Nine-day chick embryos were injected with a β-xyloside and their sternal cartilage was examined 3 days and a week later. Sterna from 16-day embryos showed a reduction in size as compared to controls, with little or no change in the fraction of extracellular space, and a significant decrease in tensile strength. At the ultrastructural level, collagen fibrils in control sterna were dispersed evenly in the interstitial space, with few contacts between adjacent fibrils. In sterna from treated embryos, almost all collagen fibrils were aggregated into clumps and arrays throughout the interstitial space, with fibril-free areas in between. No abnormalities could be detected in the morphology of individual fibrils or in the ultrastructure of the chondrocytes. The changes in spatial distribution of collagen were fully evident 3 days after drug administration.

The hydroxyproline/DNA ratio was the same in control and treated sterna, and no change was observed in the type of collagen. The uronic acid/DNA ratio was reduced by 14% 3 days after drug administration and by 40% after a week. The degree of sulfation of chondroitin sulfate was reduced from 80% in control sterna to 40% in treated sterna; almost all of this chondroitin sulfate was attached to peptide and the sedimentation pattern of the proteo-glycan resembled that of normal cartilage proteoglycan.

The function of chondroitin sulfate in embryonic cartilage is discussed in terms of our results and others. It is suggested that a major physiological role of the proteoglycan is to control the spatial distribution of collagen fibrils as they assemble to form a cross-linked gel.

The interstitial matrix of cartilage contains a three-dimensional network of fibrils of Type-II collagen held together by apparently random covalent cross links (Lane & Weiss, 1975; Miller, 1976,1977). Chemically, the cross links are of several types, all of which probably result from reactions of the aldehyde function of aliysine or hydroxy-allysine (Tanzer, 1976). Interspersed in the network of collagen fibrils are molecules of proteoglycan aggregate, each consisting of a large number of molecules of proteochondroitin sulfate held by non-covalent bonds to a long strand of hyaluronic acid, and stabilized by one or more ‘link’ proteins (Comper & Laurent, 1978). The proteoglycan aggregate may be held in place by entrapment within the cartilage network (Fessler, 1957; Hamerman & Schubert, 1962) or by non-covalent interaction with collagen (Lee-Owen & Anderson, 1976; Toole, 1976).

The physiological function of the collagen network is reasonably well understood. The network probably provides most of the tensile strength of adult cartilage (Kempson, Muir, Pollard & Tuke, 1973). The overall morphology of adult cartilage is retained when it is subjected to rather stringent extraction procedures, which remove more than 80% of the proteoglycans but leave cross-linked collagen (Sajdera & Hascall, 1969). Agents that interfere with the cross linking of collagen in vivo, such as lathyrogens, drastically reduce the tensile strength of cartilage and cause abnormalities of its morphology if applied during development (Barrow, Simpson & Miller, 1974). From studies of this type it is likely that the collagen network forms the structural framework of the cartilage (Sokoloff, 1969), in embryos and in adults.

The function of the proteoglycan aggregate is less well defined. In load-bearing cartilages, such as occur at adult joints, the proteoglycan is probably largely responsible for the ability of the cartilage to withstand deformation and to recover its shape after local pressure has been applied (Kempson, Muir, Swanson & Freeman, 1970). These properties of adult cartilage would not be expected to be important in embryos. In spite of much speculation, no fully satisfactory physiological role has been assigned to the proteoglycan in embryonic cartilage. However, the existence of the genetic abnormalities nanomelia in chicks, in which extremely stunted growth is associated with a large reduction in the synthesis of the major cartilage proteoglycan (Landauer, 1965; Pennypacker & Goetinck, 1976), and brachymorphy in mice, in which reduced growth is associated with undersulfation of cartilage chondroitin sulfate (Lane & Dickie, 1968; Orkin, Pratt & Martin, 1976), does suggest that correct embryonic growth requires the presence in cartilage of a full complement of fully sulfated chondroitin sulfate.

In this study, we have used the teratological syndrome induced by administration of β-D-xylosides to investigate the effects of altering the structure of chondroitin sulfate on the ultrastructure of cartilage. Both in vitro and in vivo, synthetic β-xylosides prime the synthesis of chains of chondroitin sulfate (Fukunaga et ai. 1975; Galligani, Hopwood, Schwartz & Dorfman, 1975; Gibson & Segen, 1977; Gibson, Segen & Doller, 1979). When β-xylosides are administered to 9-day chick embryos, there is an increase in the total embryonic synthesis of chondroitin sulfate and a decrease in the average degree of sulfation of chondroitin sulfate, from about 80% to 40% (Gibson, Doller & Hoar, 1978; Gibson et al. 1979). The major morphological changes in the embryos are edema of all soft tissues and marked dwarfism; however, the skeleton shows no morphological abnormalities and appears to ossify normally (Gibson et al. 1978). Since there is probably little interference with other biochemical pathways, the xyloside-induced syndrome appears to be an excellent model for investigating the role of chondroitin sulfate in the growth and development of cartilage. In. this paper, we present the results of an ultrastructural and chemical analysis of sterna from 12- and 16-day chick embryos that had been treated with a β-xyloside at 9 days of age. The sternum was chosen because it does not undergo ossification in the chick until the embryo reaches term (Romanoff, 1960).

Nine-day chick embryos (stage 34–35 of Hamburger & Hamilton, 1951), from an inbred block of White Leghorns, were obtained from Spring Lake Farms, Wyckoff, NJ. Eggs were injected once with a suspension of 10 mg 4-methylumbelliferyl β-D-xyloside in 0·1 ml sterile corn oil (Gibson et al. 1978, 1979); control eggs received corn oil only. Eggs were maintained in a humidified incubator at 37°C until sacrifice.

Sterna were removed from the embryos, dissected free of adhering muscle and fixed for 1 h in cold (4°C) glutaraldehyde/formaldehyde fixative, buffered with 0·2 M sodium cacodylate at pH 7·3. Post-fixation in 2% OsO4, buffered with 0·144 M sodium cacodylate buffer, at room temperature was followed by dehydration in an ascending series of graded acetone (50–100%) and propylene oxide before embedding in Epon 812 (Ladd Research Industries, Inc., Burlington VT). Sectioning was carried out with an LKB-8800 A Ultramicrotome III. Thin, sections were mounted on uncoated 200 mesh copper grids and stained with lead citrate and uranyl acetate (5% in absolute ethanol). Tissue examination was carried out on a Jeol 100B electron microscope at original magnifications of 2000–20000.

For determination of their gross chemical composition, seven or eight pooled sterna (wet weight 200–250 mg) were homogenized in 2 ml ice-cold water, using a Brinkman Polytron homogenizer operated at full speed. Portions of the homogenates were used for the determination of DNA (Burton, 1956) with calf thymus DNA (Calbiochem., LaJolla, Calif.) as standard, total protein (Lowry, Rosebrough, Farr & Randall, 1951) with bovine serum albumin (Sigma Chemical Co., St Louis, Mo.) as standard, and total hydroxyproline (Kivirikko, Laitinen & Prockop, 1967). Uronic acid was determined by the carbazole reaction (Bitter & Muir, 1962) with glucuronic acid as standard, after digestion of the homogenates with papain (Gibson et al. 1979). The degree of sulfation of chondroitin sulfate was determined by subjecting crude glycosaminoglycans, prepared from these papain digests, to digestion with chondroitin lyase AC (EC 4.2.2.5) followed by paper chromatography as described (Gibson et al. 1979). Crude glycosaminoglycans were also analyzed by electrophoresis in 0·15 M-ZnSO4 on cellulose acetate strips (Gibson et al. 1979).

The distribution of chain lengths of chondroitin sulfate, and the extent to which it was attached to peptide or to the unnatural aglycone, were determined by cleavage with alkaline [3H]NaBH4 according to Hopwood & Robinson (1973) with modifications. Six sterna were suspended in 1 ml 0·05 M-[3H]NaBH4 (19 mCi/mmol)−0·2 M-NaOH and kept at 4°C for 7 days. Acetone (50 μl) was then added, followed after 1 h by 50 μl glacial acetic acid; the precipitates were removed by centrifugation and the supernatants were lyophilized. The residues were dissolved in 0·15 ml water and applied to a column (0·8×93 cm) of Sepharose 6B (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) equilibrated with 0·2 M-NaCl at 4°C. The column was developed with 0·2 M-NaCl at a flow rate of 2 m1/h. Fractions of 0·49 ml were collected with a 2112 Redirac fraction collector (LKB Instruments, Inc., Hicksville, NY). Portions (0·1−0·2 ml) of each fraction were used for estimating total glycosaminoglycan by precipitation with Alcian blue (Whiteman, 1973), radioactivity expressed as nmol by comparison with a standard of [3H]sorbitol prepared with the same batch of [3H]NaBH4 (Audhya, Segen & Gibson, 1976), and methylumbelliferone by fluorimetry after acid hydrolysis (Gibson & Segen, 1977).

Proteochondroitin sulfate was extracted by stirring groups of six to eight sterna with 15−20 vol of 4 M guanidinium chloride–0·05 M sodium acetate (pH 5·7) at 4°C for 24 h. The extraction was repeated once. Cesium chloride (0·59 g/g of extract) was added to the combined extracts, and the solutions were centrifuged at 40000 rev/min in the 65 rotor of a Spinco L2-65B ultracentrifuge for 48 h at 10°C. The contents of the tubes were split into five approximately equal fractions, which were analyzed for uronic acid. Appropriate fractions (see Fig. 13) were combined, dialyzed exhaustively against water and lyophilized. The residues were dissolved in 0·15 ml 4 M guanidinium chloride buffered with sodium acetate and layered on 5–20% linear gradients of sucrose in buffered 4 M guanidinium chloride (Kimata, Okayama, Oohira & Suzuki, 1974), The gradients were centrifuged at 38000 rev/min in a SW40 rotor of a Spinco L2-65B ultracentrifuge at 5°C, until the ω2t value was 9 × 1011 rad2/sec (approx. 16 h). Fractions of 12 drops were collected by upward displacement and analyzed for proteoglycan and glycosaminoglycan by precipitation with Alcian blue (Whiteman, 1973).

For determination of prolyl hydroxylase (EC 1.14.11.2), extracts of pooled sterna were prepared and assayed according to Hutton, Tappel & Udenfriend (1966). We are grateful to Dr A. Fallon for performing these assays. For comparison of the type of collagen, lyophilized sterna (1–3 mg) were suspended in 70% formic acid containing a 15-fold excess of cyanogen bromide and kept at 30°C for 4 h with occasional vigorous shaking (Orkin et al. 1976). Excess cyanogen bromide was removed by addition of ice-cold water followed by lyophilization. The residues were analyzed by electrophoresis on 11% polyacrylamide gels (0·5 × 6·5 cm) in the presence of sodium dodecyl sulfate, using the buffer system of Neville (1971) with 0·4244 M Tris–0·0308 N-HCI (pH 9·18) in the resolvent gel. The gels were fixed and stained according to Furthmayr & Timpl (1971) and scanned in a Gilford Model 210 spectrophotometer equipped with a Linear Transport system and a Honeywell recorder, using a wavelength of 545 nm and a slit width of 0-1 mm. Samples of Type-I and Type-TI collagen from lathyritic chick cartilage, prepared by the method of Trelstad, Kang, Toole & Gross (1972), were subjected to the same treatment.

For determination of sorbitol space, up to 10 sterna were incubated for 4 h at 37°C in 2 ml of a synthetic medium (Audhya et al. 1976) containing 0·1 nmol [3H]sorbitol (19 mCi/mmol, prepared by reducing glucose with [3H]NaBH4 according to Audhya et al. (1976)). The sterna were rapidly and thoroughly blotted, weighed and dissolved by incubation in 1 ml 90% formic acid for 48 h at 37°C. Portions (50 μl) of the incubation medium were treated in the same way. Radioactivity was determined after addition of 10 ml Aquasol and the percentage sorbitol space of each cartilage was calculated from its wet weight and radioactivity and the radioactivity of the medium.

Load-extension curves of sternal fragments from 16-day embryos were measured in an Instron Universal Testing machine. The posterior two thirds of a sternum was excised and the keel was cut off under a dissecting microscope, leaving a 7–9 mm long fragment of cartilage of very nearly constant width (1·3–1·7 mm, depending on the individual cartilage) and thickness (0·2–0·3 mm). Specimens were stretched at a rate of 10 cm/min until fracture and load was recorded on a continuously moving chart. After fracture the samples were examined for evidence of slip; if this had occurred the recording was discarded. Work-to-fracture was calculated by graphical integration of the load-extension curve.

Administration of β-xylosides to 9-day chick embryos results in a significant decrease in skeletal size with little change in the relative proportions of the calcified skeletal elements (Gibson et al. 1978). The same changes were seen in sterna from these embryos. The overall appearance of sterna from 16-day xyloside-treated embryos was indistinguishable from that of control sterna, but their size was clearly decreased. This is expressed quantitatively in Table 1, in which the lengths of the ventral edge of the keels of several sterna from 16-day control and treated embryos are compared. Treatment with the xyloside induced an 11·5% decrease in this linear dimension, corresponding to a 31% decrease in total volume of the sternum (Table 1). The wet weight/dry weight ratio had the same value (1·1–1·2) in sterna of both types, indicating that the large relative increase in the water content of whole embryos treated with β-xylosides (Gibson et al. 1979) is not reflected in the cartilage. The wet weight/DNA ratio was also the same in sterna of both types, suggesting that there was no great change in the ratio of extracellular to intracellular space. This contrasts with the situation in nanomelia, in which there is an obvious decrease in the space between chondrocytes (Pennypacker & Goetinck, 1976). Further evidence that the volume of extracellular space is not decreased in sterna from xyloside-treated embryos comes from a determination of sorbitol space, which was slightly greater in the treated sterna than in the control group (Table 1). Thus in contrast to the edema of the soft tissues of xyloside-treated embryos, which is associated with a preferential increase in the extracellular space (Gibson et al. 1979), the dwarfism is accompanied by little change in the relative volume of the extracellular space of cartilage.

Table 1

Properties of sterna from 16-day embryos

Properties of sterna from 16-day embryos
Properties of sterna from 16-day embryos

Sterna from xyloside-treated embryos were noticeably more fragile than control sterna during dissection and handling, although their fragility was not as marked as that of lathyritic sterna. To quantitate the change in tensile strength, we made use of the fact that the posterior two thirds of the sternum of a 16-day embryo has a nearly constant width. Fragments of sterna were prepared by removing the keel from the posterior two thirds of the sternum and stretched in a tensitometer until fracture. Plots of load versus extension for three representative control sterna and three sterna from treated embryos are shown in Fig. 1. The shape of the curves is similar in both cases, with fracture occurring at 2–3 mm extension (from a nominal initial length of 1·5 mm). However, the load at fracture of the treated sterna was much smaller than that of the control sterna. Values of work-to-fracture were obtained by integrating plots like those in Fig. 1 for nine control sterna and eight sterna from xyloside-treated embryos. The value for one treated sternum fell within the range for the control sterna, while the remainder were below the lower limit. The mean work for the xyloside-treated was 27% of the mean for the control sterna (Table 1). Since the fragments of cartilage used were approximately rectangular and of constant width, the work-to-fracture values are reasonably representative of the actual tensile strengths of the cartilages. Allowing for the fact that the cross-sectional area of the fragments from the xyloside-treated embryos was on the average 78% of that of the controls, the difference in work-to-fracture was significantly lower after xyloside treatment (Table 1).

Fig. 1

Load versus extension curves for 16-day sterna. Cartilage fragments were prepared as described and stretched in a tensitometer to fracture. C1–C3, curves for three representative control cartilage fragments; E1–E3; curves for three xyloside-treated fragments.

Fig. 1

Load versus extension curves for 16-day sterna. Cartilage fragments were prepared as described and stretched in a tensitometer to fracture. C1–C3, curves for three representative control cartilage fragments; E1–E3; curves for three xyloside-treated fragments.

An electron micrograph of a section of sternum from a normal 16-day chick embryo is shown in Fig. 2. Chondrocytes displaying ultrastructural features that have been described in detail by others (Anderson, Chacko, Abbott & Holtzer, 1970; Pennypacker & Goetinck, 1976) are surrounded by a uniform extra-cellular matrix composed of randomly oriented, evenly spaced fibrils and ‘electron-dense granules’. In general, the chondrocytes are not completely surrounded by well-differentiated lacunae, and the matrix shows approximately the same density of fibrils and granules throughout. In accordance with many published studies (Martin, 1954; Anderson, et al. 1970; Lane & Weiss, 1975; Pennypacker & Goetinck, 1976), we intrepret the fibrils as being composed of Type-11 collagen. The nature of the electron-dense granules is less certain. Some of these may be cross sections of collagen fibrils; however, most workers consider the larger granules at least to be composed of proteochondroitin sulfate (Matukas, Panner & Orbison, 1967; Anderson et al. 1970; Kochhar, Aydelotte & Vest, 1976; Pennypacker & Goetinck, 1976).

Fig. 2

Low-power electron micrograph of sternum from a control 16-day chick embryo. The interstitial space contains a network of evenly spaced fibrils and ‘electron-dense granules’. Nu, nucleus, er; endoplasmic reticulum. The bar represents 1 μm.

Fig. 2

Low-power electron micrograph of sternum from a control 16-day chick embryo. The interstitial space contains a network of evenly spaced fibrils and ‘electron-dense granules’. Nu, nucleus, er; endoplasmic reticulum. The bar represents 1 μm.

Comparison of the extracellular matrix in Fig. 2 to that of a sternum from a 16-day embryo injected on day 9 with 10 mg of 4-methylumbelliferyl β-D-xyloside (Fig. 3) reveals striking differences. Almost all collagen fibrils in the treated cartilage are aggregated into bundles or arrays throughout the extra-cellular space. The bundles are in general well separated from each other and make infrequent contact. Large areas devoid of fibrils are prominent. The number of electron-dense granules may be somewhat decreased relative to the control cartilage. Examination of many similar sections from control and xyloside-treated sterna indicated that the average distance between chondrocytes may have been slightly lower in the treated than in control cartilage, but the difference was not great. The cells are very much less crowded than in nanomelic chick embryo sternum, which exhibits almost total loss of electrondense granules (Pennypacker & Goetinck, 1976).

Fig. 3

Low-power electron micrograph of sternum from a xyloside-treated 16-day chick embryo. Fibrils in the interstitial space are aggregated into bundles (arrows). Nu, nucleus. The bar represents 1 μm.

Fig. 3

Low-power electron micrograph of sternum from a xyloside-treated 16-day chick embryo. Fibrils in the interstitial space are aggregated into bundles (arrows). Nu, nucleus. The bar represents 1 μm.

Figures 46 are electron micrographs of control and xyloside-treated cartilage at higher magnifications. Despite the differences in the extracellular matrix, chondrocytes from control and xyloside-treated embryos are ultrastructurally identical (Figs. 4 and 5). In the control matrix, collagen fibrils often exhibit a nearly parallel alignment, but there are almost always electron-lucent spaces between neighboring fibrils (Fig. 4). There is a marked difference from the longitudinally associated bundles of fibrils which frequently adjoin chondrocytes in the xyloside-treated cartilage (Fig. 5) but are also seen in areas of the extracellular space that appear to be at some distance from cells (Fig. 6). Similarly oriented tracts of fibrils, which however are well spaced from one another, can be discerned in corresponding locations of control cartilage. Except for the difference in distribution, the collagen fibrils of xyloside-treated cartilage are not distinguishable from those of the control.

Fig. 4

Electron micrograph of sternum from a control 16-day embryo. A tract of fibrils with nearly parallel orientations crosses the field from left to right, and is spanned by the double arrow. The bar represents 1 μm.

Fig. 4

Electron micrograph of sternum from a control 16-day embryo. A tract of fibrils with nearly parallel orientations crosses the field from left to right, and is spanned by the double arrow. The bar represents 1 μm.

Fig. 5

Electron micrograph of sternum from a xyloside-treated 16-day chick embryo. A long aggregate of fibrils (f) separates two chondrocytes. The bar represents 1 μm.

Fig. 5

Electron micrograph of sternum from a xyloside-treated 16-day chick embryo. A long aggregate of fibrils (f) separates two chondrocytes. The bar represents 1 μm.

Fig. 6

Electron micrograph of sternum from a xyloside-treated 16-day chick embryo A large bundle of fibrils (f) is at least 1 μm from the nearest cell process (p). The bar represents 1 βm.

Fig. 6

Electron micrograph of sternum from a xyloside-treated 16-day chick embryo A large bundle of fibrils (f) is at least 1 μm from the nearest cell process (p). The bar represents 1 βm.

The structure of the fibrillar network is altered after only 3 days of xyloside treatment. Cartilage from a 12-day-old control embryo shows a fairly homogeneous distribution of fibrils (Fig. 7), while that of a xyloside-treated embryo contains only clumps and arrays of fibrils (Fig. 8). In general, chondrocytes in the control sternum have a uniformly distributed matrix within 0·5 μm of the cell surface, and isolated collagen fibrils can be observed at all distances from the cell (Fig. 7). In the xyloside-treated cartilage, almost all fibrils are associated into bundles, whether they are close to the cell surface or at some distance from it (Fig. 8). This suggests that collagen fibrils aggregate into bundles as soon as they are formed.

Fig. 7

Electron micrograph of sternum from a control 12-day chick embryo. Most fibrils in the extracellular space are well separated from each other. Nu, nucleus; er, endoplasmic reticulum; m, mitochondrion. The bar represents 1 μm.

Fig. 7

Electron micrograph of sternum from a control 12-day chick embryo. Most fibrils in the extracellular space are well separated from each other. Nu, nucleus; er, endoplasmic reticulum; m, mitochondrion. The bar represents 1 μm.

Fig. 8

Electron micrograph of sternum from a xyloside-treated 12-day chick embryo. Almost all extracellular fibrils are gathered into bundles (arrows), m, Mito-chondrion. The bar represents I μm.

Fig. 8

Electron micrograph of sternum from a xyloside-treated 12-day chick embryo. Almost all extracellular fibrils are gathered into bundles (arrows), m, Mito-chondrion. The bar represents I μm.

Chemical analysis of chick embryo sternal cartilage after administration of xyloside is shown in Table 2. There was a slightly lower protein/DNA ratio in cartilage from treated embryos relative to control embryos. In spite of the marked ultrastructural changes in the organization of the collagen component of the extracellular matrix, no consistent difference in the hydroxyproline/DNA ratio could be detected. There was also no difference in prolyl hydroxylase (EC 1.14.11.2) activity of sterna from control and xyloside-treated 16-day embryos. Sterna from 16-day control and xyloside-treated embryos were treated with cyanogen bromide and analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate (Fig. 9). The same major peptides were present in both cases. The electrophoretic pattern showed major bands in positions corresponding to the CNBr peptides of Type-II collagen, supporting the ultrastructural evidence which indicates that the major collagen in xyloside-treated sterna, as in control sterna, was Type II.

Table 2

Chemical composition of sterna

Chemical composition of sterna
Chemical composition of sterna
Fig. 9

Cyanogen bromide cleavage of sterna. Sterna from 16-day embryos were cleaved with CNBr and the resulting peptides separated by electrophoresis on SDS polyacrylamide gels. The gels were stained and scanned at 545 nm. The positions of the major CNBr peptides from Type-I and Type-II chick collagen are shown at the top of the figure.

Fig. 9

Cyanogen bromide cleavage of sterna. Sterna from 16-day embryos were cleaved with CNBr and the resulting peptides separated by electrophoresis on SDS polyacrylamide gels. The gels were stained and scanned at 545 nm. The positions of the major CNBr peptides from Type-I and Type-II chick collagen are shown at the top of the figure.

The uronic acid content of xyloside-treated sterna was 14% less than that of control sterna 3 days after drug administration and 40% less in 16-day embryos (Table 2). This contrasts with the analyses of whole 16-day embryos, which show increased accumulation of uronic acid after xyloside treatment (Gibson et al. 1979). Electrophoresis of sternal glycosaminoglycans revealed a polydispersity of charge in the xyloside-treated sterna, suggesting varying degrees of sulfation of the predominant glycosaminoglycan, chondroitin sulfate; whereas the charge density of the chondroitin sulfate from control sterna was more nearly constant (Fig. 10). Quantitation of the disaccharides released by chondroitin lyase digestion showed a marked undersulfation in the xyloside-treated sterna (Table 2). Sixty per cent of all chondroitin disaccharides were unsulfated in xyloside-treated cartilage, while only 20–25% were unsulfated in control cartilage. Undersulfation was fully evident at day 12 and continued through day 16.

Fig. 10

Electrophoresis of sternal glycosaminoglycans from control and xyloside-treated embryos. Standards of chondroitin 6-sulfate (1), dermatan sulfate (2), heparan sulfate (3) and hyaluronic acid (4) were applied to the left-hand strip; glycosaminoglycans from 16-day control (C) or xyloside-treated (X) sterna were applied to the center strip; and glycosaminoglycans from 12-day control (C) or xyloside-treated (X) sterna to the right-hand strip. The arrow indicates the point of application; the direction of migration is towards the top.

Fig. 10

Electrophoresis of sternal glycosaminoglycans from control and xyloside-treated embryos. Standards of chondroitin 6-sulfate (1), dermatan sulfate (2), heparan sulfate (3) and hyaluronic acid (4) were applied to the left-hand strip; glycosaminoglycans from 16-day control (C) or xyloside-treated (X) sterna were applied to the center strip; and glycosaminoglycans from 12-day control (C) or xyloside-treated (X) sterna to the right-hand strip. The arrow indicates the point of application; the direction of migration is towards the top.

Gibson et al. (1979) reported that chondroitin sulfate extracted from whole 16-day embryos treated with methylumbelliferyl β-xyloside was markedly undersulfated and shorter in chain length, with 75% of the chains being linked to the fluorescent aglycone. To determine whether this was true for cartilage also, control and treated sterna were reduced with sodium borotritide to simultaneously cleave chondroitin chains from the core protein and stoichiometrically radiolabel the freed chondroitin chains (Hopwood & Robinson, 1973). When analyzed on a gel filtration column, chondroitin sulfate prepared in this way from control or xyloside-treated 16-day embryos migrated in an identical manner (Fig. 11). The amount of fluorescence associated with the chondrotin sulfate peak was insignificant (Fig. 11, lower panel), indicating that very little methylumbelliferyl chondroitin sulfate was present. These findings indicate that the chondroitin sulfate chains of cartilage from xyloside-treated embryos are linked predominantly to a core peptide and have the same distribution of chain length as the chondroitin sulfate chains from control cartilage.

Fig. 11

Gel-filtration chromatography of end-labeled sternal chondroitin sulfate. End-labeled chondroitin sulfate was produced by cleavage in alkaline [3H]NaBH4 and analyzed on a column of Sepharose 6B. The void volume and total volume of the column were at 13·6 and 31·6 ml respectively. Upper panel, chondroitin sulfate from control 16-day sterna; lower panel, chondroitin sulfate from xyloside-treated 16-day sterna.

Fig. 11

Gel-filtration chromatography of end-labeled sternal chondroitin sulfate. End-labeled chondroitin sulfate was produced by cleavage in alkaline [3H]NaBH4 and analyzed on a column of Sepharose 6B. The void volume and total volume of the column were at 13·6 and 31·6 ml respectively. Upper panel, chondroitin sulfate from control 16-day sterna; lower panel, chondroitin sulfate from xyloside-treated 16-day sterna.

Proteoglycans were extracted from control 16-day sterna and subjected to isopycnic centrifugation in a ‘dissociative’ cesium chloride gradient. Nearly 90% of the uronic acid from control sterna was found at the bottom of the gradient (Fig. 12, upper left panel). When this material was analyzed by zone sedimentation, most of the proteoglycan migrated well into the gradient (Fig. 12, right panel, CIT), indicating that it had a large molecular weight (Kimata et al. 1974). There was also a minor peak near the top of the sucrose gradient, which may correspond to the ‘ubiquitous’ proteoglycan found in chick embryo cartilage as well as other tissues (Palmoski & Goetinck, 1972; Okayama, Pacifici & Holtzer, 1976). The very small amount of uronic acid at the top of the original CsCl gradient did not correspond to any high molecular weight proteoglycan when analyzed by zone sedimentation (Fig. 12, right panel, Cl). A different picture was obtained when proteoglycan from xyloside-treated sterna was subjected to isopycnic centrifugation in a CsCl gradient. Only 75% of the uronic acid was in the bottom three fifths of the gradient, while the remainder banded at a density close to 1·44g/ml (Fig. 12, lower left panel). Each of these fractions was subjected to zone sedimentation and gave a pattern very similar to that shown by the proteoglycan from control sterna (Fig. 12, right panel, El and EH). Thus the proteoglycan from xyloside-treated sterna had approximately the same sedimentation properties as that of control sterna, but at least 25% of it had a much lower buoyant density. These data are compatible with the presence of a normal core peptide together with a change in the degree of sulfation. Essentially the same results were obtained with proteoglycans extracted from 12-day control and xyloside-treated sterna.

Fig. 12

Isopycnic centrifugation and zone sedimentation of proteoglycans from sterna. Proteoglycans were extracted from 16-day sterna and subjected to isopycnic centrifugation in dissociative CsCl gradients (left panel). The gradients were cut into five fractions, which are numbered from top to bottom. Fractions were combined as shown and subjected to zone sedimentation in a linear 5–20% sucrose gradient under dissociative conditions (right panel).CI and CII, proteoglycans from control 16-day sterna; El and EH, proteoglycans from xyloside-treated 16-day sterna. Cl and El represent the combined material from the upper two fifths of the CsCl gradients in the left panel; CII and EII represent the combined material from the lower three fifths of those gradients. The direction of sedimentation was towards the right.

Fig. 12

Isopycnic centrifugation and zone sedimentation of proteoglycans from sterna. Proteoglycans were extracted from 16-day sterna and subjected to isopycnic centrifugation in dissociative CsCl gradients (left panel). The gradients were cut into five fractions, which are numbered from top to bottom. Fractions were combined as shown and subjected to zone sedimentation in a linear 5–20% sucrose gradient under dissociative conditions (right panel).CI and CII, proteoglycans from control 16-day sterna; El and EH, proteoglycans from xyloside-treated 16-day sterna. Cl and El represent the combined material from the upper two fifths of the CsCl gradients in the left panel; CII and EII represent the combined material from the lower three fifths of those gradients. The direction of sedimentation was towards the right.

From a morphological point of view, administration of β-xylosides produces two major changes in the cartilage of chick embryos. Macroscopically, there is a decrease in size of the cartilage with no significant change in its shape, and at the ultrastructural level there is a marked distortion in the spatial distribution of collagen fibrils. Dwarfism seems to be a common feature of all syndromes in which the structure or synthesis of proteochondroitin sulfate is specifically impaired. The most marked effect occurs in nanomelia (Landauer, 1965), a recessive genetic abnormality in which the synthesis of the major cartilage proteoglycan is greatly reduced (Pennypacker & Goetinck, 1976). A less marked change is seen in brachymorphy (Lane & Dickie, 1968), which is characterized by undersulfation of cartilage chondroitin sulfate (Orkin et al. 1976). The dwarfism in these conditions is not accompanied by extreme skeletal defects. Dwarfism is also a feature of the action of the glutamine analog 6-diazo-5-oxonorleucine (DON), both in vivo (Greene & Kochhar, 1975) and in mouse limb-bud cultures in vitro (Aydelotte & Kochhar, 1975). Among other metabolic effects, DON inhibits the synthesis of chondroitin sulfate by interfering with the formation of glucosamine (Ghosh, Blumenthal, Davidson & Roseman, 1960; Telser, Robinson & Dorfman, 1965). The skeletal malformations seen in embryos treated with DON probably result from interference with other metabolic pathways, whereas the dwarfism can be reversed by administration of glucosamine (Aydelotte & Kochhar, 1975; Greene & Kochhar, 1975).

The proteoglycan aggregate in the extracellular space of cartilage exerts a significant Donnan osmotic pressure (Ogston, 1970; Comper & Laurent, 1978), which should influence the total volume of the interstitial fluid. A decrease in the rate of secretion of the proteoglycan or its glycosaminoglycan sidechains will lead to a lower Donnan osmotic pressure and less interstitial fluid per chondrocyte. This can account for part of the dwarfism observed in nanomelia, where there is a clear reduction in the relative volume of interstitial space (Pennypacker & Goetinck, 1976). Since administration of β-xylosides led to some decrease in the glycosaminoglycan content of the cartilage (Table 2), a similar but smaller reduction in interstitial space might be expected to occur under these conditions. However, our observations seem to indicate little or no decrease in the relative volume of interstitial space (Table 1), and suggest that the dwarfism induced by β-xylosides is due to a reduction in the rate of cell division of chondrocytes. The effect may be rather specific for cartilage, since there is little reduction in the total DNA content of 16-day xyloside-treated embryos (Gibson et al. 1979). A decrease in the rate of cell division of chondrocytes is a likely contributory factor in the other cases of dwarfism associated with abnormal proteoglycan structure or content discussed above. Little is known about the influence of the components of the extracellular matrix on the growth of differentiated embryonic cartilage, although both collagen and proteochondroitin sulfate promote the differentiation of chondrogenic tissue to cartilage in vitro (Kosher, Lash & Minor, 1973; Kosher & Church, 1975).

The changes in spatial distribution of collagen fibrils induced by administration of β-xylosides resemble changes seen in nanomelia and in limb-bud cultures incubated in the presence of DON. In nanomelia, the synthesis and type of cartilage collagen are normal, but the fibrils in the interstitial matrix are closely packed in long arrays resembling those in Fig. 5 (Pennypacker & Goetinck, 1976). In DON-treated cultures, collagen fibrils in the interstitial matrix of cartilage are gathered into bundles and clumps (Kochhar et al. 1976). The fibrils tend to be thicker than usual and show some periodicity, a feature that is not normally obvious in Type-II collagen in chick embryo cartilage (Martin, 1954). We have not been able to document changes in periodicity of individual fibrils in cartilage from xyloside-treated chick embryos, but the changes in spatial distribution of collagen fibrils (Figs. 5, 6 and 8) are very similar to those induced by DON.

Obvious mechanisms by which changes in proteoglycan structure could influence the spatial distribution of collagen fibrils include alterations in the primary structure of collagen, interference with cross linking and changes in the rate of synthesis or secretion. Morphologically, the fibrils in xyloside-treated cartilage closely resemble those in control cartilage, and are very different from, for instance, the fibers of Type-I collagen which abound in the perichondria on the same sterna. In addition, cyanogen bromide cleavage experiments indicate that the major type of collagen is the same in control and xyloside-treated cartilage. Thus changes in the structure of the matrix are not accompanied by a significant shift in the type of collagen produced by the chondrocytes. Interference with cross linking has not been examined directly. However, cross-link-deficient cartilage, produced by treating chick embryos with β-aminopropionitrile, shows a distribution of collagen fibrils that is different from that found in control or xyloside-treated cartilage. While the ultrastructure and spatial distribution of most of the collagen fibrils resemble normal cartilage, the interstitial matrix contains many individual thin and twisted collagen fibrils that are not cross linked to other fibrils (J. T. Hjelle and K. D. Gibson, unpublished). Since no such fibrils are seen in xyloside-treated cartilage, we infer that the xyloside probably does not directly affect the mechanism for forming cross links. Administration of xyloside does not change the rate of synthesis or deposition of collagen in sterna in short-term in vitro incubations (J. T. Hjelle and K. D. Gibson, unpublished). Further, the amount of hydroxyproline per mg DNA was the same in sterna from xyloside-treated and control embryos 3 days and a week after drug administration (Table 2), indicating that in vivo the rate of synthesis of collagen per cell was unchanged. Thus the changes in spatial distribution of collagen fibrils described here are probably not secondary consequences of changes in chemical structure or rate of synthesis of collagen, but result from a direct influence of proteochondroitin sulfate on the formation of the fibrillar network.

Some insight into the physiological significance of the changes in collagen spacing induced by β-xylosides or DON may be obtained by noting that there is a strong similarity between the formation of a cross-linked collagen network in the interstitial space of cartilage and the formation of a synthetic network polymer in the presence of a diluent. When a synthetic network polymer such as moderately cross-linked polystyrene is formed in the presence of a good solvent, polymer chains and monomer subunits remain freely dispersed in the solution, at all stages of the polymerization and the final network is homogeneous, with all polymer chains evenly distributed in space (Millar, Smith, Marr & Kressman, 1963). If the solvent is replaced by a non-solvating diluent, for which the polymer chains and monomers have a low affinity, the reaction mixture tends to separate locally into two phases, one enriched in polymer and monomer and the other consisting mainly of diluent. The polymer chains become linked into a heterogeneous, macroporous network, in which areas of tightly packed polymer are enmeshed with pores that are free of polymer (Millar, Smith & Kressman, 1965). Macroporous gels formed in the presence of a non-solvent have reduced tensile strength and elasticity as compared to homogeneous gels with the same chemical composition (Millar et al. 1965). Both differences are due to the existence within the network of many surfaces that are crossed by only a few polymer chains; such surfaces offer weak spots at which fracture will occur readily. It should be emphasized that the weakness of these networks is not due to deficiencies in the number of cross links, but results from the erratic spatial distribution of the polymer chains.

The analogy with the geometry of the collagen network in control and xyloside-treated cartilage is evident, although the underlying physical causes may be quite different. The distribution of collagen fibrils in control cartilage resembles the distribution of polymer chains in a homogeneous network polymer whereas the collagen network in treated cartilage resembles a macroporous network. The reduced tensile strength of the treated cartilages (Table 1) is consistent with the propertes of macroporous networks, although as discussed above, the possibility that cross linking is abnormal in these cartilages has not been ruled out. The increase in sorbitol space of the treated cartilages is also consistent with macroporosity, since the excluded volume associated with the macromolecular components of the extracellular space (Ogston, 1958; Comper & Laurent, 1978) will be partly compensated by the presence of freely permeable pores.

Based on this analogy, and taking into account the macroscopic and ultra-structural changes seen in cartilages in which the structure or synthesis of proteochondroitin sulfate is defective, we propose that a major function of the proteoglycan in the interstitial space of embryonic cartilage is to ensure that collagen fibrils are well dispersed before they assemble to form a cross-linked gel. Load-bearing cartilages require adequate tensile strength and elasticity, and these properties are intimately connected with the geometry and topology of the collagen network (Sokoloff, 1969; Kempson et al. 1973). If a homogeneous network with optimal mechanical properties is to be formed, not only must there be the correct number of cross links, but the collagen fibrils must be evenly dispersed in the interstitial matrix at the time that they are assembled into the network. We suggest that the spatial distribution of newly formed collagen fibrils is determined by the proteoglycan aggregate, probably through non-covalent binding of Type-II collagen and cartilage proteochondroitin sulfate (Lee-Own & Anderson, 1976; Toole, 1976). Correct spacing of collagen fibrils in the cartilage of the young or adult animal will thus depend on the correct distribution of proteochondroitin sulfate within the cartilage during embryonic growth; if this is impaired because of abnormalities in the chemical composition or amount of the proteoglycan, collagen fibrils may be permanently assembled into a mechanically deficient network, such as the macroporous networks induced by β-xylosides or DON. This will have serious consequences for the viability of the animal, and may, in the case of embryos treated with β-xylosides, be partly responsible for the observation that they are more fragile than control embryos (Gibson et al. 1978).

It is a pleasure to acknowledge the advice and assistance of Dr M. Boublik and Mr F. Jenkins in performing the electron microscopic studies. We are also grateful to Dr A. Fallon for the determinations of prolyl hydroxylase activity.

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