- Email: [email protected]
The fine structure of some cytoplasmic inclusions of oxyntic cells
Experimental
488
THE
(1957)
FINE STRUCTURE OF SOME CYTOPLASMIC INCLUSIONS OF OXYNTIC CELLS C. E. CHALLICE,’
National
Cell Research 13, US-492
and D. B. SCOTT
S. BULLIVANT
Institute of Arthritis and Metabolic Diseases, Bethesda, Md., U.S.A., St. Mary’s Hospital Medical School, London, England, and National Dental Research, Bethesda, Md., U.S.A.
Physics Department, Institute of
Received April 4, 1957
(or parietal) cells have been studied by a number of workers. Lim and Ma [6] showed that the granules found in these cells could be stained with Janus green B and thus they presumed them to be mitochondria, and to contain lipid. Menzies [7] showed that the granules contain a phospholipine, and also another lipid, together with a nonlipoidal component. He did not, however, assume the granules to be identifiable with mitochondria, although they clearly have some of their characteristics. In an abstract of some observations on the electron microscopic appearance of parietal cells, Sedar [lo] mentioned the presence of numerous mitochondria, which are presumably the objects referred to in the present work as oxyntic granules. The electron microscopical observations of Palade [8] and others (e.g. [ 131) have shown that mitochondria have a characteristic internal structure which appears always to be present, although with slight variations between the cell types. This structure consists of a number of parallel pairs of membranes, the pairs often being nearly parallel to one another. It was thought to be of interest to know if oxyntic granules have a structure similar to this, and it was believed that an electron microscopical study would show whether or not this is the case, and in addition give further information on the cytology of the oxyntic cell. OXYNTIC
EXPERIMENTAL
For optimal preservation of cellular detail it is necessary to initiate fixation within a tissue in the shortest possible time following the disturbance of normal conditions. The method found most successful was a perfusion technique, in which the fixative 1 Permanent address: The Wright-Fleming Institute Hospital Medical School, London W.2., England. Experimental
Cell Research 13
and Physics Department,
St. Mary’s
Structure of cytoplasmic inclusions
489
is injected into the left ventricle of the heart of a living anaesthetized animal, allowed to perfuse through the entire body, and finally to escape via the severed pulmonary artery. The use of this procedure resulted in good fixation of almost all the tissues. Twenty to thirty g male mice were used in this study, and the fixative was osmium tetroxide in buffered potassium dichromate [2]. Immediately after perfusion, small pieces of gastric mucosa were dissected out, and placed directly in the mixture for final fixation. The fixed tissue was dehydrated in alcohol and embedded in a mixture of methyl and butyl methacrylate according to standard procedures. Sections were cut on a Spencer 820 microtome modified for automatic retraction according to NylCn (in the press), adapted for thermal advance [3], and equipped with glass knives. The sections were mounted either on evaporated carbon films or on formvar membranes reinforced with a thin layer of carbon. Examination was carried out in an R.C.A. E.M.U. 2 electron microscope. OBSERVATIONS
Sections of the region of the gastric mucose which contain the oxyntic cells were difficult to cut owing to the hardness of the granules. Internal structure of a type similar to that found in mitochondria was, however, observed (Figs. 1 and 2). Double membranes, separated by an average distance of 80 A and of thickness approximately 40 h; were seen, the pairs often being closely parallel with one another. The granules generally were observed to be denser than mitochondria hitherto observed by electron microscopy, and the double membranes were more closely packed than in most mitochondria. In addition, small dense particles, similar to those observed in the mitochondria of other vertebrate cells, were seen within the oxyntic granules. Intracellular canaliculi were observed in the cytoplasm of the oxyntic cells, the canaliculi being bordered by many irregular protrusions or microvilli. Sometimes these microvilli gave a similar appearance to the endoplasmic reticulum described by Porter [9]. Multiple membranes within the cytoplasm, features of the inclusion described as having some of the morphological Golgi apparatus in other cells (e.g. [j]) h ave been observed. These will be described elsewhere. DISCUSSION
It is of interest to find that, in addition to having attributes of mitochondria, the oxyntic granules have ultrastructure which has come to be associated almost chondria. In the abstract of Sedar [lo] it is stated that
some of the chemical also the morphological exclusively with mitothe mitochondria show Experimental
Cell Research 13
490
C. E. Challice, S. Bullivant
and D. B. Scott
an unusually high proportion of closely approximated cristae. Thus, Sedar appears to have no hesitation in calling these granules mitochondria. From the present work it would appear that they are sufficiently close in structure and cytochemical properties to previously observed mitochondria to be classed as such, although they are denser and the internal structure more closely spaced than has been observed so far in the mitochondria of other cells. Some workers claim to have shown a classical Golgi apparatus in the oxyntic cells [4,1], while others [ll, 121 state that the Golgi element does not exist in these cells. Menzies (personal communication) states that no typical Golgi element is to be seen in any oxyntic cells that he has examined using ‘classical’ or ‘modern’ methods and suggests that what Kolster, Beams and King claim to have observed is the lipid envelopes of the oxyntic granules being shed during functional activity, and distorted during fixation. Although inclusions having an appearance similar to that which has been described as Golgi apparatus in previous electron microscopical studies on other secretory cells [5, 141 have been observed in other cells of the gastric mucosa, in the present work structures bearing an unequivocal resemblance to this inclusion have not yet been observed in the oxyntic cell. Sedar, however, describes groupings of tightly packed membranes and associated vesicles resembling the structure described as the Golgi zone by other authors. Sedar further describes a system of vesicles comparable to the endoplasmic reticulum of other cell types, in addition to intracellular canaliculi lined with numerous microvilli or linger-like cytoplasmic projections. The latter observation has been made in the present study, and is evident in Fig. 1. Sections have been observed, however, which have been cut in such a way that the villi or finger-like projections also give an appearance similar to that described as endoplasmic reticulum by Porter [9]. So far, insufficient evidence has been obtained to postulate the existence of endoplasmic reticulum as distinct from the duct convolutions. It is of interest to establish this point one way or the other, and further work is in progress to this end. One of the authors (C. E. C) wishes to thank the Wright-Fleming Institute and St. Mary’s Hospital Medical School for leave of absence to take up a short term appointment as a Visiting Scientist at the National Institutes of Health, Bethesda, Maryland. U.S.A. He also wishes to thank Dr. R. W. G. Wyckoff for the hospitality of his laboratory and Dr. A. J. Dalton of the National Cancer Institute for helpful suggestions regarding the perfusion technique. We also thank Drs. Menzies and Foster of St. Mary’s Medical School for helpful discussions. Experimental
Cell Research 13
Structure of cytoplasmic inclusions
1:ig. 1..-Section through the cytoplasm of an osyntic cell of a mouse showing granules intracellular duct, with villar processes protruding from the cytoplasm. r 14,000.
and an
REFERENCES 1. I~IXMS, H. \\‘. and KISG, R. I,., Anaf. Rec. 53, 31 (1932). 2. I)ALTOP*', A. .J. and FELIX, M., Fine Structure of Cells, p. 271. Noordhoff, Groningen, 1956. 3. EDES, >I., ~'HATT, A. \V. and KAHLER, II., Rev. Sci. Jnsfr. 21, 802 (1950). 4. Kij~smn, R. l’erhandl. dnaf. Gcs. Anal. Ges. 27, 1924 (1913). 5. IACY, I). and CIIALLICE, C. L, .J. J3io/‘hys. and Hiochent. C
G. IT., Anat.
Rec. 144, 427 (1952).
C. E. Challice, S. Bullivant
Fig. 2.--Section in mitochondria
9. 10. 11. 12. 13. 14.
and D. B. Scott
through oxyntic granules showing lamellar and also typical small dense particles within
structure similar to that the granules. x 37,000.
PORTER, I<. R., .I. Hisfochem. and Cyfochem. 2, 346 (1954). SF.DAR, A. W., Anat. Rec. 121, 365 (1955). SIIIRASKA, M., Folia anat. japon. 8 (1930). (Quoted from Beams and King, SIRASKA, S., Anat. Rec. 53, 31 (1932). SJ~STRAND, Ii. S. and H~szolu, V., Erpt[. Cell Research 7, 393 (1954). ~ ibid. 7, 415 (1954).
One of the authors (S. B.) wishes to thank the Medical Research grant over the time during which this work was responded.
Present address of C. E. Challice: Experimental
Cell Research 13
University
of Alberta
in Calgary,
Council
Alberta,
observed
1932.)
for a maintenance
Canada,
488
THE
(1957)
FINE STRUCTURE OF SOME CYTOPLASMIC INCLUSIONS OF OXYNTIC CELLS C. E. CHALLICE,’
National
Cell Research 13, US-492
and D. B. SCOTT
S. BULLIVANT
Institute of Arthritis and Metabolic Diseases, Bethesda, Md., U.S.A., St. Mary’s Hospital Medical School, London, England, and National Dental Research, Bethesda, Md., U.S.A.
Physics Department, Institute of
Received April 4, 1957
(or parietal) cells have been studied by a number of workers. Lim and Ma [6] showed that the granules found in these cells could be stained with Janus green B and thus they presumed them to be mitochondria, and to contain lipid. Menzies [7] showed that the granules contain a phospholipine, and also another lipid, together with a nonlipoidal component. He did not, however, assume the granules to be identifiable with mitochondria, although they clearly have some of their characteristics. In an abstract of some observations on the electron microscopic appearance of parietal cells, Sedar [lo] mentioned the presence of numerous mitochondria, which are presumably the objects referred to in the present work as oxyntic granules. The electron microscopical observations of Palade [8] and others (e.g. [ 131) have shown that mitochondria have a characteristic internal structure which appears always to be present, although with slight variations between the cell types. This structure consists of a number of parallel pairs of membranes, the pairs often being nearly parallel to one another. It was thought to be of interest to know if oxyntic granules have a structure similar to this, and it was believed that an electron microscopical study would show whether or not this is the case, and in addition give further information on the cytology of the oxyntic cell. OXYNTIC
EXPERIMENTAL
For optimal preservation of cellular detail it is necessary to initiate fixation within a tissue in the shortest possible time following the disturbance of normal conditions. The method found most successful was a perfusion technique, in which the fixative 1 Permanent address: The Wright-Fleming Institute Hospital Medical School, London W.2., England. Experimental
Cell Research 13
and Physics Department,
St. Mary’s
Structure of cytoplasmic inclusions
489
is injected into the left ventricle of the heart of a living anaesthetized animal, allowed to perfuse through the entire body, and finally to escape via the severed pulmonary artery. The use of this procedure resulted in good fixation of almost all the tissues. Twenty to thirty g male mice were used in this study, and the fixative was osmium tetroxide in buffered potassium dichromate [2]. Immediately after perfusion, small pieces of gastric mucosa were dissected out, and placed directly in the mixture for final fixation. The fixed tissue was dehydrated in alcohol and embedded in a mixture of methyl and butyl methacrylate according to standard procedures. Sections were cut on a Spencer 820 microtome modified for automatic retraction according to NylCn (in the press), adapted for thermal advance [3], and equipped with glass knives. The sections were mounted either on evaporated carbon films or on formvar membranes reinforced with a thin layer of carbon. Examination was carried out in an R.C.A. E.M.U. 2 electron microscope. OBSERVATIONS
Sections of the region of the gastric mucose which contain the oxyntic cells were difficult to cut owing to the hardness of the granules. Internal structure of a type similar to that found in mitochondria was, however, observed (Figs. 1 and 2). Double membranes, separated by an average distance of 80 A and of thickness approximately 40 h; were seen, the pairs often being closely parallel with one another. The granules generally were observed to be denser than mitochondria hitherto observed by electron microscopy, and the double membranes were more closely packed than in most mitochondria. In addition, small dense particles, similar to those observed in the mitochondria of other vertebrate cells, were seen within the oxyntic granules. Intracellular canaliculi were observed in the cytoplasm of the oxyntic cells, the canaliculi being bordered by many irregular protrusions or microvilli. Sometimes these microvilli gave a similar appearance to the endoplasmic reticulum described by Porter [9]. Multiple membranes within the cytoplasm, features of the inclusion described as having some of the morphological Golgi apparatus in other cells (e.g. [j]) h ave been observed. These will be described elsewhere. DISCUSSION
It is of interest to find that, in addition to having attributes of mitochondria, the oxyntic granules have ultrastructure which has come to be associated almost chondria. In the abstract of Sedar [lo] it is stated that
some of the chemical also the morphological exclusively with mitothe mitochondria show Experimental
Cell Research 13
490
C. E. Challice, S. Bullivant
and D. B. Scott
an unusually high proportion of closely approximated cristae. Thus, Sedar appears to have no hesitation in calling these granules mitochondria. From the present work it would appear that they are sufficiently close in structure and cytochemical properties to previously observed mitochondria to be classed as such, although they are denser and the internal structure more closely spaced than has been observed so far in the mitochondria of other cells. Some workers claim to have shown a classical Golgi apparatus in the oxyntic cells [4,1], while others [ll, 121 state that the Golgi element does not exist in these cells. Menzies (personal communication) states that no typical Golgi element is to be seen in any oxyntic cells that he has examined using ‘classical’ or ‘modern’ methods and suggests that what Kolster, Beams and King claim to have observed is the lipid envelopes of the oxyntic granules being shed during functional activity, and distorted during fixation. Although inclusions having an appearance similar to that which has been described as Golgi apparatus in previous electron microscopical studies on other secretory cells [5, 141 have been observed in other cells of the gastric mucosa, in the present work structures bearing an unequivocal resemblance to this inclusion have not yet been observed in the oxyntic cell. Sedar, however, describes groupings of tightly packed membranes and associated vesicles resembling the structure described as the Golgi zone by other authors. Sedar further describes a system of vesicles comparable to the endoplasmic reticulum of other cell types, in addition to intracellular canaliculi lined with numerous microvilli or linger-like cytoplasmic projections. The latter observation has been made in the present study, and is evident in Fig. 1. Sections have been observed, however, which have been cut in such a way that the villi or finger-like projections also give an appearance similar to that described as endoplasmic reticulum by Porter [9]. So far, insufficient evidence has been obtained to postulate the existence of endoplasmic reticulum as distinct from the duct convolutions. It is of interest to establish this point one way or the other, and further work is in progress to this end. One of the authors (C. E. C) wishes to thank the Wright-Fleming Institute and St. Mary’s Hospital Medical School for leave of absence to take up a short term appointment as a Visiting Scientist at the National Institutes of Health, Bethesda, Maryland. U.S.A. He also wishes to thank Dr. R. W. G. Wyckoff for the hospitality of his laboratory and Dr. A. J. Dalton of the National Cancer Institute for helpful suggestions regarding the perfusion technique. We also thank Drs. Menzies and Foster of St. Mary’s Medical School for helpful discussions. Experimental
Cell Research 13
Structure of cytoplasmic inclusions
1:ig. 1..-Section through the cytoplasm of an osyntic cell of a mouse showing granules intracellular duct, with villar processes protruding from the cytoplasm. r 14,000.
and an
REFERENCES 1. I~IXMS, H. \\‘. and KISG, R. I,., Anaf. Rec. 53, 31 (1932). 2. I)ALTOP*', A. .J. and FELIX, M., Fine Structure of Cells, p. 271. Noordhoff, Groningen, 1956. 3. EDES, >I., ~'HATT, A. \V. and KAHLER, II., Rev. Sci. Jnsfr. 21, 802 (1950). 4. Kij~smn, R. l’erhandl. dnaf. Gcs. Anal. Ges. 27, 1924 (1913). 5. IACY, I). and CIIALLICE, C. L, .J. J3io/‘hys. and Hiochent. C
G. IT., Anat.
Rec. 144, 427 (1952).
C. E. Challice, S. Bullivant
Fig. 2.--Section in mitochondria
9. 10. 11. 12. 13. 14.
and D. B. Scott
through oxyntic granules showing lamellar and also typical small dense particles within
structure similar to that the granules. x 37,000.
PORTER, I<. R., .I. Hisfochem. and Cyfochem. 2, 346 (1954). SF.DAR, A. W., Anat. Rec. 121, 365 (1955). SIIIRASKA, M., Folia anat. japon. 8 (1930). (Quoted from Beams and King, SIRASKA, S., Anat. Rec. 53, 31 (1932). SJ~STRAND, Ii. S. and H~szolu, V., Erpt[. Cell Research 7, 393 (1954). ~ ibid. 7, 415 (1954).
One of the authors (S. B.) wishes to thank the Medical Research grant over the time during which this work was responded.
Present address of C. E. Challice: Experimental
Cell Research 13
University
of Alberta
in Calgary,
Council
Alberta,
observed
1932.)
for a maintenance
Canada,