Ultrastructural study of the semicircular canal cells of the frog Rana esculenta

June 30, 2017 | Autor: Olivier Oudar | Categoria: Biological Sciences, Animals, Epithelium
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THE ANATOMICAL RECORD 220:328-334 (1988)

Ultrastructural Study of the Semicircular Canal Cells of the Frog Rana esculenta OLIVIER OUDAR, EVELYNE FERRARY, AND GERARD FELDMANN Laboratoire de Biologie Cellulaire, Ddparternent de Physiologie and INSERM U.251, Facultd de Mkdecine Xavier Bichat, Uniuersitt! Paris 7, 75018 Paris, France

ABSTRACT The ultrastructure of the nonsensory cells (dark cells, transitional cells, and undifferentiated cells) of the frog semicircular canal was studied by using transmission electron microscopy in a n attempt to correlate the structure with the functions of these epithelial cells. All the nonsensory cells were linked by tight junctions and desmosomes; this suggested that there is little paracellular ionic transport from perilymph to endolymph. In the dark cell epithelium, the apical intercellular spaces were dilated; in the basal part, numerous basolateral plasma membrane infoldings, containing mitochondria, delimited electron-lucent spaces. The undifferentiated cells and the transitional cells were devoid of any basal membrane infolding. Surrounding the semicircular canal, very flattened and interdigitated mesothelial cells constituted a thin multilayer tissue which limited the perilymphatic space. The morphological aspect of the dark cells suggests that they may play a role in the secretion and/or in the reabsorption of endolymph, which bathes the apical pole of these cells. The undifferentiated and transitional cells can play a role in the maintenance of the endolymphatic ionic composition because of their apical tight junctions and desmosomes. The inner ear is divided by the membranous labyrinth into two extracellular compartments, one containing endolymph and the other perilymph. The endolymph resembles a n intracellular positively polarized fluid whereas the perilymph is similar to a plasma ultrafiltrate (Sellick and Johnstone, 1975; Sterkers et al., 1984; Bernard et al., 1986). The ionic composition of endolymph plays a n important role in transduction processes localized in the sensory cells. The endolymph originates from perilymph by a Naf,K+-ATPase-dependent mechanism (Simon et al., 1973; Bernard et al., 1986). The vestibular part of the inner ear is composed of the saccule, the utricule, and the three semicircular canals. The canal is formed by the ampulla, with its sensory crista ampullaris, and by the duct. The epithelium of the canal consists of three cell types: the transitional cells and the dark cells located in the ampulla, and the undifferentiated cells present in both ampulla and duct. Whereas most of the studies have been performed on the sensory cells and their neural elements (Wersall et al., 1965; Harada, 1972, 1973; Gleisner et al., 1973; Wever, 1973; Hoshino and Komada, 1976; Flock et al., 1977), there are few studies of the nonsensory epithelium. In the mammalian vestibule, the dark cells were studied by light microscopy (Iwata, 1924) and electron microscopy (Smith, 1956; Kimura et al., 1964; Kimura, 1969; Watanuki et al., 1970). These studies suggested that the dark cells have secretory and perhaps also absorptive functions. In the frog, the nonsensory cells of the ampulla have only been studied by scanning electron microscopy (Harada, 1972, 19731, and their fine structure has not been investigated, at least to our knowledge. 0 1988 ALAN R. LISS, INC.

In the present study, we investigated the structure of the nonsensory cells of the semicircular canal of the frog Rana esculenta by using transmission electron microscopy in a n attempt to correlate the structure with the possible functions of these epithelial cells. MATERIALS AND METHODS

Rana esculenta frogs (Elevage d'Ardenay, France), unselected with regard to sex and kept a t 4°C until the experimentation, were used. Frogs were anesthetized with percutaneous urethane (50 g/liter) (Sigma, St. Louis, MO) and killed by decapitation. The lower jaw was removed and each half-head was pinned, ventral side up, onto a dissecting tray filled with a solution similar to the frog perilymph (NaCl 96 mM, KC1 2.5 mM, NaHC03 20 mM, NaH2P04 0.17 mM, CaClz 1.8 mM, MgC12 1.2 mM) and bubbled with 95% 0 2 and 5% C02 and with a pH adjusted to 7.4 as previously described (Bernard et al., 1986). The membranous labyrinth was exposed by removing successively the mucous membrane and the cartilage overlying the inner ear. Only the posterior vertical canal was cut off and immersed in the fixative for 2 hours at 4°C. Two fixation procedures were used: 1)a 1.25% solution of glutaraldehyde (TAAB Laboratories, Berks, UK) buffered with 0.05 M phosphate buffer (pH 7.4), with a n osmolality of 220 mOsm; 2) a mixture of 5% paraformaldehyde (Merck Received June 1, 1987; accepted August 18, 1987. Address reprint requests to Olivier Oudar, Laboratoire de Biologie Cellulaire, Facult6 de MQdecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France.

ULTRASTRUCTURE OF FROG SEMICIRCULAR CANAL CELLS

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Fig. 1. Light microscopy. Semithin section along the long axis of an ampulla of a semicircular canal. The dark cells (Dc) are located on each side of the crista ampullaris, characterized by its sensory cells (Sc). A zone composed of transitional cells (Tc) is visible between dark and

sensory cells. Some capillaries (c) and melanocytes (m) are present under the connective tissue (ct). U: undifferentiated cells; mc: mesothelial cells; double arrows: nerve fibers; E: endolymph; P: perilymph. Toluidine blue. X 120.

Laboratories, Darmstadt, RFA) and 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer (pH 7.4), with an osmolality of 1,850 mOsm. The semicircular canal was then washed in 0.1 M phosphate buffer (2 baths of 15 minutes each) and was postfixed (30 minutes) in a 1% solution of osmium tetroxide buffered with 0.1 M phosphate buffer (pH 7.4). The canal was dehydrated in a graded series of ethanol and embedded in epoxy resin (Resin Epikote 812, Agar Aids, Stanted, UK). Semithin sections, stained with toluidine blue, were observed by light microscopy for orientation. Ultrathin sections, stained with uranyl acetate and lead citrate, were examined with a Siemens Elmiskop IA electron microscope.

minal face of the connective tissue was bordered by three types of epithelial cells: the transitional cells, the dark cells, and the undifferentiated cells, all of them forming a monocellular layer (Fig. 1).The transitional cells, located on each side of the crista ampullaris, were cuboidal. These cells were superceded by two well-defined dark cell areas. The dark cells, cylindrical in shape, were present in the ampulla but were absent in the duct. Undifferentiated epithelial cells, which were flattened and elongated, bordered the remainder of the luminal face of the ampulla and the whole duct. By electron microscopy, the most obvious cytological characteristics of the dark cells were the scanty cytoplasm and the presence of numerous basolateral membrane infoldings occupying about half of the cell height. At low magnification (Figs. 2, 31, the cytoplasm was essentially localized at the apical part of the cell and was very electron dense. An irregularly shaped nucleus was generally visible in the apical part (Fig. 2). Numerous mitochondria were present in the infoldings and in the apical cytoplasm. Electron-lucent spaces were distributed in the dark cell epithelium (Fig. 2). Their size changed from one cell to another, giving to the epithelium a very variable appearance. At higher magnification (Figs. 3,4), the apical plasma membrane of the dark cells showed few microvilli and invaginations. These invaginations were probably implicated in endocytosis

RESULTS

Whatever the fixative used, glutaraldehyde or the mixture of paraformaldehyde and glutaraldehyde, the appearance of the epithelial cells was similar. Consequently a single description will suffice. By light microscopy (Fig. 1)the wall of the semicircular canal consisted of a loose connective tissue with a few capillaries and melanocytes localized externally to this connective tissue. In the ampulla, several capillaries were also visible in the connective tissue facing the crista ampullaris near the nerve fibers. The capillaries were formed of tightly sealed endothelial cells. The lu-

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since some endocytotic vesicles were observed. A small number of cisternae of rough endoplasmic reticulum and saccules of Golgi apparatus could be observed. In the apical zone, the intercellular spaces were dilated and some membranous folds were present (Figs. 3, 4). The dark cells were linked in this zone by tight junctions and desmosomes. Whereas it was easy to see the junctions in the apical zone (Fig. 41,it was impossible to determine the cell limits in the middle and the lower parts of the dark cells because of the numerous plasma membrane infoldings limiting electron-lucent cavities. In the basal part (Fig. 51, numerous elongated mitochondria were arranged along the vertical axis of the cells. Very little cytoplasm was present between the outer mitochondria membranes and the plasma membrane. The transition from dark to transitional cells was abrupt (Fig. 1). The transitional cells were cuboidal and the nucleus occupied most of the volume of the cell (Fig. 6). The apical plasma membrane presented short and sparse microvilli. In these cells, small vesicles of varying size were visible. Some mitochondria and cisternae of rough endoplasmic reticulum were present in the cytoplasm. Transitional cells were linked by tight junctions at their luminal margin. Several interdigitations between two adjacent transitional cells were observed. The undifferentiated cells formed a monocellular layer except on the top of the ampulla, in front of the crista ampullaris, where two or three cells overlapped. This region could be considered as the site of attachment of the cupula (Hillman, 1974). The undifferentiated cells were flat and their nuclei occupied most of the length of the cell (Fig. 7). Mitochondria, vacuoles, and some cisternae of the rough endoplasmic reticulum could be observed in these cells. Undifferentiated cells were linked by tight junctions and desmosomes both in the apical and basal parts. A multilayer of flattened and interdigitated mesothelial cells externally covered the semicircular canal (Fig. 8).The nuclei of these cells were flattened. In the cytoplasm, a few rough endoplasmic reticulum cisternae, small vesicles, and filaments were observed. These cells were linked by numerous desmosomes (Fig. 9).

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to determine which could best preserve this tissue. Anniko and Lundquist (1977)and Park (1984)tested glutaraldehyde fixatives for mammalian and bird inner ears and concluded that the glutaraldehyde and the Karnovsky’s fixative, i.e., a mixture of paraformaldehyde and glutaraldehyde, were satisfactory. They obtained a better result with a hyperosmolar fixative solution (> 1,500 mOsm) than with a hypoosmolar one. In our study, the morphological aspect of the dark cells varied from one cell to another and from one frog to another. This was explained by the variation in the size of the electron-lucent spaces. To eliminate a possible fixation artefact, two fixative solutions were used-the 1.25% glutaraldehyde solution (220 mOsm), and the mixture of a 5% paraformaldehyde and 2.5% glutaraldehyde (1,850 mOsm). Similar appearances were observed with the two solutions, without modification of the size of the electron-lucent spaces. This result suggests that these spaces do not represent fixation artefacts but rather physiological effects. These spaces could be involved in water and electrolyte transports. Diamond and Tormey (1966)indicated that the intercellular fluid spaces in the rabbit gallbladder were opened during solute-linked water transport and closed when ionic transport was stopped by ouabain. Similarly, large fluid spaces could indicate a secretion and/or a resorption of endolymph, and in contrast, small spaces could indicate an exit of water to another compartment. As compared to the cells of renal tubules (Rhodin, 1958; Kriz and Kaissling, 1985), the cells of choroid plexus (Maxwell and Pease, 19561, and the marginal cells of stria vascularis (Hinojosa and Rodriguez-Echandia, 19661, the dark cells could be involved in electrolyte and water transport between perilymph and endolymph. This function is suggested by the presence of numerous basal infoldings and mitochondria. The apical tight junctions might limit paracellular exchanges. The localization of Na+,K+-ATPaseactivity in dark cell basolateral membranes may support the hypothesis that these cells are involved in endolymph formation (Burnham and Stirling, 1984). Such a role was also suggested by other authors (Dohlman, 1964;Kimura et al., 1964;Nakai and Hilding, 1968; Kimura, 1969; Kawamata et al., 1986). The structure of the other nonsensory cells suggests DISCUSSION that these cells are not involved in ionic transports (NaIn order to study the epithelium lining the ampulla of kai et al., 1986) but that they can play a role in the the semicircular canal, we tested two different fixatives maintenance of the endolymph composition because of the tight junctions which form a barrier between endolymph and perilymph. Indeed, vacuoles formed by diFig. 2. Electron microscopy. In this dark cell, the apical zone, with lated intercellular spaces between adjacent transitional an irregular-shaped nucleus (N) and a scanty cytoplasm, is visible. The cells could indicate fluid movements. basal zone of the cell is characterized by numerous long plasma memThe perilymphatic space was bordered by a thin and brane infoldings (arrows) containing mitochondria (m). Electron-lucent spaces (asterisks) are visible and extend almost up to the luminal cell tight mesothelial tissue which has not previously been junction. E: endolymph; c t connective tissue. ~3,500. described in the literature to our knowledge. It consists of many layers of extremely flat cells which could act as Fig. 3. Electron microscopy. Aspect of two adjacent dark cells. Note the presence of very dilated intercellular spaces (IC). ct: connective a barrier between the perilymphatic space and the cartilaginous capsule. Nevertheless, the presence of endotissue; E: endolymph; m: mitochondria. ~6,000. cytic vesicles in the cytoplasm is interpreted as indiFig. 4. Electron microscopy. Large magnification of the apical zone cating fluid movements. As this tissue was very thin of two dark cells. Note the thin lateral membranous folds (F) and the dilated intercellular spaces (IC). E: endolymph; Tj: tight junctions. and not attached to the connective tissue, it was not always preserved during dissection. The small or virtual ~26,000. space between the connective tissue and the multilayer Fig. 5. Electron microscopy. Large magnification of the basal zone of tissue corresponds to the perilymphatic space. a dark cell. Note the ulasma membrane infoldinm (In) with mitochonIn conclusion, we studied the ultrastructure of the dria (m) occupying aimost the whole cytoplasm-bm: basement memnonsensory cells of the frog semicircular canal. The morbrane; ct: connective tissue. ~20,000.

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Fig. 8. Electron microscopy. The mesothelial cells (me) surround all the semicircular canal. They are very flattened and interdigitated, constituting a thin multilayer tissue. ct: connective tissue; E: endolymph; Fi: fibroblast; U: undifferentiated cell. x 1,600.

Fig. 9. Electron microscopy. Partial view of the mesothelial cells. In their cytoplasmic processes, filaments P i ) and small endocytotic vesicles (V) are visible. Numerous desmosomes (arrows) are present.

phologic appearances of the dark cells (numerous plasma membrane infoldings, numerous mitochondria, and large intercellular spaces) suggest that they could play an important role in the formation of endolymph. The other nonsensory cells could have an indirect role in the maintenance of endolymph composition by forming a tight barrier between endolymph and perilymph.

LITERATURE CITED

ACKNOWLEDGMENTS

We are greatly indebted to Claude Amiel and Olivier Sterkers for stimulating discussions, including criticism of the manuscript. Our thanks are also due to Alain Moreau and Edith Rogier for their very valuable advice. This work was supported by grants from INSERM, Faculte de Medecine Xavier-Bichat (Universite Paris 71, and DRET 85-209.

Fig. 6. Electron microscopy. The transitional cells are cuboidal and line the connective tissue (ct). The nuclear (N) shape is regular. Note the presence of tight junctions (Tj) at the apical part of the cells. bm: basement membrane; E: endolymph. X5,500. Fig. 7. Electron microscopy. The undifferentiated cells are flattened. ct: connective tissue; E: endolymph; Fi: fibroblast; Tj: tight junctions. ~2,700.

~82,000.

Anniko, M., and P.-G. Lundquist 1977 The influence of different fixatives and osmolarity on the ultrastructure of the neuroepithelium. Arch. Otorhinolaryngol., 21857-78. Bernard, C., E. Ferrary, and 0. Sterkers 1986 Production of endolymph in the semicircular canal of the frog Runa esculentu. J. Physiol. (Lond.), 371r17-28. Burnham, J.A., and C.E. Stirling 1984 Quantitative localization of NaK pump site in frog inner ear dark cells. Hearing Res., 13:261-268. Diamond, J.M., and J.McD. Tormey 1966 Studies on the structural basis of water transport across epithelial membranes. Fed. Roc., 25: 1458-1463.

Dohlman, G.F. 1964 Secretion and absorption of endolymph. Ann. Otol. Rhinol. Laryngol., 73r708-722. Flock, A,, B. Flock, and E. Murray 1977 Studies on the sensory hairs of receptor cells in the inner ear. Acta Otolaryngol., 83:85-91. Gleisner, L., A. Flock, and J. Wersall 1973 The ultrastructure of the afferent synapse on hair cells in the frog labyrinth. Acta Otolaryngol., 76:199-207. Harada, Y. 1972 Surface view of the frog vestibular organ with the scanning electron microscope. Acta Otolaryngol., 73:316-322. Harada, Y. 1973 The scanning electron microscopic observation of the vestibule organ and electrical activity of isolated individual semicircular canal ampullae. Adv. Otorhinolaryngol., 19r50-65. Hillman, D.E. 1974 Cupular structure and its receptor relationship. Brain Behav. Evol., 10:52-68. Hinojosa, R., and E.L. Rodriguez-Echandia 1966 The fine structure of the stria vascularis of the cat inner ear. Am. J. Anat., 118:631-664. Hoshino, T., and A. Komada 1976 A scanning electron microscopic study of the crista ampullaris in vertebrates. A.N.L., 3:41-52.

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Iwata, N. 1924 Uber das Labyrinth der Fledermaus mit besonderer Berucksichtigung des statischen Apparates. Aichi J. Exp. Med., 1:41-173. Kawamata, S., Y. Harada, and N. Tagashira 1986 Electron-microscopic study of the vestibular dark cells in the crista ampullaris of the guinea pig. Acta Otolaryngol., 102:168-174. Kimura, R., P.G. Lundquist, J. Wersall 1964 Secretory epithelial linings in the ampullae of the guinea pig labyrinth. Arch. Otolaryngol., 57:517-530. Kriz, W., and B. Kaissling 1985 Structural organization of the mammalian kidney. In: The Kidney. Physiology and Pathophysiology. D.W. Selding and G. Giebisch, eds. Raven Press, New York, Vol. 1, pp. 265-306. Maxwell, D.S., and D.C. Pease 1956 Electron microscopy of the choroid plexus. Anat. Rec., 124:331. Nakai, Y., H. Cho, Y.Miki, J.S. Cho, and A. Hakuba 1986 Epithelial linings of the human semicircular canal. A scanning and transmission electron microscopic study. Arch. Otorhinolaryngol., 243:7782. Nakai, Y., and D. Hilding 1968 Vestibular endolymph-producing epithelium. Electron microscopic study of the development and histochemistry of the dark cells of the crista ampullaris. Acta Otolaryngol., 66:120-128.

Park, J.C. 1984 Glutaraldehyde fixatives for preserving the chick’s inner ear. Acta Otolaryngol., 9332-80. Rhodin, J.A. 1958 Anatomy of kidney tubules. Int. Rev. Cytol., 7:485534. Sellick, P.M., and B.M. Johnstone 1975 Production and role of inner ear fluid. Prog. Neurobiol., 5:337-362. Simon, E.J., D.A. Hilding, and M. Kasgharian 1973 Micropuncture study of the mechanism of endolymph production in the frog. Am. J. Physiol., 225114-118. Smith, C.A. 1956 Microscopic structure of the utricule. Ann. Otol. Rhinol. Laryngol., 65:450-469. Sterkers, O., E. Ferrary, and C. Amiel 1984 Inter- and intracompartmental osmotic gradients within the rat cochlea. Am. J. Physiol., 247:602-606. Watanuki, K., K. Kawamoto, and S. Katagiri 1970 Surface structure of the ampulla of the semicircular canal in the guinea pig. Pract. Otorhinolaryngol.,32: 137-148. Wersall, J., A. Flock, and P.-G. Lundquist 1965 Structural basis for directional sensitivity in cochlear and vestibular sensory receptors. Cold Spring Harbor Symp. Quant. Biol., 30:115-132. Wever, E.G. 1973The labyrinthine sense organs of the frog. Proc. Natl. Acad. Sci. USA, 70:498-502.

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