- Email: [email protected]
Studies on connective tissue
Experimental
Cell Research 20, 511418
STUDIES VI.
THE
CYTOPLASM
of Pathology,
511
ON CONNECTIVE
R. D. MOORE Institute
(1960)
OF THE
TISSUE FIBROBLAST’
and M. D. SCHOENBERG2
Western Reserve University,
Cleveland, Ohio, U.S.A.
Received August 27, 1959
THE fibroblast is a polymorphic cell which is not characterized by any unique cytoplasmic or nuclear features. It is found in all connective tissues in a wide variety of forms depending on its environmental and functional state. This cell has been shown to be important in the elaboration of collagen [l, 3, 9, 12, 18, 19, 261, the ground substance polysaccharides [6, 7, 8, 211, and the synthesis of cholesterol [2, 4, 5, 153 and as a phagocyte [23, 241. This multiplicity of functions raises the question of whether the fibroblast is a single cell type capable of assuming a variety of functions under a given set of conditions or a series of similarly appearing cells, each with specific capabilities. The cytologic features are insufficient to resolve this problem. Wharton’s jelly of the human umbilical cord is a rapidly growing tissue where fibroblasts are found in a variety of forms. These cells are known to be associated with collagen production [20], the elaboration of ground substance polysaccharides [al] and the synthesis of lipids [21]. Using Wharton’s jelly as a prototype of connective tissue, the fibroblasts were studied cytochemically and by the electron microscope to relate structural features of the cytoplasm to their functional character.
MATERIALS
AND
METHODS
Cytochemical procedures.-Wharton’s jelly from fresh human umbilical cords was smeared on glass slides and fixed immediately in 10 per cent neutral (phosphate) buffered formalin and in Bouin’s solution for the examination of lipids. Cords from fetuses of six weeks of age to term were used in this study. The hematoxylin and eosin (H and E) stain was the routine stain for general cytologic features. The periodic acid-Schiff method (PAS) [ll] and Sudan Black staining (171 were used before and after treatment of the cell preparations with 1 This work was supported in part by a grant from the National States Public Health Service. 2 Research Fellow, American Cancer Society, Cuyahoga County
Institutes
of Health,
United
Ohio Unit. Experimental
Cell Research 20
512
R. D. Moore and M. D. Schoenberg
each of the following: alpha amylase,l beta glucuronidase,l and testicular hyaluronidase’ and pyridine extraction of the Bouin’s fixed cells for 24 hours at 60°C [17]. Sudan IV staining was done after ethanol and pyridine extraction. Metachromatic staining was carried out with aqueous solutions of toluidine blue as a function of pH and ionic strength [13]. With this method the following procedures were employed on the fixed tissue and all preparations using conditions previously reported [13, 221. The cell smears and tissue sections were washed and equilibrated in the appropriate buffer. Procedures.-Control tissue section and cell smear; testicular hyaluronidase; sulfate esterification; hyaluronidase digestion followed by sulfation; alpha amylase digestion; alpha amylase digestion followed by sulfation; beta glucuronidase digestion; beta glucuronidase digestion followed by sulfation; beta glucuronidase digestion then hyaluronidase; beta glucuronidase digestion followed by hyaluronidase and then sulfated; hyaluronidase digestion then beta glucuronidase; and hyaluronidase digestion followed.by beta glucuronidase and then sulfate esterification. Sulfate esterification was carried out by the method previously described [14]. Electron microscopy.-Tissue from fresh Wharton’s jelly was fixed in buffered osmium tetroxide (pH 7.2-7.4), dehydrated and embedded in methacrylate. Successive thin and thick sections (2 mp) were cut for electron and light microscopy with a Porter-Blum microtome. The thin sections were mounted on carbon supported grids and examined with an RCA EMU 2-D electron microscope. The thick sections were treated with the periodic acid-Schiff method [15] and compared to the thin sections. RESULTS
Cytologic and cytochemical features of the fibroblasts.-The fibroblasts were clearly polymorphic. Some of the cells had elongated nuclear and cytoplasmic outlines (Fig. l), others large, oval or irregular and folded nuclei surrounded by an abundant but usually poorly defined cytoplasm (Fig. 2). Within the cytoplasm of most of the cells were numerous vacuoles (Figs. 2 and 3). These were found in the fibroblasts of the youngest cords and were more numerous and prominent as the fetal age of the tissue increased. They frequently were located in only a portion of the cytoplasm. When the cell smears and the thick, osmium tetroxide fixed sections were subjected to the PAS reaction, many packets of PAS-positive material were present in these areas. The number of these packets varied from cell to cell in the same cord, but in general more of this material was present in the fibroblasts obtained from immature cords. Some of the PAS-positive material in the cell preparations was a substrate for alpha amylase and some resistant to digestion with this enzyme as determined with the PAS reaction. Amylase digestion removed most of the 1 Nutritional Experimental
Biochemicals Cell Research 20
Corporation,
Cleveland,
Ohio.
Studies on connective tissue. VI
Fig. l.-Fibroblasts from forms. H and E x 1500.
the human
umbilical
cord with
elongated
nuclear
Fig. Z.-Fibroblasts from the human umbilical cord with folded and irregular cytoplasm and clusters of vacuoles in the cells. H and E x 1600.
and cytoplasmic nuclei,
abundant
Fig. 3.-Fibroblast from the human umbilical cord with vacuoles present in the cytoplasm. Small granules and the margins of the vacuoles are intensely PAS positive. PAS method. x 1600. Fig. 4.-Fibroblasts from the human umbilical cord which have been treated beta glucuronidase. There is no evidence of PAS positive material associated in the cytoplasm..PAS.method. x 1600. 34 - 60173253
Experimental
with amylase and with the vacuoles
Cell Research 20
R. D. kfoore and 44. D. Schoenberg PAS-positive material from the cytoplasm of the fibroblasts of the immature cords. With increasing fetal age of the tissue, less material was removed by amylase digestion so that the cells derived from term cords had an abundance of PAS-positive material resistant to alpha amylase digestion. In all cases subsequent treatment with beta glucuronidase removed all of the PASpositive material. Testicular hyaluronidase did not affect the PAS reaction. Pyridine extraction of Bouin’s fixed material had no effect on the distribution and number of PAS-positive packets in the cell or on their response to the enzymes used. In no instance was metachromatic material found in the fibroblasts over sulfate a pH range of 2. 5 to 7.5 at various ionic strengths. Following esterification some metachromatic granules were found, the number of which increased with fetal age [21]. Amylase and hyaluronidase digestion before sulfate esterification had no ell’ect on the metachromatic propertics oC these granules. \Vhen beta glucuronidase digestion was made prior to sulfation, no metachromatic packets could be demonstrated in the cytoplasm of the fibroblasts. This is similar to the en’ect of this enzyme on the PAS-positive material. In addition, there were some Sudan Black-positive granules in the cgtoplasm of these cells. Generally, the structures which stained positive with Sudan Black did not parallel those which were PAS positive before amylase digestion. They tended to correspond to the distribution of granules which c,ould be made metachromatic by sulfate esterification or which were still PAS positive after amylase digestion. Here again, the granules were not When the cells were treated affected by alpha amylase or hyaluronidase. with beta glucuronidase, the Sudan Black staining was markedly diminished so that only rarely could a Sudan Black positive granule be found. Pyridine extraction of these cells (Bouin’s fixed) had no et’fect on the Sudan Black material [17]. Sudan IV-positive material was occasionally present in fibroblasts from term cords. Extraction of the cell smears with ethanol or pyridine for one hour completely removed the Sudan IV material. Electron microscopic features of the fibroblast.-Electron microscopic study of these cells showed cytoplasmic structures in some of the cells, which were Fig. 5.-Portion of a fibroblast from the human umbilical cord which contains numerous vacuoles or channels. These have a dense limiting border with numerous thickenings and irregularities. x 15,000. Fig. 6.-Cytoplasmic vacuoles or channels within the cytoplasm umbilical cord. They contain an amorphous osmiophilic material. Experimental
Cell Research 20
of a fibroblast x 23,000.
from
human
Sfudies on connective fissue. VI
Experimental
510
Cell Research 20
516
R. D. Moore and M. D. Schoenberg
similar to those seen by light microscopy (Fig. 5). These vacuoles or channels had a limiting border with numerous thickenings and irregularities (Fig. 5). The lumen contained an amorphous osmiophilic material in which a distinct structure could not be resolved (Fig. 6). It is unlikely that these structures represent swollen mitochondria, and they are distinct from the endoplasmic reticulum. hleasurements of the size and location of these structures compared favorably with those determined optically. These were the only structures found by electron microscopy which resembled those described by light microscopy. In view of the fact that there mere differently reacting particles on a cytochemical basis an exhaustive study was made for cytoplasmic structures of different morphologic characteristics. While some differences in size, shape and osmiophilia were found, there was no feature that distinguished the vacuoles from one another. DISCUSSION
Many of the tibroblasts of the human umbilical cord contain a vacuole or channel system in their cytoplasm that is a constant feature of the cell independent of the age of the cord from which they are obtained. While the cytochemical procedures indicate that there may be several substances associated with these structures, the response of these structures to the enzymatic procedures used reveal some common features. From the partial sensitivity to alpha amylase some of the vacuoles contain glycogen. In the cells from term cords the material which remains after alpha amylase digestion is a substrate for beta glucuronidase. In all cases, the PAS reaction, the Sudan Black method and the metachromatic staining procedure after sulfate estertication are negative when preceded by treatment with beta glucuronidase. Testicular hyaluronidase had no effect on any of the cytochemical procedures employed. It is difficult from only a few enzymatic and cytochemical procedures to arrive at definitive conclusions concerning the nature of the material or materials within the vacuoles. Direct isolation procedures are prohibitive. From the sensitivity of the material in the vacuoles to beta glucuronidase some features of the substrate can be suggested. It seems certain that glucureaction ronic acid is present in the moiety. The absence of a metachromatic after beta glucuronidase and subsequent sulfation suggests a small molecule. The formation of a metachromatic complex depends upon available, regularly spaced, anionic groups, with at least four such groups in sequence Experimental
Cell Research 20
Studies on connective tissue. VI [25]. The selective attack of @-glucuronidase places glucuronic acid as the terminal non-reducing sugar [lo]. Consequently, with only the end glucuronic acid affected, the rest of the chain would remain intact, and metachromasy would result with sulfate esteritication if the original chain were greater than a tetrasaccharide. Sudan Black is non-specific for lipids unless carried out with extraction procedures. Since the latter had no effect on the Sudan Black substrate, it is unlikely that it is lipid [ 171. In a previous communication [al], it has been suggested that the substrate for beta glucuronidase is a possible precursor in the synthetic scheme for hyaluronic acid. It is attractive to suggest that some of the packets are the synthetic site or storage depot for the precursor of hyaluronic acid. The vacuole or channel system present in the cytoplasm of the tibroblast obtained from Wharton’s jelly of the human umbilical cord may represent a high degree of development of this structure in this particular tissue site. It is not confined to cells derived from Wharton’s jelly, as it has also been noted in the fibroblasts of human synovia, another tissue where there is abundant mucopolysaccharide production. From work reported previously [20] collagen tibrils are present in Wharton’s jelly before acidic polysaccharides can be demonstrated histochemically in the extracellular space, and before any significant amount of carbohydrate other than glycogen can be demonstrated in the cell cytoplasm. Since the number and type of cells is limited in Wharton’s jelly, we must assume that the same cells are capable of producing both carbohydrate and fiber material, or that diflerent cells of the same type carry out each of these functions. From our results it would appear that some of the cells are concerned with the production or storage of a substrate for beta glucuronidase, which may be the precursor of hyaluronic acid. This does not, as yet, answer the question of simultaneous production of polysaccharides and collagen by a libroblast, but would seem to indicate that not all cells designated as fibroblasts in this tissue contain the cytoplasmic structure which is apparently associated with mucopolgsaccharide production. SUMMARY
A vacuole or channel system is present of Wharton’s jelly of the human umbilical pattern as revealed by cytochemical and It may be associated with the production tissue polysaccharides.
in the cytoplasm of the tibroblasts cord. This system has a constant electron microscopic techniques. and/or storage of the connective
Experimental
Cell Research 20
518
R. D. AZoore and &f. D. Schoenberg REFERENCES
1. BANG, F. B. and GEY, G. O., Proc. Sot. Exptl. Biol. and Med. 69, 86 (1948). 2. BIESELE, J. J. and GOLDHABER, P., Cancer Research 15, 767 (1955). 3. BLOOM, W., Physiot. Reu. 17, 589 (1937). 4. BOUCEK, R. J. and NOBLE, N. L., Circulation Research 5, 27 (1957). Research 3, 519 (1955). 5. BOUCEK, R. J., NOBLE, N. L. and KAO, K. Y. T., Circulation 6. GROSSFELD, H., Proc. Sot. Exptl. Biot. and Med. 96, 144 (1957). 7. GROSSFELD, H., MEYER, K. and GODMAX, G., Proc. Exptl. Biol. and Med. 88, 31 (1955). 8. GROSSFELD, H., MEYER, K., GODMAN, G. and LINKER, A., J. Biophys. Biochem. Cytol. 3, 391 (1957). 9. JACKSON, S. F. and SMITH, R. H., J. Biophys. Biochem. CytoI. 3, 897 (1957). 10. LINKER, A., MEYER, K. and WEISSMANN, B., J. Biol. Chem. 213, 237 (1955). 11. McM~ws, J. F. A., Stain Technot. 23, 99 (1948). 12. MERCHANT, D. J. and KAHN, R. H., Proc. Sot. Exptl. Biol. Med. 97, 359 (1958). 13. MOORE, R. D. and SCHOENBERG, II. D., Am. Med. Ass. Arch. Pathol. 64, 39 (1957). 14. -Stain Technot. 32, 245 (1957). 15. MOSES, M. J., J. Biophys. Biochem. Cytol. 2, No. 4 Supplement, 397. 16. NOBLE, N. L. and BOUCEK, R. J., Circulation Research 3, 344 (1955). 17. PEARSE, A. G. E., Histochemistry: Theoretical and Applied. Boston, Little, Brown and Company, 1953. 18. PORTER, K. R., Trans. Macy Con/. Conn. Tissue, New York 2, 126 (1951). 19. PORTER, K. R. and PAPPAS, G. D., J. Biophys. Biochem. Cytol. 5, 153 (1959). 20. SCHOENBERG, M. D., HINRIAN, A. and MOORE, R. D., Laboratory Znuestigation. In press. 21. SCHOENBERG, M. I>. and MOORE, R. D., Am. Med. Ass. Arch. Pathol. 65, 115 (1958). 22. -ibid. 64, 167 (1957). 23. SMITH, D. E. and LEWIS, Y. S., Anat. Record 93, 132 (1958). 24. TAYLOR, H. E. and SAUNDERS, A. i\il., Am. J. Pathol. i3, 5i5 (1957). 25. WALTON, Ii. W. and RICKETTS, C. R., Brit. J. Exptl. Pathol. 35, 227 (1954). 26. WYCKOFF, R. W. G., Trans. Macy Conf. Corm. Tissue, Xew York, 3, 38 (1952.)
Experimental
Cell Research 20
Cell Research 20, 511418
STUDIES VI.
THE
CYTOPLASM
of Pathology,
511
ON CONNECTIVE
R. D. MOORE Institute
(1960)
OF THE
TISSUE FIBROBLAST’
and M. D. SCHOENBERG2
Western Reserve University,
Cleveland, Ohio, U.S.A.
Received August 27, 1959
THE fibroblast is a polymorphic cell which is not characterized by any unique cytoplasmic or nuclear features. It is found in all connective tissues in a wide variety of forms depending on its environmental and functional state. This cell has been shown to be important in the elaboration of collagen [l, 3, 9, 12, 18, 19, 261, the ground substance polysaccharides [6, 7, 8, 211, and the synthesis of cholesterol [2, 4, 5, 153 and as a phagocyte [23, 241. This multiplicity of functions raises the question of whether the fibroblast is a single cell type capable of assuming a variety of functions under a given set of conditions or a series of similarly appearing cells, each with specific capabilities. The cytologic features are insufficient to resolve this problem. Wharton’s jelly of the human umbilical cord is a rapidly growing tissue where fibroblasts are found in a variety of forms. These cells are known to be associated with collagen production [20], the elaboration of ground substance polysaccharides [al] and the synthesis of lipids [21]. Using Wharton’s jelly as a prototype of connective tissue, the fibroblasts were studied cytochemically and by the electron microscope to relate structural features of the cytoplasm to their functional character.
MATERIALS
AND
METHODS
Cytochemical procedures.-Wharton’s jelly from fresh human umbilical cords was smeared on glass slides and fixed immediately in 10 per cent neutral (phosphate) buffered formalin and in Bouin’s solution for the examination of lipids. Cords from fetuses of six weeks of age to term were used in this study. The hematoxylin and eosin (H and E) stain was the routine stain for general cytologic features. The periodic acid-Schiff method (PAS) [ll] and Sudan Black staining (171 were used before and after treatment of the cell preparations with 1 This work was supported in part by a grant from the National States Public Health Service. 2 Research Fellow, American Cancer Society, Cuyahoga County
Institutes
of Health,
United
Ohio Unit. Experimental
Cell Research 20
512
R. D. Moore and M. D. Schoenberg
each of the following: alpha amylase,l beta glucuronidase,l and testicular hyaluronidase’ and pyridine extraction of the Bouin’s fixed cells for 24 hours at 60°C [17]. Sudan IV staining was done after ethanol and pyridine extraction. Metachromatic staining was carried out with aqueous solutions of toluidine blue as a function of pH and ionic strength [13]. With this method the following procedures were employed on the fixed tissue and all preparations using conditions previously reported [13, 221. The cell smears and tissue sections were washed and equilibrated in the appropriate buffer. Procedures.-Control tissue section and cell smear; testicular hyaluronidase; sulfate esterification; hyaluronidase digestion followed by sulfation; alpha amylase digestion; alpha amylase digestion followed by sulfation; beta glucuronidase digestion; beta glucuronidase digestion followed by sulfation; beta glucuronidase digestion then hyaluronidase; beta glucuronidase digestion followed by hyaluronidase and then sulfated; hyaluronidase digestion then beta glucuronidase; and hyaluronidase digestion followed.by beta glucuronidase and then sulfate esterification. Sulfate esterification was carried out by the method previously described [14]. Electron microscopy.-Tissue from fresh Wharton’s jelly was fixed in buffered osmium tetroxide (pH 7.2-7.4), dehydrated and embedded in methacrylate. Successive thin and thick sections (2 mp) were cut for electron and light microscopy with a Porter-Blum microtome. The thin sections were mounted on carbon supported grids and examined with an RCA EMU 2-D electron microscope. The thick sections were treated with the periodic acid-Schiff method [15] and compared to the thin sections. RESULTS
Cytologic and cytochemical features of the fibroblasts.-The fibroblasts were clearly polymorphic. Some of the cells had elongated nuclear and cytoplasmic outlines (Fig. l), others large, oval or irregular and folded nuclei surrounded by an abundant but usually poorly defined cytoplasm (Fig. 2). Within the cytoplasm of most of the cells were numerous vacuoles (Figs. 2 and 3). These were found in the fibroblasts of the youngest cords and were more numerous and prominent as the fetal age of the tissue increased. They frequently were located in only a portion of the cytoplasm. When the cell smears and the thick, osmium tetroxide fixed sections were subjected to the PAS reaction, many packets of PAS-positive material were present in these areas. The number of these packets varied from cell to cell in the same cord, but in general more of this material was present in the fibroblasts obtained from immature cords. Some of the PAS-positive material in the cell preparations was a substrate for alpha amylase and some resistant to digestion with this enzyme as determined with the PAS reaction. Amylase digestion removed most of the 1 Nutritional Experimental
Biochemicals Cell Research 20
Corporation,
Cleveland,
Ohio.
Studies on connective tissue. VI
Fig. l.-Fibroblasts from forms. H and E x 1500.
the human
umbilical
cord with
elongated
nuclear
Fig. Z.-Fibroblasts from the human umbilical cord with folded and irregular cytoplasm and clusters of vacuoles in the cells. H and E x 1600.
and cytoplasmic nuclei,
abundant
Fig. 3.-Fibroblast from the human umbilical cord with vacuoles present in the cytoplasm. Small granules and the margins of the vacuoles are intensely PAS positive. PAS method. x 1600. Fig. 4.-Fibroblasts from the human umbilical cord which have been treated beta glucuronidase. There is no evidence of PAS positive material associated in the cytoplasm..PAS.method. x 1600. 34 - 60173253
Experimental
with amylase and with the vacuoles
Cell Research 20
R. D. kfoore and 44. D. Schoenberg PAS-positive material from the cytoplasm of the fibroblasts of the immature cords. With increasing fetal age of the tissue, less material was removed by amylase digestion so that the cells derived from term cords had an abundance of PAS-positive material resistant to alpha amylase digestion. In all cases subsequent treatment with beta glucuronidase removed all of the PASpositive material. Testicular hyaluronidase did not affect the PAS reaction. Pyridine extraction of Bouin’s fixed material had no effect on the distribution and number of PAS-positive packets in the cell or on their response to the enzymes used. In no instance was metachromatic material found in the fibroblasts over sulfate a pH range of 2. 5 to 7.5 at various ionic strengths. Following esterification some metachromatic granules were found, the number of which increased with fetal age [21]. Amylase and hyaluronidase digestion before sulfate esterification had no ell’ect on the metachromatic propertics oC these granules. \Vhen beta glucuronidase digestion was made prior to sulfation, no metachromatic packets could be demonstrated in the cytoplasm of the fibroblasts. This is similar to the en’ect of this enzyme on the PAS-positive material. In addition, there were some Sudan Black-positive granules in the cgtoplasm of these cells. Generally, the structures which stained positive with Sudan Black did not parallel those which were PAS positive before amylase digestion. They tended to correspond to the distribution of granules which c,ould be made metachromatic by sulfate esterification or which were still PAS positive after amylase digestion. Here again, the granules were not When the cells were treated affected by alpha amylase or hyaluronidase. with beta glucuronidase, the Sudan Black staining was markedly diminished so that only rarely could a Sudan Black positive granule be found. Pyridine extraction of these cells (Bouin’s fixed) had no et’fect on the Sudan Black material [17]. Sudan IV-positive material was occasionally present in fibroblasts from term cords. Extraction of the cell smears with ethanol or pyridine for one hour completely removed the Sudan IV material. Electron microscopic features of the fibroblast.-Electron microscopic study of these cells showed cytoplasmic structures in some of the cells, which were Fig. 5.-Portion of a fibroblast from the human umbilical cord which contains numerous vacuoles or channels. These have a dense limiting border with numerous thickenings and irregularities. x 15,000. Fig. 6.-Cytoplasmic vacuoles or channels within the cytoplasm umbilical cord. They contain an amorphous osmiophilic material. Experimental
Cell Research 20
of a fibroblast x 23,000.
from
human
Sfudies on connective fissue. VI
Experimental
510
Cell Research 20
516
R. D. Moore and M. D. Schoenberg
similar to those seen by light microscopy (Fig. 5). These vacuoles or channels had a limiting border with numerous thickenings and irregularities (Fig. 5). The lumen contained an amorphous osmiophilic material in which a distinct structure could not be resolved (Fig. 6). It is unlikely that these structures represent swollen mitochondria, and they are distinct from the endoplasmic reticulum. hleasurements of the size and location of these structures compared favorably with those determined optically. These were the only structures found by electron microscopy which resembled those described by light microscopy. In view of the fact that there mere differently reacting particles on a cytochemical basis an exhaustive study was made for cytoplasmic structures of different morphologic characteristics. While some differences in size, shape and osmiophilia were found, there was no feature that distinguished the vacuoles from one another. DISCUSSION
Many of the tibroblasts of the human umbilical cord contain a vacuole or channel system in their cytoplasm that is a constant feature of the cell independent of the age of the cord from which they are obtained. While the cytochemical procedures indicate that there may be several substances associated with these structures, the response of these structures to the enzymatic procedures used reveal some common features. From the partial sensitivity to alpha amylase some of the vacuoles contain glycogen. In the cells from term cords the material which remains after alpha amylase digestion is a substrate for beta glucuronidase. In all cases, the PAS reaction, the Sudan Black method and the metachromatic staining procedure after sulfate estertication are negative when preceded by treatment with beta glucuronidase. Testicular hyaluronidase had no effect on any of the cytochemical procedures employed. It is difficult from only a few enzymatic and cytochemical procedures to arrive at definitive conclusions concerning the nature of the material or materials within the vacuoles. Direct isolation procedures are prohibitive. From the sensitivity of the material in the vacuoles to beta glucuronidase some features of the substrate can be suggested. It seems certain that glucureaction ronic acid is present in the moiety. The absence of a metachromatic after beta glucuronidase and subsequent sulfation suggests a small molecule. The formation of a metachromatic complex depends upon available, regularly spaced, anionic groups, with at least four such groups in sequence Experimental
Cell Research 20
Studies on connective tissue. VI [25]. The selective attack of @-glucuronidase places glucuronic acid as the terminal non-reducing sugar [lo]. Consequently, with only the end glucuronic acid affected, the rest of the chain would remain intact, and metachromasy would result with sulfate esteritication if the original chain were greater than a tetrasaccharide. Sudan Black is non-specific for lipids unless carried out with extraction procedures. Since the latter had no effect on the Sudan Black substrate, it is unlikely that it is lipid [ 171. In a previous communication [al], it has been suggested that the substrate for beta glucuronidase is a possible precursor in the synthetic scheme for hyaluronic acid. It is attractive to suggest that some of the packets are the synthetic site or storage depot for the precursor of hyaluronic acid. The vacuole or channel system present in the cytoplasm of the tibroblast obtained from Wharton’s jelly of the human umbilical cord may represent a high degree of development of this structure in this particular tissue site. It is not confined to cells derived from Wharton’s jelly, as it has also been noted in the fibroblasts of human synovia, another tissue where there is abundant mucopolysaccharide production. From work reported previously [20] collagen tibrils are present in Wharton’s jelly before acidic polysaccharides can be demonstrated histochemically in the extracellular space, and before any significant amount of carbohydrate other than glycogen can be demonstrated in the cell cytoplasm. Since the number and type of cells is limited in Wharton’s jelly, we must assume that the same cells are capable of producing both carbohydrate and fiber material, or that diflerent cells of the same type carry out each of these functions. From our results it would appear that some of the cells are concerned with the production or storage of a substrate for beta glucuronidase, which may be the precursor of hyaluronic acid. This does not, as yet, answer the question of simultaneous production of polysaccharides and collagen by a libroblast, but would seem to indicate that not all cells designated as fibroblasts in this tissue contain the cytoplasmic structure which is apparently associated with mucopolgsaccharide production. SUMMARY
A vacuole or channel system is present of Wharton’s jelly of the human umbilical pattern as revealed by cytochemical and It may be associated with the production tissue polysaccharides.
in the cytoplasm of the tibroblasts cord. This system has a constant electron microscopic techniques. and/or storage of the connective
Experimental
Cell Research 20
518
R. D. AZoore and &f. D. Schoenberg REFERENCES
1. BANG, F. B. and GEY, G. O., Proc. Sot. Exptl. Biol. and Med. 69, 86 (1948). 2. BIESELE, J. J. and GOLDHABER, P., Cancer Research 15, 767 (1955). 3. BLOOM, W., Physiot. Reu. 17, 589 (1937). 4. BOUCEK, R. J. and NOBLE, N. L., Circulation Research 5, 27 (1957). Research 3, 519 (1955). 5. BOUCEK, R. J., NOBLE, N. L. and KAO, K. Y. T., Circulation 6. GROSSFELD, H., Proc. Sot. Exptl. Biot. and Med. 96, 144 (1957). 7. GROSSFELD, H., MEYER, K. and GODMAX, G., Proc. Exptl. Biol. and Med. 88, 31 (1955). 8. GROSSFELD, H., MEYER, K., GODMAN, G. and LINKER, A., J. Biophys. Biochem. Cytol. 3, 391 (1957). 9. JACKSON, S. F. and SMITH, R. H., J. Biophys. Biochem. CytoI. 3, 897 (1957). 10. LINKER, A., MEYER, K. and WEISSMANN, B., J. Biol. Chem. 213, 237 (1955). 11. McM~ws, J. F. A., Stain Technot. 23, 99 (1948). 12. MERCHANT, D. J. and KAHN, R. H., Proc. Sot. Exptl. Biol. Med. 97, 359 (1958). 13. MOORE, R. D. and SCHOENBERG, II. D., Am. Med. Ass. Arch. Pathol. 64, 39 (1957). 14. -Stain Technot. 32, 245 (1957). 15. MOSES, M. J., J. Biophys. Biochem. Cytol. 2, No. 4 Supplement, 397. 16. NOBLE, N. L. and BOUCEK, R. J., Circulation Research 3, 344 (1955). 17. PEARSE, A. G. E., Histochemistry: Theoretical and Applied. Boston, Little, Brown and Company, 1953. 18. PORTER, K. R., Trans. Macy Con/. Conn. Tissue, New York 2, 126 (1951). 19. PORTER, K. R. and PAPPAS, G. D., J. Biophys. Biochem. Cytol. 5, 153 (1959). 20. SCHOENBERG, M. D., HINRIAN, A. and MOORE, R. D., Laboratory Znuestigation. In press. 21. SCHOENBERG, M. I>. and MOORE, R. D., Am. Med. Ass. Arch. Pathol. 65, 115 (1958). 22. -ibid. 64, 167 (1957). 23. SMITH, D. E. and LEWIS, Y. S., Anat. Record 93, 132 (1958). 24. TAYLOR, H. E. and SAUNDERS, A. i\il., Am. J. Pathol. i3, 5i5 (1957). 25. WALTON, Ii. W. and RICKETTS, C. R., Brit. J. Exptl. Pathol. 35, 227 (1954). 26. WYCKOFF, R. W. G., Trans. Macy Conf. Corm. Tissue, Xew York, 3, 38 (1952.)
Experimental
Cell Research 20