Ultrastructural demonstration of proteoglycans in adult rat cornea

TISSUE & CELL 1988 20 (4) 567-575 @ 1988 Longman Group UK Ltd

ANGEL VELASCO and JOSEFINA HIDALGO

ULTRASTRUCTURAL DEMONSTRATION OF PROTEOGLYCANS IN ADULT RAT CORNEA

Keywords: Proteogylcans, rat cornea, cuprolinic blue, glycosaminoglycans, protein A-gold

ABSTRACT. Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl,, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in cornea1 basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by cornea1 cells.

Introduction

The adult rat cornea consists of three different cellular layers: an epithelium at its anterior surface, an intermediate stroma and an endothelium at the posterior surface. In addition, a basal membrane is situated under the epithelium, separating this from the anterior region of the stroma. The latter, at its posterior region, is also separated from the endothelium by a thick basement membrane, usually called Descemet membrane. The stroma, the thickest portion of cornea, is a highly specialized connective tissue com- posed of an orthogonal network of tightly packed type I collagen fibres, embedded in an interfibrillary matrix where proteoglycans are particularly abundant (Trelstad and Coulombre, 1971).

Stromal proteoglycans are produced by fibroblastic cells (stromacytes) and are thought to be responsible for the stability and orderly packing of the collagen network which in turn accounts for the transparency of the cornea (Hassell et al., 1980a; Dische et

Department of Cellular Biology, Faculty of Biology, University of Sevilla, E-41012 Sevilla.

Received 1 February 1988.

al., 1985). Proteoglycans so far described in the stroma of different species belong to two classes: keratan sulphate proteoglycan and chondroitin-dermatan sulphate proteoglycan (Meyer et al., 1953; Axelsson and Heinegard, 1975, 1978, 1980; Speziale et al., 1978; Hassell et al., 1979; Gregory et al., 1982). In addition, heparan sulphate proteoglycans have been detected by immunofluorescence in both cornea1 basement membranes (Hassell et al., 1980b). They could be synthesized by cornea1 epithelial and endothelial cells, which also seem to produce other glycosaminoglycans (Yue et al., 1976; Hart, 1978; Yonekura et al., 1982; Robinson and Gospodarowicz, 1983; Wang et al., 1985).

Despite the above mentioned biochemical studies, the in situ distribution of different cornea1 proteoglycans in the whole organ is uncertain. At present there are several histo- chemical methods for the specific detection of proteoglycans. One that has gained widespread use in ultrastructural studies involves staining with a cationic dye such as alcian blue, safranin 0, ruthenium red or cuprolinic blue (Thyberg et al., 1973; Shepard and Mitchell, 1976; Scott and Orford, 1981; Reale et al., 1983; Chen and

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Wight, 1984; Van Kuppevelt et al., 1985; Volker et al., 1986). For instance, in the presence of a critical electrolyte concentration of 0.3 M MgC&, cuprolinic blue (CB) selectively stains polysulphated proteoglycans but not other tissular polyanions (Scott, 1985). The proteoglycan-CB complexes have charac- teristic shapes, sizes and electron density; this along with their sensitivity to various degrading enzymes makes possible the characterization of precipitates. Another approach makes use of antibodies raised against anti- genie determinants of the core protein or glycosaminoglycan chains of proteoglycans. It has been particularly useful in the localization of minor molecular species such as small chondroitin, dermatan or heparan sulphate proteoglycans (Hassell et al., 1980b; Mang- kornkanok-Mark et al., 1981; Aquino et al., 1984; Couchman et al., 1984; Longas and Fleischmajer, 1985; Voss et al., 1986). In the present study both methods have been applied to cornea1 tissue and the results compared for a better comprehension of the topographical distribution of proteoglycans in this organ.

Materials and Methods

Antibodies and reagents

The mouse monoclonal antibodies used in this study were generously donated by Dr B. Caterson (West Virginia Medical Center). The properties of these antibodies have been previously described (reviewed by Caterson et al., 1985); they were raised against cartilage proteoglycan monomers previously treated with chondroitinase ABC. The 5-D-4 antibody recognizes an antigenic determi- nant present in keratan sulphate chains from both cartilage and cornea1 proteoglycans. The antibodies 2-B-4,3-B-3 and l-B-5 recog- nize disaccharide units (corresponding to chondroitin-4-sulphate, chondroitin-6-sulphate and unsulphated chondroitin respectively) which remain linked to the core protein after chondroitinase ABC digestion of proteoglycans. A rabbit antimouse IgG polyclonal antibody was obtained from E-Y Laboratories and used in immunocytochemical demonstration as a secondary antibody in order to overcome the low affinity of protein A for mouse immunoglobulins. Protein A (Pharmacia) was complexed to 15 nm colloi- dal gold particles according to Roth (1982).

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The colloid was freshly prepared by reducing a O-01% tetrachloroauric acid (Merck) solution with 1% sodium citrate (Frens, 1973). Chondroitinase ABC and keratanase were from Seikagaku, cuprolinic blue (CB) from BDH Chemicals, and Lowicryl K4M resin from Lowi.

Tissue source

Female Sprague-Dawley rats (250-300 g) with clear corneas were anaesthetized by injection of a chloral hydrate aqueous solution. Both eyes were removed and the central part of the corneas (above the iris) was sectioned to obtain 1x2 mm tissue blocks.

Immunocytochemistry

Fixation was carried out at room temperature in 1% glutaraldehyde in O-1 M phosphate buffered saline, pH 7.4, (PBS) for 1 hr. Speci- mens were then washed with 6x10 min changes of PBS and incubated for 2 hr with 50 mM NH&I in PBS at room temperature to block free aldehyde groups. After washing again six times with PBS, they were dehydra- ted in graded ethanols and embedded in Lowicryl K4M resin according to Altman et al. (1984).

Thin cross-sections containing the three layers of cornea were cut on a Reichert ultra- microtome and recovered on Formvard- carbon-coated nickel grids. These were float- ed on a PBS drop for 5 min and then treated with 1% bovine serum albumin in PBS (PBS- BSA) for 10 min. Monoclonal antibody 5-D-4 to keratan sulphate was diluted 1:20,000 with PBS-BSA and used without any previous treatment. To demonstrate chondroitin sulphate, sections had to be digested for 1 hr at 37°C with chondroitinase ABC (1 unit/ml of PBS) before being incubated with the antibodies; a mixture of equal parts of the antibodies 2-B-4, 3-B-3 and l-B-5 was used for immunolabellings after being diluted 1:20,000 with PBS-BSA. Incubation with monoclonal antibodies, in both cases, was for 2 hr. Grids were then rinsed in PBS (2x5 min) and PBS-BSA (10 min) before being incubated for 1 hr with rabbit antimouse IgG polyclonal antibody (dilution 1:200 in PBS- BSA). After washing again with PBS and PBS-BSA, they were treated with protein A-gold solution (diluted with PBS-BSA to give A5Z5nm=0.4) for 1 hr. Following this incubation, the grids were washed with PBS

PROTEOGLYCANS IN ADULT RAT CORNEA

(3x5 min) and distilled water (2x5 min). They were finally counterstained with uranyl acetate (10 min) and lead citrate (5 min) before observation in a Philips 300 electron microscope. The whole immunolabelling procedure was carried out at room temperature and incubations with antibodies and protein A-gold complexes were performed in moist chambers. The incubation step with monoclonal antibodies was omitted for some control grids.

Cuprolinic blue (CB) staining

The critical electrolyte concentration method described by Scott and Orford (1981) was followed with modifications. Briefly, tissue blocks were fixed for 12 hr at room temperature in 0.025M sodium acetate buffer, pH 5.6, containing 2.5% glutaraldehyde, 0.2% CB and 0.3 M MgC&. Specimens were then rinsed (3x5 min) with buffer lacking CB and treated with 1% sodium tungstate in distilled water (3x 10 min). Dehydration was carried out through graded ethanols; the 30 and 50% ethanol steps also containing 1% sodium tungstate. Tissue was embedded in Epon; sectioning and counterstaining was done as commented for Lowicryl blocks.

In addition, other pieces of tissue were fixed without CB for 2 hr and treated for 24 hr at 37°C with either chondroitinase ABC (2.5 units/ml) or keratanase (5 units/ml) or both sequentially. Enzymes were dissolved in 0.25 M Tris buffer, pH 8.0, containing 0.5 mg/ml BSA, 0.33 M sodium acetate and 0.5 NaCl. Soybean trypsin inhibitor (1.5 mg/ml), EDTA (5mM) and benzamidine (2.5 mM) were added to this solution in order to inhibit proteolysis. Fragments incubated in buffer without enzyme were also processed as con- trols. Both kinds of specimens were there- after stained with CB as described.

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are short filaments of about 16-20 nm length and 9-11 nm width; they have a fuzzy aspect and account for the majority of the precipitates in the stroma. Type II is formed by longer and more electron-dense filaments which are about 50-70 nm long and 11-22 nm thick (Fig. 2). Both kinds appear intermixed, irregularly arranged, throughout the stroma, although a clear association with the collagen bundles was evident for both types. Thin filaments were also seen in the basal membranes of both epithelium (Fig. 1) and endothelium (Fig. 3), but these as the cellular details could hardly be distinguished in non-osmicated tissue incubated with CB solution.

Using the 5-D-4 monoclonal antibody to detect keratan sulphate by immunocytochemistry the distribution of this glycosaminoglycan could be judged more precisely. In particular, gold particles were frequently seen in the epithelium. They were located in the contacts and interdigitations between cells of the basal and medial layers of the epithelium (Fig. 4). On the contrary, the cytoplasm and nucleus of these epithelial cells as well as the basal membrane of the epithelium were not labelled. Labelling was specially intense in the stroma where the collagen fibres appeared decorated with gold particles along their length (Figs 4, 16). No differences in reactivity were seen between different parts (anterior, middle and posterior) of the stroma. However, some round or oval areas with no label at all were often detected throughout cornea1 stroma (Fig. 5). Labelling was also absent from those collagen bundles which were in close contact with the cell surface of fibroblasts (Fig. 6), and that most probably represent newly secreted fibres. Interestingly, some specific reaction with 5-D-4 antibody was routinely observed in Descemet membrane and endothelium (Fig. 7). These observations were confirmed when the proteoglycans bearing keratan sulphate chains were selectively stained with CB after treatment of the tissue with chondroitinase ABC (Figs 8-10). Thus type I filaments were identified with keratan sulphate proteoglycans. They occupy the whole stroma (Figs 8, 9), in association with collagen, except at the ‘clear’ areas commented on above (Fig. 9).

When a mixture of the monoclonal antibodies 2-B-4,3-B-3 and l-B-5 was used only a slight immunocytochemical staining of the

Results

The whole population of cornea1 proteoglycans was selectively revealed in the tissue stained with CB in the presence of 0.3 M MgCl? (Figs l-3). In the stroma (Figs 1,2) CB precipitates, representing proteoglycan monomers, appeared highly concentrated. They have a roundish profile in cross-section but when seen lying in the plane section they show a filamentous appearance. Two kinds of filaments were detected. Type I precipitates

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PROTEOGLYCANS IN ADULT RAT CORNEA 571

cornea1 stroma was obtained (Figs 11-13). Reaction was more intense at posterior (Fig. 11) and anterior (Fig. 12) regions of the stroma as compared with the middle part (Fig. 13). Thus protein A-gold complexes were seen accumulated at the boundaries between stromal tissue and basement membranes, especially Descemet membrane (Fig. 11). The latter, as the epithelium basement membrane however, did not show strong immunoreactivity with antibodies to chondroitin or chondroitin sulphate disaccharides.

Proteoglycans which were sensitive to chondroitinase ABC treatment but not to keratanase degradation, tentatively identified as chondroitin-dermatan sulphate proteoglycans, correspond to type II filaments in the CB technique. These were readily discer- nible in the collagen bundles which run along the plane of the section (Figs 14, 15), appear- ing like roundish granules or points at those cross-sectioned bundles. Within the collagen bundles most of the type II filaments showed a transfibrillar orientation, being linked to adjacent fibres by their ends (Fig. 17). In spite of the results obtained with the immunocytochemical approach, no signifi- cant differences were apparent in the distribution of type II filaments between anterior, middle and posterior parts of the stroma.

The cornea1 stroma which was sequentially treated with chondroitinase ABC and keratanase (Fig. 19) did not show any kind of stained filaments after CB treatment. On the contrary, in these preparations, as in those

which had been incubated with either chondroitinase ABC (Figs 8, 10) or keratanase (Figs 14, 15), stained but ill- defined filaments remained in both epithelial and endothelial basement membranes.

Incubation of the tissue-in buffer solution without any degrading enzyme did not alter .or-reduce the number;distribution and shape of the CB precipitates. On the other hand, no immunocytochemical reaction was observed in those sections which were not exposed to monoclonal antibodies (Fig. 18).

Discussion

Stromal proteoglycans of the adult rat cornea stained with CB according to the critical electrolyte concentration method (Scott and Orford, 1981; Scott, 1985) appear as filamentous precipitates of two types. We have identified these two types with molecular species of proteoglycans previously described in the stroma of other animals as keratan sulphate- rich proteoglycans and chondroitin-dermatan sulphate-rich proteoglycans (Axelsson and Heinegird, 1975,1978,1980; Speziale et af., 1978; Hassell et al., 1979; Gregory et al., 1982). This conclusion was supported by the following observations: (a) filaments were sensitive to either chondroitinase ABC or keratanase treatment; (b) no other CB precipitates were found in the stroma of corneas previously incubated with both enzymes in sequence; and (c) at least for type I filaments, their distribution in the stroma was corrobor- ated by immunocytochemical demonstration of keratan sulphate with a monoclonal anti-

Figs. 1-3. Rat cornea incubated in CB solution containing 0.3 M M&I, without previous enzymatic treatment. Fig. 1, anterior region; Fig. 2, middle part of the stroma; Fig. 3, posterior region. Figs 1,2, precipitates fill the stroma (St) where type I (encircled) and type II (arrowhead) filaments are found; Figs 1,3, other filaments (arrows) are located in the basal membrane under the epithelium (Ep) and in Descemet membrane under the endothelium. Fig. 1. ~37,000; Fig. 2, x81,ooO, Fig. 3, x~O,GQO.

Figs 410. Detection of keratan sulphate proteoglycans by immunocytochemical demonstration of keratan sulphate chains (Figs 4-7). and precipitation with CB after chondroitinase ABC treatment (Figs b10). Fig. 4, epithelial cell showing labelling (arrow); Figs 5, 6, 9, areas (*) lacking reaction in the stroma; Fig. 7, immunolabelling in Descemet membrane; Figs b10, localization of proteoglycans in the stroma (arrowheads) and basal membranes (arrows). Ep, epithelium; N, nucleus; En, endothelium; F, fibroblast. Fig. 4, x31.200; Fig. 5. x30,ooO; Fig. 6, X12,600; Fig. 7, ~18,000; Fig. 8, ~55,000; Fig. 9, ~32,000; Fig. 10, x20,tHO.

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Figs 11-15. Presence of chondroitin-dermatan sulphate proteoglycans in anterior (Figs 12,14). middle (Fig. 13) and posterior (Figs 11,lS) regions of rat cornea. Figs 11-13, immunodemonstra- tion with monoclonal antibodies; Figs 14, 15, CB staining after keratanase treatment. Ep, epithelium; arrows. CB staining in Descemet membrane. Fig. 11. ~24,ooO; Fig. 12, X 16.ooO: Fig. 13, x20,ooO; Fig. 14, x25,KW; Fig. 15, x22.ooO.

Figs 16, 17. Association of stromal proteoglycans with collagen fibres as shown with both methods of detection, Fig. 16, keratan sulphate revealed by protein A-gold immunocytochemistry; Fig. 17, chondroitin-dermatan sulphate proteoglycans in cornea stained with CB after keratanase treatment. Fig. 16, X33,m; Fig. 17, x43,ooO.

Figs 18.19. Absence of reaction in control tissue. Fig. 18, section non-incubated with primary (monoclonal) antibody during the immunocytochemical procedure; Fig. 19. cornea digested with both chondroitinase ABC and keratanase enzymes in sequence before CB staining. Fig. 18. ~17,000; Fig. 19, x23,CNIO

body. Therefore, we conclude that type I and geneously distributed throughout the cornea1 type II filaments induced by CB dye repre- stroma. However, a clear relationship sent respectively keratan sulphate and between filaments and collagen fibres was chondroitin-dermatan sulphate proteoglycan evident; that is, both types were mostly asso- monomers. They appear intermixed, homo- ciated at intervals along the fibres with only

PROTEOGLYCANS IN ADULT RAT CORNEA

scanty precipitates isolated in the interfibril- lar spaces.

The association of rabbit cornea1 proteoglycans with type I collagen fibres has been recently studied by Scott and Haigh (1985), who described that keratan sulphate proteoglycans stained with CB are linked to the ‘a’ and ‘c’ bands, whereas chondroitinase ABC-sensitive filaments (supposedly chondroitin-dermatan sulphate proteoglycans) are linked to the ‘d’ or ‘e’ bands from the gap zone. It was also shown in this study that whereas chondroitinase ABC-sensitive filaments are long enough to establish bridges between fibres within a bundle, keratanase- sensitive filaments are shorter but more regularly spaced along the fibres (Scott and Haigh, 1985). The interest for proteoglycan- collagen interactions comes from studies where the stability and organization of the collagen network has been shown to be dra- matically altered by the loss of proteoglycans (Dische er al., 1985) or by changes in their production and molecular characteristics (Hassell et al., 1980a, 1983). Taken together these results have suggested that by interact- ing with collagen proteoglycans control the spacing and packing of the fibres and, at the same time, the degree of hydratation of the stroma; so, transparency and rigidity are attained (Hassell et al., 1983; Dische et al., 1985).

The results described in the present article are consistent with these previous observations since we have detected a predominant association of stromal proteoglycans with collagen, but also demonstrate that areas exist in the stroma where proteoglycans, particularly keratan sulphate proteoglycans, are absent. The possibility of these areas being an arte- fact is excluded by the fact that they could be seen with both methods, protein A-gold immunocytochemical procedure and CB staining. Although not conclusively we con- sider that these regions represent newly secreted collagen bundles, since we occa- sionally observed some of them in proximity to stroma fibroblasts. This raises the problem of considering how the production and secre- tion of both proteoglycans and collagen are coordinated in the fibroblastic cell, and also which are the factors that determine the proteoglycan-collagen interactions in the stroma.

Gregory et al. (1982) described the exis-

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tence of two kinds of keratan sulphate proteoglycans and two kinds of chondroitin- dermatan sulphate proteoglycans in rabbit cornea1 stroma. We could not detect those differences with the ultrastructural techniques here used. Neither a gradient in keratan sulphate distribution, as reported for bovine cornea (Bettelhfim and Goetz, 1976), was evident. Moreover, both the immunocytochemical procedure and the CB method were consistent with the existence of keratan sulphate as a single-type of proteoglycan, homogeneously distributed throughout anterior, middle and posterior regions of the stroma. We have also used a mixture of monoclonal antibodies, directed towards sulphated and unsulphated chondroitin sulphate disaccharides, to detect cornea1 chondroitin-dermatan sulphate proteoglycans after chondroitinase ABC treatment of the sections. With this approach we have observed that anterior and posterior regions of the stroma, in contact with basement membranes, are more reactive than the middle part. This could be considered as a proof for molecular heterogeneity existing within a population of proteoglycans which are visualized as a single type of filaments (type II) by the CB method. Alternatively, epi- topes recognized by these antibodies could be differently masked in the three regions of the stroma.

Another interesting observation of the present study was the finding that Descement membrane, the basal membrane of the endothelium, contains immunocyto- chemically detectable keratan sulphate glycosaminoglycan. Previously only heparan sulphate proteoglycan was considered to be present in both cornea1 basement membranes (Hassell et al., 1980b). Heparan sulphate is also the major glycosaminoglycan synthesized by cornea1 epithelial and endothelial cells, which also produce chondroitin and dermatan sulphates (Hart, 1978; Yonekura et al., 1982; Robinson and Gospodarowicz, 1983; Wang et al., 1985). In fact, it is com- monly believed that cornea1 keratan sulphate is exclusively produced by stromal fibroblasts (Hay, 1980) although other reports indicate that endothelial cells also have the ability to do it (Yue et al., 1976; Hart, 1978). In our study with CB dye we have observed thin filaments in both cornea1 basement membranes that persisted after chondroitinase

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ABC and/or keratanase treatments. We think that they mostly represent heparan sulphate proteoglycans and possibly also chondroitin sulphate proteoglycans, although only scanty labelling was obtained in these locations with antibodies to this latter glycosaminoglycan. On the other hand, the presence of keratan sulphate in the rat cor- neal epithelium is intriguing since there is no previous report on this fact. This, along with the presence of keratan sulphate in Descemet membrane, should be confirmed by using other specific antibodies such as those which have been recently applied to study the

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appearance of this kind of proteoglycans during cornea1 development (Funderburgh er al., 1986; Hydahl er af., 1986; Sundarraj et al.. 1986).

Acknowledgements

The authors are thankful to Dr Bruce Cater- son for providing the monoclonal antibodies, to Dr E. Rafel for his continuous support, and to Mrs R. Garcia and Mr P. Moya for their technical assistance. This work was supported by grant GR85-0064 from CAICYT.

References

Altman, L. G., Schneider, B. G. and Papermaster, D. S. 1984. Raprd embedding of tissues in Lowicryl K4M for

immunoelectron microscopy. J. Histochem. Cytochem., 32, 1217-1223. Aquino, D. A., Margolis, R. U. and Margolis, R. K. 1984. Immunocytochemial localization of a chondroitin sulfate

proteoglycan in nervous tissue. I. Adult brain. retina, and peripheral nerve. J. Ceil Biol., 99, 1117-1129.

Ax&son, I. and Heinegbrd, D. 1975. Fractionationofproteoglycansfrom bovine cornea1 stroma. Biochem. J., 145,491-

500.

Axelsson, I. and Heineg&d, D. 1978. Characterization of the keratan sulphate proteoglycans from bovine cornea1

stroma. Biochem. J., 169, 517-530.

Axelsson, I. and Heinegird, D. 1980 Characterization of chondroitin sulphate rich proteoglycans from bovine cornea1

stroma. Expl Eye Res.. 30, 57-66.

Bettelhfim, F. A. and Goetz, D. 1976. Distribution of hexosammes in bovine cornea. Invesr. Ophthohnol.. 15,301-304.

Caterson, B., Christner, .I. E., Baker, J. R. and Couchman. J. R. 1985. Production and characterization ofmonoclonal antibodies directed against connective tissue proteoglycans. Fe‘Ed. Proc.. 44, 3X6-393.

Chen. K. and Wight, T. N. 1984. Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemlcal

analysis. J. Htitochem. Cytochem., 32, 347-357. Couchman, .I. R., Caterson. B., Christner. J. E. and Baker. J. R. 1984. Mapping by monoclonal antibody detectIon ot

glycosaminoglycans in connective tissues. Narure, 307, 65&652.

Dische, 2.. Cremer-Bartels, G. and Kaye, G. I. 1985. Differences between two fractions of glycosaminoglycans of the

cornea1 stroma in their structural relation to collagen. Proc. natl. Acad. Sci. U.S.A., 82, 76G764. Frens, G. 1973. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature

(Phy. Sci.), 421, 2&22.

Funderburgh, J. L., Caterson. B. and Conrad, G. W. 1986. Keratan sulfate protcoglycan during embryonic development

of the chicken cornea. Devl. Biol 116,267-277.

Gregory. .I. D., Cbster. L. and Damle. S. P. 1982. Proteoglycans of rabbit cornea1 stroma. Isolation and partial

characterization. J. Biol. Chem.. 257, 6965-6970.

Hart, G. W. 1978. Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea. &v/

Biol., 62, 78-98.

Hassell, .I. R.. Newsome. D. A. and Hascall, V. C. 1979 Characterlzatlon and biosynthesis of proteoglycans of cornea1

stroma from Rhesus monkey. J. Rio/. Chern.. 254, 1234612354. Hassell, J. R., Newsome, D. A.. Krachmer, J. H. and Rodrigues, M. M. 1980a. Macular cornea1 dystrophy: Failure to

synthesize a mature keratan sulfate proteoglycan. Proc. natL Acad. Sri. U.S.A., 77, 3705-3709. Hassell, J. R., Robey, P. G., Barrach, H. J., Wilczek, J., Rennard, S. I. and Martin, G. R. 1980b. Isolation of a hcparan

sulfate-containing proteoglycan from basement membrane. Proc natl. Acad. Sci. U.S.A.. 77, 4494-4498. Hassell, J. R., Cintron, C.. Kublin, C. and Newsome, D. A. 1983. Proteoglycan changes during restoration of

transparency in cornea1 scars. Arch Biochem. Biophys.. 222,362-369.

Hay. E. D. 1980. Development of the vertebrate cornea. ht. Rev. C~~iol., 63, 263-322.

Hydahl, L.. Aspinall, R. and Watt, F. M. 1986. Immunolocalization of keratan sulphate in the human embryonic cornea

and other human foetal organs. J. Cell Sci., 80, 180-191.

Longas. M. 0. and Fleischmajer, R. 1985. lmmunoelectron microacopq of proteodcrmatan sulfate in human mid-

dermis. Connect. Tin Res.. 13, 117-125.

PROTEOGLYCANS IN ADULT RAT CORNEA 515

Mangkornkanok-Mark, M., Eisenstein, R. and Bahu, R. M. 1981. Immunologic studies of bovine aortic and cartilage proteoglycans. 1. Histochem. Cytochem., 29,547-552.

Meyer, K., Linker, A., Davidson, E. A. and Weissmann, B. 1953. The mucopolysaccharides of bovine cornea. J. Biol. Chem., 205, 611616.

Reale, E., Luciano, L. and Kuhn, K. W. 1983. Ultrastructural architecture of proteoglycans in the glomerular basement membrane: a cytochemical approach. J. Histochem. Cytochem., 31, 662-668.

Robinson, J. and Gospodarowicz, D. 1983. Glycosaminoglycans synthesized by cultured bovine cornea1 endothelial cells. J. Cd Physiol., 117, 36%376.

Roth, J. 1982. The protein A-gold (pAg) technique-a qualitative and quantitative approach for antigen localization of thin sections. In Techniques in Immunocytochemistry (eds G. R. Bullock and P. Petruz), Vol. 1, pp. 107-133. Academic Press, London.

Scott, J. E. 1985. Proteoglycan histochemistry-a valuable tool for connective tissue biochemists. Collagen Res. Rel., 5, 541-575.

Scott, J. E. and Haigh, M. 1985. ‘Small’-proteoglycan: collagen interactions: keratan sulphate proteoglycan associates with rabbit cornea1 collagen fibrils at the ‘a’ and ‘c’ bands. Biosci. Rep., 5, 765-774.

Scott, J. E. and Orford, C. R. 1981. Dermatan sulphate-rich proteoglycan associates with rat tail-tendon collagen at the d band in the gap region. Biochem. J., 197,21?-216.

Shepard, N. and Mitchell, N. 1976. The localization of proteoglycan by light and electron microscopy using safranin 0. A study of epiphyseal cartilage. J. Ulrrastntct. Res., 54, 451-460.

Speziale, P., Speziale, M. S., Galligani, L. and Balduini, C. 1978. Interactions between different cornea1 proteoglycans. Biochem. J., 173,935939.

Sundarraj, N., Chao, J., Gregory, J. D. and Damle, S. P. 1986. Ocular distribution of keratan sulfates during pre- and post-natal development in rabbits. J. Histochem. Eytochem., 34,971-976.

Thyberg, J., Lohmander, S. and Friberg, U. 1973. Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage. J. UNrastruct. Res., 45,407-427.

Trelstad, R. L. and Coulombre, A. J. 1971. Morphogenesis of the collagenous stroma in the chick cornea. J. Cell Biol., 50, 840-858.

Van Kuppevelt, T. H. M. S. M., Cremers, F. P. M., Domen, J. G. W., Van Beuningen, H. M., Van Den Brule, A. J. C. and Kuyper, C. M. A. 1985. Ultrastructural localization and characterization of proteoglycans in human lung alveoli. Eur. J. Cell Biol., 36, 74-80.

Vblker, W., Schmidt, A. and Buddecke, E. 1986. Compartmentation and characterization of different proteoglycans in bovine arterial wall. J. Histochem. Cytochem., 34, 1293-1299.

Voss, B., Glossl, J., Cully, Z. and Kresse, H. 1986. Immunocytochemical investigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human. J. Histochem. Cytochem., 34, 10131019.

Wang, 2. W., Irimura, T., Nakajima, M., Belloni, P. N. and Nicolson, G. L. 1985. Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine cornea1 endothelial cells in culture. Eur. J. Biochem., 153, 125-130.

Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y. and Okayama, M. 1982. Isolation and partial characterization of sulfated glycoproteins synthesized by cornea1 epithelium. J. Biol. Chem., 257, 11166-11175.

Yue, B. Y. J. T., Baum, J. L. and Silbert, J. E. 1976. The synthesis of glycosaminoglycans by cultures of rabbit cornea1 endothelial and stromal cells. Biochem. J., 158, 567-573.

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Ultrastructural demonstration of proteoglycans in adult rat cornea