THE JOURNAL OF Bromo~ca~ CHEMISTRY Vol. 249. No. 5,Issue of March 10, pp. X37-1645, 1974

P&led in U.S.A.

A Correlation between Synthetic Activities for Matrix

Macromolecules and Specific Stages of Cyto-

differentiation in Developing Cartilage*

(Received for publication, July 26, 1973)

ATSUHIKO OOHIRA, I<OJI IIInwIA, AND SAKARU SUZUKI$

From the Department of Chemistry, Faculty of Science, Nagoya University, Nagoya 464, Japan

KENZO TAKATA

From the Biological Institute, Faculty of Science, Nagoya University, Nagoya 464, Japan

IKUO SUZUKI AND MUNEMITSU HOSHINO

From the Aichi Cancer Center Research Institute, Nagoya 464, Japan

SUMMARY

Light and electron microscopic studies were made on the structures of cells (chondroblasts or chondrocytes) and matrices of developing epiphysial cartilage of U-day-old chick embryo. Four zones composed of cells with different morphological features were characterized; Zone 1 desig- nated small, round cells; Zone 2, flattened cells; Zone 3 and Zone 4, enlarging cells characterized by an increasing vacuo- lated cytoplasm.

Synthetic activities of DNA, chondroitin sulfate, and collagen were investigated in the different zones by means of radioautographic techniques. The results indicated that a close relation exists between variations of the three synthetic activities and changes in the ultrastructure of cells or in the stage of cytodifferentiation.

The chemical nature of the newly synthesized collagens in the different zones was examined to see if any structural heterogeneity may exist in the collagens. Exposure of each zone from the proline-labeled cartilages to 4 M guanidine hydrochloride resulted in the solubilization of more than 80% of the labeled collagen in the zones, there being little difference in the extractability of the labeled collagens among the four zones. The labeled collagens from the four zones showed little change in the degree of hydroxyla- tion of the proline moieties. Further characterization of the collagen samples indicated that all the samples comprised two forms, one corresponding to the known crl(I1) chain and the other a precursor form of the otl(I1) chain. It was shown from pulse-chase experiments that the precursor form was rapidly converted to cul(I1) with a reduction of molecular size: the apparent rates of this conversion in- creasing gradually in going from Zone 1 to Zone 4.

* This research was supported in part by research grants from the Ministry of Education of Japan.

$ To whom requests for reprints should be addressed.

In recent years, rapid studies have been made in elucidating the fine structures of collagen and proteoglycan, the main com- ponents of connective tissue matrix. Accompanying these ad- vances, it has become evident that the macromolecules can appear with varying degrees of structural modifications. Some ex- amples of such variations in skeletal tissues are: the occurrence in chick cartilaginous sterna of two types of collagen, [orl(II)]a and [al(I)]*2, which differ from each other both in amino acid sequence of the a chains and in their distribution within the triple helical molecule (l-4) ; the age-related changes in the extent of hydroxylation of lysyl residues of chick bone collagen (5) ; the occurrence in laryngeal cartilage of proteoglycan species with different number of polypeptide and carbohydrate chains (6) ; the variations of the chain length of chondroitin sulfates found in different portions of rat costal cartilage (7); the age- related changes in the extent and location of sulfate substitution of cartilage chondroitin sulfate chains (8, 9); the introduction of dermatan sulfate element into chondroitin-6-sulfate chain by fibrous (collagen-rich) cartilage (10) ; and the difference in the type of oversulfation found among the chondroitin sulfates and dermatan sulfates encountered in skeletal tissues of phylogeneti- tally distant animals (11-15).

Cartilage, as the skeletal tissue with the most abundant matrix macromolecules, would appear to be the most favorable object in which to study the format,ion and secretion of matrix macro- molecules relat,ed to cellular phenomena, especially during the period of their most active production in the embryo. Previous reports 011 the morphology of cartilage in the limbs of chick embryo (for example, see Ref. 16 and 17) have described that developing epiphysis undergoes the whole course of differentiation and maturation from a mass of mesenchyme cells at 2 or 3 days to a condyle consisting of art.icular cartilage, epiphysial plate, diaphysis, and marrow cavities by 20 or 21 days. One may pos- tulate then that some of the heterogeneities of collagen and proteoglycan are implicated, either directly or indirectly, in the process of cell specialization (cytodifferentiation) to produce particular types of matrix macromolecules, and it was for this reason that further studies of the correlation between synthetic

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activities for matris macromolecules and specific stages of cyto- differentiation have beeu carried out. l‘he primary purpose of this and the accompanying paper (18) is to describe the results of such comparative studies using 12.day-old chick embryo.

The study has also result.ed in our unespcctcd finding that the cartilage cells may syuthesize, in addition to the k~lown al(I1) chain, a precursor form of al(H). In this paper, therefore, a brief comment is made on the experiment that leads to this fhd-

ing.

EXP1~RI&lWWAL PROCEIKIRl’:

Naterials

Chondroitiilase-AUC, choudroitiuase-AC, chondro-4-sulfatase, and chontlro-6-sulfatase were prepared by the method of Yama- gata et al. (19), and acid-soluble collagen from rat skiu by the met.hod of Ilicz et al. (20). We are grateful to Dr. H. IJtiyama, Kyoto University, and Dr. K. ~Iihashi, Nagoya Universit.y, for helpful suggestioris as to the preparation of the soluble coIl:~gx~~~

A highly purified preparation of bact,crial collagenase (from Clostridium hisfolyticum) was kindly giver1 by l)r. 1’. Ohya, Amano J’harmaceutical Co., Ltd., Nagoya. A criterion of the specificity of the collager~asc is that it tlitl uot, digest, 13.‘*(‘I- tryptopllarr-cot~taiili~i~ proteins isolated from chick embryo epiphgsis (collugc~~ dots Ilot eoiit,aiu this amiuo acid).

The following commercial materials wcrc usctl: [ttu2thyl-311]- thymidine, 17.8 Ci per rnmole, and L-[ V14C]prolinc, 205 m(:i per mmole, from New Ihglard Nuclear; L-[3-‘4C]tryptopllItll, 58 mCi per mmole, from The Radiochemical Ccut,re, Amersham, Ihg-

land; carrier-free Na3%04 from Radioisotope Association, Tokyo, Japan; Sakura emulsion, type NR-312, alid lionidol-X developer from Konishiroku Co., Ltd., Tokyo; carbosgmethyl (CM) cellu- lose, Whatrnun CAI-32, microgranular, capacity 1 .O meq per g, from II. Reeve Augel & Co., Loudon; lsio-Gel A-5m, 200 to 400 mesh, from l<io-Rsd Laboratories; and Diaflo membrane l’hI-10 from Amicon Corporation, Lesingt~on. All of the ot.hcr chemicals were of the highest purit,y of commercially available reagents.

Methods

Incubation of Bpiphysial Cartilage-Chick embryos (White Leghorii) were incubated at 38” for 12 days. Tibias ai~d femurs were dissected free of adherent connectJive tissue and muscle, and the cart.ilagenous portious of the boues were separated from the bony shaft.

Each incubation misturc? coiisisted of two segrneuts (about 8 mg) of cartilage. The tissues were prcincubuted at 37” for 20 min with gentle shakiiig iti either of the following solutions: 1 ml

of Krcbs’ solution (21) plus 50 ~(g of ascorbic acid; 1 ml of iIlgSOd- free Krcbs’ solut,iou plus 50 fig of ascorbic acid Jilus 243 pg of MgC12.61-120. ISithcr 5 PC1 of [rr2ethyl-311]tllylr~i~litle or 1 &i of [ CPC:]prolinc was added to the samples prciucubatcd iii the former solution, and 100 &i of Na3%04 to the samples iii the latter solution. After iiicubation for the periods iudicated iu individual csperiments, the t.issucs were either t.akcu for chase esperimerns or radioaut~ographs.

For chase incubatious, the tissues which had been iucubated with 3eS042- or [IPC]proliue were washed twice with about 5 ml of “chase medium” coutaiuiug 10 rnM NasSOd or 4 mM

L-proline, rcspcctivcly, iu the Krcbs-ascorbate solution, arid then replaced in fresh chase medium at 37” for various t.imes of nou- radioactive chase.

Preparation of Radioautograph-At the end of incubation in radioactive media, tissues were washed thoroughly with chilled

Krebs’ solutiou (for Wlabelcd tissues) or either of the chase media (for %- aud i4C-labeled tissues, set above), and immcdi- ately placed in 2cyti perchloric acid at 0”. After 30 min, t,lie t,issues were fixed in et.hanol-at&c acid-chloroform (6 :3 : 1) at 0” for 2 hours, dehydrated in ethanol, embedded in paraffin blocks, aud 5.pm-thick sections were prepared iii usual manners. After removal of the parafliu iii xyleue, the sectious were coated with Sakura emulsion, type IiR-M2. After exposure at 4” for the periods iudicated in individual espcrimeuts, the ratlioauto- graphs were developed for 4 min with Kouidol-X developer arid t,hen fixed. The sections u-ere stained wit.h a mixture of 1 part. 0.1 y0 toluidiue blue iu 60% ethanol and 2 parts 0.05 M sodium phosphate buffer, pH 6.8.

Prior to the cmulsiou coating, some sections were incubated for 60 min at 37” iu a mixture containing 5 units of chondroi- tinase-ABC, 0.5 unit of choutlro-4-sulfatase, 0.5 uuit of choudro-6- sulfat.ase, 1 mg of bovine strum albumin, and 0.5 mmole of sodium acetate in 10 ml of 0.05 M TrisHCl, pI1 8.0. At, t.he end of iucubatiou, the se&oils were washed successively with 0.05 111 ‘Iris-HCl ($1 8.0), 0.1 M N&04, am1 distilled water. After dellv(lratioii in ethanol, the sectiotis were coated with Snkura emulsion 3s tlcseribed above.

The possibility t,hat the use of these enzymes nlay rrsult in tlcgradatiot~ of Gssue proteiils (it~clutling eollager~ :urtl t,hc prot.cin moiet~g of J)rotoogl~catrs) has beeu ruled out by our control espcrimcuts iti which the niisturc of c’liolltlroitiii2lse aricl choiitlro- sulfatases was iucubatetl at 37” for 60 mill wit,11 crude “C-protein prcpnratioiis obtained from chick embryo tibias aud femurs lab&d with [ U-%]proliue or [3-iT]tryptophau. The result,s iudicated that, even at the four times higher concent.ration, the enzyme mixture could uot release more than 4.2uj, of the tot’al radioactivities associated with the proteins.

Electron Jlicroscopy-The tissues were fixed in glutaraldchyde, washed with isotonic phosphate buffer (pH 7.4)) fised in osmiurn tctroside, dehydrated, and embedded in Epou in t.he usual man- ner. Sections were cut at approximately 60 nm on an LKD microtome, picked up on carbon-coated 200.mesh grids, and subjected to double stainiug with urauyl acetate and lead hy- tlrosidc (22) for esaminatiou iu a Hitachi HIJ 11-C electrou microscope.

Anatytical Jfethods-The following conipouuds were deter- mined by t.he indicated methods: I>NA by the method of Burton (23); urouic acid by t.hc method of Wter and Muir (24); hy- drosyproliue by the method of l’rockop aud Udcnfriend (25) ; i4C-labclcd hydrosyproliuc by the method of ,Juva t~11d l’rockop (26) ; 3”S-labelctl cholidroititi sulfate by the method of Saito, Yamagatu, :ml Suzuki (27) ; am1 protein by the method of Lowry et al. (28) or by t.he measurement of absorbance at 230 111~~.

Jleasuremenl of Radioactivity-Samples were counted in :I IIoriba liquid scintillutioli spectrometer, model M-500, with the solveut system recommcudcd by the rnauufacturer (IIoriba Scisakusho, Kyoto).

RESULTS

XorphologicaE Charaeleristics of CelEs and Natrices in Various Zones of Epiphysial Cartilage

In epiphyses from the tibia and femur of 12-day-old chick embryo, four different zones could be identified by light micros- copy (Fig. 1) as well as by electron microscopy (Fig. 2). These lie along the long axis of the bone: t.he distal zone1 composed of

1 At the articulsr surface, there is a narrow zone consisting of fibroblast-like cells surrounded by very small quantities of inter-

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ZONE 1 ZONE 2 ZONE 3 ZONE 4

FIG. 1. Light microscopic appearance of four cellular zones of a tibia1 cartilage of 12-day-old chick embryo. Sections of a tibia1 cartilage on the side adjacent to the metatarsus are shown. See Fig. 3 for the location of each zone.

small, round cells (Zone 1) which are characterized by high nucleocytoplasmic ratios and underdeveloped intracellular mem- brane systems (Fig. 2~) ; the zone of flattened cells (Zone 2) which differ from the cells of Zone 1 in having greater volumes of cyto- plasm and more complex profiles of the intracellular membrane systems (Fig. 2b) ; the zone of enlarging cells (Zone 3) in which the rough-surfaced endoplasmic reticulum with dilated cisternae is more prominent (Fig. 2c) than that seen in cells of the two pre- vious zones; and the proximal zone consisting of the most enlarged and hypertrophied cells (Zone 4) some of which show degenerative changes characterized by the absence of well defined organelles (Fig. 2d). Mitotic figures are frequently found in Zones 1 and 2, but only occasionally in Zones 3 and 4. It should also be noted that there is no sharp line of division between the four zones which are all connected by transitional elements.

Corresponding differences in the appearance of intercellular matrices were also noticed. The volumes of matrices and the intensities of metachromasia increase in going from Zone 1 to Zone 4 (Fig. 1). All the matrices contain numerous microfibrils (20 nm in diameter) and dense granules (20 to 70 nm in diameter) but the granules in the matrix of Zone 1 are smaller in size than those present in the other zones (Fig. 2).

Distribution of DNA, Chondroitin Sulfates, and Collagen among

Four Zones

The distribution of DNA, chondroitin sulfates, and collagen was measured among the four histologically segregated zones of cartilage (Table I). The chemical analyses indicated that Zone 1 through Zone 4 contain these macromolecules, with the DNA content (on a wet weight basis) progressively decreasing and the ratio of chondroitin sulfates or collagen to DNA increasing. Since the microscopic observation (Fig. 1) has indicated that the cell density decreases gradually, from Zone 1 to Zone 4, with a concomitant increase in the volume of matrix, it is apparent that the concentrations of DNA may mirror the cell densities, while the ratios of chondroitin sulfates or collagen to DNA are par- alleled with the matrix quantities per unit number of cells.

cellular matrix. This zone is described by some histologists as a special zone for “articular cartilage.” However, the fibroblastic appearance of cells as well as the well developed bundles of matrix collagen make this zone readily discernible from the zones of chondrocytes. In the present study, this articular surface zone was eliminated during the manipulation for tissue sampling.

Synthetic Activities for DNA. Chondroitin Sulfates, and Collagen in DiJerent Zones

The histological and chemical observations described above suggest that the differentiation of cell type and tissue morphology may be correlated with changes in cellular activities for the syn- thesis of DNA, chondroit,in sulfate, and collagen. To obtain more direct evidence supporting the suggested correlation, the rates of synthesis in the different zones of the cartilage were determined by radioautography taking advantage of the fact that cartilage segments from 12-day-old embryo allow heavy labeling of DNA, chondroit,in sulfates, and collagen during a short pulse with [methyl-3H]thymidine, Y504*-, and [ U-14C]proline, respectively. Under the conditions described under “Methods,” the tissues were almost uniformly labeled throughout the thick- ness of the specimens, suggesting that t.he thickness of the speci- mens does not present a barrier to the penetration of label.

DNA--In medium containing [methyl-3H]thymidine, uptake of label (as judged by the number of radioautographically labeled cells per unit number of cells) proceeded actively for 90 mm, presumably at the expense of the endogenous supply of the other nucleosides in the specimen. After 90 min, incorporation grad- ually decreased.

Electron microscopic examination of the cartilages which had been incubated for 2 hours in the media revealed almost normal cytological details: the elements of the nucleus, the Golgi appara- tus, and the rough-surfaced endoplasmic reticulum appeared unchanged in morphology and location relative to one another.

In all of the specimens t,aken within 90 min after isotope ad- ministration, the histological aspect of the radioautographs was similar in that the cells in Zone 3 and Zone 4 had litt’le overlying grains. The number of labeled cells (per unit number of cells) increased progressively toward Zone 1, with the highest popula- tion of labeled cells in a central part of Zone 1 (Fig. 3). Since int’racellular incorporation of [methyPH]thymidine is considered as an index of DNA synthesis, the data appear to indicate that cell division occurs most actively in Zone 1 and ceases in Zone 3 and Zone 4.

C/zondroitin Sulfates-In the cartilage segment exposed to 3sS042- for 5 min, the radioautographic grains were already detectable in all the four zones except some of the degenerative cells in Zone 4 that had little overlying grains. At t’his time, most

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FIG. 2. Electron micrograph of cells and matrices in the four different zones. a, small, round cell in Zone 1; b, flattened cell in Zone 2; c, enlarging cell in Zone 3; and d, hypertrophied cell with degenerative figures in Zone 4. See Fig. 3 for the location of each zone. Key: N, nucleus; GA, Golgi apparatus; REII, rough endoplasmic reticulum; GG, glycogen granules; F, fibrils; DG, dense granules; and L, lacunar area X 7000.

of the label was over the cells2 and, when the label‘was chased with carrier sulfate, a large amount of label appeared over the extracellular matrix. The number of grains per unit number of cells (calculated from the radioautographs of the pulse-labeled tissues) indicates that the cells in Zone 2, especially those lying immediately adjacent to Zone 3, are the site of highest activity (Fig. 3). Relative activities decrease gradually in going to the extreme zones.

The foIlowing criteria have established that sulfation of chon- droitin sulfates actually takes place in the cell and most of the radioactivity detected in radioautographs emanates from chon- droitin sulfates: (a) radioautographic examination of a control specimen, which had been placed in the pulse medium at 0” for

z Most of the grains were shown by electron microscopic radio- autography to overlie the Golgi apparatus (29).

15 min, indicated that the number of grains was less than 1 y0 of the number of grains found in the specimen incubated at 37” for 5 min; (b) if the specimen labeled at 37” for 5 min was pretreated with a mixture of chondroitinase-ABC, chondro-4sulfatase, and chondro-6sulfatase, the number of radioautographic grains was reduced to 5 to 10% of that in the untreated sample with a con- comitant decrease in intensity of metachromasia over the inter- cellular matrix; and (c) extraction of the labeled specimen with 4 M guanidine hydrochloride (37”, 24 hours) permitted a solubili- zation of almost all of the radioactive substances, of which about 95 y0 were susceptible to degradation with either chondroitinase- ABC or chondroitinase-AC.

Since our previous study (29) has indicated that the intracellu- lar proteoglycan fraction contains two components of different molecular-weights and sulfate-location (designated as Compo-

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TABLE 1 T~BLIC II

Dislribulion of DNA, chondroilin suljale, and collagen in four Analysis of [W]proline-labeled materials in jou~ zones

zones of epiphysial cartilage Epiphysial cartilages (about 1 g, wet weight) were incubated in Epiphyses of tibias and femurs from 30 embryos were placed in

chilled 270 perchloric acid for 30 min and then in 50% ethanol for 30 min. Each cartilage was dissected into the four zones, and like zones were combined, weighed, and homogenized in water. The resulting homogenates were dialyzed against running tap water and assayed for DNA, chondroitin sulfate, and collagen.

3 ml of Krebs-ascorbate containing 3 &i of [W4CJproline and washed with the carrier solution as described in the text. The tissues were then placed successively in 29; perchloric acid and in 505% ethanol as described in the note for Table I. Each cartilage was dissected into the four zones, like zones were combined, and each zone was homogenized in water. The homogenates were di- alyzed against running tap water. Aliquots of the dialyzed homogenates were assayed for W-labeled hydroxyproline. The remaining portions were thoroughly washed by repeating the pre- cipitation with 757;, ethanol containing l%;, potassium acetate and then assayed for collagenase-digestible material.

Total wet weight”

mg

381 272 247

164

I

-

DNAC Chondroitin sulfate”

~“~;f”,“,/,“” ny~s,/,g nmoler/ nmole DNA

1.49 19.5 13.1 1.15 28.7 25.0 0.431 38.9 88.1

0.151 33.0 219

a See Fig. 3. * Total weight of each zone from 30 embryos. c Expressed as dAMP (23). d Expressed as glucuronic acid (24). e Expressed as hydroxyproline (25).

COllagene

amoles/mg nmoles/ lissue nmole DNA

8.80 5.90

10.4 9.05 12.2 28.3 12.2 80.7

(Al . .

* FEMUR

“% CARTILAGE ,” ,....; “‘:“,

0 5 10 (mm)

(a)

ZONE NO. 1 2 3 4

r 1 0 1 2 3 4 5

DISTANCE FROM DISTAL END (mm)

DISTANCE FROM DISTAL END ( mm)

FIG. 3. A, schematic representation of longitudinal section of a tibia of a la-day-old chick embryo. B, schematic representation of the epiphysial cartilage of the tibia divided into four cellular zones. C, the synthetic activities for DNA, chondroitin sulfate, and collagen of chondrocytes in different zones. The number of cells over a 3800 pm2 area (x- - -x) was counted under a micro- scope at a magnification of 1,500: each point indicating the mean value of cell numbers of ten different fields within the indicated zone. The relative activities for isotope incorporation (O--O, (methyL3HJthymidine; O---O, %04’; A---A, [U-Wjproline) were calculated from the number of 3H-labeled cells per unit number of total cells (in the case of [methyL3H]thymidine incor- poration) or from the number of grains per unit number of total cells (in the cases of 3%0,* and [U-“Clproline incorporation). Values obtained with the most active zones are set as 1OO7o ac- tivity.

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zonea

1 2

3 4

Total radioactivity per unit number of

Radioactivity

cell* digestible with

collagenaseC

Radioactivity of hydroxyprolined

CM

686 905

2260 1721

I

%

54.4 25.7

63.9 29.3 71.1 34.0 68.3 20.2

0 See Fig. 3. b Total radioactivity (cpm) per nmole of DNA (as dAMP).

c The amount of radioactivity appearing in the supernatant after incubation with collagenase followed by ethanol precipita- tion was used as a measure of collagen degradation.

d Radioactivity of hydroxyproline (cpm) X 100 per total radio- activity (cpm).

nent H and Component L), it is desirable to characterize further the labeled materials in terms of these structural parameters. Indeed, closer examination of the labeled proteoglycan samples

from the different zones have shown that there is considerable heterogeneity among them which might relate to the mechanism of cytodifferentiation. Details of these experiments will be de- scribed in the accompanying paper (18).

Collagen-Incorporation of [ U-14C]proline into proteins pro- ceeded actively for 30 min and, at the end of the incubation, radioautographic grains were concentrated over the cells with a

few grains overlying the extraceIlular matrix. When the labeled tissue was chased with carrier proline, the matrix began t.o accu- mulate label and after 60 min was highly labeled.

To determine how much proportion of the radioautographic grains may represent collagenous substance, the four zones of the cartilage which had been incubated with [U-W]proline for 30

min were separately homogenized in water, dialyzed against water, and then subjected to digestion with bacterial collagen- ase.3 Labeled hydroxyproline in the homogenates was also deter- mined by the method of Juva and I’rockop (26). Data from the four zones are shown in Table II. There are significant differ- ences in the proportion of collagenase-sensitive radioactivity of

four zones; Zone 1 being lowest in the proportion of collagenase- sensitive radioact,ivity (54.4 y0 of the total radioactivity). Irre- spective of these differences, radioactive hydroxpproline accounts

for approximately 50% of the collagenase-sensitive radioactivity in all the specimens from four zones, indicating that the collagen- ase-sensitive fractions are similar in t,hat. they have a hydroxy- proline to proline ratio of about 1: 1. This ratio is in accordance with the previous findings of Trelstad et al. (2) and Miller (3)

a Since direct treatment of the labeled specimens with bacterial collagenase caused damage to the tissues, histochemical identifica- tion of the grains has not succeeded.

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FIG. 4. Chromatography on Bio-Gel A-5m of the extract from Zone 1 of epiphysial cartilage incubated with [U-“Clproline for 30 min in vitro. The elution profiles of the extracts from the other zones were very similar to the profile of Zone 1 sample, hence those are not shown here. About 5 ml each of the guanidine hydrochloride extracts (see the text) was fractionated in a Bio-Gel A-5m column (2.1 X 140 cm), equilibrated with 2 M guanidine hydrochloride containing 50 mM Tris-HCl, pH 7.5, at 20”. Frac- tions of 5 ml were collected and assayed for both radioactivity and glucuronic acid (24). For the measurement of glucuronic acid, proteoglycan was precipitated with 3 volumes of ethanol contain- ing 1.3yc potassium acetate. The void volume of the column was in Fraction 34, and the column volume was in Fraction 98. Solid bar above curve indicates the fractions corresponding to a-chains, which were subsequently pooled and subjected to chromatography on CM-cellulose (see Fig. 5).

which indicate that the component of chick cartilage collagen, cul(II), has a hydroxyproline to proline ratio of approximately 1:1.2.

Fig. 3C illustrates the relative concentrations of radiocarbon (calculated from the radioautographic grains) over different zones of the specimen incubated with [U-W]proline for 30 min. The zone of the highest activity (per unit number of cells) comprises the site of transition of enlarging cells (Zone 3) to hypertrophied cells (Zone 4). The essential conclusion is not altered if the data are corrected for the radioactivities corresponding to noncollage- nous proteins (see Table II). In the cells over Zone 1 and Zore 2 which are synthesizing DNA and chondroitin sulfates, respec- tively, at a maximum rate, the collagen synthesis proceeds at one- seventeenth to one-twentieth the rate occurring in the cells over the most active zone.

In order to characterize further the labeled material, the four zones of the cartilage which had been incubated with [ UJ4C]pro- line for 30 min were separately suspended in 15 volumes of 4 M

guanidine hydrochloride (containing 50 mM Tris-HCl, pH 7.5) and heated in a boiling water bath for 5 min. The suspensions were then stirred for 40 hours at room temperature. ‘The cartilage residues were removed by centrifugation. From each zone, more than 80% of the labeled material could be brought into solution by this treatment, there being little difference in the extractability among the four zones. The endogenous (unlabeled) collagen, on the other hand, was much more resistant to this extraction as assessed by measurement of unlabeled hydroxyproline; about 90 y0 of the total hydroxyproline remaining insoluble in each case. The difference in extractability between the labeled material and the unlabeled collagen appears to reflect differences in the degree of intermolecular cross-linking of intra- and extracellular collagens.

The labeled materials extracted from the four zones of cartilage were chromatographed separately on a column of Bio-Gel A-5m in 2 M guanidine hydrochloride containing 50 mM Tris-HCl, pH 7.5. Fig. 4 shows a typical elution profile of Zone 1 sample. The

Y El0 c ZONE 2 , :~ ,

a

E CL’0

t

ZONE3, :: ,

” i?

- 5- -.. _( ,.\

12

- 0.1

1 ,;// . ! .:;,,,, :' i, ~.,_/...,

0 _x Ly 0 /

ZONE 4 3

2 I 0.1

Frc. 5. CM-cellulose elution patterns of the ‘%-labeled a-corn- ponents (t.he fractions indicated by the bar above curve in Fig. 4) obtained from four zones of epiphysial cartilage incubated with [U-“Clproline for 30 min. Each of the fractions obtained by Bio- Gel chromatography (Fig. 4) was concentrated to 5 ml on a L)iaflo PM-10 membrane. To this solution, 3 mg of acid-soluble collagen from rat skin were added, and the sample was dialyzed against five l-liter changes of 4 M urea containing 0.04 M sodium acetate, pH 4.8, at 4” for 2 days. The sample was heated to 40” for 10 min and immediately applied to a column (1.6 X 3 cm) of CM-cellulose (Whatman microgranular CM 32) that had been previously equili- brated at 40” with the urea-acetate buffer above. The column was developed at 40” by use of a linear salt gradient of NaCl from 0 to 0.072 M in the equilibrating buffer. This procedure is a modifica- tion of that described (30). Fractions of 2 ml were collected and assayed for both radioactivity and absorbance at 230 nm.

samples from the other zones also gave a similar elution pattern with a large peak eluted at or near the predict.ed position for single chain components ((2 chains). Very little double chain (p) or triple chain (y) component was detected as could be expected for an intracellular nascent collagen (for the molecular size of the samples, see below). The profile of uranic acid-react,ing material indicates t,hat the bulk of proteoglycan was separated from the major collagenous component by this chromatography.

The radioactive fractions eluting in the cr region were pooled, concentrated, and, after mixed with acid-soluble collagen from rat skin (internal marker), dialyzed at, 4’ against five l-liter changes of 4 M urea cont,aining 0.04 M sodium acetate buffer, pH 4.8. Each of the resulting solutions was heated to 40” for 10 min and chromatographed on a CM-cellulose column (Fig. 5). In all the zones there were t.wo major comp0nent.s whose elution posi- tions were distinct from either al(I) or cu2 of rat skin. The first component. (designated as “X”) was elut.ed slightly faster than al (I) and presumably identical with the crl (II) chain previously found in t.he cartilage of lathyritic chick (l-4). The elut,ion position of the second component (designated as “Y”) was char- acteristic enough to exclude from consideration all the known (Y

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TABLE III

Analysis of two collagenous components purijied from chick embryo cartilage

To the pooled fractions of Component X (total 48 ml) and Com- ponent Y (total 48 ml), guanidine hydrochloride was added to give a final concentration of 2 M. Each of the solutions was concen- trated to 3 ml on a Diaflo PM-10 membrane. From these solu- tions, collagenous material was precipitated and washed with ethanol as described in the note for Table II. Aliquots (1000 cpm each) of the samples were assayed for collagenase-digestible material and for labeled hydroxyproline.

Component” Radioactivity digestible with collagenas?

Radioactivity of hydroxyprolineC

GJm

X 921

I

442 Y 895 445

a See Fig. 5. * The amount of radioactivity appearing in the supernatant

after incubation with collagenase followed by ethanol precipita- tion was used as a measure of collagen hydrolysis.

c Measured by the method of Juva and Prockop (26).

FRACTION NUMBER

FIG. 6. CM-cellulose elution pattern of the r4C-labeled a-com- ponents obtained from Zone 1 of epiphysial cartilage incubated with [U-“Clproline for 30 min and then chased with carrier proline for 60 min. The elution profiles of the samples from the other zones were similar to the profile of Zone 1 sample, hence those are not shown here. The experimental methods for extraction from tissues, purification on Bio-Gel A-5m, and chromatography on CM-cellulose were essentially the same as those described in the legends for Fig. 4 and Fig. 5. The Bio-Gel elution profiles (not shown) indicated that there was only slight increase in the radio- activities corresponding to p and y components during the chase incubation.

components including al(I), crl(II), cr2, and so-called procolla- gens found in tendon, bone, and skin systems (30-34).

Both Component X and Component Y were susceptible t.o degradation with bacterial collagenase and were shown to have a hydroxyproline to proline ratio of about 1: 1 (Table III). It was furt.her possible to demonstrate a rapid decrea.se in r4C eluting with Component Y by incubat.ion of the pulse-labeled cartilages in a chase medium cont,aining unlabeled proline (Fig. 6; the samples from the other zones gave a similar pattern). Thus, the chromatograms of the 60.min chase samples indicate a composi- tion predominantly of Component X. These data can be inter- preted as suggesting that Component Y is a precursor or a trans- port form which may be convert,ed to t.he al(H) chain. If t.his hypothesis is tenable, the variations in the ratio of X to Y in the various zones (Fig. 5) may indicate that (rl(I1) is synthesized via

Component Y at higher rates by hypertrophied cells (Zone 4) than by flattened or round cells (Zones 1 and 2).

Aliquots of the fractions of Component X and Component Y (see Table III) were dissolved in 1 M CaClz containing 0.05 M

Tris-HCl, pH 7.5, and applied to a Bio-Gel A-5m column (1 X 68 cm, void volume, 20 ml, 40 fractions; total volume, 53 ml, 106 fractions). The column was eluted with the same CaClg solution at room t,emperature. Almost all the radioact,ivity of Compo- nent X eluted as a sharp peak (elution volume, 31 ml, 62 frac- tions), coinciding with (~1 (I) chain of the skin carrier. The radio- activity of Component Y, on the other hand, eluted at signifi- cantly faster rate (elution volume, 29 ml, 58 fractions).4 The apparent size of Component Y, estimated from these data by the met.hod of Yiez (35), was approximately 125,000, suggesting the existence of an extra peptide of about 30,000 molecular weight.

DISCUSSION

The primary purpose of the present paper has been to show the existence of a close relationship between changes in synthetic ac- t,ivitics for matrix macromolecules and cytological modifications occurring in chondroeytes of the developing epiphysis. It. is evi- dent that the cartilage of 12.day-old chick embryo is formed of chondrocytcs displaying different st,ructurc and metabolic activ- ity. Thus, in agreement with the microscopic observations that round cells in Zone 1 have a high nucleocytoplasmic ratio and show mitotic figures with high frequency, est.ensive labeling of cells in Zone 1 with [methyl-3H]thymidine has been observed (Fig. 3). Zone 2 is composed of flattened cells which display a prominent Golgi apparatus. The radioaut.ographic dat.a have indicated that the grains of 35Smlabeled chondroit,in su1fat.e were particularly concentrat,ed in this zone (Fig. 3), a result. which sup- ports the current knowledge that the elongation and sulfation of chondroitin sulfate occur in association w&h Golgi membranes (29, 36-39). Enlarging cells in the adjacent area (Zone 3) cease to divide and display a dilated, well developed endoplasmic reticulum. Those cells closer to the ossifying region are more highly vacuolated and degenerat’e in appearance (Zone 4). The difference in these cells may relate to t’he observed change in the rate of collagen synt,hesis; i.e. cells in Zone 3 showed a progressive increase in t,he rat,e of collagen synbhesis as they approach the site of kansition to Zone 4, beyond which sit.e an abrupt decrease was observed (Fig. 3).

These phenomena could be interpret,ed as indicat,ing t.hat the biosynthetic activities for matrix macromolecules are induced during the mitotic cycles of “primitive cells” in Zone 1 and suc- cessively expressed in “progeny cells” between Zone 2 and Zone 4. The idea that the production of a specialized substance is pre- cluded in proliferating cells was supported by n’ameroff and Holtzer (40). These authors showed that dividing chondrocytes did not synthesize chondroitin sulfate, and that cells making chondroit.in sulfate did not synt,hesize DNA. There are many contradictory examples, however, showing that the processes of differentiation and proliferation are not mutually exclusive. Davies et al. (41) demonstrated that collagen synthesis was not repressed in 3T3 and PR105 cells during chromosomal replicat.ion. Coon (42) described that, under clonal cult,ure conditions, chon- drocytes continued to divide and maintained the production of metachromat.ic matrix substance. The present study has af-

4 When the column was eluted with 2 M guanidine hydrochloride containing 50 rnM Tris-HCl, pH 7.5, the elution volumes of Com- ponents X and Y were 27 ml (54 fractions) and 25 ml (50 fractions), respectively, indicating a general trend toward a decrease in the elution volumes with the guanidine hydrochloride solution.

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forded new evidence indicating that, although the three synthetic processes tend to be carried out in alternating waves, those are not strictly exclusive from one another in a growing epiphysis. The radioautographic grains of %-labeled chondroitin sulfate, for example, have been observed in all of the cells including those in proliferation (Zones 1 and 2) or in active production of collagen (Zone 3). There is no reason, therefore, to believe that the cells in Zone 1 would be primitive in regard to the synthetic activity for matrix macromolecules.

According to Fell (16) and our own histological studies, it is highly probable that the heterogeneous structure of the cartilage in la-day-old chick embryo is a prerequisite for the later develop- ment of the epiphysis which may be characterized by the forma- tion of more sharply demarcated zones (e.g. articular cartilage, epiphysial plate, diaphysis, and marrow cavities). Therefore, the variations in the synthetic activities in different zones of the immature epiphysis suggest the existence of a plan of specializa- tion of matrix macromolecules to fabricate these sharply defined parts of cartilage with different physiological functions. It is possible that this plan includes not only changes in metabolic rates of proteochondroitin sulfate and collagen, but also struc- tural modifications of the matrix macromolecules. In the accom- panying paper (18), some evidence is presented to prove that there are indeed structural differences among the proteochon- droitin sulfates produced by the cells in different zones.

Earlier studies on collagen extracted from various tissues of the lathyritic chick (adult) demonstrated that collagen derived from both sternal and epiphysial cartilages contains a genetically distinct type of crl chain, al(II), which is not present in collager! extracted from chick bone or skin (I). Subsequent work on the lathyritic long bones (43) indicated that the (~1 to (~2 ratios in the diaphysial collagen and the growth (epiphysial) plate collagen are consistent with the composition [(~l(I)]~a2 and [cul(II)]a, respec- tively, but the ratio for the junction (metaphysial) collagen cor- responds to a mixture of the two forms. These results with adult cartilages raised the question as to whether the transition of col- lagen type may occur in the diaphysial region (Zone 4) of develop- ing cartilage. The results of the present study indicate clearly that there is no detectable activity for [cul(I)lza2 synthesis throughout the cartilagenous regions of the developing epiphysis. It is thus conceivable that the cells in the cartilagenous region are more or less specialized elements whose function is the secretion of the cartilage matrix consisting of [crl (11)13 and they are sharply discriminated in this respect from t.he cells in the ossifying region whose function is the secretion of the bone matrix consisting of bl(I)lzcr2.

An additional finding worthy of comment is that incubation of the epiphysis for short times in the presence of radioactive proline revealed the existence of a collagen fraction (Component Y) which differed from the known a-chains of collagen, cul (I), al (II), and cr2, in its position of elution from CM-cellulose (Fig. 5). This new component appears to be larger in molecular size than cul(II), as assessed by Bio-Gel A-5m chromatography. The chase experiments (Fig. 6) indicate that radioactivity in this peak was decreased at a strikingly high rate. A compensatory in- crease in the radioactivity of crl(I1) has been demonstrated by our recent pulse-chase experiments5 Indeed, this figure bears a striking resemblance with respect to the time course to the radio- autographic profiles in which the intracellular space was the site of high radioactivity which moved to the extracellular field after 60 min of chase incubation. All these facts support the interpre-

6 Unpublished data.

2

3 4 5

6

7.

8.

9.

10.

11. 12. 13.

14.

15.

16. 17.

18.

19.

20.

21. 22. 23. 24. 25.

26. 27.

28.

29.

30.

31.

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M. J. (1967) J. Biol. Chem. 242, 5481-5489 TSIGANOS, C. P., HAILDINGH,\M, T. 15., ,\ND MUIR, 1-I. (1971)

Biochim. l$ophys. Acta 229, 529-534 W~STESON, A., LINDHAL, U., AND HAL&N, A. (1972) Biochem.

J. 130, 729-738 MATHEWS, M. B., AND GLBGOV, S. (1966) J. C&in. Invest. 46,

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348-352 HAINJCHI, H., YAMAGATA, T., IWATA, H., AND SUZUKI, S.

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J. Biol. Chem. 249. 1646-1663 YRMAGATA, T., SAITO; H., ITANJ~HI, O., AND SUZUKI, S. (1968)

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chemistry 2, 58-66 Knrcus, H. A. (1950) Biochim. Biophys. Acta 4, 249-269 SATO, T. (1968) J. Electron Microsc. 1’7, 158-159 BUILTON, K. (1956) Uiochem. J. 62, 315-323 BITTER, T., ‘(ND MUII~, H. M. (1962) Anal. Biochem. 4,33(f334 PI~OCKOP, 1). J., ,~ND UDENFNEND, S. (1960) Anal. Biochem. 1,

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After the completion of this study, the synthesis and secretion -

of procollagen were reported by Dehm and Prockop (46) using freshly isolated cells from sternal cartilage of 17-day chick embryo.

tation that Component Y is a biosynthetic precursor of cul(I1). Using tendon, skin, and bone systems, it has been demonstrated that crl (I) and ar2 are derived from the corresponding precursors, pro-al(I) and pro-a2 (for example, see Ref. 30-34). Further- more, a procollagen peptidase activity, which is able to convert t,he procollagens to normal cy chains, has been detect.ed in various connective tissues including cartilage (44,45). The results cited in the present paper strongly suggest t,hat an analogous mecha- nism may operate for the biosynthesis of [al (II)h in the epiphy- sis.6

Acknowledgment-We are indebted to Dr. T. Hama for his valuable discussions and for extending to us the hospitality of his laboratory for preparation of the tissue sections for histology.

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Munemitsu HoshinoAtsuhiko Oohira, Koji Kimata, Sakaru Suzuki, Kenzo Takata, Ikuo Suzuki and

Specific Stages of Cytodifferentiation in Developing CartilageA Correlation between Synthetic Activities for Matrix Macromolecules and

1974, 249:1637-1645.J. Biol. Chem. 

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