Histochemical detection of glycogen and glycoconjugates in the inner ear with modified concanavalin...

Histochemical Journal 26, 437-446 (1994)

Histochemical detection of glycogen and glycoconjugates in the inner ear with modified concanavalin A-horseradish peroxidase procedures M A K O T O I T O , S A M U E L S. S P I C E R and B R A D L E Y A. S C H U L T E

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 17I Ashley Avenue, Charleston, South Carolina 29425 USA

Received 29 June 1993 and in revised form 17 December 1993

Summary

Inner ears from neonatal and adult Mongolian gerbils were examined to determine developmental changes in the content of glycogen and glycoconjugates as shown by histochemical application of the jack bean lectin, concanavalin A (con A). Sections of fixed paraffin-embedded inner ears were stained using the con A-horseradish peroxidase sequence in conjunction with prior treatments including periodate oxidation with or without subsequent reduction and diastase digestion. In adult inner ear, brief periodate oxidation followed by reduction and con A-horseradish peroxidase staining demonstrated abundant glycogen in Deiters' cells and in fibrocytes of the spiral ligament and submacular plaque. This procedure also detected diastase-resistant glycoprotein, probably containing N-linked complex-type saccharides, in the basal and marginal regions of the rectorial membrane and in the otolithic membrane. During morphogenesis and maturation, various cochlear cells showed changes in their glycogen content possibly related to stage-specific energy requirements. Cellular glycogen storage reached adult levels by postnatal day 14. The tectorial membrane gradually acquired con A reactivity during the first postnatal week. Thus, application of modified con A staining procedures has provided further knowledge for comparison with data from previous biochemical and histochemical studies of carbohydrate-rich components in the inner ear.

Introduction

The structure and distribution of glycoconjugates in the inner ear have been studied with a variety of histochemical techniques. Among these, the periodic acid-Schiff reaction was employed to demonstrate sugars with vicinal-hydroxyls (Wislocki & Ladman, 1955; B61anger, 1956; Falbe-Hansen & Thomsen, 1963; Falbe-Hansen, 1967; Ishii eta]., 1969; Lim & Rueda, 1990). In addition, cationic dyes such as Alcian Blue (Santi & Anderson, 1987), colloidal iron (Prieto et al., 1990b), high-iron diamine (Munyer & Schulte, 1991) and Ruthenium Red (Slepecky & Chamberlain, 1985) were used to delineate negatively-charged glycoconjugates associated with cellular or extracellular matrix constituents.

The introduction of lectins as histochemical probes has enabled more detailed characterization of glycoconjugates in situ (for review, see Spicer & Schulte, 1992a).

0018-2214 �9 1994 Chapman & Hall

Recent lectin histochemical studies have provided infor- mation concerning presence of oligosaccharides with specific terminal sugars or internal sugar linkages in various cell types and extracellular structures in the inner ear (Gil-Loyzaga et al., I985a, b, 1990; Lim& Rueda, 1990; Prieto et al., 1990a; Sugiyama et d., 1991a, b, I992; Yamashita & Bagger-Sj6b/ick, 1992). The histochemical observations on gelatinous membrane glycoconjugates were in good accordance with those obtained by analyti- cal biochemistry (Khalkhali-Ellis et al., 1987; Richardson et al., 1987; Khan & Drescher, 1990; Suzuki et al., 1992).

Among many available lectins, the jack bean lectin, concanavalin A (con A), has been shown to have a wide range of binding sites among inner ear tissues. Con A binds to terminal ~-mannose and s-glucose (GibLoyzaga et al., 1985a, b, 1990; Yamashita & Bagger-Sj6b/ick, 1992) and perhaps more preferentially to internal sugars in N-linked complex-type side-chains as do other mitogenic

438 ITO, SPICER and SCHULTE

lectins (Osawa & Tsuji, 1987). However, con A generally imparts diffuse staining in tissue sections with little selectivity for specific sites. A modified procedure em- ploying chemical alteration of tissue carbohydrates with vicinal-glycol oxidation and reduction, prior to exposure to con A (Katsuyama & Spicer, 1978) has been shown to stain intensely certain epithelial mucins whose precise structure has not been identified (Katsuyama et al., 1985). This modified con A-horseradish peroxidase procedure offers in addition a sensitive means of visualizing sites of glycogen storage.

We report here that modified con A staining discloses glycogen stores that are not otherwise detected in mammalian inner ear and which change markedly during neonatal development. Moreover, this procedure demon- strates diastase-resistant, con A-reactive N-glycosides in restricted regions of the gelatinous membrane.

Materials and methods

Tissue processing Inner ears were collected from quiet-reared Mongolian gerbils (Meriones unguiculatus) aged 3-6 months and also from neonates ranging from postnatal day 2 to day 24. The animals were anesthetized by intraperitoneal injection of urethane (1.0 g kg -I) and perfused transcardially with saline containing 0.1% sodium nitrite, followed immediately by one of the following fixative solutions: (1) 4% freshly-depolymerized paraformaldehyde in 0.1 M phosphate buffer, pH 7.2; (2) 10% formalin in saline containing 0.5% zinc dichromate (pH 5.0); and (3) Carnoy's solution. After opening the bulla, removing the middle ear ossicles and perforating the round window, fixative solution was perfused gently through the oval window. The length of exposure to fixative during perfusion approximated 30 rain, and was carried out at room temperature. The temporal bone was then removed and briefly rinsed in 0.1 M phosphate- buffered saline (PBS) at pH 7.2, prior to decalcification in 0.12 M

EDTA (pH 7.4) for 48 h or in 8 N formic acid (pH 3.3) overnight with gentle stirring, at room temperature. Following decalcifi-

cation, the inner ears were trimmed, dehydrated through a series of graded ethanols, deared in Histoclear (National Diagnostics, Manville, NJ) and embedded in Paraplast Plus (Curtin Matheson, Marietta, GA). Mid-modiolar 5 ~m-thick sections were cut and mounted onto chrome alum-subbed slides.

Lectin histochemistry Deparaffinized and rehydrated sections were incubated in 0.1 M

PBS containing 0.1 mM CaC12, MgC12, and MnC12 (PBSC). Some sections were digested with diastase in flesh saliva or an o~-amylase solution (Type IX-A from human saliva- Sigma, St Louis, MO) freshly reconstituted with PBS (5 mg ml -~) for 30 rain at 37~ The modified con A-horseradish peroxidase staining was carried out as described previously (Katsuyama & Spicer, 1978). Sections were first pretreated by one or more of the following methods: (1) oxidation for 10 or 60 min with fleshly-prepared 1% aqueous periodic acid (PA) or 1% lead tetraacetate (PbOAc 4) (Shimizu & Kumamoto, 1952); (2) reduction for 2 min with 0.2% sodium borohydride (NaBH 4) in 1% NazHPO 4 solution; or (3) aldehyde block (3-4 h) with 0.2% N,N'-dimethyl-meta-phenylenediamine (m-diamine) in 1% NaHPO 4 solution adjusted to pH 5.0 by I M Na2HPO 4 solution (Spicer, 1965). The pretreated sections were rinsed in PBSC and then incubated for 30 min at room temperature with unconju- gated con A (Sigma, St Louis, MO), diluted at Img ml -~ in PBSC. After rinsing in PBSC, the sections were flooded for 20 min with 0.01 mg ml -I horseradish peroxidase (type IV, Sigma) in 0.1 M phosphate buffer. Following final washes with buffer, the binding sites were visualized by incubating sections with 0.05% 3,3'-diaminobenzidine hydrochloride and 0.006% H202 in 0.1 M phosphate buffer for 5 min.

Results

Con A reactivity in aldehyde-fixed specimens exceeded that in Carnoy-fixed tissues. Staining of aldehyde-fixed specimens was stronger after formic acid than after EDTA decalcification. The following results pertain to aldehyde- fixed specimens that unless noted otherwise were decalcified with formic acid.

Figs 1-11 illustrate adult gerbil inner ears fixed with paraformaldehyde and decalcified with formic acid, unless otherwise noted.

Fig. 1. Cochlear cells and stroma show diffuse coloration. Unmodified con A method, x375. Fig. 2. In the middle turn of the cochlear duct three rows of Deiters' cells (DC) stain throughout their cytoplasm. The cover net (short arrow), marginal band (long arrow) and basal layer (arrowheads) of the tectorial membrane stain more intensely than the fibrous layer (FL) and limbal layer (LZ). Outer hair cells (OHC) and osseous spiral lamina (SL) are weakly positive. PA-NaBH4-con A stain, x400. Fig. 3. Type I fibrocytes in the lateral wall between the stria vascularis (SV) and otic capsule (C) show coloration. PA-NaBH4-con A method, x 600. Fig. 4. In macula vestibuli, the otolithic membrane (OM), including otoliths and their associated gelatinous meshwork, is strongly positive. Fibrocytes in the fibrous plaque beneath the macula are stained. PA-NaBH4-con A stain, x385. Fig. 5. Tectorial membrane in the hook region resembles that of the middle turn (Fig. 2), except for appearing narrower due to reduced fibrous and limbal layers. PA-NaBH4-con A stain, x510. Fig. 6. Deiters' cells in the hook region resemble those of the middle turn (Fig. 2) except for their smaller size. PA-NaBH4-con A staining, x600. Fig. 7. The staining of Deiters' cells equals that of Fig. 2 but reactivity of tectorial membrane appears weaker. PA-m-diamine-con A stain, x460.

439

Figs 1-7


Fig. 8. Deiters' cells lose affinity for con A after diastase digestion, whereas rectorial membrane remains positive. PA-NaBH4-con A method (a) without diastase; (b) after diastase digestion. (a) x280. (b) x370. Fig. 9. Deiters' cells lose their weak reactivity whereas rectorial membrane and spiral osseous lamina retain strong staining after diastase digestion. Periodic acid-Schiff reaction (a) without diastase; (b) after diastase digestion, x350. Fig. 10. Type I fibrocytes become negative after diastase digestion. PA-NaBH4-con A procedure (a) without and (b) following diastase digestion. (a) x 560. (b) x 420. Fig. 11. Con A reactivity of otolithic membrane resists diastase digestion, whereas submacular fibrocytes lose their reactivity (cf Fig. 4). Diastase PA-NaBH4-con A sequence, x375.

Adult inner ear

The con A-horseradish peroxidase sequence without prior oxidation or reduction imparted diffuse coloration to inner ear tissues, including both epithelial and stromal

components (Fig. 1). Periodic acid (PA) oxidation for 10 rain and subsequent NaBH 4 reduction substantially decreased the stromal binding of con A except in the gelatinous membranes. In contrast, the reactivity persisted or increased in selected sites (Figs 2-6). The

Modified con A reactivity in inner ear 44I

PA-reduction-con A procedure imparted intense staining to Deiters' cells (Figs 2 and 6), type I fibrocytes in the spiral ligament (Fig. 3) and certain submacular fibrocytes (Fig. 4). Outer hair cells occasionally showed light positivity (Fig. 2). Omitting the reduction step with NaBH 4 after periodate oxidation yielded similar results but with less selectivity. The method using 2rain oxidation with PbOAc4 gave comparable results. The staining pattern appeared similar using a 5-h blockage of periodate-engendered aldehydes with m-diamine instead of NaBH4 reduction (Fig. 7). The con A reactivity in Deiters' cells and fibrocytes was abolished by prior diastase digestion and was therefore attributable to glycogen (Figs 8-11).

On the other hand, the gelatinous membranes exhib- ited PA-reduction-con A reactivity resistant to diastase digestion (Figs 8b and 11). The rectorial membrane stained intensely in the basal layer, marginal band, and cover net but only faintly in the limbal zone and fibrous layer (Figs 2, 5, 7 and 8). Otoliths and their associated meshwork showed rather uniform binding (Figs 4 and 11). Notably, the PA-con A reactivity of otolithic membrane and the fibrous layer of rectorial membrane was signifi- cantly impaired by imposing aldehyde blockage with m-diamine between PA and con A.

To assess lability to prolonged oxidation, sections were exposed 60 min to periodate and stained with con A. With such oxidation none of the above described con A reactivities were observed. Moreover, subsequent reduction failed to affect the con A reactivity. Thus, the type of con A affinity observed here can be interpreted as that of the labile type III reactivity (Katsuyama et aI., 1984).

Developing inner ear In the earliest stage of postnatal development, glycogen appeared widespread in the immature inner ear as demonstrated by the PA-reduction-con A procedure. Overall content of glycogen gradually diminished and reached the adult level by day 14.

Between postnatal days 2 and 4, heavy glycogen deposits were observed in the inner portion of greater

epithelial ridge cells as well as in precursors of tunnel pillar and other supporting cells (Figs 12 and 17a). Strial marginal cells, spiral ligament fibrocytes and cells of Reissner's membrane also stained heavily (Fig. I2). In addition, chondrocytes within the developing otic capsule (Fig. 13), mesenchymal cells underneath the macula (Fig. 14) and neural tissues contained abundant glycogen.

By postnatal day 10 when the inner sulcus had become hollowed out and the tunnel of Corti had opened, glycogen-rich cells of the greater epithelial ridge disap- peared (Figs 16 and 18a). At that time, glycogen persisted in some supporting cells showing a tendency to greater abundance in Deiters' and inner pillar cells. Staining of spiral ligament fibrocytes, however, exceeded adult levels until day 12 except in the region populated by type II fibrocytes (Fig. 15).

In the rectorial membrane, glycoprotein demonstrated by the diastase-PA-reduction-con A sequence was already obvious and confined to the basal layer at postnatal day 2 (Fig. 17). The area of reactivity expanded and became localized on the basal layer and marginal band by postnatal day 10 (Fig. 18).

D i s c u s s i o n

Glycogen was first detected in outer hair cells with the periodate-Schiff reaction (B61anger, 1956). Since that time, the distribution of glycogen in the cochlea has been studied at the light microscope level exclusively by this method combined with diastase digestion (Falbe- Hansen & Thomsen, 1963; Falbe-Hansen, 1967; Ishii eta]., 1969; Stack & Webster, 1971a). More recently, electron microscopical examination of tissue fixed with osmium tetroxide and potassium ferricyanide (Duvall & Hukee, 1976; Hilding et al., 1977; Qvortrup & Rostgaard, 1990) or treated with periodic acid and thiosemicarbazide followed by osmium tetroxide (Ishii eta]., 1969) or silver proteinate (Prieto et al., 1990) has enabled glycogen deposition to be detected at the ultrastructural level.

The present study affirmed the PA-con A procedure as a highly sensitive method for demonstrating glycogen

Figs 12-16 illustrate inner ear of gerbil neonates. Sections were taken from zinc formalin-fixed and EDTA-decalcified specimens.

Fig. 12. Reactive sites in cochlea include the inner greater epithelial ridge cells (arrowhead), pillar cells (PC), presumed precursors of the Hensen, Claudius, Boettcher cell group, strial marginal cells (SV), spiral ligament fibrocytes (asterisk), and Reissner's membrane (RM). Tectorial membrane (TM) lacks affinity for the lectin. Postnatal day 2. PA-rn-diamine con A stain, x375. Fig. 13. Chondrocytes in premature otic capsule stain heavily. Postnatal day 2. PA-m-diamine con A stain, x375. Fig. 14. Submacular fibrocytes show dark coloration which is diastase labile and attributable to glycogen. Postnatal day 2. PA-m-diamine con A stain, x 375. Fig. 15. Spiral ligament fibrocytes except in the type II fibrocyte area (asterisk) disclose staining attributable to glycogen. Postnatal day 10. PA-m-diamine-con A method, x240. Fig. I6. Cells of the inner greater epithelial ridge have regressed and the organ of Corti has differentiated into the mature form. Staining is prominent in the inner (arrow) but not outer pillar cell, Deiters' cells (open arrow), Boettchers cells (arrowheads) and tectorial membrane (TM). Postnatal day 10. PA-m-diamine con A stain, x460.

442

Figs 12-16

Modified con A reactivity in inner ear 443

Fig. 17. All epithelial cells stained without prior diastase shown here and in Fig. 12 lose reactivity after diastase digestion and contain glycogen. The basal layer of rectorial membrane appears selectively positive after digestion and contains glycoproteins. Postnatal day 2. PA-m-diamine-con A stain. (a) without diastase; (b) after diastase digestion, x470. Fig. 18. Epithelial cells lose reactivity after diastase digestion in contrast to rectorial membrane which selectively retains lectin affinity. Postnatal day 10. PA-m-diamine-con A stain. (a) without diastase; (b) after diastase digestion, x350.

(Katsuyama & Spicer, 1978). To our knowledge, this is the first report describing the distribution of glycogen in the gerbil inner ear. The data showed abundant stores of glycogen in Deiters' cells of the adult gerbil confirming results in other species (B61anger, 1956; Falbe-Hansen & Thomsen, i963; Ishii et al., 1969; Lira & Rueda, I990). Outer hair cells in the mature gerbil cochlea were found to contain only small amounts of glycogen similar to those of the mouse and cat (Ishii et al., 1969). In contrast, rat outer hair cells have been reported to contain abundant glycogen (B61anger, 1956; Falbe-Hansen & Thomsen, 1963; Falbe-Hansen, 1967; Hilding et al., 1977; Prieto et aI., 1990). In addition, the method allowed detection of glycogen in sites in the mature inner ear where it has previously not been reported to occur such as in spiral ligament and submacular fibrocyt:es.

Glycogen in Deiters' cells provides a store for meeting their high energy requirement implied by content of

numerous mitochondria (Spicer & Schulte, 1993) and abundant creatine kinase (Spicer & Schulte; 1992b). However, glycogen storage relates not only to energy need, but also to the type of metabolism and access to substrate. Thus, the most metabolically active cells of the inner ear, the spiral ganglion and strial marginal cells (Marcus et al., 1978), lacked histochemically demonstrable levels of glycogen in the gerbil. Apparently the latter cells rely less on glycogen storage and more on a constant supply of glucose. Their closer association compared with Deiters' cells and fibrocytes to local blood supply agrees with this consideration favouring aerobic metabolism. Moreover, recent immunolocalization of the erythroid/brain type of glucose transporter (GLUT1) in the strial basal cells and satellite cells surrounding the spiral ganglion cells in the gerbil (Ito et al., 1993) indicates that ganglion and strial marginal cells have access to a continuous supply of glucose and rely on uptake and not


storage to support a high energy requirement. Interest- ingly, spiral ganglion cells in the adult guinea pig have been shown to contain moderate amounts of glycogen (Falbe-Hansen & Thomsen, 1963) and satellite cells in this species contained no demonstrable GLUT 1 (Ito et al., 1993). This finding further correlates low glucose uptake capacity with increased glycogen storage and points to species differences in the mechanisms developed by specialized cochlear cell types for taking up and metabolizing glucose.

The marked differences in glycogen content of outer hair cells between species also indicates variability among animals in the hair cell's ability to take up and utilize glucose from perilymph. We have recently shown the presence of abundant glucose transporter (GLUT 5) on the basolateral membrane of the gerbil's outer hair cells (unpublished observations) a finding possibly related to the low glycogen stores in the gerbil's hair cells. Presumably outer hair cells containing abundant glycogen stores in species such as the rat have less well developed systems to facilitate glucose uptake and rely more on anaerobic metabolic processes supported by glycogen stores. Recognizing such variability among animals leads to considering whether differences in susceptibility to acoustic trauma and transient ischaemic events could be related to differences among species in the contribution of aerobic versus anaerobic metabolism to inner ear cell function.

Previous studies have reported developmental changes in glycogen content in fetal and neonatal rat cochleas (Falbe-Hansen, 1967; Hilding et al., 1977). Types of cell which first gain and then lose glycogen stores in the developing rat include epithelial cells lining Reissner's membrane, strial marginal cells, inner pillar cells, Deiters' cells and outer sulcus cells. In addition, conditions that lead to disturbances in glycogen metabolism such as chemical sympathectomy induced by 6-hydroxydopamine (Ross, 1978) or hypothyroidism induced by propyl- thiouracil (Prieto et al., 1990a) lead to abnormal glycogen accumulation or depletion in developing cells. As in the gerbil, glycogen stores in the rat reach adult levels near the time of onset of auditory function.

The presence of copious glycogen in many developing cochlear cells is not surprising, since glycogen generally fuels the anaerobic metabolism of embryonic cells. Glyco- gen may be essential as well to meet metabolic energy demands during postnatal cochlear morphogenesis (in altricial mammals like the gerbil). Conversion of glycogen-rich cells of the neonatal cochlea into those observed in the adult proceeds with the maturation of the organ of Corti. Notably, glycogen-rich cells of the inner greater epithelial ridge convert during the first postnatal week to inner sulcus cells. These cells participate in genesis of the rectorial membrane and hence glycoprotein secretion, suggesting a relation between their especially abundant glycogen and such a secretory process.

The PA-reduction-con A reactivity of the gelatinous

membranes was not abolished by diastase digestion, and was therefore attributable to glycoprotein. The type II collagen present in tectorial membrane (Richardson et al., 1987; Thalmann et al., 1987; Slepecky et al., 1992) cannot account for this lectin binding since cartilage matrix rich in type II collagen lacks PA-con A positivity. Moreover glycosaminoglycans present in the tectorial membrane (Munyer & Schulte, 1991) generally lack lectin affinity and are unlikely to explain the PA-con A staining (Sugiyama et al., 1991b; Spicer & Schulte, 1992a). Through comparison with data available for the carbohydrate composition of the gelatinous membranes analysed by a battery of lectins in the adult gerbil (Sugiyama et al., 1991b, 1992) and developing rat (Prieto et aI., 1990a), the PA-reduction-con A reactive sites in rectorial membrane can be considered attributable to N-linked bi- and tri-antennary glycosides containing internal GlcNAc[31,2Man chains. Thus Phaseolus vulgaris agglutinins E and L (PHA-E and L) yielded a staining pattern like that with the PA-reduction-con A method in rectorial membrane as well as the otolithic membrane. The latter structure differs somewhat in carbohydrate composition from rectorial membrane (Sugiyama, 1991b) but could possess certain glycoproteins in common.

The PA-reduction-con A staining reactivity, however, does not fully agree with that shown by PHA-E and L in that strial marginal cells lacked the PA-reduction- con A reactivity but reacted strongly with PHA-L and E (Sugiyama et al., 1991a). Possibly this discrepancy reflects microheterogeneity of glycosylation among in- dividual cell type-specific glycoproteins (Osawa & Tsuji, 1987).

N-linked complex-type glycoproteins are thought possibly to function as recognition/adhesion molecules between the surface of hair cell stereocilia and gelatinous membranes in the manner of neural cell adhesion molecules. One such glycoconjugate possesses the HNK- 1 epitope, which is a sulphated N-linked glycoside reactive with PHA-L and E (Mikol et al., 1988). Dynamic changes occur during development in the secretion and glycosylation of rectorial membrane glycoproteins (Lim, 1987; Rueda & Lim, 1988; Prieto et al., 1990b; Yamashita & Bagger-Sj6b/ick, 1992). A gradual postnatal increase in the PA-reduction-con A reactivity of rectorial membrane, noted here, appears consistent with establish- ing a loose contact between cochlear sensory hairs and rectorial membrane early in postnatal development. Identification and further characterization of presumed adhesive glycoconjugates in rectorial membrane and stereocilia offers a prospect of gaining insight into interactive mechanisms between the rectorial membrane and hair cells.

Acknowledgements The authors express appreciation to Mrs Sharon Munyer and Mrs Leslie Harrelson for valued technical and

Modif ied con A react iv i ty in inner ear 445

secretarial assistance. This work was suppor ted b y research grants DC00422 and DC00713 from the Nat ional Insti tutes of Health.

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Histochemical detection of glycogen and glycoconjugates in the inner ear with modified concanavalin...

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