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. 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