult, there is a continuous turnover of otoconial calcium of 4% per day in
gerbils (Preston et al., 1975).In rats the uptake
of radioactive Ca” by otoconia follows a time
course comparable to bone, but on a much
smaller scale (Ross, 1979).
Several questions are raised by the occurrence of calcium turnover coupled with a constant otoconial mass. Are new otoconia being
continuously generated and old ones decomposed, or does calcium exchange occur within
individual otoconia? Furthermore, where and
how are the otoconia generated?
The chick embryo is an ideal organism for
studying the genesis of otoconia due to its
rapid rate of development during which otoconia are initially produced. The development
of otoconia in chick embryos has been investigated with light microscopic methods by Bal-
0003-276X/82/2041-00&3$02.000 1982 ALAN R. LISS, INC.
samo et d. (1969). In addition, several studies
of mammalian embryos have been carried out
using transmission and scanning electron microscopy. Veenhof (1969) examined the development of otoconia in embryonic mice; Nakahara and Bevelander (1979)and Salamat et al.
(1980) investigated otoconial genesis in the
fetal rat.
As a first step in studying otonconial growth
in the chick we have investigated the forms of
otoconia found in embryos using scanning electron microscopy and have attempted to organize these morphological forms into a possible
growth sequence.
For the sake of comparison and as a control
for our histological and dissection techniques
we also examined a few specimens of embryonic rat utriculii which have been studied previously by Salamat et al. (1980).
MATERIALS AND METHODS
Embryos of white leghorn chickens, Cornell
Strain K, were removed from the egg at various stages of development and decapitated.
We examined the otoconia from 15 six-day embryos, one 7-day, four g-day, two 11-day embryos, and seven newly hatched chicks (21-day
embryos). Utricular organs were separated
from the semicircular canals, fixed for 1hour in
4% glutaraldehyde buffered to p H 8 with
sodium phosphate buffer, and run through an
acetone dehydration series. Utricular organs
from 19-day rat embryos were treated in the
same manner. The specimens were then dried
in carbon dioxide with a Sorvall critical-point
drying apparatus. Dried specimens we;e
placed on stubs and coated with a 200-300-A
Received December 4. 1981; accepted June 17, 1982.
84
J. BALLARINO AN D H.C. HOWLAND
layer of gold. Specimens were observed under
an AMR 1000 scanning electron microscope at
20 keV and at various magnifications. Measurements of the size of otoconia in scanning
electron micrographs were made only on those
otoconial diameters orthogonal to the direction
of view. “Giant”otoconiawere measured with a
light microscope on a micrometer stage accurate to 1 pm.
OBSERVATIONS
Mature otoconia
In Figure 1 we show mature otoconia from
21-day chick embryos. The otoconia from a
single otolithic organ vary in size from 3 to 30
pm. In Figure 1A it may be seen that otoconia
on the otolithic membrane occur in groups of
similar sizes. Those at the right-hand side of
the figure have an average length of 10.0 + 2.5
(S.D.)pm (n = 42) and those at the left-hand
side have an average length of 18.9 + 5.0 (S.D.)
pin (n = 52). The difference between the average lengths of the two groups is highly significant (P < 0.001, t = 10.3).The otoconia of the
newly hatched chick have the normal calcite
structure of otoconia found in birds and mammals (Carlstrom, 1963; Lim, 1974) with rough,
rounded sides and hexagonal, faceted ends
(Fig. 1B).The facets at each end are at angles
of 120” to each other and are rotated 60” with
respect to the facets of the opposite end, thus
forming a crystal with a “screw” axis (Hurlbut,
1971). Also found in the 21-day embryos are
“shrunken”otoconia in which the midsections
are wrinkled as shown in Figure 1C. These rest
on the otoconial membrane above the hair
cells.
Double-fluted o toconia
In Figure 2A we show “double-fluted” otoconia of a 6-day embryo. Measurements of
several fields of such double-fluted otoconia
yielded an average length of 1.9 0.5 (S.D. pm
(n = 20). The fins at each end of the otoconia
make an angle of 120” to each other. Each set
of fins is rotated 60” with respect to those of
the opposite end, just as are the facets of the
mature otoconia. Some of these otoconia are
bent at the the center.
In Figure 2B are shown double-fluted otoconia from a 6-day embryo which appear more
“filled out.” Measurements of several fields of
such filled out double-fluted otoconia yielded
1.3 (S.D.)pm (n =
an average length of 7.6
37). A few appear to be collapsed and bent in
shape. We have also observed these forms on
the sensory epithelium of a 21-day embryo
whose otoconial membrane had been removed.
*
Fig. 1. Scanning electron micrographs of otoconia from
2 1 d a y chick embryos. A) Otoconia showing a size range
3-30 pm in length. R ) Otoconia exhibiting faceted ends a t
120” angles to each other. Cj “Shrunken” otoconia with
faceted ends.
Figure 2C shows otoconia from an 11-day
chick embryo. These otoconia range in size
from 4 to 14 pm; their average length is 6.5 +
2.3 (S.D.)pm (n = 53). At the ends of some fins
one can begin to see facets (arrow).Several of
85
OTOCONIAL MORPHOLOGY
Fig. 2. Double-fluted otoconia. A ) “Skeletal”otoconia of
&day embryos. B) Double-fluted otoconia of 6-day embryos
which show beginnings of facets on the fins. C) Otoconia of
11-day embryos exhibiting a more “filled out” appearance.
Small facets may again be seen on the fins (arrow). D) Otoconia of 21-day chick embryo exhibiting the remnants of fins
(arrow).
these otoconia appear to be growing together.
Figure 2D depicts otoconia of a 21-day embryo
which approach in form the mature otoconia of
Figure 1; they exhibit rough, rounded sides,
but possess the remains of a fin structure
(arrow).These range in length from 4 to 20 pm
(average = 12.2 + 4.9 S.D. pm, n = 11).
“Giant’’ otoconia
In one newly hatched chick we observed
“giant” otoconia whose large size altered the
gross morphological appearance of the mass of
otoconia from the normal white powdery appearance to that of apearly translucent one. In
approximately 200 dissections we observed
such otoconia only once in one side of the head
of a chick. These otoconia ranged in size from
25 to 140 p m in length (average = 77.8 + 30.6
S.D. pm, n = 16).
Fetal rat otoconia
The mature otoconia of 19-day fetal rats
(Fig. 3) are very similar in appearance to those
of embryonic chicks and are the most predominant form of otoconia that we observed in our
preparations. The same treatment which frequently gave us very clean preparations of
chick otoconia often resulted in the formation
of a beadlike precipitate on the fetal rat otoconia (Fig. 3A). A few “dumbbell-shaped,”trigonal,” and “multifaceted” otoconia (Salamat
e t al.,1980)were also observed (Fig. 3B).
DISCUSSION
Mature otoconia
The mature otoconia of newly hatched chicks
(21-day embryos) have the same structure as
otoconia from adult pigeons, guinea pigs, cats,
and squirrel monkeys (Lim, 1973,1974).These
otoconia have smooth, faceted ends and rough,
86
J. BALLARINO AND H.C. HOWLAND
those reported by Lim in guinea pig utricle
(Lirn, 1973). Lim interpreted such “collapsed”
otoconia as being resorbed by a process mediated by the dark cells.
The shrunken otoconia we observed indicate
that there exist otoconia with fully formed
facets which are not completely mineralized.
The association of these otoconia with the otoconial membrane rather than with dark cells
may signify that they were in a process of
growth rather than degradation; however, this
cannot be determined from micrographs alone
Fig. 3. Otoconia from a 19-day rat fetus. A) Note mature
otoconia with round sides and planar ends (I).joined otoconia ( 2 ) ,and ”multifaceted”otoconia (3).The rnucopolysaccharide membrane appears to have precipitated on some of
the mature otoconia (4). B) Dumbbell-shaped otoconium exhibiting “multifaceted” structure (1).“Spindle-shaped” otoconium ( 2 ) with “beadlike protuberances” which in this micrograph appear to he a precipitate of otoconial membrane.
convex sides, and exhibit the general shape of
the scalenohedron common to calcite (Hurlbut,
1971). As noted by Ross and Peacor (1975),the
faceted ends of the mature otoconia are rhombohedral and are rotated 60” with respect to
each other.
In general, mature otoconia range in size
from 3 to 30 pm in length. However, the observation of “giant”otoconiain one newly hatched
chick indicates that there is some mechanism
that normally regulates otoconial size which
was not functioning in this case.
Shrunken otoconia
The shrunken otoconia we observed are located on the otoconial membrane and are not
found in association with dark cells, as are
Double-fluted otoconia
Double-fluted otoconia were seen most frequently in 6- and 11-day embryos, in which we
observed whole fields of this form on the otoconial membrane. They do not appear on the
upper levels of the otoconial membranes of 21day embryos. However, since we observed a
few of these double-fluted forms on the sensory
epithelium of a 21-day embryo after the otoconial membrane had been removed, a systematic search might reveal their presence at
lower levels of the membrane in older animals.
Skeletal otoconia (Fig. 2A) were occasionally
observed to be in a bent configuration. The
bending may simply be an artifact of dehydration coupled with incomplete mineralization.
The double-fluted forms which we have observed may correspond to the “dumbbell” otoconia found in 13-day mouse embryos by
Veenhof (1969)using the technique of microincineration. Dumbbell-shaped otoconia were
also observed by Salamat et al. (1980)in 18-day
fetal rats using transmission and scanning
electron microscopy (SEM). Under SEM the
otoconia of their preparations exhibited a regular array of “beadlike protuberances.” These
protuberances were interpreted by Salamat et
al. (1980) as rows of subunits comparable to
rows of fibrous filaments that they found in
otoconia using transmission electron microscopy (TEM).We did not observed beadlike protuberances on any chick otoconia.
The dumbbell-shaped otoconia we found in
fetal rats were similar to those termed “multifaceted” by Salamat et al. (1980)but also did
not have beadlike protuberances (Fig.3B). We
did find some otoconia in our fetal rat preparations which superficially resembled the “trigonal” otoconia of Salamat et al. (1980) and
which did have protuberances (Fig. 3B).
Growth sequence
An otoconial developmental sequence can be
constructed in which the skeletal form of Figure 2A is the youngest, the double-fluted forms
of Figure 2B and C are intermediate stages,
and Figure 2D is the penultimate stage. The
87
OTOCONIAL MORPHOLOGY
mature otoconia of Figure 1B represents the
final growth form. In this growth model the
facets of the mature otoconia develop on the
ends of the fins of the skeletal forms. All of
these forms show the 120” separation between
fins or facets and a 60 rotation with respect to
the opposite end.
The large range of sizes of mature otoconia
(3-30 pm) may initially appear to present a
problem with this growth sequence, since some
of the mature otoconia are smaller than some
of the double-fluted forms. There are at least
two possible explanations for the large range
in sizes of dumbbell-shaped and mature otoconia which are consistent with this postulated
growth sequence. One possibility is that all
otoconia may not pass through this sequence
at the same rate. The process of “filling out”
from the skeletal form to the mature form may
be short in some and long in others, resulting in
different sizes of double-fluted and mature otoconia. Alternatively, it is possible that a variable initial amount of organic matrix is incorporated into the otoconia and dictates the size of
the double-fluted forms and ultimately of the
mature otoconium.
In summary, we have found in the embryonic chick a variety of morphological forms of
otoconia which are not seen in the mature animal, and have organized these into a possible
growth sequence. Underlying this sequence
are a series of events leading to normal otoconial growth. These include 1)the production
of organic matrices which may act as nucleation seeds; 2 ) the crystallization of calcium
carbonate and incorporation of matrix material to form the double-fluted morphology; 3)
the final growth into calcite as influenced by
the physical chemistry of the endolymph; and
4) the inhibition of crystal growth which
usually limits the size of otoconia to 30 pm or
less in length through regulation of the ionic
content of the endolymph to a saturated state
or through the action of inhibitors of calcite
crystal growth. The mechanisms behind these
events must first be understood if we are to
understand the pathological conditions leading to the absence of otoconia, reduction of otoconial numbers (Lim and Erway, 1974;Wright
et al., 1979), or formation of giant otoconia.
Knowledge of the morphological forms which
lead to the formation of mature otoconia provides a foundation for future investigations of
both the regulatory mechanisms behind normal growth and the conditions leading to abnormal growth.
O
SUMMARY
1) The utricular otolithic organs of embryo
chicks were dissected out and studied using
scanning electron microscopy.
2) In 21-day embryos the otoconia had
rounded sides and faceted ends and resembled
those normally found in adult birds and
mammals.
3) In six-day embryos otoconia were found
which were thin in the middle and had fluted
ends.
4) Eleven-day embryos exhibited otoconia
which were rounded in the middle but had
facets at the end of fins. This form may represent a transitional stage between the 6-day and
21-day otoconia.
ACKNOWLEDGMENTS
We thank M. Howland for assistance with
drafting, K. Pueschel for Figure l A , Dr. M.V.
Parthasarathy and M.K. Campenot for help
and technical assistance with the SEM, and
Drs. W. Basset, E. Brothers, B. Halpern, K.
Skinner, S. Reif, D. Dussourd, and an
anonymous referee for comments on the manuscript.
LITERATURE CITED
Balsamo, G.. M. de Vincentiis. and F. Marmo (1969)The effect of tetracycline on the processes of calcification of the
otoliths in the developing chick embryo. J. Embryol. Exp.
Morphol., 22(3):327-332.
Carlstrom, D. (19631 A crystallographic study of vertebrate otoliths. Biol. Bull.. 125444-463.
Hurlbut. C.S. (1971) Dana’s Manual of Mineralogy. John
Wiley and Sons, Inc.. New York.
Lim, D.J. (1973) Formation and fate of otoconia. Ann. Otol.
Rhinol. Laryngol., i?2:23-35.
Lim, D.J. 11974) The statoconia of the non-mammalian
species. Brain Behav. Evol., 10:37-51.
Lim, D.J., and L.C. Erway (1974) Influence of manganese on
genetically defective otolith. Ann. Otol. Rhinol.
Laryngol., 83:565-581.
Marmo, F., and G. Balsamo (1977-1978) Ulteriori osservazioni, in microscopia elettronica a scansione, sulla natura e
I’accrescimento degli otoconi nell’embrione di pollo. Annuario Instituto e Museo di Zoologia dell’ Universita di
Napolia, XXll:lO9-127.
Nakahara, H.. and G . Bevelander (1979) An electron microscope study of crystal calcium carbonate formation in the
mouse otolith. Anat. Rec., 19.912):233-242.
Preston, R.E., L.B. Johnsson, J . H . Hill, and J. Schancht
(1975) Incorporation of radioactive calcium into otolithic
membranes and middle ear ossicles of the gerbil. Acta
Otolaryngol.. 80:269-275.
Ross, M.D. (1979) Calcium ion uptake and exchange in otoconia. Adv. Oto Rhino Laryngol., 25:26-33.
Ross, M.D.. and D. Peacor 11975) The nature and crystal
growth of otoconia. Ann. Otol. Khinol. Laryngol., 84:2236.
Salamat, M.S., M.D. Ross, and D.R. Peacor (1980)Otoconial
formation in the fetal rat. Ann. Otol. Rhinol. Laryngol.
X.W.229-238.
Veenhof. V.B. (1969)The development of statoconia in mice.
Verhandelingen der Koninklijke Nederlandse Akademie
van Wetenschappen. Afd. Natuurkunde Tweede Reeks Dee1 LVIII. 4:l-49.
Wright, C.G., D.G. Hubbard. and J.W. Graham (1979) Ahsence of otoconia in a human infant. Ann. Otol. Rhinol.
Laryngol., 88:779-783.
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