TISSUE
& CELL
1976 8 (I) 159-162
Published by Longman
Croup Ltd. Printed in Great Britain
A. F. BARADI
and S. N. RAO
A SCANNING ELECTRON MICROSCOPE STUDY OF MOUSE PERITONEAL MESOTHELIUM ABSTRACT. As seen in the scanning electron microscope, peritoneal mesothelial cells of the mouse diaphragm, anterior abdominal wall and intestinal serosa carry numerous microvilli. These microvilli are absent over certain areas of the cell surface and are. sometimes, interlocked in meshwork patterns or coronal formations. The apical cell membranes of the mesothelium at the base of the microvilli, are invaginated by many plasmalemmal vesicles and vacuoles and carry a number of protruding spherical structures. Deep circular craters, giving the impression of stomata, are also visible.
Introduction
fine structure of mesothelium has been elucidated by transmission electron microscopy in rats, rabbits, mice and humans (Odor, 1954; Felix, 1961; Fukuta, 1963; Baradi and Hope, 1974; Kluge and Hovig, 1967a, b). To our knowledge the only scanning electron microscope study of mesothelium dealt, solely with the ultrastructural morphology of mesothelial microvilli (Andrews and Porter, 1973). The present report deals with several surface features which were not included in Andrews and Porter’s report and further amplifies these authors’ observations on the surface microvilli.
THE
Materials and Methods A total of eight Swiss-Webster
mice of both sexes ranging in weight from 18 to 20 g were anaesthetized with ether. Each animal received an intraperitoneal injection of 1 mg of isotonic tubocurarine B.P. followed 30 set later by 2 ml of a 2.5% gluteraldehyde in 0.15 M cacodylate buffer at room temperature. Pieces of tissue from the anterior abDepartments of Anatomy and Pathology, University of Otago Medical School, Dunedin, New Zealand. Received 27 March 1975. Revised 5 September 1975. II
dominal wall, the diaphragm and the small intestine were removed 5 min after the in vioo fixation. They were postfixed in 1% osmium tetroxide for I hr, dehydrated in ascending grades of acetone and dried by carbon dioxide substitution. The dried specimens were mounted on aluminium stubs, carbon-gold coated and examined in a Siemens Autoscan at 20 kV and at various magnifications. Curare was used prior to fixation to produce muscle relaxation and thus minimize shrinkage of the peritoneal surface. Observations
The mesothelia of the anterior abdominal wall, the diaphragm and the intestinal serosa appear essentially similar. The observations reported here pertain, generally, to all three peritoneal sites. It is evident that the free surface of mesothelium is covered with numerous microvilli, and that there are significant differences in the density of microvilli over that surface (Fig. I ). In most cases mesothelial cell outlines are concealed by the microvilli (Fig. I) and only rarely do they become discernible. Microvilli occasionally interlock in meshwork patterns (Fig. 2) and rarely in unusual coronal formations (Fig. 3). Details of the apical cell membranes of the mesothelium at the base of the microvilli are 159
BARADI
160
revealed by a close examination of the bare areas. The cell surface is closely pitted by minute circular shallow depressions of near uniform diameter (about 800 A) (Fig. 4) and is randomly invaginated by larger shallow depressions of variable diameter measuring as much as 6000 A (Fig. 6). Numerous small spherical structures of almost uniform diameter (about 800 A) protrude from the surface (Fig. 5) as well as occasional large globular formations which may reach a diameter of 4000 A (Fig. 6). Deep circular craters reaching a diameter of up to about 3000 A and giving the impression of stomata or fenestrae are visible (Fig. 6).
AND
RAO
that this arrangement protects the mesothelium from surface friction and ensures its integrity against adhesion formation. The unusual entanglement of microvilli in meshwork patterns and in coronal formations seen in the present study may be morphological evidence for the existence of such compartments. In several light microscope publications, including some dating back to the nineteenth century (references in Odor, 1956), authors have speculated on the existence of intercellular pore-like structures in mesothelium. Physiological studies on the mechanism of solute transfer have indicated that lipidinsoluble molecules traverse the mesothelium by passive diffusion. This phenomenon could be theoretically accounted for by the presence of pores occupying up to 0.6% of mesothelial surface area (Boen, 1961; Brendt and Gosselin, 1961a, b, 1962; Gosselin and Brendt, 1962). Our observations suggest the occurrence of intracellular mesothelial stomata or fenestrae. Admittedly further proof is needed to demonstrate that these structures do indeed span the height of the cell from the apical surface to the basal surface. Certain studies with electron-opaque tracers (Odor, 1956; Fukuta, 1963; Staubesand, 1963) have suggested that transport across mesothelium is carried out mainly by micropinocytotic vesicles and by vacuoles, a finding which is not entirely consistent with the process of passive diffusion. More recent in vitro and in vivo experiments performed with improved tracer techniques strongly favour intercellular over intracellular trans-
Discussion The consistent and characteristic distribution of microvilli over the mesothelial free surface suggests that the regularly disposed bare areas are true features of mesothelial morphology rather than preparation artefacts. Andrews and Porter (1973) are also of the opinion that these denuded areas may exist in vivo. Previous transmission electron microscope studies, in several species, have shown that microvilli have an uneven distribution along the mesothelial surface (Baradi and Hope, 1964; Kluge and Hovig, 1967a). Andrews and Porter (1973) suggested that the peritoneal serous exudate becomes entrapped in small compartments created by adjacent microvilli being held together by the strong water-binding capabilities of the negatively charged acid glycosaminoglycans of microvillous glycocalyx. They postulated
Fig. 1. Mesothelial surface showing numerous Cell outlines are not apparent. x 2500. Fig. 2. Microvilli
in meshwork
Fig. 3. Microvilli
in coronal
patterns.
formation.
microvilli
and occasional
bare areas.
x 5750. x 5750
Fig. 4. A bare area pitted by small circular
shallow depressions.
Fig. 5. A bare area showing
protruding
small spherical
structures.
x 12,500. x 12,500.
Fig. 6. A bare area demonstrating what appear to be stomata (arrows), depressions (arrowhead), and large globular formations. x 23,000.
large shallow
BARADI
162
port routes (Casley-Smith, 1967; Cotran and Majno, 1967; Cotran and Karnovsky, 1968; Kluge, 1969). The small and the large circular shallow depressions seen by us at the mesothelial free surface probably represent plasmalemmal vesicles and vacuoles in communication with the cell membrane. If these structures are not engaged in some form of intracellular transport phenomena then their functional significance is still open to question. We are unable to comment on the nature and/or functional significance of the small protruding spherical structures sometimes
AND
RAO
seen at the mesothelial free surface. Surface globular membranous formations termed ‘extrusion vacuoles’ were noted in association with normal and necrotic mesothelium (Raftery, 1973). If the large globular formations in our micrographs are in fact ‘extrusion vacuoles’, this perhaps signifies cell death involved in a process of mesothelial turnover. Acknowledgement
The authors thank Professor W. D. Trotter for reading the manuscript and Mr B. T. Partridge for technical help.
References ANDREWS, P. M. and PORTER, K. R. 1973. Theultrastructure morphology and possible functional significance of mesothelial microvilli. Anat. Rec., 177, 409-426. BARADI, A. F. and HOPE, J. 1964. Observations on ultrastructure of rabbit mesothelium. Expl. Cell Res., 34, 33-44. BOEN, S. T. 1961. Kinetics of peritoneal dialysis. Medicine, 40, 243-287. BRENDT, W. 0. and GOSSELLN,R. E. 1961a. Physiological factors influencing radiorubidium flux across isolated rabbit mesentery. Am. J. Physiol., 200, 454458. BRENDT, W. 0. and GOSSELIN, R. E. 1961b. Rubidium and creatinine transport across isolated mesentery. Biochem. Pharmac., 8, 359-366. BRENDT, W. 0. and GOSSELIN, R. E. 1962. Differential permeability of the mesentery to rubidium and phosphate. Am. J. Physiol., 202, 761-767. CALSEY-SMITH,J. R. 1967. An electron microscopic study of the passage of ions through endothelium of lymphatic and blood capillaries and through mesothelium. Q. Jl exp. Physiol., 52, 105-I 13. COTRAN, R. S. and MAJNO, G. 1967. Studies on intercellular junctions of mesothelium and endothelium. Protoplasma, 63,45-51. COTRAN, R. S. and KARNOVSKY, M. J. 1968. Ultrastructural studies on the permeability of mesothelium to horseradish peroxidase. J. Cc/[ Biol., 37, 123-l 37. FELIX, M. D. 1961. Observations on the surface cells ofthe mouse omentum as studied with phase contrast and electron microscopes. J. natn. Cancer Inst., 27, 713-745. FUKUTA, H. 1963. Electron microscopic study on normal rat peritoneal mesothelium and its changes in absorption of particular iron dextran complex. Acta Path. Jop., 13, 309-325. GOSSELIN,R. E. and BRENDT, W. 0.1962. Diffusional transport of solutes through mesentery and peritoneum. J. theor. Biol., 3, 487-495. KLUGE, T. 1969. The permeability of mesothelium to horseradish peroxidase. Acfapath. microbio/. wand., 75, 251-269. KLUGE, T. and Hov~ti, T. 1967a. The ultrastructure of human and rat pericardium. 1. Parietal and visceral mesothelium. Acta path. mirrobiol. stand., 71, 529-546. KLUGE, T. and HOVIG, T. 1967b. The ultrastructure of human and rat pericardium. 2. Intercellular spaces and junctions. Acta path. microbial. stand.. 71, 547-563. ODOR, D. L. 1954. Observations of the rat mesothelium with the electron and phase microscopes. Am. J. Anat., 95, 433-465. ODOR, D. L. 1956. Uptake and transfer of particulate matter from the peritoneal cavity of the rat. J. biophys. biochem. Cytol., 2 (Suppl.), 105-107. RAFTERY, A. T. 1973. Mesothelial cells in peritoneal fluid. J. Anat., 115, 237-253. STAUBESAND,J. 1963. Zur histophysiologie des Herzbeutels. II. Mitteilung. Elektronmikroskopische Untersuchungen tiber die Passage von Metall-solen durch mesotheliale Membranen. Z. Zellforsch. mikrosk. Anot., 58,915-952.
& CELL
1976 8 (I) 159-162
Published by Longman
Croup Ltd. Printed in Great Britain
A. F. BARADI
and S. N. RAO
A SCANNING ELECTRON MICROSCOPE STUDY OF MOUSE PERITONEAL MESOTHELIUM ABSTRACT. As seen in the scanning electron microscope, peritoneal mesothelial cells of the mouse diaphragm, anterior abdominal wall and intestinal serosa carry numerous microvilli. These microvilli are absent over certain areas of the cell surface and are. sometimes, interlocked in meshwork patterns or coronal formations. The apical cell membranes of the mesothelium at the base of the microvilli, are invaginated by many plasmalemmal vesicles and vacuoles and carry a number of protruding spherical structures. Deep circular craters, giving the impression of stomata, are also visible.
Introduction
fine structure of mesothelium has been elucidated by transmission electron microscopy in rats, rabbits, mice and humans (Odor, 1954; Felix, 1961; Fukuta, 1963; Baradi and Hope, 1974; Kluge and Hovig, 1967a, b). To our knowledge the only scanning electron microscope study of mesothelium dealt, solely with the ultrastructural morphology of mesothelial microvilli (Andrews and Porter, 1973). The present report deals with several surface features which were not included in Andrews and Porter’s report and further amplifies these authors’ observations on the surface microvilli.
THE
Materials and Methods A total of eight Swiss-Webster
mice of both sexes ranging in weight from 18 to 20 g were anaesthetized with ether. Each animal received an intraperitoneal injection of 1 mg of isotonic tubocurarine B.P. followed 30 set later by 2 ml of a 2.5% gluteraldehyde in 0.15 M cacodylate buffer at room temperature. Pieces of tissue from the anterior abDepartments of Anatomy and Pathology, University of Otago Medical School, Dunedin, New Zealand. Received 27 March 1975. Revised 5 September 1975. II
dominal wall, the diaphragm and the small intestine were removed 5 min after the in vioo fixation. They were postfixed in 1% osmium tetroxide for I hr, dehydrated in ascending grades of acetone and dried by carbon dioxide substitution. The dried specimens were mounted on aluminium stubs, carbon-gold coated and examined in a Siemens Autoscan at 20 kV and at various magnifications. Curare was used prior to fixation to produce muscle relaxation and thus minimize shrinkage of the peritoneal surface. Observations
The mesothelia of the anterior abdominal wall, the diaphragm and the intestinal serosa appear essentially similar. The observations reported here pertain, generally, to all three peritoneal sites. It is evident that the free surface of mesothelium is covered with numerous microvilli, and that there are significant differences in the density of microvilli over that surface (Fig. I ). In most cases mesothelial cell outlines are concealed by the microvilli (Fig. I) and only rarely do they become discernible. Microvilli occasionally interlock in meshwork patterns (Fig. 2) and rarely in unusual coronal formations (Fig. 3). Details of the apical cell membranes of the mesothelium at the base of the microvilli are 159
BARADI
160
revealed by a close examination of the bare areas. The cell surface is closely pitted by minute circular shallow depressions of near uniform diameter (about 800 A) (Fig. 4) and is randomly invaginated by larger shallow depressions of variable diameter measuring as much as 6000 A (Fig. 6). Numerous small spherical structures of almost uniform diameter (about 800 A) protrude from the surface (Fig. 5) as well as occasional large globular formations which may reach a diameter of 4000 A (Fig. 6). Deep circular craters reaching a diameter of up to about 3000 A and giving the impression of stomata or fenestrae are visible (Fig. 6).
AND
RAO
that this arrangement protects the mesothelium from surface friction and ensures its integrity against adhesion formation. The unusual entanglement of microvilli in meshwork patterns and in coronal formations seen in the present study may be morphological evidence for the existence of such compartments. In several light microscope publications, including some dating back to the nineteenth century (references in Odor, 1956), authors have speculated on the existence of intercellular pore-like structures in mesothelium. Physiological studies on the mechanism of solute transfer have indicated that lipidinsoluble molecules traverse the mesothelium by passive diffusion. This phenomenon could be theoretically accounted for by the presence of pores occupying up to 0.6% of mesothelial surface area (Boen, 1961; Brendt and Gosselin, 1961a, b, 1962; Gosselin and Brendt, 1962). Our observations suggest the occurrence of intracellular mesothelial stomata or fenestrae. Admittedly further proof is needed to demonstrate that these structures do indeed span the height of the cell from the apical surface to the basal surface. Certain studies with electron-opaque tracers (Odor, 1956; Fukuta, 1963; Staubesand, 1963) have suggested that transport across mesothelium is carried out mainly by micropinocytotic vesicles and by vacuoles, a finding which is not entirely consistent with the process of passive diffusion. More recent in vitro and in vivo experiments performed with improved tracer techniques strongly favour intercellular over intracellular trans-
Discussion The consistent and characteristic distribution of microvilli over the mesothelial free surface suggests that the regularly disposed bare areas are true features of mesothelial morphology rather than preparation artefacts. Andrews and Porter (1973) are also of the opinion that these denuded areas may exist in vivo. Previous transmission electron microscope studies, in several species, have shown that microvilli have an uneven distribution along the mesothelial surface (Baradi and Hope, 1964; Kluge and Hovig, 1967a). Andrews and Porter (1973) suggested that the peritoneal serous exudate becomes entrapped in small compartments created by adjacent microvilli being held together by the strong water-binding capabilities of the negatively charged acid glycosaminoglycans of microvillous glycocalyx. They postulated
Fig. 1. Mesothelial surface showing numerous Cell outlines are not apparent. x 2500. Fig. 2. Microvilli
in meshwork
Fig. 3. Microvilli
in coronal
patterns.
formation.
microvilli
and occasional
bare areas.
x 5750. x 5750
Fig. 4. A bare area pitted by small circular
shallow depressions.
Fig. 5. A bare area showing
protruding
small spherical
structures.
x 12,500. x 12,500.
Fig. 6. A bare area demonstrating what appear to be stomata (arrows), depressions (arrowhead), and large globular formations. x 23,000.
large shallow
BARADI
162
port routes (Casley-Smith, 1967; Cotran and Majno, 1967; Cotran and Karnovsky, 1968; Kluge, 1969). The small and the large circular shallow depressions seen by us at the mesothelial free surface probably represent plasmalemmal vesicles and vacuoles in communication with the cell membrane. If these structures are not engaged in some form of intracellular transport phenomena then their functional significance is still open to question. We are unable to comment on the nature and/or functional significance of the small protruding spherical structures sometimes
AND
RAO
seen at the mesothelial free surface. Surface globular membranous formations termed ‘extrusion vacuoles’ were noted in association with normal and necrotic mesothelium (Raftery, 1973). If the large globular formations in our micrographs are in fact ‘extrusion vacuoles’, this perhaps signifies cell death involved in a process of mesothelial turnover. Acknowledgement
The authors thank Professor W. D. Trotter for reading the manuscript and Mr B. T. Partridge for technical help.
References ANDREWS, P. M. and PORTER, K. R. 1973. Theultrastructure morphology and possible functional significance of mesothelial microvilli. Anat. Rec., 177, 409-426. BARADI, A. F. and HOPE, J. 1964. Observations on ultrastructure of rabbit mesothelium. Expl. Cell Res., 34, 33-44. BOEN, S. T. 1961. Kinetics of peritoneal dialysis. Medicine, 40, 243-287. BRENDT, W. 0. and GOSSELLN,R. E. 1961a. Physiological factors influencing radiorubidium flux across isolated rabbit mesentery. Am. J. Physiol., 200, 454458. BRENDT, W. 0. and GOSSELIN, R. E. 1961b. Rubidium and creatinine transport across isolated mesentery. Biochem. Pharmac., 8, 359-366. BRENDT, W. 0. and GOSSELIN, R. E. 1962. Differential permeability of the mesentery to rubidium and phosphate. Am. J. Physiol., 202, 761-767. CALSEY-SMITH,J. R. 1967. An electron microscopic study of the passage of ions through endothelium of lymphatic and blood capillaries and through mesothelium. Q. Jl exp. Physiol., 52, 105-I 13. COTRAN, R. S. and MAJNO, G. 1967. Studies on intercellular junctions of mesothelium and endothelium. Protoplasma, 63,45-51. COTRAN, R. S. and KARNOVSKY, M. J. 1968. Ultrastructural studies on the permeability of mesothelium to horseradish peroxidase. J. Cc/[ Biol., 37, 123-l 37. FELIX, M. D. 1961. Observations on the surface cells ofthe mouse omentum as studied with phase contrast and electron microscopes. J. natn. Cancer Inst., 27, 713-745. FUKUTA, H. 1963. Electron microscopic study on normal rat peritoneal mesothelium and its changes in absorption of particular iron dextran complex. Acta Path. Jop., 13, 309-325. GOSSELIN,R. E. and BRENDT, W. 0.1962. Diffusional transport of solutes through mesentery and peritoneum. J. theor. Biol., 3, 487-495. KLUGE, T. 1969. The permeability of mesothelium to horseradish peroxidase. Acfapath. microbio/. wand., 75, 251-269. KLUGE, T. and Hov~ti, T. 1967a. The ultrastructure of human and rat pericardium. 1. Parietal and visceral mesothelium. Acta path. mirrobiol. stand., 71, 529-546. KLUGE, T. and HOVIG, T. 1967b. The ultrastructure of human and rat pericardium. 2. Intercellular spaces and junctions. Acta path. microbial. stand.. 71, 547-563. ODOR, D. L. 1954. Observations of the rat mesothelium with the electron and phase microscopes. Am. J. Anat., 95, 433-465. ODOR, D. L. 1956. Uptake and transfer of particulate matter from the peritoneal cavity of the rat. J. biophys. biochem. Cytol., 2 (Suppl.), 105-107. RAFTERY, A. T. 1973. Mesothelial cells in peritoneal fluid. J. Anat., 115, 237-253. STAUBESAND,J. 1963. Zur histophysiologie des Herzbeutels. II. Mitteilung. Elektronmikroskopische Untersuchungen tiber die Passage von Metall-solen durch mesotheliale Membranen. Z. Zellforsch. mikrosk. Anot., 58,915-952.
