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Lectin staining of saccharides in the normal and hypothyroid developing organ of Corti
Developmental Brain Research, 52 (1990) 141-149 Elsevier
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Lectin staining of saccharides in the normal and hypothyroid developing organ of Corti Jorge J. Prieto, Joaquin Rueda, Maria L. Sala and Jaime A. Merchan Department of Histology, Faculty of Medicine, University of Alicante, Alicante (Spain) (Accepted 26 September 1989) Key words: Organ of Corti; Hypothyroidism;Lectin; Saccharide
Lectin staining has been used to detect mono- and oligosaccharidesin normal and hypothyroid developing organs of Corti in the rat. Eight developmental stages were studied (1, 5, 8, 10, 15, 20, 50 and 60 days after birth). Congenital hypothyroidism was induced by oral administration of propylthyouracil to pregnant rats. Labelling of the tectorial membrane with 3 lectins, Ulex europaeus agglutinin-I (UEA-I), Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin-I (RCA-I) showed no significant differences between normal and hypothyroid animals. Staining with peanut agglutinin (PNA) showed that the hypothyroid adult tectorial membrane (but not the normal one) possesses the disaccharide galactose+N-acetyl galactosamine. Phaseolus vulgaris agglutinin-L (PHA-L) labels the whole tectorial membrane in both groups of animals, but the staining is more intense in the hypothyroid one for a narrow band of oligosaccharide located just between the tectorial membrane and the underlying organ of KOlliker. Both soybean agglutinin (SBA) and succinylated wheat germ agglutinin (WGA) stain the tectorial membrane as well as the cytoplasm of the cells constituting the inner portion of the organ of Kflliker; this latter feature disappears in the normal animals about the 8th postnatal day, but it is abnormally preserved until the 60th postnatal day in the hypothyroid ones. In the adult hypothyroid animals, 3 of the lectins (LCA, PHA-L and WGA) stain extracellular conglomerates located under the synaptic pole of the outer hair cells. INTRODUCTION For a long time the association of hearing loss with 3 forms of thyroid disease has been observed: myxedema in the adult, Pendred's syndrome and endemic cretinism 24. An experimental model of the influence of congenital hypothyroidism on the development of the auditory receptor has been performed on chick embryos 1, and newborn mice 2'3 and rats 25'26. The administration of propylthyouracil (PTU) to pregnant rats results in severe abnormalities both in the morphology of the organ of Corti and in the cochlear potentials of the pups. The structural alterations consist of: (1) the abnormal persistence of the organ of K611iker (OK)25; (2) the distortion of the tectorial membrane (TM)25; (3) the absence of synaptogenesis between the efferent fibers and the outer hair cells27; and (4) a poor development of both the supporting cells and the large fluid spaces surrounding them (tunnel of Corti and Nuel's spaces) 4. In congenitally hypothyroid animals, the TM is of enormous size and remains abnormally attached to the underlying OK. It has been suggested 25 that the distortion of the TM could be due to the abnormal persistence of the OK (which seems to be involved in the secretion
of the TM in early developmental stages). Both structural alterations lead to an anomalous functional development, characterized by a threshold elevation in the VIIIth nerve compound action potential and the broadening of the action potential tuning curves 28. Based on the fact that thyroxine plays a key role in protein synthesis 23, Deol 3 suggested that the derangement of the hypothyroid TM could be due to abnormalities in its protein composition. This hypothesis was ruled out by a chemical analysis of hypothyroid TMs using SDS polyacrylamide gel electrophoresis 21, which failed to show any difference between them and the normal ones. As the TM is composed by a network of protein fibrils embedded in a saccharide matrix 22, an anomalous secretion of this latter component could possibly be the explanation for the morphological findings. For the demonstration of saccharides, lectins (proteins and glycoproteins of non-immune origin capable of binding specifically to different mono- and oligosaccharides) have been used several times in different systems e°, including the organ of Corti 5'6,18. Several authors 25-z7 have shown a collection of extracellular amorphous material surrounding the synaptic poles of hypothyroid adult outer hair cells, thus proposing that this material could be involved in the lack of
Correspondence: J.J. Prieto, Department of Histology, Faculty of Medicine, University of Alicante, 03690, Alicante, Spain. 0165-3806/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 contact between outer hair cells and nerve fibers. The chemical composition of this material is not known. though it has been suggested that it could be a mucopolysaccharide 25-27. The aim of this work was to determine if hypothyroidism induces the O K to maintain a secretory activity of saccharide c o m p o n e n t s far from the normal period, that could explain the abnormalities of the TM, and if this secretion is different from that of the normal animals. In addition, we also tried to verify if the amorphous material interposed between the outer hair cells and the nerve fibers in adult hypothyroid animals is composed of saccharides. MATERIALS AND METHODS Animals Forty-three rat pups ranging from birth to 60 days old were used in this study. Fifteen of them served as controls, and the other 28 were rendered hypothyroid by daily feeding the dams with PTU (50 mg/day) by gastric intubation. The PTU treatment was carried out from the 17th gestational day until the end of lactation, about the 30th postnatal day (PND). The day of birth was considered as the 1st PND. Animals were scacrified at PNDs 1, 5, 8, 10, 15, 20, 50 and 60. Histological procedure The animals were anesthetized by an intraperitoneal injection of chloral hydrate (0.3 g/kg b. wt.), prior to decapitation. After opening the bullae, the cochleae were removed and fixed in 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.35, for 3 h at 4 °C. Afterwards, the specimens were washed in the same buffer overnight, postfixed in ice-cold 2.0% osmium tetroxyde for 2 h, dehydrated through graded ethanol and embedded in Epon. The blocks were then cut, obtaining smaller ones, each one containing a single coil of the cochlea. In all cases, the medial coil of the cochlea was chosen, and cut in a Reichert Ultracut ultramicrotome. One-micrometer-thick sections were mounted on glass slides, on which the rest of the experimental procedures were carried out. Lectin labelling The biotinylated lectins employed were: soybean agglutinin (SBA), succinylated-wheat germ agglutinin (succ-WGA), Ulex europaeus agglutinin-I (UEA-I), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA) and Phaseolus vulgaris agglutinin-L (PHA-L). All of them were obtained from Vector Laboratories, Burlingame, CA, U.S.A. Following Hsu and Raine8, the sections were etched in sodium
ethoxide and then immersed in a 7.5'; tl,O,
TABLE I List of lectins used, specificity and tested optimal concentrations Lectin Soybean agglutinin Succinylated-wheat germ agglutinin Ulex europaeus agglutinin-I Lens culinaris agglutinin Ricinus communis agglutinin Peanut agglutinin Phaseolus vulgaris agglutinin-L
(SBA) (succ-WGA) (UEA-I) (LCA) (RCA) (PNA) (PHA-L)
Specificity
Optimal concentration 6uglml)
N-acetyl galactosamine N-acetyl glucosamine fucose mannose galactose, N-acetyl galactosamine galactose + N-acetyl galactosamine oligosaccharide
10 10 50 10 5 5(I 50
143 to the incubation of the sections, similar results were
Labelling of organ of K611iker and tectorial membrane
o b t a i n e d (Fig. 2H). T h e cellular nucleus was never labelled by any of the tested lectins. T h e slight staining that the nuclei show in the p h o t o m i c r o g r a p h s p r e s e n t e d here is due to the hematoxylin.
Disregarding the e x p l a i n e d T M d e f o r m a t i o n of the t r e a t e d animals, there was not any significant difference b e t w e e n the n o r m a l and the h y p o t h y r o i d TMs concerning the distribution of the m o n o s a c c h a r i d e s m a n n o s e (stained by the L C A ) , fucose (stained by the U E A - I ) and
Fig. 1. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with SBA. A,C,E,G: control animals; B,D,F,H,I,J: hypothyroid animals. A and B: 1st PND; C and D: 5th PND; E and F: 8th PND; G and H: 20th PND; I: 50th PND and J: 60th PND. Arrows point to the labelled region of the organ of K611iker. SL, spiral limbus; SV, stria vascularis; LT, limbal portion of the rectorial membrane; MT, major tectorial membrane; mT, minor tectorial membrane. Scale bar = 10 ~m.
144 galactose (stained by the RCA-I). The main findings concerning the secretion of the TM and its coupling with the OK were those obtained with three lectins: SBA and succinylated W G A on one side, and PHA-L on the other. Besides, the PNA showed qualitative differences between the normal and the hypothyroid TMs. To describe regional differences, the spiral limbus (Fig. 1A, SL) will be considered as inner, and the stria vascularis (Fig. 1A, SV) as outer.
SBA labelling (Fig. 1) In the 1st PND, both normal (Fig. 1A) and hypothyroid (Fig. 1B) TMs are intensely labelled by SBA in their 3 main portions: limbal zone (Fig. 1A, LT), major TM
(Fig. 1A, MT) and minor TM (Fig. i A, roT). The developing organ of Corti is not stained, except for the apical surface of hair cells, under the minor TM. The cells of the inner half of the OK are slightly labelled, from the basal portion up to the apical surface, in contact with the TM. The nuclei are not labelled, thus appearing as small, rounded clear areas. In the 5th PND, the staining of the hypothyroid TMs (Fig. 1D) remains similar to that of the normal ones (Fig. 1C), although the outer portion of the major TM is not labelled. The staining of the OK, much more intense than in the previous stage, has been reduced to, approximately, its inner third. In the 8th PND, the tall columnar ceils of the OK of
Fig. 2. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with succ-WGA. A,C,E,G,H: control animals; B,D,F: hypothyroid animals. A and B: 5th PND; C and D: 10th PND; E and F: 20th PND. Arrows point to the labelled region of the organ of Krlliker. Controls are made by either omitting the incubation in the lectin solution (G, normal animal, 10th PND) or incubating the sections with a solution of 2 M N-acetyl glucosamine, prior to the lectin incubation step (H, normal animal, 20th PND). Scale bar = 10/.tin.
145 normal animals (Fig. 1E) have begun to degenerate, appearing as cuboidal cells (lacking any cytoplasmic labelling), and the inner spiral sulcus becomes open. The labelling of the TM is reduced to the limbal zone, the inner part of its main body and the cover net. Strikingly, in the hypothyroid organ of Corti (Fig. 1F) the OK has not regressed, its inner portion is still intensely labelled by SBA and the anomalous TM remains attached to it. As the development proceeds, the staining pattern of the 8th PND persists unchanged both in the normal (Fig. 1G) and in the hypothyroid animals (Fig. 1H). In the latter case, prominent staining of the inner portion of the OK is present as far as the 50th PND (Fig. 1I), although the cells constituting it have diminished in height. Even at the 60th PND (Fig. 1J) it is possible to observe a small group of cells in the OK, whose cytoplasm is still labelled.
Succinylated-WGA labelling (Fig. 2) The staining pattern of both TM and OK by succW G A is quite similar to that obtained with SBA, although some differences can be stated: first, the labelling of the TM is homogenous, and all its portions are intensely stained from birth to adulthood, both in the
normal and in the hypothyroid animals, Second, the staining of the hypothyroid OK with succ-WGA is lighter than that observed with SBA, although the zone involved is similar (Fig. 2B,D,F). Controls of staining (Fig. 2G) and specificity of the lectin (Fig. 2H) show no labelling (compare Fig. 2G with 2C and 2H with 2E).
PHA-L labelling (Fig. 3) In all the studied stages, PHA-L labels uniformly both the normal and the hypothyroid TMs. In the latter case, however, from the 8th PND (in which the normal TM becomes detached by the regression of the OK, Fig. 3A), a more intense staining of the lower surface, in contact with the OK, became apparent until the 50th PND (Fig. 3E). This band is present only in the area of contact between both structures, being absent from a small inner region in which they become separated in the 20th PND.
PNA labelling (Fig. 4) Until the 5th PND (Fig. 4A,B) the staining of the TM with PNA is very weak, except for the cover net, the minor TM, and a narrow band located over the most
D
i
Fig. 3. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with PHA-L. A,C: control animals; B,D,E: hypothyroid animals. A and B: 8th PND; C and D: 20th PND; E: 50th PND. Arrowheads point to the more intensely labelled band between the OK and the tectorial membrane. Scale bar = 10/zm.
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B
Fig. 4. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with PNA, A,C: control animals; B,D,E: hypothyroid animals. A and B: 5th PND; C and D: 20th PND; E: 50th PND. Arrows point to the labelled surface of the OK; arrowheads point to the labelled region of the tectorial membrane. Scale bar = 10 #m. inner cells of the OK. This latter band is prolonged over the spiral limbus' interdental cells. In the normal animals, such labelling entirely disappears from all the cited locations in the 8th PND, thus being absent from this stage until adulthood. On the other hand, the staining of the cover net and minor TM is preserved in hypothyroid animals. When a small separation between the TM and the inner region of the OK takes place (approximately on the 20th PND, Fig. 4D), it is shown that the labelling in this zone is restricted to the surface of the OK, stopping at the point where the TM is not detached from it. Between the 20th and the 50th PNDs (Fig. 4E), the staining of the TM involves the lower portion of its limbal region, the cover net, the minor TM and the outer edge of the major TM. In the latter location, the labelling stops at the point of contact with the OK.
Labelling of saccharides at the basal pole of outer hair cells Three of the tested lectins, LCA (Fig. 5A), PHA-L (Fig. 5B) and W G A (Fig. 5C), densely stain extracellular conglomerates located below the outer hair cells and between adjacent Deiters' cells in the hypothyroid
animals, but not in the normal ones. This labelling becomes apparent at the 50th PND, The labelling is also present in the cup-like depression that the Deiters' cells form to enclose the synaptic pole of the o u t e r hair cells (Fig. 5B). It is not present, however, at the synaptic pole of inner hair cells. DISCUSSION In the early stages of the development of the auditory receptor, the TM is thought to be secreted by the spiral limbus' interdental cells 12, the supporting cells of the organ of Corti (pillars and Deiters' cells) i4 and, mainly, by the OK 11'13. In the rat, until the 8th PND the OK appears as a pseudostratified epithelium constituted by tall, prismatic cells. At this stage the cells of the OK begin to reduce their height progressively, in such a way that the inner spiral sulcus, located between the spiral limbus and the organ of Corti, is opened. From this stage onwards, the maintenance of the TM seems to be due to the secretion of the spiral limbus' interdental cells, exclusively. At the ultrastructural level, the cells of the O K display a great number of microvilli in their apical surface,
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Fig. 5. Light photomicrographs of the medial coil of hypothyroid cochleas (50th PND), sained with several lectins. A: LCA; B: PHA-L; C: WGA. Densely labelled conglomerates (arrows) are apparent between adjacent Deiters' cells (D), below the outer hair cells (O). On the left, the tunnel of Corti (TC) lined by the two pillar cells. Scale bar = 10 pm. connected with the TM by a network of fine filaments; their cytoplasm shows plenty of rough endoplasmic cisterns and contains a prominent Golgi apparatus, facts which suggest that they may be involved in active secretory processes 7'1°. This secretory role was demonstrated by Gil-Loyzaga et al. 6 using radiolabelled glucosamine, which was incorporated in the OK and, subsequently, in the TM, after its systemic injection. However, until now it was generally assumed that all the cells in the O K had the same secretory properties, as their morphology is similar. Our results show that there are regional differences between different parts of the OK, in terms of secretion of saccharides: until the regression of the OK on the 8th PND, the distribution of N-acetyl glucosamine and N-acetyl galactosamine become circumscribed from the
inner half of the OK at birth, to the inner third of it, at the 5th PND. This peculiar feature cannot be interpreted as reflecting a degeneration process which would take place from inner hair cells to the spiral limbus, as it has been previously demonstrated that the involution within the normal OK proceeds in the opposite direction 2526. The rest of tested lectins failed to label the OK, but not the TM, a fact that could be due to the inability of lectins to bind their specific sugars if the latter are 'masked' in a polysaccharide chain inside the cell. Once out of the cell, in the TM, the sugars would be 'unmasked', either by a change in the 3-dimensional structure of the polysaccharide chain, or by its fragmentation. On the other hand, N-acetyi glucosamine and N-acetyl galactosamine would be accessible to SBA and succ-WGA, respectively, both prior to their secretion and after it has
148 taken place. Thus, it can be suggested that these two saccharide residues are principally secreted by the most inner cells of the OK. With respect to the secretion of both sugars, the hypothyroid OK is similar to the normal one from birth to the 5th PND. Although this secretion obviously stops in normal animals on the 8th PND, it is abnormally maintained in the hypothyroid ones as far as the 60th PND. It has been previously reported that, after ceasing the PTU treatment on the 30th PND, there is a partial recovery of auditory function 26. The main morphological finding that has been related to this fact is the restarting of maturational processes of the OK, leading to an incomplete opening of the inner spiral sulcus 26. However, from the results of the present study it seems that, though the cells of the OK reduce their height after the cessation of the antithyroid treatment, they still display very active secretory properties, at least until the 50th PND, much more than it has been previously suggested from the ultrastructural appearance of the cells of the OK (approximately until the 20th PND). In a recent work on the polypeptide composition of the TM 17 it was demonstrated that the normal, adult TM is composed of half collagen proteins (types II, V and IX), and half non-collagenous, high-molecular weight polypeptide material. These authors found that N-acetyl glucosamine and N-acetyl galactosamine residues were associated to the latter compound. Thus, it seems reasonable to assume that, if both saccharides are secreted linked to this material, the lectin labelling pattern would reflect a pathologically prolonged secretion of this compound in the hypothyroid animals. Another difference between normal and hypothyroid TMs is revealed by the PHA-L labelling. PHA-L binds to an oligosaccharide, not yet identified biochemically. Although PHA-L stains all the regions of the TM, as succ-WGA, RCA-I and LCA do, it is the unique lectin which, in the hypothyroid animals, labels more intensely a narrow band located only between the TM and the OK, from the 8th to the 60th PND. In fact, both LCA and RCA-I stain the whole apical surface of the organ of Corti (and even that of the OK), but only the PHA-L labelling is restricted to the area of contact between the OK and the TM. Actually, the apical surface of the inner region of the OK, where a little detachment of the TM takes place on the 20th PND, lacks any staining. To explain a tentative role for this oligosaccharide in the hypothyroid organ of Corti, it must be taken into account that carbohydrates have been implied several times in a number of processes of cellular adhesion in different systems, as in fertilization, cellular migration, specific intercellular recognition, neurite fasciculation, etc. (see references in Hughes and Pena9). Then, the precise
localization of the labelling between the OK and the TM~ together with the fact that it only appears in the hypothyroid animals from the stage in which normal TM becomes detached from the OK, suggest that the abnormal persistence of this band of oligosaccharides could be responsible for the anomalous attachment of the TM to the underlying OK. PNA is the unique lectin whose labelling pattern of the TM reveals qualitative differences between the normal and the hypothyroid animals. Actually, although the staining features are similar in both experimental groups until the 5th PND, from the 8th PND on only the TMs of the hypothyroid animals contain the disaccharide galactose+N-acetyl galactosamine. Nevertheless, its location in the limbal zone of the TM, the cover net and the minor TM cannot account for any explanation of the structural distortion of the TM. The lectin labelling of large conglomerates at the basal pole of the outer hair cells can be related to the extraceUular amorphous material that has been previously reported in TEM studies of the hypothyroid organ of Corti 25-27. In these studies, the authors suggested that this substance could correspond to acid polysaccharides, since the existence of an acidophilic precipitate in the cochlear duct of hypothyroid adult animals had been previously described 15"16'19. From our results, it can be stated that this substance is composed of an oligosaccharide (PHA-L-positive) which contains mannose (LCApositive) and sialic acid (WGA-positive and succ-WGAnegative). This oligosaccharide could be either a normal constituent of the glycocalix of the cell membranes at this location, whose secretion is abnormally maintained in hypothyroid animals (in a way similar to that of the secretion of the TM by means of the OK), or an extraneous collection not related to the normal composition of the glycocalix of these cells. It has been suggested that the accumulation of this substance may act avoiding the contact between the nerve fibers and the basal pole of the outer hair cells; this process could lead to the mechanical separation of both afferent and efferent fibers from the synaptic pole of the outer hair ceils27 (but not from inner hair cells, which lack these conglomerates, and whose innervation is not altered by hypothyroidism). From the results obtained with lectin staining of saccharide residues in normal and hypothyroid developing organs of Corti, it can be stated that thyroxine plays a key role in the carbohydrate metabolism in the auditory receptor, and that the hypothyroidism leads both to an abnormal persistence of some developmental processes (secretion of saccharides by the OK, which are incorporated to the TM), and to pathological changes (attachment of the TM to the OK and dense conglomerates
149 u n d e r the outer hair cells). Regarding the saccharide composition of the T M , there is evidence of qualitative differences b e t w e e n the hypothyroid and the normal ones, since the disaccharide galactose+N-acetyl galactosa m i n e is only present in the hypothyroid TMs. Besides, it is possible that the a b n o r m a l persistence of the secretion of some saccharide residues, and not of others,
Acknowledgements. The authors wish to thank Mrs. Mercedes Garcfa and Mr. Emilio Guti6rrez for the photographic technical assistance, Mrs. Maria Dolores Segura for sectioning of the specimens, and Miss M.J. Pujante for processing the hypothyroid specimens. The authors are also grateful to Dr. Enrique Alcaraz for the style corrections of the manuscript and to the referee for useful comments. This work was supported by grants from the Spanish Goverment (FISS 1646/87, FISS 1652/87 and CUCYT PB 86-0277) and a personal fellowship from the FISS (J.J.P.).
would lead to a different relative proportion a m o n g them in the hypothyroid TMs. REFERENCES 1 Bargman, G.J. and Gardner, L.I., Otic lesions and congenital hypothyroidism in the developing chick, J. Clin. Invest., 46 (1967) 1828-1839. 2 Deol, M.S. An experimental approach to the understanding and treatment of hereditary syndromes with congenital deafness and hypothyroidism, J. Med. Gen., 10 (1973) 235-242. 3 Deol, M.S., The role of thyroxine in the differentiation of the organ of Corti, Acta Otolaryngol., 81 (1976) 429-435. 4 Gabrion, J., Legrand, C., Mercier, B., Harricane, M.C. and Uziel, A., Microtubules in the cochlea of the hypothyroid developing rat, Hearing Res., 13 (1984) 203-207. 5 Gil-Loyzaga, P., Gabrion, J. and Uziel, A., Lectins demonstrate the presence of carbohydrates in the tectorial membrane of mammalian cochlea, Hearing Res., 20 (1985) 1-8. 6 Gil-Loyzaga, P. and Brownell, W.E., Wheat germ agglutinin and Helix pomatia agglutinin lectin binding on cochlear hair cells, Hearing Res., 34 (1988) 149-156. 7 Hinojosa, R., A note on development of Corti's organ, Acta Otolaryngol., 84 (1977) 238-251. 8 Hsu, S.M. and Raine, L., Lectin and Avidin-Biotin-Peroxydase complex for localization of carbohydrate in tissue sections, J. Histochem. Cytochem., 30 (1982) 157-161. 9 Hughes, R.C. and Pena, S.D.J., The role of carbohydrates in cellular recognition and adhesion. In Carbohydrate Metabolism and its Disorders, Vol. 3, Academic, London, 1981, pp. 374-412. 10 Kikuchi, K. and Hilding, D., The development of the organ of Corti in the mouse, Acta OtolaryngoL, 60 (1965) 207-222. 11 Lim, D.J., Fine morphology of the tectorial membrane. Its relationship to the organ of Corti, Arch. Otolaryngol., 96 (1972) 199-215. 12 Lim, D.J., Fine morphology of the tectorial membrane. Fresh and developmental. In M. Portmann and J.M. Aran (Eds.), Les Colloques de l'Institut National de la Sant~ et de la Recherche M6dicale, INSERM, 1977, pp. 47-60. 13 Lira, D.J. and Anniko, M., Correlative development of sensory cells and overlying membrane of the inner ear: micromechanical aspects. In R.W. Ruben (Ed.), The Biology of Change in Otolaryngology, Elsevier, Amsterdam, 1986, pp. 55-69. 14 Lim, D.J. and Rueda, J., Localization of glycoconjugates in the developing organ of Corti, Abstracts of the XXIV Workshop on Inner Ear Biology, Nijmegen, 1987, p. 2.
15 Meyerhoff, W.L., Hypothyroidism and the ear: electrophysiological, morphological and chemical considerations, Laryngoscope, 89 (1980) Suppl. 19. 16 Poulsen, H., Thyrotrophic and thyroid hormone control of the inner ear with special reference to myxoedema and M6ni~re's disease. In G. Asboe-Hansen (Ed.), Hormones and Connective Tissue, Williams, Baltimore, 1966, pp. 239-257. 17 Richardson, G.P., Russell, I.J., Duance, V.C. and Bailey, A.J., Polypeptide composition of the mammalian tectorial membrane, Hearing Res., 25 (1987) 45-60. 18 Rueda, J. and Lim, D., Possible transient stereociliary adhesion molecules expressed during cochlea development: a preliminary study. In M. Ohyama and T. Muramatsu (Eds.), Glycoconjugates in Medicine, Professional Postgraduate Services, Tokyo, 1988, pp. 338-350. 19 Sch/itzle, W. and Haubrich, J., Histochemical changes in the guinea-pig cochlea in the experimental hypothyroidism, Arch. Klin. Exp. Ohren- Nasen- Kehlkopfheilkd., 188 (1967) 224-231. 20 Schrevel, J., Gros, D. and Monsigny, M., Cytochemistry of cell glycoconjugates, Prog. Histochem. Cytochem., 14 (1981) 1-269. 21 Steel, K., The proteins of normal and abnormal tectorial membranes, Acta Otolaryngol., 89 (1980) 27-32. 22 Steel, K., The tectorial membrane of mammals, Hearing Res., 9 (1983) 327-359. 23 Tata, J.R., Cell structure and biosynthesis during hormonemediated growth and development. In M. Hamburg and E.J.W. Barrington (Eds.), Hormones in Development, Appleton-Century-Crofts, New York, 1971, pp. 19-49. 24 Trotter, W.R., The association of deafness with thyroid dysfunction, Brit. Med. Bull., 16 (1960) 92-98. 25 Uziel, A., Gabrion, J., Ohresser, M. and Legrand, C., Effects of hypothyroidism on the structural development of the organ of Corti in the rat, Acta Otolaryngol., 92 (1981) 469-480. 26 Uziel, A., Legrand, C., Ohresser, M. and Marot, M., Maturational and degenerative processes in the organ of Corti after neonatal hypothyroidism, Hearing Res., 11 (1983) 203-218. 27 Uziel, A., Pujol, R., Legrand, C. and Legrand, J., Cochlear synaptogenesis in the hypothyroid rat, Dev. Brain Res., 7 (1983) 295-301. 28 Uziel, A., Rabie, A. and Marot, M., The effects of hypothyroidism on the onset of cochlear potentials in developing rats, Brain Res., 182 (1980) 172-175.
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BRESD 51031
Lectin staining of saccharides in the normal and hypothyroid developing organ of Corti Jorge J. Prieto, Joaquin Rueda, Maria L. Sala and Jaime A. Merchan Department of Histology, Faculty of Medicine, University of Alicante, Alicante (Spain) (Accepted 26 September 1989) Key words: Organ of Corti; Hypothyroidism;Lectin; Saccharide
Lectin staining has been used to detect mono- and oligosaccharidesin normal and hypothyroid developing organs of Corti in the rat. Eight developmental stages were studied (1, 5, 8, 10, 15, 20, 50 and 60 days after birth). Congenital hypothyroidism was induced by oral administration of propylthyouracil to pregnant rats. Labelling of the tectorial membrane with 3 lectins, Ulex europaeus agglutinin-I (UEA-I), Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin-I (RCA-I) showed no significant differences between normal and hypothyroid animals. Staining with peanut agglutinin (PNA) showed that the hypothyroid adult tectorial membrane (but not the normal one) possesses the disaccharide galactose+N-acetyl galactosamine. Phaseolus vulgaris agglutinin-L (PHA-L) labels the whole tectorial membrane in both groups of animals, but the staining is more intense in the hypothyroid one for a narrow band of oligosaccharide located just between the tectorial membrane and the underlying organ of KOlliker. Both soybean agglutinin (SBA) and succinylated wheat germ agglutinin (WGA) stain the tectorial membrane as well as the cytoplasm of the cells constituting the inner portion of the organ of Kflliker; this latter feature disappears in the normal animals about the 8th postnatal day, but it is abnormally preserved until the 60th postnatal day in the hypothyroid ones. In the adult hypothyroid animals, 3 of the lectins (LCA, PHA-L and WGA) stain extracellular conglomerates located under the synaptic pole of the outer hair cells. INTRODUCTION For a long time the association of hearing loss with 3 forms of thyroid disease has been observed: myxedema in the adult, Pendred's syndrome and endemic cretinism 24. An experimental model of the influence of congenital hypothyroidism on the development of the auditory receptor has been performed on chick embryos 1, and newborn mice 2'3 and rats 25'26. The administration of propylthyouracil (PTU) to pregnant rats results in severe abnormalities both in the morphology of the organ of Corti and in the cochlear potentials of the pups. The structural alterations consist of: (1) the abnormal persistence of the organ of K611iker (OK)25; (2) the distortion of the tectorial membrane (TM)25; (3) the absence of synaptogenesis between the efferent fibers and the outer hair cells27; and (4) a poor development of both the supporting cells and the large fluid spaces surrounding them (tunnel of Corti and Nuel's spaces) 4. In congenitally hypothyroid animals, the TM is of enormous size and remains abnormally attached to the underlying OK. It has been suggested 25 that the distortion of the TM could be due to the abnormal persistence of the OK (which seems to be involved in the secretion
of the TM in early developmental stages). Both structural alterations lead to an anomalous functional development, characterized by a threshold elevation in the VIIIth nerve compound action potential and the broadening of the action potential tuning curves 28. Based on the fact that thyroxine plays a key role in protein synthesis 23, Deol 3 suggested that the derangement of the hypothyroid TM could be due to abnormalities in its protein composition. This hypothesis was ruled out by a chemical analysis of hypothyroid TMs using SDS polyacrylamide gel electrophoresis 21, which failed to show any difference between them and the normal ones. As the TM is composed by a network of protein fibrils embedded in a saccharide matrix 22, an anomalous secretion of this latter component could possibly be the explanation for the morphological findings. For the demonstration of saccharides, lectins (proteins and glycoproteins of non-immune origin capable of binding specifically to different mono- and oligosaccharides) have been used several times in different systems e°, including the organ of Corti 5'6,18. Several authors 25-z7 have shown a collection of extracellular amorphous material surrounding the synaptic poles of hypothyroid adult outer hair cells, thus proposing that this material could be involved in the lack of
Correspondence: J.J. Prieto, Department of Histology, Faculty of Medicine, University of Alicante, 03690, Alicante, Spain. 0165-3806/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
142 contact between outer hair cells and nerve fibers. The chemical composition of this material is not known. though it has been suggested that it could be a mucopolysaccharide 25-27. The aim of this work was to determine if hypothyroidism induces the O K to maintain a secretory activity of saccharide c o m p o n e n t s far from the normal period, that could explain the abnormalities of the TM, and if this secretion is different from that of the normal animals. In addition, we also tried to verify if the amorphous material interposed between the outer hair cells and the nerve fibers in adult hypothyroid animals is composed of saccharides. MATERIALS AND METHODS Animals Forty-three rat pups ranging from birth to 60 days old were used in this study. Fifteen of them served as controls, and the other 28 were rendered hypothyroid by daily feeding the dams with PTU (50 mg/day) by gastric intubation. The PTU treatment was carried out from the 17th gestational day until the end of lactation, about the 30th postnatal day (PND). The day of birth was considered as the 1st PND. Animals were scacrified at PNDs 1, 5, 8, 10, 15, 20, 50 and 60. Histological procedure The animals were anesthetized by an intraperitoneal injection of chloral hydrate (0.3 g/kg b. wt.), prior to decapitation. After opening the bullae, the cochleae were removed and fixed in 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.35, for 3 h at 4 °C. Afterwards, the specimens were washed in the same buffer overnight, postfixed in ice-cold 2.0% osmium tetroxyde for 2 h, dehydrated through graded ethanol and embedded in Epon. The blocks were then cut, obtaining smaller ones, each one containing a single coil of the cochlea. In all cases, the medial coil of the cochlea was chosen, and cut in a Reichert Ultracut ultramicrotome. One-micrometer-thick sections were mounted on glass slides, on which the rest of the experimental procedures were carried out. Lectin labelling The biotinylated lectins employed were: soybean agglutinin (SBA), succinylated-wheat germ agglutinin (succ-WGA), Ulex europaeus agglutinin-I (UEA-I), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA) and Phaseolus vulgaris agglutinin-L (PHA-L). All of them were obtained from Vector Laboratories, Burlingame, CA, U.S.A. Following Hsu and Raine8, the sections were etched in sodium
ethoxide and then immersed in a 7.5'; tl,O,
TABLE I List of lectins used, specificity and tested optimal concentrations Lectin Soybean agglutinin Succinylated-wheat germ agglutinin Ulex europaeus agglutinin-I Lens culinaris agglutinin Ricinus communis agglutinin Peanut agglutinin Phaseolus vulgaris agglutinin-L
(SBA) (succ-WGA) (UEA-I) (LCA) (RCA) (PNA) (PHA-L)
Specificity
Optimal concentration 6uglml)
N-acetyl galactosamine N-acetyl glucosamine fucose mannose galactose, N-acetyl galactosamine galactose + N-acetyl galactosamine oligosaccharide
10 10 50 10 5 5(I 50
143 to the incubation of the sections, similar results were
Labelling of organ of K611iker and tectorial membrane
o b t a i n e d (Fig. 2H). T h e cellular nucleus was never labelled by any of the tested lectins. T h e slight staining that the nuclei show in the p h o t o m i c r o g r a p h s p r e s e n t e d here is due to the hematoxylin.
Disregarding the e x p l a i n e d T M d e f o r m a t i o n of the t r e a t e d animals, there was not any significant difference b e t w e e n the n o r m a l and the h y p o t h y r o i d TMs concerning the distribution of the m o n o s a c c h a r i d e s m a n n o s e (stained by the L C A ) , fucose (stained by the U E A - I ) and
Fig. 1. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with SBA. A,C,E,G: control animals; B,D,F,H,I,J: hypothyroid animals. A and B: 1st PND; C and D: 5th PND; E and F: 8th PND; G and H: 20th PND; I: 50th PND and J: 60th PND. Arrows point to the labelled region of the organ of K611iker. SL, spiral limbus; SV, stria vascularis; LT, limbal portion of the rectorial membrane; MT, major tectorial membrane; mT, minor tectorial membrane. Scale bar = 10 ~m.
144 galactose (stained by the RCA-I). The main findings concerning the secretion of the TM and its coupling with the OK were those obtained with three lectins: SBA and succinylated W G A on one side, and PHA-L on the other. Besides, the PNA showed qualitative differences between the normal and the hypothyroid TMs. To describe regional differences, the spiral limbus (Fig. 1A, SL) will be considered as inner, and the stria vascularis (Fig. 1A, SV) as outer.
SBA labelling (Fig. 1) In the 1st PND, both normal (Fig. 1A) and hypothyroid (Fig. 1B) TMs are intensely labelled by SBA in their 3 main portions: limbal zone (Fig. 1A, LT), major TM
(Fig. 1A, MT) and minor TM (Fig. i A, roT). The developing organ of Corti is not stained, except for the apical surface of hair cells, under the minor TM. The cells of the inner half of the OK are slightly labelled, from the basal portion up to the apical surface, in contact with the TM. The nuclei are not labelled, thus appearing as small, rounded clear areas. In the 5th PND, the staining of the hypothyroid TMs (Fig. 1D) remains similar to that of the normal ones (Fig. 1C), although the outer portion of the major TM is not labelled. The staining of the OK, much more intense than in the previous stage, has been reduced to, approximately, its inner third. In the 8th PND, the tall columnar ceils of the OK of
Fig. 2. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with succ-WGA. A,C,E,G,H: control animals; B,D,F: hypothyroid animals. A and B: 5th PND; C and D: 10th PND; E and F: 20th PND. Arrows point to the labelled region of the organ of Krlliker. Controls are made by either omitting the incubation in the lectin solution (G, normal animal, 10th PND) or incubating the sections with a solution of 2 M N-acetyl glucosamine, prior to the lectin incubation step (H, normal animal, 20th PND). Scale bar = 10/.tin.
145 normal animals (Fig. 1E) have begun to degenerate, appearing as cuboidal cells (lacking any cytoplasmic labelling), and the inner spiral sulcus becomes open. The labelling of the TM is reduced to the limbal zone, the inner part of its main body and the cover net. Strikingly, in the hypothyroid organ of Corti (Fig. 1F) the OK has not regressed, its inner portion is still intensely labelled by SBA and the anomalous TM remains attached to it. As the development proceeds, the staining pattern of the 8th PND persists unchanged both in the normal (Fig. 1G) and in the hypothyroid animals (Fig. 1H). In the latter case, prominent staining of the inner portion of the OK is present as far as the 50th PND (Fig. 1I), although the cells constituting it have diminished in height. Even at the 60th PND (Fig. 1J) it is possible to observe a small group of cells in the OK, whose cytoplasm is still labelled.
Succinylated-WGA labelling (Fig. 2) The staining pattern of both TM and OK by succW G A is quite similar to that obtained with SBA, although some differences can be stated: first, the labelling of the TM is homogenous, and all its portions are intensely stained from birth to adulthood, both in the
normal and in the hypothyroid animals, Second, the staining of the hypothyroid OK with succ-WGA is lighter than that observed with SBA, although the zone involved is similar (Fig. 2B,D,F). Controls of staining (Fig. 2G) and specificity of the lectin (Fig. 2H) show no labelling (compare Fig. 2G with 2C and 2H with 2E).
PHA-L labelling (Fig. 3) In all the studied stages, PHA-L labels uniformly both the normal and the hypothyroid TMs. In the latter case, however, from the 8th PND (in which the normal TM becomes detached by the regression of the OK, Fig. 3A), a more intense staining of the lower surface, in contact with the OK, became apparent until the 50th PND (Fig. 3E). This band is present only in the area of contact between both structures, being absent from a small inner region in which they become separated in the 20th PND.
PNA labelling (Fig. 4) Until the 5th PND (Fig. 4A,B) the staining of the TM with PNA is very weak, except for the cover net, the minor TM, and a narrow band located over the most
D
i
Fig. 3. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with PHA-L. A,C: control animals; B,D,E: hypothyroid animals. A and B: 8th PND; C and D: 20th PND; E: 50th PND. Arrowheads point to the more intensely labelled band between the OK and the tectorial membrane. Scale bar = 10/zm.
146
?
B
Fig. 4. Light photomicrographs of the medial coil of the cochlea at several developmental stages, stained with PNA, A,C: control animals; B,D,E: hypothyroid animals. A and B: 5th PND; C and D: 20th PND; E: 50th PND. Arrows point to the labelled surface of the OK; arrowheads point to the labelled region of the tectorial membrane. Scale bar = 10 #m. inner cells of the OK. This latter band is prolonged over the spiral limbus' interdental cells. In the normal animals, such labelling entirely disappears from all the cited locations in the 8th PND, thus being absent from this stage until adulthood. On the other hand, the staining of the cover net and minor TM is preserved in hypothyroid animals. When a small separation between the TM and the inner region of the OK takes place (approximately on the 20th PND, Fig. 4D), it is shown that the labelling in this zone is restricted to the surface of the OK, stopping at the point where the TM is not detached from it. Between the 20th and the 50th PNDs (Fig. 4E), the staining of the TM involves the lower portion of its limbal region, the cover net, the minor TM and the outer edge of the major TM. In the latter location, the labelling stops at the point of contact with the OK.
Labelling of saccharides at the basal pole of outer hair cells Three of the tested lectins, LCA (Fig. 5A), PHA-L (Fig. 5B) and W G A (Fig. 5C), densely stain extracellular conglomerates located below the outer hair cells and between adjacent Deiters' cells in the hypothyroid
animals, but not in the normal ones. This labelling becomes apparent at the 50th PND, The labelling is also present in the cup-like depression that the Deiters' cells form to enclose the synaptic pole of the o u t e r hair cells (Fig. 5B). It is not present, however, at the synaptic pole of inner hair cells. DISCUSSION In the early stages of the development of the auditory receptor, the TM is thought to be secreted by the spiral limbus' interdental cells 12, the supporting cells of the organ of Corti (pillars and Deiters' cells) i4 and, mainly, by the OK 11'13. In the rat, until the 8th PND the OK appears as a pseudostratified epithelium constituted by tall, prismatic cells. At this stage the cells of the OK begin to reduce their height progressively, in such a way that the inner spiral sulcus, located between the spiral limbus and the organ of Corti, is opened. From this stage onwards, the maintenance of the TM seems to be due to the secretion of the spiral limbus' interdental cells, exclusively. At the ultrastructural level, the cells of the O K display a great number of microvilli in their apical surface,
147
I
I
~,
Fig. 5. Light photomicrographs of the medial coil of hypothyroid cochleas (50th PND), sained with several lectins. A: LCA; B: PHA-L; C: WGA. Densely labelled conglomerates (arrows) are apparent between adjacent Deiters' cells (D), below the outer hair cells (O). On the left, the tunnel of Corti (TC) lined by the two pillar cells. Scale bar = 10 pm. connected with the TM by a network of fine filaments; their cytoplasm shows plenty of rough endoplasmic cisterns and contains a prominent Golgi apparatus, facts which suggest that they may be involved in active secretory processes 7'1°. This secretory role was demonstrated by Gil-Loyzaga et al. 6 using radiolabelled glucosamine, which was incorporated in the OK and, subsequently, in the TM, after its systemic injection. However, until now it was generally assumed that all the cells in the O K had the same secretory properties, as their morphology is similar. Our results show that there are regional differences between different parts of the OK, in terms of secretion of saccharides: until the regression of the OK on the 8th PND, the distribution of N-acetyl glucosamine and N-acetyl galactosamine become circumscribed from the
inner half of the OK at birth, to the inner third of it, at the 5th PND. This peculiar feature cannot be interpreted as reflecting a degeneration process which would take place from inner hair cells to the spiral limbus, as it has been previously demonstrated that the involution within the normal OK proceeds in the opposite direction 2526. The rest of tested lectins failed to label the OK, but not the TM, a fact that could be due to the inability of lectins to bind their specific sugars if the latter are 'masked' in a polysaccharide chain inside the cell. Once out of the cell, in the TM, the sugars would be 'unmasked', either by a change in the 3-dimensional structure of the polysaccharide chain, or by its fragmentation. On the other hand, N-acetyi glucosamine and N-acetyl galactosamine would be accessible to SBA and succ-WGA, respectively, both prior to their secretion and after it has
148 taken place. Thus, it can be suggested that these two saccharide residues are principally secreted by the most inner cells of the OK. With respect to the secretion of both sugars, the hypothyroid OK is similar to the normal one from birth to the 5th PND. Although this secretion obviously stops in normal animals on the 8th PND, it is abnormally maintained in the hypothyroid ones as far as the 60th PND. It has been previously reported that, after ceasing the PTU treatment on the 30th PND, there is a partial recovery of auditory function 26. The main morphological finding that has been related to this fact is the restarting of maturational processes of the OK, leading to an incomplete opening of the inner spiral sulcus 26. However, from the results of the present study it seems that, though the cells of the OK reduce their height after the cessation of the antithyroid treatment, they still display very active secretory properties, at least until the 50th PND, much more than it has been previously suggested from the ultrastructural appearance of the cells of the OK (approximately until the 20th PND). In a recent work on the polypeptide composition of the TM 17 it was demonstrated that the normal, adult TM is composed of half collagen proteins (types II, V and IX), and half non-collagenous, high-molecular weight polypeptide material. These authors found that N-acetyl glucosamine and N-acetyl galactosamine residues were associated to the latter compound. Thus, it seems reasonable to assume that, if both saccharides are secreted linked to this material, the lectin labelling pattern would reflect a pathologically prolonged secretion of this compound in the hypothyroid animals. Another difference between normal and hypothyroid TMs is revealed by the PHA-L labelling. PHA-L binds to an oligosaccharide, not yet identified biochemically. Although PHA-L stains all the regions of the TM, as succ-WGA, RCA-I and LCA do, it is the unique lectin which, in the hypothyroid animals, labels more intensely a narrow band located only between the TM and the OK, from the 8th to the 60th PND. In fact, both LCA and RCA-I stain the whole apical surface of the organ of Corti (and even that of the OK), but only the PHA-L labelling is restricted to the area of contact between the OK and the TM. Actually, the apical surface of the inner region of the OK, where a little detachment of the TM takes place on the 20th PND, lacks any staining. To explain a tentative role for this oligosaccharide in the hypothyroid organ of Corti, it must be taken into account that carbohydrates have been implied several times in a number of processes of cellular adhesion in different systems, as in fertilization, cellular migration, specific intercellular recognition, neurite fasciculation, etc. (see references in Hughes and Pena9). Then, the precise
localization of the labelling between the OK and the TM~ together with the fact that it only appears in the hypothyroid animals from the stage in which normal TM becomes detached from the OK, suggest that the abnormal persistence of this band of oligosaccharides could be responsible for the anomalous attachment of the TM to the underlying OK. PNA is the unique lectin whose labelling pattern of the TM reveals qualitative differences between the normal and the hypothyroid animals. Actually, although the staining features are similar in both experimental groups until the 5th PND, from the 8th PND on only the TMs of the hypothyroid animals contain the disaccharide galactose+N-acetyl galactosamine. Nevertheless, its location in the limbal zone of the TM, the cover net and the minor TM cannot account for any explanation of the structural distortion of the TM. The lectin labelling of large conglomerates at the basal pole of the outer hair cells can be related to the extraceUular amorphous material that has been previously reported in TEM studies of the hypothyroid organ of Corti 25-27. In these studies, the authors suggested that this substance could correspond to acid polysaccharides, since the existence of an acidophilic precipitate in the cochlear duct of hypothyroid adult animals had been previously described 15"16'19. From our results, it can be stated that this substance is composed of an oligosaccharide (PHA-L-positive) which contains mannose (LCApositive) and sialic acid (WGA-positive and succ-WGAnegative). This oligosaccharide could be either a normal constituent of the glycocalix of the cell membranes at this location, whose secretion is abnormally maintained in hypothyroid animals (in a way similar to that of the secretion of the TM by means of the OK), or an extraneous collection not related to the normal composition of the glycocalix of these cells. It has been suggested that the accumulation of this substance may act avoiding the contact between the nerve fibers and the basal pole of the outer hair cells; this process could lead to the mechanical separation of both afferent and efferent fibers from the synaptic pole of the outer hair ceils27 (but not from inner hair cells, which lack these conglomerates, and whose innervation is not altered by hypothyroidism). From the results obtained with lectin staining of saccharide residues in normal and hypothyroid developing organs of Corti, it can be stated that thyroxine plays a key role in the carbohydrate metabolism in the auditory receptor, and that the hypothyroidism leads both to an abnormal persistence of some developmental processes (secretion of saccharides by the OK, which are incorporated to the TM), and to pathological changes (attachment of the TM to the OK and dense conglomerates
149 u n d e r the outer hair cells). Regarding the saccharide composition of the T M , there is evidence of qualitative differences b e t w e e n the hypothyroid and the normal ones, since the disaccharide galactose+N-acetyl galactosa m i n e is only present in the hypothyroid TMs. Besides, it is possible that the a b n o r m a l persistence of the secretion of some saccharide residues, and not of others,
Acknowledgements. The authors wish to thank Mrs. Mercedes Garcfa and Mr. Emilio Guti6rrez for the photographic technical assistance, Mrs. Maria Dolores Segura for sectioning of the specimens, and Miss M.J. Pujante for processing the hypothyroid specimens. The authors are also grateful to Dr. Enrique Alcaraz for the style corrections of the manuscript and to the referee for useful comments. This work was supported by grants from the Spanish Goverment (FISS 1646/87, FISS 1652/87 and CUCYT PB 86-0277) and a personal fellowship from the FISS (J.J.P.).
would lead to a different relative proportion a m o n g them in the hypothyroid TMs. REFERENCES 1 Bargman, G.J. and Gardner, L.I., Otic lesions and congenital hypothyroidism in the developing chick, J. Clin. Invest., 46 (1967) 1828-1839. 2 Deol, M.S. An experimental approach to the understanding and treatment of hereditary syndromes with congenital deafness and hypothyroidism, J. Med. Gen., 10 (1973) 235-242. 3 Deol, M.S., The role of thyroxine in the differentiation of the organ of Corti, Acta Otolaryngol., 81 (1976) 429-435. 4 Gabrion, J., Legrand, C., Mercier, B., Harricane, M.C. and Uziel, A., Microtubules in the cochlea of the hypothyroid developing rat, Hearing Res., 13 (1984) 203-207. 5 Gil-Loyzaga, P., Gabrion, J. and Uziel, A., Lectins demonstrate the presence of carbohydrates in the tectorial membrane of mammalian cochlea, Hearing Res., 20 (1985) 1-8. 6 Gil-Loyzaga, P. and Brownell, W.E., Wheat germ agglutinin and Helix pomatia agglutinin lectin binding on cochlear hair cells, Hearing Res., 34 (1988) 149-156. 7 Hinojosa, R., A note on development of Corti's organ, Acta Otolaryngol., 84 (1977) 238-251. 8 Hsu, S.M. and Raine, L., Lectin and Avidin-Biotin-Peroxydase complex for localization of carbohydrate in tissue sections, J. Histochem. Cytochem., 30 (1982) 157-161. 9 Hughes, R.C. and Pena, S.D.J., The role of carbohydrates in cellular recognition and adhesion. In Carbohydrate Metabolism and its Disorders, Vol. 3, Academic, London, 1981, pp. 374-412. 10 Kikuchi, K. and Hilding, D., The development of the organ of Corti in the mouse, Acta OtolaryngoL, 60 (1965) 207-222. 11 Lim, D.J., Fine morphology of the tectorial membrane. Its relationship to the organ of Corti, Arch. Otolaryngol., 96 (1972) 199-215. 12 Lim, D.J., Fine morphology of the tectorial membrane. Fresh and developmental. In M. Portmann and J.M. Aran (Eds.), Les Colloques de l'Institut National de la Sant~ et de la Recherche M6dicale, INSERM, 1977, pp. 47-60. 13 Lira, D.J. and Anniko, M., Correlative development of sensory cells and overlying membrane of the inner ear: micromechanical aspects. In R.W. Ruben (Ed.), The Biology of Change in Otolaryngology, Elsevier, Amsterdam, 1986, pp. 55-69. 14 Lim, D.J. and Rueda, J., Localization of glycoconjugates in the developing organ of Corti, Abstracts of the XXIV Workshop on Inner Ear Biology, Nijmegen, 1987, p. 2.
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