J. Embryol. exp. Morph. Vol. 23, 1, pp. 169-184, 1970 169 Printed in Great Britain

Electron microscopic studies on developing

cartilage

I. The membrane system related to the synthesis and secretion of extracellular materials

By S. C. GOEL 1

From the Institute of Animal Genetics, West Mains Road, Edinburgh 9

The present communication describes the changing developmental pattern of the cellular organelles concerned with the synthesis and transfer of the extra­cellular materials during the process of chondrogenesis in chick limb-buds.

Cartilage consists of a large amount of extracellular phase interspersed with chondrocytes. Chemically it is well established (Eastoe, 1961) that the main con­stituents of the extracellular phase are collagen and protein-polysaccharides. The latter are made up of a non-collagenous protein and acid mucopolysac­charides. The acid mucopolysaccharides are chondroitin sulphate A and C (Godman & Porter, 1960; Jackson, 1964).

In electron micrographs, the extracellular phase is seen as an amorphous electron-translucent ground substance interlaced by fibrous material overlaid with electron-dense granules. The chemical interpretation of this ultrastructure is still dubious (Matukas, Panner & Orbison, 1967). It is usually considered that the amorphous ground substance consists of the acid mucopolysaccharides, the noncollagenous proteins and tropocollagen, which is a soluble form of collagen (Godman & Porter, 1960).

From studies on developing cartilage and other tissues (Godman & Porter, 1960; Sheldon & Kimball, 1962; Revel & Hay, 1963; Caro & Palade, 1964; Jackson, 1964; Porter, 1964; Jacob & Jurand, 1965; Fawcett, 1966; Kessel, 1966; Perry & Waddington, 1966; Jamieson & Palade, 1967; Henrikson & Matoltsy, 1968; Silberberg, 1968; and others) the following general pattern of events explaining the synthesis and secretion of extracellular materials has been formulated. The proteins needed for intracellular use are believed to be synthesized by polysomes, lying free in the cytoplasm (Karasaki, 1964; Porter, 1964; Fawcett, 1966). The proteins required for transfer to extracellular sites, on the other hand, are considered to be synthesized by ribosomal groups that are attached to the endoplasmic reticulum. The product so synthesized is

1 Authors address: Department of Zoology, University of Rajasthan, Jaipur, India.

170 S. C. GOEL

transferred from the cisternae of the rough endoplasmic reticulum to the Golgi apparatus. The vacuoles of the Golgi apparatus migrate to the cortical parts of the cell, their membranes fuse with the plasmalemma and eventually form an opening or stoma with the extracellular space where they secrete their contents by way of merocrine secretion. The addition of the carbohydrate moiety to the protein, if necessary, takes place in the area of the Golgi apparatus. However, the details of various processes and various other ways that possibly may be used by the cell to elaborate the extracellular substances are still under discussion (Revel & Hay, 1963; Porter, 1964; Ross & Benditt, 1965; Cooper & Prockop, 1968; Reith, 1968; Ross, 1968).

MATERIALS AND METHODS

Chick embryos of the Brown Leghorn variety were incubated for varying times in a humid atmosphere at 38 °C and were staged according to Hamburger & Hamilton (1951).

For studies on cartilage developed in vivo the embryos were taken out of the eggs in Hanks's balanced salt solution and fixed in 2 % osmium tetroxide in veronal buffer at 4°C for 45-60 min : the fixation of chondrocytes at stage 39 is improved by a prior removal of perichondrium from cartilage. During dehydra­tion in graded alcohols the hind limb-buds were excised and then embedded in Araldite. The silver to gold coloured sections obtained from a Porter-Blum ultramicrotome using glass knives were mounted on carbonized collodion-coated grids. After staining with uranyl nitrate (20-30 min) and lead citrate

EXPLANATION OF FIGURES

Abbreviations, cen, centromere; ch, chondrogen granule; cs, cytosomes; dg, electron-dense granule of extracellular phase; dnl, electron-dense part of nucleolus; er, endoplasmic reticulum profiles; ƒ, fibres of the extracellular phase; G, Golgi appar­atus; gv, vacuoles of Golgi apparatus; ics, intercellular space; M, mitochondrion; m, matrix of extracellular phase; N, nucleus; nl, nucleolus; nm, nuclear membrane; np, nuclear pore; p, polysomes; pin, pinocytotic vesicles; pi, plasmalemma.

All electron micrographs are from tissues fixed in 2 % Os04 in veronal buffer, em­bedded in Araldite and stained with uranyl acetate and lead citrate.

FIGURE 1

(A) Mesodermal cell from chick limb-bud stage 23 ( x 25700). Note the fragmentary nature of plasmalemma {pi) and the paucity of profiles of rough endoplasmic reticulum (er). (B) Mesodermal cell from chick limb-bud stage 23 ( x 20700). Note the blebbing of outer nuclear membrane (arrow), and characteristic appearance of the Golgi appara­tus (G). (C) Prechondrogenic cell from chick limb-bud stage 26 (x 31400). Notice the con­tinuity of the plasmalemma (pi), presence of rough endoplasmic reticulum profiles (er) and the electron-translucent intercellular space (ics).

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(20 min), the sections were viewed in AEI EM 6 or Phillips 75 electron micro­scope (see Goel & Jurand, 1968, for details of various procedures).

For studies on cartilage developed in vitro, hind limb-buds from 4-day-old embryos were used. The excised buds were incubated with calcium- and mag­nesium-free balanced salt solution (CMF) for 15 min at 38 °C. This was followed by further incubation for 7-10 min in CMF containing 1 % of trypsin (crystal­lized and lyophilized, from Worthington Biochemical Co., Freehold, New Jersey; minimum activity 150 units/mg, I.U.B. system). The buds were then carefully washed in Hanks's balanced salt solution and transferred to standard culture medium. The ectodermal jacket was removed and the chondrogenic mesoderm cut into small pieces approximately 0-75 mm3. Hanging drop cul­tures were set up and the culture medium was changed every 48 h. The medium consisted of Minimal Essential medium of Eagle (with Earle's salts) supple­mented with 10 % horse serum (from Flow Laboratories, Irvine, Scotland) and 50 % fresh chick embryo extract. The process of fixation and preparation of the material for electron microscopy was similar to that used for in vivo studies.

RESULTS

(a) Mesenchyme cells (stage 23)

These appear from the unspecialized arrangement of the cell organelles to be undifferentiated. In the photomicrographs the plasmalemma is usually either missing over large areas of cells or is fragmentary and vesiculated (Fig. 1 A, B). The mesodermal tissue gives an impression of being to some extent syncytial; however, the discontinuities of the plasmalemma are possibly preservation artifacts, because sometimes the plasmalemma is well preserved at the sites of cell-to-cell attachment while it is missing at the sites where the cell is adjacent to the extracellular phase. Tormey (1964) also reported breakdown of plasma-lemma after osmium tetroxide fixation of ciliary epithelium from eyes of albino rats; and similar observations were made on mesenchyme from mouse (author's unpublished observations). The intercellular spaces are electron-translucent and are devoid of any fibrous components.

FIGURE 2 (A) Chrondroblast from chick limb-bud stage 31 ( x 25700). The arrow indicates the vesicular profile of the endoplasmic reticulum which may well contribute to the for­mation of Golgi apparatus (G). (B) Chondroblast from chick hind limb-bud stage 31 (x 25700). Notice the profile with arrow—the lower half has attached ribosomes, while the upper half forms part of the Golgi apparatus. (C) Chondroblast from chick hind limb-bud stage 31 ( x 27800). The arrow indicates the connexion between the endoplasmic reticulum (er) and the intercellular space (ics). Also notice the electron-translucent nature of Golgi vacuoles (gv) and moderately electron-dense contents of saccular cisternae of endoplasmic reticulum.

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In the cytoplasm the ribosomes are numerous and occur either singly or in polysomal groups with up to 5-8 ribosomes in each group. The only membran­ous structures to which they are sometimes attached are the vesicles of the endoplasmic reticulum. The reticulum at this stage consists mainly of smooth-membraned vesicles usually about 100-200 nm in diameter. Their contents are hyaline or translucent. The Golgi apparatus consists of one or two groups of 3-5 lamellae and a few vesicles, about 100 nm in diameter, on either side of these lamellae (Fig. IB). The nucleus is centrally placed and very large. The nuclear envelope is usually very well preserved and its outer membrane is some­times continuous with vesicular profiles. It frequently shows nuclear pores containing very electron-dense material.

(b) Prechondrogenic cells {stage 26)

Although in the light-microscopic pictures the prechondrogenic cells do not differ from the undifferentiated mesoderm cells, the electron micrographs of these cells show definite signs of activation of protein-synthesizing apparatus. The cytoplasm is filled with free-lying polysomes. The endoplasmic reticulum profiles are elongated and their membranes studded with ribosomes (Fig. 1C). There is no noticeable change in the structure of the Golgi apparatus. The plasma-lemma is quite well defined. The extracellular spaces are still electron-translucent. There are very frequent cytoplasmic extensions forming intercellular junctions with the extensions of neighbouring cells.

(c) Chondrogenic cells {stage 31)

The chondroblasts present an ultrastructural appearance characteristic of cells engaged in synthesis of material for extracellular use. The cells have a scalloped or bayed appearance, probably due to the opening out of the vacuoles containing extracellular material (Fig. 2C).

In the cytoplasm the ribosomes are present either as granules attached to the endoplasmic reticulum or as free-lying polysomal groups. The endoplasmic reticulum is now well developed and consists mainly of interconnected elongated cisternal profiles about 60-70 nm across (Fig. 2B, C). The cisternae are often dilated, forming saccular cisternae of various sizes; some are as large as 0-3 jn across and 0-6 JLI long. The saccular cisternae are never regular in outline. The system of endoplasmic reticulum is filled with an amorphous moderately electron-dense material and its surface membranes are studded with ribosomes. The cisternae are seen to reach and fuse with the plasmalemma; but are only very infrequently seen to contact the extracellular space as in Fig. 2C. The Golgi apparatus is the most outstanding cell organelle and consists of one or two groups of flattened lamellae, an enormous number of small vesicles and a few vacuoles. In Fig. 2B (arrow) the continuity between a smooth lamella of the Golgi apparatus and a profile of rough endoplasmic reticulum is seen. Moreover the vesicles that seem to contribute to the Golgi lamellae appear to be connected

Studies on cartilage 175

with the endoplasmic reticulum (Fig. 2 A, arrow). The Golgi vesicles are usually oval or circular, smooth-membraned sacs about 70-80 nm in diameter and are at times indistinguishable from the vesicles of the endoplasmic reticulum. The Golgi vesicles as well as the lamellae contain a moderately electron-dense amorphous material similar to that in the endoplasmic reticulum. The Golgi vacuoles, on the other hand, are large and usually rounded with electron-translucent contents. They are seen towards the maturing phase of the Golgi apparatus and seem to be derived from the swelling of the Golgi lamellae (Fig. 2 A) or by the enlargement and fusion of small vesicles (Fig. 2B, C). The electron-transparency of the contents of the vacuoles is proportional to their size.

The Golgi vacuoles and the saccular cisternae of the endoplasmic reticulum, therefore, differ from each other mainly in three respects : first, the contents of the Golgi vacuoles are electron-translucent while those of the cisternae are homogeneous and moderately electron-dense; second, the vacuoles are regular in outline while the cisternae are not; third, the limiting membrane of the vacu­oles is smooth while that of the cisternae is studded with ribosomes (Fig. 2B, C). These differences persist in the chondrocytes, except that the Golgi vacuoles often acquire a central core of fibrillar material, the chondrogen granule (Figs. 3 C, 4 A). The difference between the Golgi apparatus and the system of endoplasmic reticulum is easily noticed in low-power electron micrographs, because the Golgi area, due to its vacuoles, appears hyaline.

Another interesting feature of the chondroblasts is the phenomenon of ' ecdy-sis' ('excortication ' or 'delamination'), which is noticeable in the chondro­blasts differentiated in vivo as well as in vitro. The electron-microscopic evidence for it consists of the frequent association between the cell membrane and the fibrous elements of the extracellular matrix (Fig. 3 A, B). The fibres do not penetrate deeply into the cell cytoplasm. They are either continuous with the cell plasmalemma itself or seem to go just under the plasmalemma, run parallel to it for a little distance and fade away. Moreover, the otherwise well-defined cell membrane usually appears indistinct at such points of contact between plasmalemma and fibres.

In addition, small vesicular invaginations of the plasmalemma, probably pinocytotic vesicles, are noticeable during this period (Fig. 2B). The contents of the invaginations are sometimes continuous with, and show a moderate electron opacity similar to, the cell surface-coat.

In the micrographs the matrix of the cartilage at this stage consists of an amorphous, largely electron-translucent ground substance with a fair number of short unbanded fibres embedded in it. At times some electron-dense granules are also observed on the fibres.

176 S. C. GOEL

(d) Chondrocytes (stage 39)

The cells by now have become regular in outline and have almost no con­nexions with the neighbouring cells.

The endoplasmic reticulum remains very well developed (Fig. 3C, 4A). Its saccular cisternae are much larger and more numerous; they may measure up to 5 f.L in length. The contents of cisternae are still moderately electron-dense and amorphous and their boundary membranes remain granular. They do not often seem to open to the outside but are at times in continuity with the nuclear envelope (Fig. 4B). The moderate electron-opacity of the general cytoplasm is accentuated by the presence of numerous ribosomes which are usually arranged in helical groups (Fig. 4C).

The Golgi apparatus also is very well developed (Fig. 4A) and usually occu­pies a juxtanuclear position. It differs from the chondroblastic Golgi apparatus chiefly in the relative abundance of the Golgi vacuoles and the scarcity of vesicles and lamellae. Moreover, the vacuoles almost invariably contain a chondrogen granule which, under high power, appears remarkably similar to some components of the extracellular phase (Fig. 3C); it consists of a moder­ately electron-dense amorphous substance in which are embedded fibrillar elements and small electron-opaque granules. However, both the fibrils and the granules in the vacuoles are thinner and smaller respectively, as compared to the corresponding components of the extracellular phase.

Sometimes certain vacuoles, the cytosomes, are seen; these contain either a small number of vesicles or lipid-like substances (Fig. 4 A). A variety of similar structures is very common in the cartilage cells developed in vitro (Fig. 3B). The function of these structures is not very clear.

The extracellular phase consists of an amorphous electron-translucent ground substance and a large amount of fibrous material (Fig. 4D). The 20-25 nm thick fibres are usually long and straight, each consists of two or three thin fibrils, 7-10 nm in thickness. These fibrils sometimes show a periodicity of 9 nm with a light band of 3 nm and a dark band of 6 nm. Along the course of the fibres polygonal electron-dense granules are distributed at intervals of about 30 nm. The granules usually measure 20-30 nm across, but may be as large as 45 nm.

FIGURE 3

(A) Chondroblast from chick hind limb-bud stage 31 ( x 27800). Notice the process of 'ecdysis' (arrow). (B) Cell from chick cartilage developed in tissue culture (x 20700). The arrows indicate the points of 'ecdysis'. Also notice the fibrous material (ƒ). The cytosomes (cs) are unusually prominent. (C) Chondrocyte from chick limb-bud stage 39 (x 25700). Compare the nature of the contents of the Golgi vacuoles (chondrogen granule, ch) with those of matrix (m) and the saccular cisternae of the endoplasmic reticulum (er). The proximity of Golgi vacuoles (gv) to the plasmalemma (pi) suggests that it may soon open out.

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DISCUSSION

(a) Non-collagenous proteins

The present observations indicate an ultrastructural similarity between the vesicles of the endoplasmic reticulum and the Golgi apparatus (Fig. 2 A). More­over, a direct continuity between the ribosome-studded elongated cisternal profiles of the endoplasmic reticulum and the smooth lamellae of the Golgi apparatus has also been observed (Fig. 2B).

Jamieson & Palade (1967) presented electron-microscopic autoradiographic evidence of the active transport of material from the endoplasmic reticulum to the Golgi apparatus through membrane-bound vesicles in pancreatic tissue. Caro & Palade (1964) have already presented evidence that the only site of protein synthesis in pancreatic tissue is the ribosomes.

The present observations therefore support the work of Palade and co-workers and are consistent with the assumption that the non-collagenous protein is also synthesized in the endoplasmic reticulum and then transported to the Golgi apparatus, where it is combined with the mucopolysaccharides.

(b) Mucopolysaccharides

The results reported here show that as cartilage development proceeds, most of the Golgi vacuoles lose their former hyaline appearance and acquire a 'chondrogen granule' consisting of a number of smaller electron-dense granules similar to those of the matrix. Similar structural changes can be observed in the electron micrographs in other publications, though they are not always men­tioned (Godman & Porter, 1960; Sheldon & Kimball, 1962; Revel & Hay, 1963; Matukas et al. 1967; Silberberg, 1968).

Matukas et al. (1967), by adapting colloidal iron staining to electron micro­scopy, showed that the electron-dense granules of the matrix are centres of accumulation of the protein-polysaccharide moiety. They also suggest that the granules may represent clumps formed by fixation of protein-polysaccharide chains, which in vivo may extend in the hyaline space between the fibres and

FIGURE 4 (A) Chondrocyte from chick hind limb-bud stage 39 ( x 20700). Note the difference between the contents of Golgi vacuoles (gv) and the cisternae of the endoplasmic reticulum (er). All the cellular organelles now appear more electron-dense than in early stages (compare Fig. 1). (B) Chondrocyte from chick hind limb-bud stage 39 ( x 20700). The arrow indicates the connexion between the endoplasmic reticulum (er) and the external nuclear membrane (nm). (C) Polysomal patterns (pi) from chondrocyte of chick stage 39 ( x 20700). (D) Extracellular phase of cartilage from chick embryo stage 39 ( x 91000). Notice the incipient form of periodicity in fibres (ƒ).

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granules. Although Smith, Peters & Serafini-Fracassini (1967), working with bovine articular and nasal cartilage, consider the protein-polysaccharide macro-molecules either to be lying free in the extracellular phase or to be attached around the circumference of the collagen fibre over the a and bx bands, their evidence is by no means compelling. There is also evidence of the presence and sulphation of the acid mucopolysaccharides in the Golgi apparatus (Fewer, Threadgold & Sheldon, 1964; Godman & Lane, 1964; Matukas et al. 1967).

Therefore, it seems plausible that the chondrogen granules represent the protein-poly saccharides that are ready to be released into the extracellular phase by merocrine secretion (Fig. 3C).

(c) Fibrogenesis

In the literature the fibres of the extracellular phase in cartilage are assumed to be made of collagen, though there is no cytochemical evidence for it at the ultrastructural level (Jackson, 1964). In chondroblasts, as well as in fibroblasts and odontoblasts, the site of formation of electron-microscopically visible fibres of the matrix has always been a disputed subject; cytoplasm cell surface, and extracellular space have all been suggested as the sites of fibre formation.

The two relevant observations in the present study are as follows, (a) The saccular cisternae of the endoplasmic reticulum always differ from the Golgi vacuoles in the ultrastructural appearances of their contents and may at times open to the extracellular space (Fig. 2C and the author's unpublished observa­tions on mouse limb-bud cartilage), (b) The phenomenon of 'ecdysis' is quite common in cartilage developed both in vivo and in vitro (Fig. 3 A, B).

As there is evidence that collagen is synthesized as tropocollagen molecules on the ribosomes of the granular endoplasmic reticulum (Revel & Hay, 1963; Kretsinger, Manner, Gould & Rich, 1964; Fernandez-Madrid, 1967), it is reasonable to assume that the moderately electron-dense material of the saccu­lar cisternae in the present study is mainly tropocollagen. The observation of a direct opening out of the cisternae can, therefore, be interpreted to mean that the collagen is directly secreted from the endoplasmic reticulum. Similar sug­gestions have been made in the past (Godman & Porter, 1960; Ross & Benditt, 1965; Ross, 1968), although no electron micrographs showing a direct opening of the endoplasmic reticulum to the outside have been presented. Revel & Hay (1963), on the other hand, working with amphibian cartilage, deny any direct connexion of endoplasmic reticulum to the outside, and suggest the endoplasmic reticulum-Golgi apparatus route for the release of tropocollagen.

The release of tropocollagen molecules directly from the ground cytoplasm through the cell plasmalemma has also been suggested (Reith, 1968; Cooper & Prockop, 1968). Reith (1968) suggests that the soluble tropocollagen molecule 'threads its way through the cell membrane at the expense of time, rather than passing through as a coiled globular mass, the part of the molecule that is out of the cell would aggregate with other emerging threads because of the

Studies on cartilage 181 extracellular conditions'. Related to the suggestion of Reith (1968) is the pro­cess of 'ecdysis' (Porter & Pappas, 1959; Godman & Porter, 1960; Yardley, Heaton, Gaines & Shulman, 1960; Chapman, 1961) in which the collagen is secreted directly from the ground cytoplasm by delamination of the fibres from the plasmalemma. But considering the thickness of the electron-microscopic sections (70-90 nm) and the fibre diameter (20 nm), the nature of 'ecdysis' and the role played by it in fibrogenesis cannot be properly judged till there is corroborative evidence from cytochemistry or autoradiography.

(d) Conclusions

The electron-microscopic evidence presented here indicates the following points. (1) Throughout chondrogenesis the contents of the saccular cisternae of the endoplasmic reticulum are different from those of the Golgi vacuoles (Figs. 2C, 3C, 4A). (2) The endoplasmic reticulum communicates directly with the Golgi apparatus (Fig. 2B), with the outside (Fig. 2C) and with the external nuclear membrane (Fig. 4B), suggesting that the various membrane systems are interconvertible. (3) The ultrastructure of the chondrogen granule in the Golgi vacuoles (Fig. 3C) is very similar to those components of the extracellular phase that presumably represent the protein-polysaccharides.

It is suggested that in cartilage all the proteins destined for the extracellular phase are synthesized on ribosomes attached to the endoplasmic reticulum. From here the non-collagenous protein is transferred to the Golgi apparatus, where it combines with the mucopolysaccharides to form the protein-polysaccharide complex, which is then extruded by merocrine secretion. The collagenous pro­tein, on the other hand, may well be secreted directly from the endoplasmic reticulum to the outside. The present hypothesis predicts that the main intra­cellular site of accumulation of the collagen is the endoplasmic reticulum, and not the Golgi apparatus. Further cytochemical work is therefore needed to decide the question of the synthesis of the extracellular phase.

SUMMARY

1. The developmental changes in the cellular organelles concerned with the synthesis and transfer of the extracellular material in embryonic chick limb cartilage have been studied by means of electron microscopy. Some observa­tions were also made on cartilage developed in tissue culture.

2. The results indicate that differentiating chondrogenic mesenchymal cells acquire a very well-developed endoplasmic reticulum, a juxtanuclear Golgi apparatus and numerous polysomes. The endoplasmic reticulum shows three types of profile—elongated, dilated saccular and vesicular—all filled with a moderately electron-dense amorphous material. The Golgi apparatus also shows three types of profiles: lamellar, vesicular and large vacuolar. The lamellar and the vesicular profiles have moderately electron-dense material

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within them, but the vacuolar profiles are either electron-translucent or have a chondrogen granule inside them.

3. The endoplasmic reticulum has been seen in direct continuity with the external nuclear membrane, Golgi apparatus, and plasmalemma, indicating that the various components of the membrane system are interconnected.

4. The extracellular phase consists of fibres, electron-dense granules and amorphous ground substance. The phenomenon of 'ecdysis' on the cell surface is seen in the cartilage developed in the embryo as well as in tissue culture.

5. The significance of differences between the contents of the Golgi apparatus and the saccular cisternae of endoplasmic reticulum is discussed. It is sugges­ted that in cartilage the non-collagenous protein after synthesis in the endoplas­mic reticulum is combined with the polysaccharides in the Golgi apparatus and then secreted, whereas the collagen is secreted directly out of the endoplasmic reticulum.

RÉSUMÉ

Etude ultrastructurale du cartilage au cours de sa différenciation. I. Etude du système membranaire en relation avec la synthèse et la sécrétion de la substance extracellulaire.

1. Les modifications des structures cellulaires impliquées dans la synthèse et la sécrétion du matériel extracellulaire du cartilage de membre de l'embryon de Poulet ont été étudiées en microscopie électronique. Quelques études ont été également faites sur du cartilage différencié en culture in vitro.

2. Les résultats obtenus montrent que les cellules chondrogènes du mesen­chyme en voie de différenciation acquièrent un reticulum endoplasmique très bien développé, un appareil de Golgi juxtanucléaire et de nombreux polysomes. Le reticulum endoplasmique montre trois types de structure: allongé, dilaté en vésicules et saccules. Ces structures sont remplies d'un matériel amorphe faiblement opaque aux électrons. L'appareil de Golgi montre également trois types de structure: lamellaire, vésiculaire et de larges vacuoles. Les structures lamellaires et vésiculaires contiennent du matériel moyennement opaque aux électrons. Le matériel contenu dans les structures vacuolates est soit non opaque aux électrons soit formé d'un granule chondrogène.

3. On a constaté que le reticulum endoplasmique est en continuité directe avec le membrane nucléaire externe, avec le plasmalemme et l'appareil de Golgi. Ceci montre que les différents composants du système membranaire se joindrent.

4. La phase extracellulaire est composée de fibres, de granules opaques aux électrons et d'une substance fondamentale amorphe. Le phénomène d' 'ecdysis' au niveau de la surface cellulaire peut être observé aussi bien dans du cartilage formé in vivo qu'en culture in vitro.

5. La signification des différences de composition du contenu de l'appareil de Golgi et des citernes du reticulum endoplasmique est discutée. Il est suggéré que dans le cartilage les protéines autres que le collagène sont sécrétées dans le

Studies on cartilage 183

reticulum endoplasmique, puis associées dans l'appareil de Golgi à des poly­saccharides, et enfin rejetées. Le collagène, par contre, serait synthétisé et sécrété directement par le reticulum endoplasmique.

The author wishes to express his gratitude to Dr A. Jurand for encouragement as well as for reading and discussing the manuscript. The author is also thankful to Professor C. H. Wad-dington and Dr G. G. Selman for their invaluable comments on the manuscript, and for kind interest.

Thanks are also due to Mr F. Johnston for photographic assistance.

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{Manuscript received 6 February 1969)