Ultrastructural distribution of sulfated complex carbohydrates in elastic cartilage of the young rabbit.код для вставкиСкачать
THE ANATOMICAL RECORD 207547-556 (1983) Ultrastructural Distribution of Sulfated Complex Carbohydrates in Elastic Cartilage of the Young Rabbit MINORU TAKAGI, RICHARD T. PARMLEY, FRANCIS R. DENYS, MASATO KAGEYAMA, AND HIROSHI YAGASAKI Department ofdnatomy, Nihon University School ofDentistry, Tokyo, Japan (M.T, M.K., H.Y), and the Institute ofDental Research (M. T, R.TP., ER.D.) and the Departments of Pediatrics and Pathology (R.T P ) , University ofAlabama in Birmingham, Birmingham, AL 35294 ABSTRACT Sulfated glycosaminoglycans are a n integral component of elastic cartilage. We have investigated the ultrastructural distribution of sulfated complex carbohydrates (CC) in the mature cartilage and the perichondrium of young rabbit auricles using the high iron diamine-thiocarbohydrazidesilver proteinate (HID-TCH-SP) and the tannic acid-ferric chloride (TA-Fe) methods. In the mature cartilage, HID-TCH-SP stained intracellular Golgi saccules of the mature face, secretory granules, and the extracellular matrix granules, but staining was not discernible in collagen fibrils and osmiophilic elastic fibers consisting of only amorphous elastin. The HID and TA-Fe staining were similarly observed in matrix granules, whereas the elastic fibers and collagen fibrils lacked the staining. The pericellular matrix granules had a diameter of 34 5 nm (mean SD; n = 30). Thiery’s periodate-TCH-SP (PATCH-SP) method stained vicinal glycol-containing CC in collagen fibrils but failed to stain matrix granules and elastic fibers. In the perichondrium, HIDTCH-SP staining of the organelles was less intense in the flattened chondrocytes when compared with those in large mature chondrocytes. The extracellular HID and HID-TCH-SP staining were observed in the matrix granules. The diameter of pericellular matrix granules (19 4 nm, mean k SD; n = 30) was significantly smaller when compared to those in the mature cartilage (P < 0.001). The HID-TCH-SP staining was closely associated with collagen fibrils. However, the staining was not seen in collagen fibrils and osmiophilic elastic fibers consisting of elastin and microfibrils. The PA-TCH-SP method stained collagen fibrils and microfibrils but did not stain the amorphous elastin. Thus these studies demonstrate that sulfated CC are packaged in chondrocyte secretory granules and are released into the extracellular matrix to form matrix granules, but are not incorporated into collagen fibrils and elastic fibers. Elastic cartilage contains glycosaminoglycans (GAGs), collagen fibrils, and elastic fibers (Serafini-Fracassini and Smith, 1974). The content of elastic fibers makes this cartilage tissue unique in its physiochemical properties. Biochemical studies (Wusteman and Gillard, 1977) have indicated that the GAGs of ear elastic cartilage are composed of chondroitin sulfate and hyaluronic acid, but the definitive location of each type of GAG is not known. Recent light microscopic histochemical studies (Yamada et al., 1982) have demonstrated the presence of keratanase cc) 1983 ALAN R. LISS, INC. (Pseudomonas sp.1-digestible keratan sulfate in ear elastic cartilage, in addition to chondroitinase ABC- and AC-digestible chondroitin sulfate. Several ultrastructural cytochemical methods have been utilized to localize GAGs in elastic cartilage and have included ruthenium red (Myers, 1976; Nielsen, 19761, colloidal thorium (Yamada and HoshReceived J u n e 2, 1983; accepted July 27, 1983 Address reprint requests to Dr. Minoru Takagi, Department of Anatomy, Nihon University School of Dentistry, 1-8-13,KandaSurugadai, Chiyoda-ku, Tokyo, Japan. 548 M. TAKAGI ET AL. ino, 1973),and bismuth nitrate (Serafini-Fracassini and Smith, 1974). However, more specific stains for sulfated GAGS have not been utilized. The present study of ear elastic cartilage was undertaken utilizing various specific stains for complex carbohydrates to determine the subcellular route of the sulfated complex carbohydrate synthesis and secretion, and its subsequent distribution in the extracellular space, especially in relationship to collagen fibrils and elastic fibers. MATERIALS AND METHODS Tissue Preparation Tissue from the external ear of 1.5-kg rabbits was used in this study. Specimens were taken from the tip of the ear under anesthesia. Some specimens were fixed in 2.7% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.35) for 2 hours a t 4°C or 22"C, whereas others were fixed in 2.7% glutaraldehyde in 0.1 M cacodylate buffer (pH 6.8) containing 2% tannic acid (TA) (Futaesaku et al., 1972) for 2 hours a t 22°C. Subsequently, the specimens were rinsed several times in 0.1 M cacodylate buffer (pH 7.35) containing 7% sucrose and then stained a s outlined below. described previously (Thiery, 1967; Sannes et al., 1979).The TCH-diamine and/or iron complex presumably reduces SP to form a n electron-dense stain deposit. SP background staining was eliminated by filtering (Whatman filter #2) the SP solution twice before use. Acid MgC12 controls were similarly processed. TA-Fe method The aldehyde-TA-fixed, rinsed specimens were routinely dehydrated in graded alcohols and propylene oxide and embedded in Spurr low-viscosity resin. Thin sections mounted on stainless steel grids were treated for 10 minutes with a filtered fresh TA solution, pH 2.6-2.8, containing 5 gm tannic acid (J.T. Baker Chemical Co., Phillipsburg, NJ) in 95 ml of distilled water, rinsed three times in distilled water, and treated for 1minute with a filtered fresh ferric chloride (Fe) solution (pH 1.4-1.6) which was prepared by adding 5 ml of 40% ferric chloride (Fisher Scientific Co., Fair Lawn, NJ) to 95 ml of distilled water (Sannes et al., 1978; Takagi et al., 1983).Subsequently, stained sections were rinsed six times in distilled water and examined. Control specimens were also examined without the TA-Fe treatment or after exposing only to the TA or the Fe solution. Sulfated Complex Carbohydrate Staining HID-TCH-SP method Vicinal Glycol-Containing Complex The aldehyde-fixed, rinsed specimens withCarbohydrate Staining out TA were stained for 18 hours a t 22°C in a high iron diamine (HID) solution (Spicer, PA-TCH-SP method 1965; Spicer et al., 19671, which was prepared The aldehyde-fixed, rinsed specimens withby adding 1.4 ml of 40% FeC13 (Fisher Scien- out TA were routinely dehydrated and tific Co., Fair Lawn, NJ) to a fresh diamine solution containing 120 mg of N,N-dimethylm-phenylenediamine (HC1)z (Eastman Kodak Co., Rochester, NY) and 20 mg of N,NFig. 1. In mature auricle cartilage high iron diaminedimethyl-p-phenylenediamine(HCl) (Fisher thiocarbohydrazide-silverproteinate (HID-TCH-SPjstain Scientific Co., Fair Lawn, NJ) in 50 ml of deposits are visible in chondrocyte Golgi expanded sacH20. Control specimens (for evaluation of cules (ES), condensing vacuoles (CV), intermediate vacand mature secretory granules (SG) but are uoles (IW, intrinsic density) were incubated for 18 hours not present in mitochondria (Mt), rough endoplasmic at 22°C in a MgCl2 solution, pH 1.4, pre- reticulum (ER), flattened Golgi saccules (S), distended pared by adding 1.4 ml of 40% MgC12 to 50 portions (DS), coated vesicles (arrows), and the nucleus ml of HzO and adjusting the pH with HC1. (N). Unosmicated specimen, not counterstained. X 31,250. Some specimens were then postfixed in 1% Fig. 2. HID staining of osmicated specimens can be Os04, buffered with 0.1 M cacodylate, rouin elastic cartilage matrix granules (arrows)around tinely dehydrated, and embedded in Spurr seen a mature chondrocyte (C). HID-stained matrix granules (1969) low-viscosity resin. The post-osmica- attach to the osmiophilic elastic fiber (El. Not counterstained. ~31,250. tion step was omitted for other specimens. To enhance HID staining, some thin secFig. 3. HID-TCH-SP stain deposits are observed in tions were stained (by immersion) with a thiocarbohydrazide (Eastman Kodak Co., elastic cartilage matrix granules (arrows) in this unosmicated specimen. Elastic fibers (El lack staining; howRochester, NY)-silver proteinate (strong sil- ever, the stain deposits (arrowheads) can be occasionally ver protein, Roboz Surgical Instrument Co., found within the interstices of the elastic fibers. Not Inc., Washington, DC) sequence (TCH-SP) as counterstained. x 31,250. SULFATE IN ELASTIC CARTILAGE 549 550 M. TAKA,GI ET AL. embedded in Spurr low-viscosity resin. Cytochemical localization of periodate (PA)-reactive complex carbohydrates was accomplished by staining thin sections of the unosmicated specimens according to the PA-TCHSP method of Thiery (1967). Sections were mounted on stainless steel grids, oxidized in 1%periodic acid (G. Frederick Smith Chemical Co., Columbus, OH) for 45 minutes, rinsed five times for 10 minutes each in distilled water, and treated for 40 minutes with 2% thiocarbohydrazide in 20% acetic acid. After rinsing in 10% acetic acid and four times in distilled water, they were exposed for 30 minutes to 1%silver proteinate in the dark and rinsed six times in distilled water. As a control, periodic acid oxidation was omitted from the sequence. RESULTS Ultrastructural details of ear elastic cartilage have been described previously (Sheldon and Robinson, 1958; Anderson, 1964; Serafini-Fracassini and Smith, 1974; Cox and Peacock, 1977). In this study we have mainly described ultrastructural cytochemical results which were obtained from the mature cartilage and the perichondrium. The Mature Cartilage Mature chondrocytes were round, ovoid shaped, and contained a prominent Golgi apparatus consisting of several stacks of flattened saccules (Fig. 1).Distended portions of the first saccules of the stack were seen and lacked HID-TCH-SP staining. Toward the maturing face, a few saccules appeared more expanded than the others. At close proximity to those saccules, condensing and intermediate vacuoles, which were derived from the Golgi apparatus, could be observed and demonstrated minimal or moderate HID-TCH-SP staining. Mature secretory granules demonstrated intense HID-TCH-SP staining. Mitochondria, rough endoplasmic reticulum, and the nucleus lacked HID-TCH-SP staining (Fig. 1). In the extracellular matrix, matrix granules believed to represent proteoglycan monomer(s) (Hascall, 1980; Poole et al., 1982; Takagi et al., 1982) demonstrated intense HID staining. These granules were round, ovoid, elongated, or irregularly shaped and often adhered to collagen fibrils and elastic fibers consisting of only amorphous elastin without microfibrils (Figs. 2 , 7). The HIDreactive matrix granules, which were local- ized in the pericellular matrix, appeared to represent newly synthesized and secreted proteoglycans. These pericellular matrix granules had a diameter of 34 5 nm (mean + SD; n = 30; Fig. 2). HID-TCH-SP stain deposits were observed in matrix granules but were not seen in collagen fibrils and elastic fibers. However, HID-TCH-SP-positive sulfated material, which was entrapped in elastic fibers, was found within the interstices of the elastic fibers (Figs. 3, 4). HIDTCH-SP staining was similar in unosmicated and osmicated specimens; however, elastic fibers demonstrated intense osmiophilia a s previously reported by Thyberg et al. (1979). Acid MgC12 controls of unosmicated specimens exposed to TCH-SP lacked staining, whereas some fine TCH-SP stain deposits ( < 6 nm in diameter) were observed on a few membrane structures of osmicated control specimens. Elastic fibers demonstrated intense osmiophilia in the osmicated control specimens (Fig. 5). TA-Fe-stained matrix granules but did not stain collagen fibrils and elastic fibers (Fig. 6). Unosmicated control specimens without TA-Fe treatment lacked similar density. PA-TCH-SP staining was observed in collagen fibrils whereas matrix granules and elastic fibers lacked staining. Microfibrils were not associated with the amorphous component, elastin (Fig. 7). Control specimens + Fig. 4. HID-TCH-SP stain deposits can be seen in elastic cartilage matrix granules (arrows) in this osmicated specimen whereas osmiophilic elastic fibers (El lack staining. The stain deposits (arrowhead) are occasionally seen within the interstices of the elastic fibers. Not counterstained. x 31,250. Fig. 5. In osmicated MgCla control specimens treated with the TCH-SP sequence, matrix granules, collagen fibrils, and elastic fibers in the extracellular cartilage matrix lack stain deposits. However, elastic fibers (El demonstrate intense osmiophilia. Not counterstained. ~31,250. Fig. 6. Tannic acid-ferric chloride (TA-Fe) staining of the elastic cartilage matrix in this unosmicated specimen is localized in matrix granules (arrows) which are round, ovoid, elongated, or irregularly shaped, whereas elastic fibers lack staining. Not counterstained. x 31,250. Fig. 7. Periodate (PA)-TCH-SPstains collagen fibrils (Co) in the mature cartilage matrix whereas matrix granules and elastic fibers (E) lack staining. PA-TCHSP-positive microfihrils are not associated with the elastin (cf. Fig. 12). Unosmicated specimen, not counterstained. ~31,250. SULFATE IN ELASTIC CARTILAGE 551 M. TAKAGI ET AL 552 without PA lacked TCH-SP staining of the extracellular cartilage matrix. The Perichondrium Immature chondrocytes, which were localized in the perichondrium, had a flattened shape and contained a few HID-TCH-SPreactive secretory granules (Figs. 8,9). In the extracellular cartilage matrix, HID and HID-TCH-SP methods stained the matrix granules which were localized between and on the collagen fibrils. The diameter of HID-positive matrix granules (19 k 4 nm, mean SD; n = 30),which were localized in the pericellular matrix, was significantly smaller when compared to those in the mature cartilage (P < 0.001). Most collagen fibrils with a diameter of about 50-100 nm were more densely packed when compared to those in the mature cartilage (Figs. 9-11). HID and HID-TCH-SP staining were observed in the longitudinal sections of collagen fibrils, whereas transverse sections of these fibrils always lacked staining (Figs. 10, 11).Elastic fibers in this region mainly consisted of microfibrils and also possibly amorphous elastin, and demonstrated intense osmiophilia but lacked HID-TCH-SP staining (Figs. 8,9). Acid MgC12 control specimens of unosmicated specimens exposed to TCH-SP lacked staining, whereas some fine TCH-SP stain deposits were seen on a few membrane structures of osmicated specimens in which elastic fibers demonstrated intense osmiophilia. PA-TCH-SP moderately to intensely stained microfibrils with a diameter of about 10-15 nm and collagen fibrils, whereas the central amorphous component, elastin, and matrix granules lacked staining (Fig. 12). Control specimens without PA lacked TCHSP staining in these sites. DISCUSSION The present study has utilized ultrastructural cytochemical methods to localize sulfated and vicinal glycol-containing complex carbohydrates in elastic cartilage of young rabbit auricles. The more intense intracellular HID-TCH-SP staining observed in mature cartilage compared to the perichondrium indicates a gradual increase in synthesis and secretion of sulfated complex carbohydrates during chondrocyte differentiation from the perichondrium to the mature cartilage. The staining of intracellular sulfated glycoconju- gates in Golgi saccules and secretory granules of mature chondrocytes is similar to the staining of sulfated material in these sites in the hypertrophic chondrocytes from rat epiphyseal cartilage (Takagi et al., 1981).Extracellular HID and HID-TCH-SP staining in the matrix granule is in agreement with previous ultrastructural cytochemical and immunocytochemical studies (Takagi et al., 1982) that have demonstrated chondroitin sulfate and keratan sulfate in this site in rat epiphyseal cartilage, indicating that the matrix granule may represent proteoglycan monomer(s). Similarly, previous studies suggest that the matrix granule is formed by the collapsed proteoglycan(s) after tissue fixation, staining, and dehydration (Hascall, 1980; Poole et al., 1982).The lack of PA-TCHSP staining of vicinal glycols in matrix granules suggests that the sulfated material represents a glycosaminoglycan rather than a glycoprotein, consistent with biochemical isolation of chondroitin sulfate from this tissue (Wusteman and Gillard, 1977). The larger size of HID- and HID-TCH-SPpositive matrix granules in the mature cartilage compared with those in the perichondrium suggests that chondrocytes may secrete two different (i.e., small and large) proteoglycan monomers at both sites or that each matrix granule increases its content of sulfate and proteoglycan monomers during cartilage differentiation. The possibility of two different proteoglycan monomers is supported by previous biochemical studies (Franien et al., 1981)demonstrating that the superficial and the middle zone do not contain the larger proteoglycan monomers found Fig. 8. HID-TCH-SP staining in this osmicated specimen can be seen in the presumed secretory granule (SG, and enlarged in inset) of a flattened chondrocyte in the perichondrium. The extracellular staining is localized in the matrix granules but is not discernible in the collagen fibrils (Co) and the elastic fibers (El. Not counterstained. ~ 9 , 9 0 0Inset, . ~31,250. Fig. 9. In the perichondrium HID-TCH-SP stain deposits can be seen in the presumed secretory granule (SG) in an immature chondrocyte and the extracellular matrix granules (arrows). Elastic fibers (E, and enlarged in inset) mainly consisting of microfibrils and also possibly amorphous elastin lack staining but demonstrate the intense osmiophilia. Osmicated specimen, not counterstained. ~31,250.Inset, ~64,000. SULFATE IN ELASTIC CARTILAGE 553 554 M. TAKAGI ET AL in the deeper zone of articular cartilage. These results indicate that smaller proteoglycans probably contain less chondroitin sulfate (Franien et al., 1981). On the other hand, previous ultrastructural cytochemical studies indicate that each matrix granule represents one or several proteoglycan monomers (Hascall, 19801, and conceivably smaller matrix granules, such as those in the perichondrium, could contain fewer monomers than the larger matrix granules in mature cartilage. The lack of HID and HID-TCH-SP staining in elastic fibers, including amorphous elastin and/or microfibrils, is in contrast to previous light microscopic studies (Spicer, 1965; Gad and Sylven, 1969) demonstrating HID reactivity of elastic fibers. Our findings are in agreement with biochemical studies of elastic fibers indicating that elastin and microfibrils do not contain sulfated material (Ross, 1973; Serafini-Fracassini and Smith, 1974). However, HID-positive material was closely associated with elastic fibers and collagen fibrils in the present ultrastructural studies. This observation suggests that distinct resolution of elastic fibers, collagen fibrils, and sulfated material may not be possible at the light microscopic level. Anionic material, which is clearly localized outside collagen fibrils, has been ultrastructurally demonstrated around these fibrils in ear elastic cartilage (Myers, 1976) and other sites (Ruggeri et al., 1975; Shepard and Mitchell, 1977; Behnke and Zelander, 1970; Takusagawa et al., 1982) using cationic reagents. Immunocytochemical studies (Poole et al., 1982) of articular cartilage have localized particulate reaction products for proteoglycan monomers in close association with collagen fibrils. In contrast, recent ultrastructural cytochemical studies (Butler and Heap, 1982)have localized Alcian blue staining inside the collagen fibrils. The HID and HID-TCH-SP methods are more specific for sulfated complex carbohydrates and clearly provide adequate stain penetration in densely packed collagen fibrils. Although HID-TCH-SP stain deposits were observed in the longitudinal sections of collagen fibrils, their transverse sections always lacked the HID-TCH-SP staining. This observation suggests that sulfated complex carbohydrates are very closely associated with collagen fibrils; however, they are localized outside collagen fibrils. This interpretation is consistent with previous biochemical results (see review by Lindahl and Hook, 1978) utilizing a variety of techniques indicating that all glycosaminoglycans except hyaluronic acid, which lacks sulfate groups, and keratan sulfate, bind to collagen by electrostatic interaction at physiological pH and ionic strength. The PA-TCH-SP staining of microfibrils and collagen fibrils may represent localization of vicinal glycol-containing glycoproteins, which have been biochemically identified in microfibrils (see review by Ross, 1973; Sear et al., 1978) as well as in collagen fibrils (Nimni, 1974).The PA-TCH-SPmethod was useful in distinguishing the positive microfibrils from the unreactive amorphous elastin in the present study. Microfibrils were predominantly localized in the perichondrium, whereas the amorphous elastin without microfibrils was present in the mature cartilage. This observation indicates that the appearance of the microfibrils precedes the appearance of the amorphous elastin during the cartilage maturation or differentiation from the perichondrium to the mature cartilage. This interpretation is consistent with the previous light and electron microscopic studies demonstrating the presence of abundant preelastic fibers or microfibrils with small foci of amorphous elastin in the perichondrium (Bradamante et al., 1975) and elastin without preelastic fibers in the ma- Fig. 10. In the perichondrium, HID staining of osmicated specimens is present in matrix granules (arrows) that are closely associated with collagen fibrils. However, HID-reactive matrix granules are not visible in transverse sections of collagen fibrils. The diameter of these granules is significantly decreased when compared to those in the mature cartilage (cf. Fig. 2). The banding of collagen fibrils is evident. Not counterstained. ~31,250. Fig. 11. HID-TCH-SP stain deposits are observed in the longitudinal sections of collagen fibrils (enlarged in right upper inset) in the perichondrium, but are not always seen in transverse sections of these fibrils (left lower inset). Osmicated specimen, not counterstained. x 31,260. Insets, ~62,500. Fig. 12. PA-TCH-SP staining is localized in collagen fibrils (Co) and microfibrils (Mf, and enlarged in inset), whereas amorphous elastin (arrowheads) and matrix granules lack staining. Unosmicated specimen, not counterstained. ~ 3 1 , 2 5 0Inset, . X62,500. SULFATE IN ELASTIC CARTILAGE 555 556 M. TAKAGI ET AL ture cartilage (Serafini-Fracassini and Smith, 1974). ACKNOWLEDGMENTS The authors thank Ms. Barbara A. Woolley €or her secretarial assistance. This work was supported in part by National Institutes of Health grant No. DE-02670. LITERATURE CITED Anderson, D.R. (1964)The ultrastructure of elastic and hyaline cartilage of the rat. Am. J. Anat., 114:403-433. Behnke, O., and T. Zelander (1970) Preservation of intercellular substances by the cationic dye Alcian blue in preparative procedures for electron microscopy. J. U1trastruct. Res., 31:424-438. Bradamante, Lj. Kostovic-KneZeviC, and A. Svajger (1975) Light- and electron-microscopicobservations on the presence of pre-elastic (oxytalan) fibers around the mature cartilage in the external ear of the rat. Experientia, 31t979-980. Butler, W.F., and P. Heap (1982)An ultrastructural study of glycosaminoglycans associated with collagen and other constituents of the cat anulus fibrosus. Histochem. J., 14t113-123. Cox, R.W., and M.A. Peacock (1977) The fine structure of developing elastic cartilage. J. Anat., I23:283-296. Franien, A,, S.Inerot, S. Hejderup, and D. Heinegird (1981) Variations in the composition of bovine hip articular cartilage with distance from the articular surface. Biochem. J., 195r535-543. Futaesaku, Y., V. Mizuhira, and H. Nakamura (1972) The new fixation method using tannic acid for electron microscopy and some observations of biological specimens. Proc. 4th Int. Cong. Histochem. Cytochem. Kyoto, pp. 155-156 (Abstract). Gad, A,, and B. Sylven (1969) On the nature of the high iron diamine method for sulfomucins. J. Histochem. Cytochem., 17:156-160. Hascall, G.K. (1980) Cartilage proteoglycans: Comparison of sectioned and spread whole molecules. J. Ultrastruct. Res., 7Ot369-375. Lindahl, U., and M. Hook (1978) Glycosaminoglycans and their binding to biological macromolecules. Annu. Rev. Biochem., 47:385-417. Myers, D.B. (1976) Electron microscopic autoradiography of 35S0Jabelled material closely associated with collagen fibrils in mammalian synovium and ear cartilage. Histochem. J., 8:191-199. Nielsen, E.H. (1976)The elastic cartilage in the normal rat epiglottis. I. Fine structure. Cell Tissue Res., 173:179-191. Nimni, M.E. (1974) Collagen: Its structure and function in normal and pathological connective tissues. Sem. Arthritis Rheum., 4.95-150. Poole, A.R., I. Pidoux, A. Reiner, and L. Rosenberg (1982) An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage. J. Cell Biol., 93.921-937. Ross, R. (1973)The elastic fiber. A review. J. Histochem. Cytochem., 21: 199-208. Ruggeri, A., C. Dell’orbo, and D. Quacci (1975) Electron microscopic visualization of proteoglycans with Alcian blue. Histochem. J., 7t187-197. z., Sannes, P.L.,T. Katsuyama, and S.S. Spicer (1978)Tannic acid-metal salt sequences for light and electron microscopic localization of complex carbohydrates. J. Histochem. Cytochem., 26:55-61. Sannes, P.L., S.S. Spicer, and T. Katsuyama (1979)Ultrastructural localization of sulfated complex carbohydrates with a modified iron diamine procedure. J. Histochem. Cytochem., 27t1108-1111. Sear, C.H.J., M.A. Kewley, C.J.P. Jones, M.E. Grant, and D.S. Jackson (1978)The identification of glycoproteins associated with elastic-tissue microfibrils. Biochem. J., 170r715-718. Serafini-Fracassini, A,, and J.W. Smith (1974)The Structure and Biochemistry of Cartilage. Churchill Livingstone, London. Sheldon, H., and R.A. Robinson (1958) Studies on cartilage: Electron microscope observations on normal rabbit ear cartilage. J. Biophys. Biochem. Cytol., 4: 40 1-406. Shepard, N., and N. Mitchell (1977) The localization of articular cartilage proteoglycan by electron microscopy. Anat. Rec., 187t463-476. Spicer, S.S. (1965) Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem., 13.211-234. Spicer, S.S., R,G. Horn, and T.J. Leppi (1967) Histochemistry of connective tissue mucopolysaccharides. In: The Connective Tissue. International Academy of Pathology Monograph. B.M. Wagner and D.E. Smith, eds. Williams and Wilkins Co., Baltimore, pp. 251-303. Spurr, A.R. (1969)A low-viscosityepoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26t31-43. Takagi, M., R.T. Parmley, and F.R. Denys (1981)Ultrastructural cytochemistry and radioautography of complex carbohydrates in secretory granules of epiphyseal chondrocytes. Lab. Invest., 44t116-126. Takagi, M., R.T. Parmley, Y.Toda, and R.L. Austin (1982) Ultrastructural cytochemistry and immunocytochemistry of sulfated glycosaminoglycans in epiphyseal cartilage. J. Histochem. Cytochem., 30:1179-1185. Takagi, M., R.T. Parmley, F.R. Denys, and M. Kageyama (1983) Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acidmetal salt methods. J. Histochem. Cytochem. 31:783790. Takusagawa, K., F. Ariji, K. Shida, T. Sato, N. Asoo, and K. Konno (1982) Electron microscopic observations on pulmonary connective tissue stained hy Ruthenium Red. Histochem. J., 14r257-271. Thiery, J.P. (1967)Mise en evidence des polysaccharides sur coupes fines en microscopie electronique. J. Microsc., 6987-1018. Thyberg, J., A. Hinek, J. Nilsson, and U. Friberg (1979) Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material. Histochem. J., 11:1-17. Wusteman, F.S., and G.C. Gillard (1977)Hyaluronic acid in elastic cartilage. Experientia, 33t721-723. Yamada, K., and M. Hoshino (1973)Digestion with chondroitinases of acid mucosaccharides in rabbit cartilages as revealed by electron microscopy. Histochem. J., 5t195-197 (Letter). Yamada, K., Y. Fujita, and S. Shimizu (1982)The effect of digestion with keratanase (Pseudomonas sp.)on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues. Histochem. J., 14:897-910.