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Ultrastructure of ovine ruminal epithelium and localization of sodium in the tissue
© 1970 by Academic Press, Inc.
j. ULTRASTRUCTtJRERESEARCr~30, 385-401 (1970)
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U l t r a s t r u c t u r e of Ovine Ruminal Epithelium and Localization of Sodium in the Tissue 1 RAY C. HENRIKSONz
Division of Animal Physiology, C.S.LR. O. Prospect, N.S.W., Australia Received June 13, 1969 The rumen is lined by a keratinizing epithelium across which are transported large amounts of sodium and short-chain fatty acids. Maculae occludentes, but apparently not zonulae occludentes, are seen in the epithelium. Actively transported sodium may diffuse across the stratum corneum by way of the maculae occludentes and be pumped into the intercellular space in the midlevel of the epithelium. After appropriate fixation precipitates of sodium pyroantimonate are noted in the intercellular space and along cell membranes in the mid-epithelium. A barrier to the back diffusion of solutes may be formed by the narrow tortuous intercellular space, the polysaccharide coating on cornified cells, and the maculae occludentes. Thus, active transport of sodium across ruminal epithelium appears to have a similar morphological basis to that mechanism postulated for transport across frog epidermis. The rumen is a voluminous dilatation of the alimentary canal found in many members of the Artiodactyla such as the sheep, goat, and cow. In the adult sheep, the rumen has a volume of several liters and is partially subdivided into dorsal and ventral sacs. A keratinizing nonglandular epithelium lines the rumen (12, 17, 22, 28) and the epithelium bears some histological similarity to epidermis. The surface area of the rumen is increased considerably by the presence of papillae which vary in shape and size (27) and contain a richly vascularized connective tissue core (6, 29). Considerable research has been done on the physiology of the rumen (see reviews in H a n d b o o k of Physiology, Sect. 6, Vol. V, 1968). Ingested plant material is passed into the rumen, where it is fermented by microorganisms. Short-chain fatty acids (acetic, propionic, and butyric) are the main products of fermentation, several moles of fatty acids are absorbed from the rumen per day (4), and the fatty acids represent 70-80 % of the total energy intake of the animal (1). The fatty acids diffuse from the rumen into the plasma. Acetic acid passes from the tureen to the plasma without z Some of the research included in this publication was presented to the Anatomical Society of Australia and New Zealand, May 1969, in a paper entitled "Ruminal Epithelium: Transport of Solutes in the Absence of Tight Junctions (Zonulae Occludentes)," aT. Anat., London, in press. 2 Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York.
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being extensively metabolized by the lining epithelium whereas propionic acid and to a greater extent butyric acid are metabolized by the epithelium before reaching the blood (2). Sodium is transported, against its electrochemical gradient, from the interior of the rumen into the plasma (10, 16). The transport of sodium from the rumen is of considerable physiological significance; an amount of sodium equivalent to half that contained in the extracellular space of the animal is transported per day from the rumen to the plasma (11). The rate of sodium transport across ruminal epithelium is similar to that observed across frog skin (10). A mechanism for sodium transport across amphibian epidermis has been suggested by Koefoed-Johnsen and Ussing (20) and recently has been modified by Farquhar and Palade (15). The salient features include inward diffusion of sodium across the cornified layer by way of areas of membrane fusion, pumping of sodium into the intercellular space at the mid-level of the epithelium, and prevention of back diffusion by zonulae occludentes. In studying ruminal epithelium with the electron microscope Hyddn and Sperber (17) thought that water and solutes diffuse through an open intercellular space to the level of basal cells, clearly a different process from that suggested by Farquhar and Palade (15). In another study of the ultrastructure of ruminal epithelium, Schnorr and Vollmerhaus (28) make little attempt to view the results within the framework of existing knowledge of the structural basis of transport across epithelial tissues. The aim of the present study was to obtain further structural information on ruminal epithelium in the sheep and in addition to use the pyroantimonate reaction for the localization of sodium. The results have been used to make an assessment of the transport function of ruminal epithelium in the light of recent research on the gall bladder, frog skin, and kidney. MATERIALS AND METHODS
Removal of tissues. Rumen lining from various regions of the organ was removed from six sheep (Ovis aries; Merino or Merino-English Leicester breeds) either at autopsy, within a few minutes after death, or by biopsy through a rumen fistula. The latter technique proved to be more satisfactory since it afforded the possibility of making sequential observations on an animal. Chevalier-Jackson specimen forceps (length 29 cm) with basket punch jaws were found to be a satisfactory instrument for biopsying the rumen wall. The instrument was inserted through a fistula to the opposite lateral wall of the rumen. A biopsy of rumen surface, about 10 square millimeters, could be cut from the wall, apparently with little discomfort to the animal. Another biopsy technique which worked well was to reach into the rumen through a large (10 cm diameter) fistula and to biopsy the wall with curved surgical scissors. This approach offered the advantage of being able to see the biopsy site.
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FIG. 1. Surface view of a whole-mount of the wall of the rumen. Large, paddle-shaped papillae are seen toward the left of the field and they contrast with the much smaller papillae at the right. × 1.7. FIG. 2. Section, approximately 3 mm thick, through the tureen wall showing papillae in lateral view. x2.3. FIo. 3. Section of rumen wall showing entire thickness of the lining epithelium and some subepithelial connective tissue, stained with Alcian blue in 0.1 N HCI. Horny cells are sharply outlined by stained material (arrows). x 250. FrG. 4. An area similar to that shown in Fig. 3, but stained by the PAS reaction. Horny cells are clearly outlined by stained material (arrows). The swollen appearance of many of the surface cells is demonstrated, x 250.
Tissue fixation and microscopy. Ruminal epithelium was fixed in osmium tetroxide buffered by Veronal acetate, collidine, or phosphate. Glutaraldehyde buffered by cacodylate was used in some instances. Tissues were dehydrated in ethanol and embedded in Araldite or Maraglas. Generally, thin sections were stained with saturated aqueous uranyl acetate followed by lead citrate. The electron microscope used was a Hitachi HU-11C. For light microscopy, tissues were fixed in phosphate-buffered formalin or in Bouin's or Helly's solutions. Paraffin sections were stained by hematoxylin and eosin, periodic acidSchiff (PAS) reaction (diastase control), Alcian blue in 0.1 NHC1, or Feulgen reaction. Pyroantimonate technique. Fixatives containing pyroantimonate were used to locate sodium by precipitating it in the epithelium as sodium pyroantimonate (21, 35). After exposure to these fixatives, the tissues were processed for electron microscopy as described above. OBSERVATIONS
Light microscopy Papillae cover the l u m i n a l surface of the rumen. They vary f r o m low, knob-like p r o t r u s i o n s less t h a n 1 m m high, to p e n d u l o u s extensions of the surface, approximately 5 m m i n length (Figs. 1 a n d 2).
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A sequence of differentiation similar to that noted in mammalian epidermis is seen in the epithelium lining the rumen. Basal cells in ruminal epithelium are the germinative cells, as evidenced by their ability to incorporate tritiated thymidine (Henrikson, unpublished). The remainder of the Malpighian layer varies considerably in thickness but generally is formed proximally (relative to the basal lamina) by cells surrounded by conspicuous intercellular spaces and distally by cells containing keratohyalin-like granules. The epithelial cells which topographically correspond to the stratum corneum of the epidermis have a variable morphology. Some cells forming the surface layers are flattened, whereas others are swollen by a large vacuole. Material stained by the PAS reaction and by the Alcian blue technique, presumably a polysaccharide, sharply outlines the horny cells, but is absent from the more proximal layers of the epithelium (Figs. 3 and 4). Some regional differences are noted by light microscopy in the structure of the epithelium. Vacuolated horny cells generally are absent from the tips of the papillae, probably because of abrasion of the surface by ingesta. Vacuolated surface cells are regularly found, however, along the more protected lateral surfaces of the papillae and on the interpapillary epithelium. A second regional difference is seen when comparing epithelium from the dome of the dorsal sac which normally is exposed to a gas space, above the fermenting ruminal contents, with the more ventral aspects of the surface. Those parts of the rumen in contact with the gas space are covered by a thicker layer of horny cells. After staining by the Feulgen reaction, nuclei are very distinct in deeper epithelial cells. Some Feulgen-positive material is seen in the proximal cells of the stratum corneum, but no reaction is apparent in the most superficial cells.
Electron microscopy There were no indications of regional differences in the fine structure of a given type of epithelial cell from papillae or interpapillary areas, or in cells from the dorsal and ventral sacs. Also, no consistent morphological differences were encountered it1 sequential biopsies from a single animal or in epithelium from different animals. Basal cells. The surface of the basal cell abutting the connective tissue is often covered by an irregular, microvillus-like array of cell processes (Figs. 5-7). Maculae FIG. 5. Electron micrograph of the entire thickness of ruminal epithelium. Part of a blood vessel (BV) is seen in transverse section near the base of the epithelium. Cells in the deep and mid-layers of the epithelium contain dense populations of mitochondria. (*). Most cells at this level are surrounded by a relatively wide intercellular space. More superficially, the epithelial cells begin to flatten and dense granules, resembling keratohyalin (K), differentiate in the cytoplasm. The surface cells lack the numerous organelles seen more proximally in the epithelium. Some surface cells are dense and flattened whereas others contain a large vacuole (V). Some bacteria are seen on the luminal surface. x 5300.
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FIG. 6. Proximal surface of ruminal epithelium, sectioned obliquely. The deeply indented basal surface is characteristic of this epithelium, x 20,000.
occludentes a n d desmosomes occasionally j o i n adjacent processes a n d h e m i d e s m o somes often are seen at the tips of the processes, adjacent to the basal lamina. Organelles such as m i t o c h o n d r i a are n o t f o u n d in the processes. A typical b a s a l l a m i n a separates the epithelium f r o m the connective tissue; however, the basal l a m i n a does n o t follow the irregularities of the cell surface (Fig. 7). Instead, the l a m i n a follows a p l a n e demarcated by the tips of the processes. I n rare instances the base of the epithelium presents a relatively s m o o t h surface to the u n d e r l y i n g connective tissue. I n the cytoplasm of the basal cells, distal to the basal processes, is f o u n d a dense FIG. 7. Example of the elaborate microvilli on the proximal surface of a basal cell. Flocculent material is seen in the intercellular space. A basal lamina is seen toward the bottom of the field, x 30,000. FIG. 8. Distal cytoplasm of a cell containing a small keratohyalin granule (1<2). Note the clustering of ribosomes around the forming granule. Other smaller and more sharply defined granules are seen in this area of the cytoplasm (arrow). Their size and distribution are suggestive of membrane-coating granules. The intercellular space at this level of the epithelium is relatively narrow. × 35,000. F~G. 9. Cytoplasm of a cell in the mid-epithelium showing a typical concentration of mitochondria. x 50,000.
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FIG. 10. Junction between several cells in the mid-epithelium. The complex intercellular channel is prominent (*) and several desmosomes and a distinct macula occludens (-~) are seen along the surfaces, x 60,000. population of mitochondria (Fig. 5). Golgi membranes and elements of the ergastoplasm are seldom encountered. The intercellular space surrounding the distal and lateral aspects of basal cells is dilated but the apposition of cell processes at the basal surface closes off the intercellular space, to varying degrees, from the basal lamina and connective tissue (Figs. 5-7). Mid-epithelial cells. The thickness of this layer varies considerably due to the corrugated nature of the epithelium. The proximal cells, bordering the basal layer, are nearly spherical and many finger-like processes project from their surfaces. The processes found at this level in the epithelium are shorter and less numerous than those covering the proximal surface of basal cells. Desmosomes and maculae occludentes are seen where processes from adjacent cells make contact (Fig. 10). The intercellular space is prominent at this level. Unusually high concentrations of mitochondria are found in cells from the midlayers of the epithelium (Figs. 5 and 9). The mitochondria, other than containing
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Fie. 11. Junction between two cornified cells at the surface of the epithelium. A point of fusion between adjacent plasma membranes, a macnla occludens, is seen (-+). × 100,000. very dense granules in their matrices, are not atypical. In general, mitochondria are clustered deep in the cytoplasm and are not found in the ectoplasm. Mitochondria in these cells, as well as in deeper cells, are not oriented in any particular plane (Fig. 5). In the more distal level of the mid-epithelium cells become more compactly arranged and the width of the intercellular space is drastically reduced (Figs. 5, 8). Also often noted in the cells are structures which resemble keratohyalin granules. The granules are first recognizable as small electron-opaque masses surrounded by ribosomes (Fig. 8). The relationship between the granules and ribosomes is maintained as the granules increase in size. Areas of the epithelium do exist, however, where keratohyalin granules are absent. Spherical or ovoid granules 1200-1500 N in diameter are regularly observed in the cytoplasm of cells in the mid-epithelium, especially near the distal cell surface (Fig. 8). These organelles are similar in size and distribution to membrane-coating granules (23). Similar granules are described by Schnorr and Vollmerhaus (28) in ruminal epithelium from goats and cattle. They consider the granules to be phospholipid. Surface cells. The distal layers of cells show a variable morphology which probably is related to their stage of differentiation (Fig. 5). The entire layer is comparable to the stratum corneum of epidermis. Flattened, electron-opaque cells generally are found nearer the keratohyalin-containing cells, and vacuolated cells are situated
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more superficially. In some areas, however, the two kinds of cells may intermingle, the vacuolated horny cells being more proximal. The vacuole, which is formed by what appears to be a homogeneous fibrillar material, is not bounded by a membrane, and it is always separated from the plasma membrane by a denser layer of cytoplasm (Fig. 15). In the more opaque cytoplasm surrounding the vacuole may be identified remains of organelles, but nuclei or their remnants are not usually apparent in ruminal horny cells. The "keratin pattern" (5) is not seen in ruminal horny cells. A bilaminar cell membrane surrounds horny cells (Figs. 12-14). Forming the cytoplasmic side of the membrane is an electron-opaque surface 130-180 ]t thick separated by a light space from the outer lamina which is 30-50 A thick. The outer lamina is covered by fine projections (Figs. 12-14). Deposits of fibrillar material are seen between horny cells, and this material is sharply demarcated from the fibrils associated with the cell membrane (Figs. 13 and 14). Desmosomes and maculae occludentes connect horny cells (Fig. 11). No zonulae occludentes are seen. Lysosome-like structures are not found in horny cells. Vollmerhaus and Schnorr (33), studying ruminal epithelium of the goat, indicate that lysosomes are found in horny cells and have an important role in transport across the superficial layers of the epithelium. Large numbers of microorganisms are found over the surface of the epithelium (Fig. 15). In a few instances bacteria are noted in the intercellular space between the most superficial cells. Occasionally the entry of bacteria into horny cells is observed and sometimes microorganisms in horny cells are surrounded by a light zone (evidence of lysis?). Location of sodium pyroantimonate. Electron-opaque precipitates of sodium pyroantimonate are regularly noticed along the luminal surface of the epithelium. Precipitates also are found in vacuolated horny cells which suggests that these cells are no longer able to prevent the accumulation of sodium (Fig. 16). In the mid-layers of the epithelium particles of sodium pyroantimonate are noted in the intercellular space close to the cell surface (Figs. 17 and 18). It is in this part of the epithelium that the intercellular space is dilated. Throughout the mid and deeper levels of the epithelium a fine precipitate of sodium pyroantimonate is seen in the cytoplasm and also in
FIG. 12. The surface of a horny cell lining the rumen. Some blunt processes are seen projecting from the surface which is covered by a fine fibrillar material. At some points continuity of the fibrils with the outer lamina of the plasma membrane is seen (arrows). x 62,000. FtG. 13. Portions of two superficial horny cells. The intercellular space is clearly shown, x 30,000. Fro. 14. In the intercellular space of the horny layer of ruminal epithelium are seen cell processes sectioned through various planes. The bilaminar horny cell membrane is shown (arrow). A sharp junction is seen between fibrils associated with the cell membrane and material found in the intercellular space, x 58,000.
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FI~. 15. Microorganisms observed at the surface of the epithelium. Portions of two horny cells are included in the lower part of the field. The denser rim of cytoplasm (arrows) and lighter vacuoles in the center of each cell are shown, x 8000. nuclei (cf. 30, 36). In general, precipitates are less numerous around basal cells but are noted in the plasma of subepithelial blood vessels.
DISCUSSION Farquhar and Palade (15) have proposed a model for sodium transport across amphibian epidermis which is based on the electrophysiological evidence of KoefoedJohnsen and Ussing (20) and their own ultrastructural findings. One notable feature of this model is the proposal that sodium is transported through the intercellular spaces. They suggest that sodium diffuses across the stratum corneum and moves intracellularly across the epithelium by way of the maculae and zonulae occludentes. Sodium is then pumped into the intercellular space by a mechanism involving an adenosine triphosphatase located in the plasma membranes of epithelial cells. Sodium pumped into the intercellular space, at approximately the mid level of the epithelium, then diffuses along the space to the dermis. Further insight into the problem of sodium transport across other epithelia has been provided by recent studies on gall bladder epithelium by Diamond and Tormey (8), Kaye et al. (19), and Tormey and Diamond (32) and on renal tubular epithelium by Thorburn and Molyneux (31) and Schmidt-Neilsen and Davis (26). In the present study of ruminal epithelium the site of high sodium concentration, as judged by precipitates of sodium pyroantimonate, was in the widened intercellular spaces of the mid-epithelial layers. The concentration of sodium decreased in the more basal layers of the epithelium. This distribution of sodium might be anticipated if the Standing-Gradient Osmotic Flow Theory of Diamond and Bossert (9) were applicable to ruminal epithelium. The model proposes that sodium is actively trans-
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Fins. 16-18. Ruminal epithelium exposed to potassium pyroantimonate during fixation by osmium tetroxide. Fro. 16. Particles of sodium pyroantimonate are seen on the luminal surface, at the top of the field, and smaller particles are noted in the large vacuole which occupies much of the horny cell. Very little sodium pyroantimonate is observed in the peripheral rim of denser cytoplasm (arrows). Unstained. x 25,000. Fins. 17 and 18. The intercellular space (*) in the mid-epithelium. The space is relatively wide and electron-opaque deposits of sodium pyroantimonate line the cell membrane (arrows). Fig. 17 stained; Fig. 18 unstained. ×25,000.
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ported into the intercellular spaces, thereby producing a localized region of high solute concentration. The hyperosmotic region draws water into the space and as the fluid passes between the cells toward the basal lamina, more water is drawn in and the fluid leaves the space isosmotic with the intracellular fluid. Essential to these models is the presence of a barrier in the intercellular space to prevent or minimize the back diffusion of sodium from the site of high sodium concentration to the lumen. A significant back diffusion would decrease the net sodium flux and short-circuit the transepithelial potential. Such a barrier would also prevent the free movement of other solutes along the intercellular spaces from the lumen to the blood. There is evidence to suggest that the permeability of ruminal epithelium is low (34); for example, the water permeability may be calculated to be in the range of that found for frog skin. Therefore, there does appear to be an effective barrier preventing the free intercellular movement of water-soluble compounds across ruminal epithelium. Hyd~n and Sperber (17) proposed that passage of materials across ruminal epithelium is largely impeded by the microvillous processes on basal cells. Schnorr and Vollmerhaus (28) concluded that the barrier in ruminal epithelium is at the level of proximal horny cells and distal granular cells, and also that the barrier is related to the narrowness of the intercellular space at this level in the epithelium. The present study tends to support the observations of Schnorr and Vollmerhaus. However, two additional fine structural features have been described which may be of importance in limiting membrane permeability: the points of fusion between plasma membranes and the polysaccharide coating of cornified cells. The intercellular space between cells in the stratum corneum is narrow and the surfaces of these cells may be deeply infolded, although dilatations of the intercellular space are sometimes seen. Desmosomes and maculae occludentes lie along the surface. It might be expected that the flow of materials through the intercellular space would be influenced both by the maculae occludentes and the complex nature of the intercellular channel. The surface coating on keratinized cells of ruminal epithelium is morphologically similar to coatings found on a variety of other types of cells and might be considered an example of a "glycocalyx" (3). The mucopolysaccharide nature of the coating on intestinal microvilli has been demonstrated histochemically and the continuity of the fine fibrils forming the surface coat with the outer lamina of the unit membrane may be seen by electron microscopy (18). In studying the surface coat on various cells from the rat Rambourg and co-workers (24, 25) came to the conclusion that the surface coating is not part of the plasma membrane and is composed of glycoproteins and acidic residues. In the present study, the PAS and Alcian blue reactivity at the surface of keratinized cells probably is related to the surface coating or "fuzz,"
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some of which is seen by electron microscopy to be continuous with the outer lamina of the thickened cell membrane. It is likely that this surface coating of mucopolysaccharide would influence the movement of substances through the intercellular space. The possibility of a barrier existing more proximally in ruminal epithelium must be considered. Recent experiments performed in this laboratory (Henrikson and Stacy, unpublished) have shown that horseradish peroxidase, infused into a small ruminal artery, penetrates the epithelium but stops at the level of proximal cornified cells. While the tortuous intercellular space, cellular processes, desmosomes, and maculae occludentes in the deeper levels of the epithelium do not function as an absolute barrier, they probably retard the transepithelial movement of solutes and fluids. In the present study of ruminal epithelium some cell-to-cell contacts are formed by fusion of the outer leaflets of the plasma membranes. The extracellular space is not seen at the point of fusion. Similar examples of this have been described by many investigators and have been called "maculae occludentes" and "zonulae occludentes" (13) and "nexuses" (7). Points of membrane fusion or maculae occludentes are found throughout ruminal epithelium from the basal layer to the superficial cornified cells. The length of the zone of fusion varies from about 300 ~ to approximately 1 #. In general, the maculae occludentes between cornified cells are smaller and less numerous than the maculae occludentes found deeper in the epithelium. The points of membrane fusion are less numerous than desmosomes. The maculae occludentes appear not to be oriented in any particular plane in the epithelium. The zones of membrane fusion between ruminal epithelial cells are punctate; there is little evidence that the maculae occludentes are extended to form "belts" or "zonulae" which are described by Farquhar and Palade (14) in the superficial layers of amphibian epidermis. This type of membrane fusion has been termed a "zonula occludens" and is thought to seal off the intercellular space of the epithelium from the external environment. It is evident that the passage of metabolites across ruminal epithelium is not dependent on the intercellular space being completely blocked by zonulae occludentes. The possibility does exist, however, that in ruminal epithelium the punctate maculae occludentes are partial barriers to diffusion through the intercellular space as well as sites of cell-to-cell diffusion of solutes. Despite indications from earlier work (17) there appears to be no need to invoke new mechanisms of sodium transport across ruminal epithelium. The basic essentials of the model discussed by Farquhar and Palade (15) appear to be present, with the exception of zonulae occludentes, the function of which is probably served by other structures. The present results with ruminal epithelium may therefore be adequately explained on the combined basis of the structural features enunciated by Farquhar 2 6 - 691822 J . Ultras~ruc~ure Research
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and Palade (15) and the theory of transport function developed by Diamond and Bossert (9). The author thanks R. T. Gemmell for assistance. REFERENCES 1. ANNISON, E. F., in DOUGHERTY, R. W. et al. (Eds.), Physiology of Digestion in the Ruminant, p. 185. Butterworths, Washington, D.C., 1965. 2. ARMSTRONG,O. G., ibid. p. 272. 3. BENNETT,H. S., J. Histochem. Cytochem. 11, 14 (1963). 4. BLAXTER,K. L., The Energy Metabolism of Ruminants. Hutchinson, London, 1962. 5. BRODY, I., J. Ultrastruct. Res. 2, 482 (1959). 6. CHEETrIAM,S. E. and STEVEN, D. H., J. Physiol. (London) 186, 56P (1966). 7. DEWEY, M. M. and BARR, L., J. Cell Biol. 23, 553 (1964). 8. DIAMOND,J. M. and TORMEY,J. McD., Nature. 210, 817 (1966). 9. DIAMOND,J. M. and BOSSERT,W. H., J. Gen. Physiol. 50, 2061 (1967). 10. DOBSON,A., J. Physiol. (London) 146, 235 (1959). 11. DOBSON,A. and PIMLLIPSON,A. T., in CODE, C. E. (Ed.), Handbook of Physiology, Sect. 6, Vol. 5, p. 2761. Am. Physiol. Soc., Washington, D.C., 1968. 12. DOBSON, M. J., BROWN, W. C. B., DOBSON,A. and PHILLIPSON,A. T., Quart. J. Exptl. Physiol. 41, 247 (1956). 13. EARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). 14. - ibid. 26, 263 (1965). 15. - - - - ibid. 30, 359 (1966). 16. HYD~N, S., Lantbrukshogskol. Ann. 27, 273 (1961). 17. HYD£N, S. and SVERBER,I., in DOUGHBRTY,R. W. et al. (Eds.), Physiology of Digestion in the Ruminant, p. 51. Butterworths, Washington, D.C., 1965. 18. ITO, S., J. CellBiol. 27, 475 (1965). 19. KAYE, G. I., WHEELER,H. O., WHITLOCK,R. T. and LANE, N., J. Cell Biol. 30, 237 (1966). 20. KOEFOED--JoHNSEN,V. and USSING, H. H., Acta Physiol. Scand. 42, 298 (1958). 21. KOMNICK,H., Protoplasma 55, 414 (1962). 22. LINDH~.,B. and SVERBER,I., Lantbrukshogskol. Ann. 25, 321 (1959). 23. MATOLTSY,A. G. and PARAKKAL,P. F., in ZELICKSON,A. S. (Ed.), Ultrastructure of Normal and Abnormal Skin, p. 76. Lea & Febiger, Philadelphia, Pennsylvania, 1967. 24. RAMBOURG,A., NEUTRA, M., and LEBLONO,C. P., Anat. Record 154, 41 (1966). 25. RAMBOURG,A., and LEBLOND,C. P., Y. Cell Biol. 32, 27 (1967). 26. SCHMIDT--NIELSEN,B. and DAVIS, L. E., Science 159, 1105 (1968). 27. SCHNORR,B. and VOLLMERHAUS,B., Zentr. Veterinaermed. A 14, 93 (1967). 28. - - - - ibid. 14, 789 (1967). 29. - ibid. 15, 799 (1968). 30. SVlCER, S. S., HARDIN, J. H. and GREENE, W. B., J. Cell Biol. 39, 216 (1968). 31. THORBURN,G. D. and MOL'zZqEUX,G. S., Bull. Post Grad. Comm. Med. Univ. Sydney, 23, 199 (1967).
RUMINAL EPITHELIUM 32. 33. 34. 35. 36.
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TORMEY,J. McD. and DIAMOND,J. M., J. Gen. Physiol. 50, 2031 (1967). VOLLMERHAUS,B. and SCHNORR,B., Zentr. Veterinaermed. A 14, 761 (1967). WARNER, A. C. I. and STACY, B. D., Brit. J. Nutr. 22, 389 (1968). ZADUNAISKY,J. A., J. Cell Biol. 31, Cll (1966). ZADUNAISKY,J. A., GENNARO, J. F., JR., BASHIRELAHI,N. and HILTON, M., Y. Gen. Physiol. 51, 290s (1968).
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U l t r a s t r u c t u r e of Ovine Ruminal Epithelium and Localization of Sodium in the Tissue 1 RAY C. HENRIKSONz
Division of Animal Physiology, C.S.LR. O. Prospect, N.S.W., Australia Received June 13, 1969 The rumen is lined by a keratinizing epithelium across which are transported large amounts of sodium and short-chain fatty acids. Maculae occludentes, but apparently not zonulae occludentes, are seen in the epithelium. Actively transported sodium may diffuse across the stratum corneum by way of the maculae occludentes and be pumped into the intercellular space in the midlevel of the epithelium. After appropriate fixation precipitates of sodium pyroantimonate are noted in the intercellular space and along cell membranes in the mid-epithelium. A barrier to the back diffusion of solutes may be formed by the narrow tortuous intercellular space, the polysaccharide coating on cornified cells, and the maculae occludentes. Thus, active transport of sodium across ruminal epithelium appears to have a similar morphological basis to that mechanism postulated for transport across frog epidermis. The rumen is a voluminous dilatation of the alimentary canal found in many members of the Artiodactyla such as the sheep, goat, and cow. In the adult sheep, the rumen has a volume of several liters and is partially subdivided into dorsal and ventral sacs. A keratinizing nonglandular epithelium lines the rumen (12, 17, 22, 28) and the epithelium bears some histological similarity to epidermis. The surface area of the rumen is increased considerably by the presence of papillae which vary in shape and size (27) and contain a richly vascularized connective tissue core (6, 29). Considerable research has been done on the physiology of the rumen (see reviews in H a n d b o o k of Physiology, Sect. 6, Vol. V, 1968). Ingested plant material is passed into the rumen, where it is fermented by microorganisms. Short-chain fatty acids (acetic, propionic, and butyric) are the main products of fermentation, several moles of fatty acids are absorbed from the rumen per day (4), and the fatty acids represent 70-80 % of the total energy intake of the animal (1). The fatty acids diffuse from the rumen into the plasma. Acetic acid passes from the tureen to the plasma without z Some of the research included in this publication was presented to the Anatomical Society of Australia and New Zealand, May 1969, in a paper entitled "Ruminal Epithelium: Transport of Solutes in the Absence of Tight Junctions (Zonulae Occludentes)," aT. Anat., London, in press. 2 Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York.
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being extensively metabolized by the lining epithelium whereas propionic acid and to a greater extent butyric acid are metabolized by the epithelium before reaching the blood (2). Sodium is transported, against its electrochemical gradient, from the interior of the rumen into the plasma (10, 16). The transport of sodium from the rumen is of considerable physiological significance; an amount of sodium equivalent to half that contained in the extracellular space of the animal is transported per day from the rumen to the plasma (11). The rate of sodium transport across ruminal epithelium is similar to that observed across frog skin (10). A mechanism for sodium transport across amphibian epidermis has been suggested by Koefoed-Johnsen and Ussing (20) and recently has been modified by Farquhar and Palade (15). The salient features include inward diffusion of sodium across the cornified layer by way of areas of membrane fusion, pumping of sodium into the intercellular space at the mid-level of the epithelium, and prevention of back diffusion by zonulae occludentes. In studying ruminal epithelium with the electron microscope Hyddn and Sperber (17) thought that water and solutes diffuse through an open intercellular space to the level of basal cells, clearly a different process from that suggested by Farquhar and Palade (15). In another study of the ultrastructure of ruminal epithelium, Schnorr and Vollmerhaus (28) make little attempt to view the results within the framework of existing knowledge of the structural basis of transport across epithelial tissues. The aim of the present study was to obtain further structural information on ruminal epithelium in the sheep and in addition to use the pyroantimonate reaction for the localization of sodium. The results have been used to make an assessment of the transport function of ruminal epithelium in the light of recent research on the gall bladder, frog skin, and kidney. MATERIALS AND METHODS
Removal of tissues. Rumen lining from various regions of the organ was removed from six sheep (Ovis aries; Merino or Merino-English Leicester breeds) either at autopsy, within a few minutes after death, or by biopsy through a rumen fistula. The latter technique proved to be more satisfactory since it afforded the possibility of making sequential observations on an animal. Chevalier-Jackson specimen forceps (length 29 cm) with basket punch jaws were found to be a satisfactory instrument for biopsying the rumen wall. The instrument was inserted through a fistula to the opposite lateral wall of the rumen. A biopsy of rumen surface, about 10 square millimeters, could be cut from the wall, apparently with little discomfort to the animal. Another biopsy technique which worked well was to reach into the rumen through a large (10 cm diameter) fistula and to biopsy the wall with curved surgical scissors. This approach offered the advantage of being able to see the biopsy site.
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FIG. 1. Surface view of a whole-mount of the wall of the rumen. Large, paddle-shaped papillae are seen toward the left of the field and they contrast with the much smaller papillae at the right. × 1.7. FIG. 2. Section, approximately 3 mm thick, through the tureen wall showing papillae in lateral view. x2.3. FIo. 3. Section of rumen wall showing entire thickness of the lining epithelium and some subepithelial connective tissue, stained with Alcian blue in 0.1 N HCI. Horny cells are sharply outlined by stained material (arrows). x 250. FrG. 4. An area similar to that shown in Fig. 3, but stained by the PAS reaction. Horny cells are clearly outlined by stained material (arrows). The swollen appearance of many of the surface cells is demonstrated, x 250.
Tissue fixation and microscopy. Ruminal epithelium was fixed in osmium tetroxide buffered by Veronal acetate, collidine, or phosphate. Glutaraldehyde buffered by cacodylate was used in some instances. Tissues were dehydrated in ethanol and embedded in Araldite or Maraglas. Generally, thin sections were stained with saturated aqueous uranyl acetate followed by lead citrate. The electron microscope used was a Hitachi HU-11C. For light microscopy, tissues were fixed in phosphate-buffered formalin or in Bouin's or Helly's solutions. Paraffin sections were stained by hematoxylin and eosin, periodic acidSchiff (PAS) reaction (diastase control), Alcian blue in 0.1 NHC1, or Feulgen reaction. Pyroantimonate technique. Fixatives containing pyroantimonate were used to locate sodium by precipitating it in the epithelium as sodium pyroantimonate (21, 35). After exposure to these fixatives, the tissues were processed for electron microscopy as described above. OBSERVATIONS
Light microscopy Papillae cover the l u m i n a l surface of the rumen. They vary f r o m low, knob-like p r o t r u s i o n s less t h a n 1 m m high, to p e n d u l o u s extensions of the surface, approximately 5 m m i n length (Figs. 1 a n d 2).
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A sequence of differentiation similar to that noted in mammalian epidermis is seen in the epithelium lining the rumen. Basal cells in ruminal epithelium are the germinative cells, as evidenced by their ability to incorporate tritiated thymidine (Henrikson, unpublished). The remainder of the Malpighian layer varies considerably in thickness but generally is formed proximally (relative to the basal lamina) by cells surrounded by conspicuous intercellular spaces and distally by cells containing keratohyalin-like granules. The epithelial cells which topographically correspond to the stratum corneum of the epidermis have a variable morphology. Some cells forming the surface layers are flattened, whereas others are swollen by a large vacuole. Material stained by the PAS reaction and by the Alcian blue technique, presumably a polysaccharide, sharply outlines the horny cells, but is absent from the more proximal layers of the epithelium (Figs. 3 and 4). Some regional differences are noted by light microscopy in the structure of the epithelium. Vacuolated horny cells generally are absent from the tips of the papillae, probably because of abrasion of the surface by ingesta. Vacuolated surface cells are regularly found, however, along the more protected lateral surfaces of the papillae and on the interpapillary epithelium. A second regional difference is seen when comparing epithelium from the dome of the dorsal sac which normally is exposed to a gas space, above the fermenting ruminal contents, with the more ventral aspects of the surface. Those parts of the rumen in contact with the gas space are covered by a thicker layer of horny cells. After staining by the Feulgen reaction, nuclei are very distinct in deeper epithelial cells. Some Feulgen-positive material is seen in the proximal cells of the stratum corneum, but no reaction is apparent in the most superficial cells.
Electron microscopy There were no indications of regional differences in the fine structure of a given type of epithelial cell from papillae or interpapillary areas, or in cells from the dorsal and ventral sacs. Also, no consistent morphological differences were encountered it1 sequential biopsies from a single animal or in epithelium from different animals. Basal cells. The surface of the basal cell abutting the connective tissue is often covered by an irregular, microvillus-like array of cell processes (Figs. 5-7). Maculae FIG. 5. Electron micrograph of the entire thickness of ruminal epithelium. Part of a blood vessel (BV) is seen in transverse section near the base of the epithelium. Cells in the deep and mid-layers of the epithelium contain dense populations of mitochondria. (*). Most cells at this level are surrounded by a relatively wide intercellular space. More superficially, the epithelial cells begin to flatten and dense granules, resembling keratohyalin (K), differentiate in the cytoplasm. The surface cells lack the numerous organelles seen more proximally in the epithelium. Some surface cells are dense and flattened whereas others contain a large vacuole (V). Some bacteria are seen on the luminal surface. x 5300.
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FIG. 6. Proximal surface of ruminal epithelium, sectioned obliquely. The deeply indented basal surface is characteristic of this epithelium, x 20,000.
occludentes a n d desmosomes occasionally j o i n adjacent processes a n d h e m i d e s m o somes often are seen at the tips of the processes, adjacent to the basal lamina. Organelles such as m i t o c h o n d r i a are n o t f o u n d in the processes. A typical b a s a l l a m i n a separates the epithelium f r o m the connective tissue; however, the basal l a m i n a does n o t follow the irregularities of the cell surface (Fig. 7). Instead, the l a m i n a follows a p l a n e demarcated by the tips of the processes. I n rare instances the base of the epithelium presents a relatively s m o o t h surface to the u n d e r l y i n g connective tissue. I n the cytoplasm of the basal cells, distal to the basal processes, is f o u n d a dense FIG. 7. Example of the elaborate microvilli on the proximal surface of a basal cell. Flocculent material is seen in the intercellular space. A basal lamina is seen toward the bottom of the field, x 30,000. FIG. 8. Distal cytoplasm of a cell containing a small keratohyalin granule (1<2). Note the clustering of ribosomes around the forming granule. Other smaller and more sharply defined granules are seen in this area of the cytoplasm (arrow). Their size and distribution are suggestive of membrane-coating granules. The intercellular space at this level of the epithelium is relatively narrow. × 35,000. F~G. 9. Cytoplasm of a cell in the mid-epithelium showing a typical concentration of mitochondria. x 50,000.
~~- ~ ,~~ii~, i~
I
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FIG. 10. Junction between several cells in the mid-epithelium. The complex intercellular channel is prominent (*) and several desmosomes and a distinct macula occludens (-~) are seen along the surfaces, x 60,000. population of mitochondria (Fig. 5). Golgi membranes and elements of the ergastoplasm are seldom encountered. The intercellular space surrounding the distal and lateral aspects of basal cells is dilated but the apposition of cell processes at the basal surface closes off the intercellular space, to varying degrees, from the basal lamina and connective tissue (Figs. 5-7). Mid-epithelial cells. The thickness of this layer varies considerably due to the corrugated nature of the epithelium. The proximal cells, bordering the basal layer, are nearly spherical and many finger-like processes project from their surfaces. The processes found at this level in the epithelium are shorter and less numerous than those covering the proximal surface of basal cells. Desmosomes and maculae occludentes are seen where processes from adjacent cells make contact (Fig. 10). The intercellular space is prominent at this level. Unusually high concentrations of mitochondria are found in cells from the midlayers of the epithelium (Figs. 5 and 9). The mitochondria, other than containing
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Fie. 11. Junction between two cornified cells at the surface of the epithelium. A point of fusion between adjacent plasma membranes, a macnla occludens, is seen (-+). × 100,000. very dense granules in their matrices, are not atypical. In general, mitochondria are clustered deep in the cytoplasm and are not found in the ectoplasm. Mitochondria in these cells, as well as in deeper cells, are not oriented in any particular plane (Fig. 5). In the more distal level of the mid-epithelium cells become more compactly arranged and the width of the intercellular space is drastically reduced (Figs. 5, 8). Also often noted in the cells are structures which resemble keratohyalin granules. The granules are first recognizable as small electron-opaque masses surrounded by ribosomes (Fig. 8). The relationship between the granules and ribosomes is maintained as the granules increase in size. Areas of the epithelium do exist, however, where keratohyalin granules are absent. Spherical or ovoid granules 1200-1500 N in diameter are regularly observed in the cytoplasm of cells in the mid-epithelium, especially near the distal cell surface (Fig. 8). These organelles are similar in size and distribution to membrane-coating granules (23). Similar granules are described by Schnorr and Vollmerhaus (28) in ruminal epithelium from goats and cattle. They consider the granules to be phospholipid. Surface cells. The distal layers of cells show a variable morphology which probably is related to their stage of differentiation (Fig. 5). The entire layer is comparable to the stratum corneum of epidermis. Flattened, electron-opaque cells generally are found nearer the keratohyalin-containing cells, and vacuolated cells are situated
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more superficially. In some areas, however, the two kinds of cells may intermingle, the vacuolated horny cells being more proximal. The vacuole, which is formed by what appears to be a homogeneous fibrillar material, is not bounded by a membrane, and it is always separated from the plasma membrane by a denser layer of cytoplasm (Fig. 15). In the more opaque cytoplasm surrounding the vacuole may be identified remains of organelles, but nuclei or their remnants are not usually apparent in ruminal horny cells. The "keratin pattern" (5) is not seen in ruminal horny cells. A bilaminar cell membrane surrounds horny cells (Figs. 12-14). Forming the cytoplasmic side of the membrane is an electron-opaque surface 130-180 ]t thick separated by a light space from the outer lamina which is 30-50 A thick. The outer lamina is covered by fine projections (Figs. 12-14). Deposits of fibrillar material are seen between horny cells, and this material is sharply demarcated from the fibrils associated with the cell membrane (Figs. 13 and 14). Desmosomes and maculae occludentes connect horny cells (Fig. 11). No zonulae occludentes are seen. Lysosome-like structures are not found in horny cells. Vollmerhaus and Schnorr (33), studying ruminal epithelium of the goat, indicate that lysosomes are found in horny cells and have an important role in transport across the superficial layers of the epithelium. Large numbers of microorganisms are found over the surface of the epithelium (Fig. 15). In a few instances bacteria are noted in the intercellular space between the most superficial cells. Occasionally the entry of bacteria into horny cells is observed and sometimes microorganisms in horny cells are surrounded by a light zone (evidence of lysis?). Location of sodium pyroantimonate. Electron-opaque precipitates of sodium pyroantimonate are regularly noticed along the luminal surface of the epithelium. Precipitates also are found in vacuolated horny cells which suggests that these cells are no longer able to prevent the accumulation of sodium (Fig. 16). In the mid-layers of the epithelium particles of sodium pyroantimonate are noted in the intercellular space close to the cell surface (Figs. 17 and 18). It is in this part of the epithelium that the intercellular space is dilated. Throughout the mid and deeper levels of the epithelium a fine precipitate of sodium pyroantimonate is seen in the cytoplasm and also in
FIG. 12. The surface of a horny cell lining the rumen. Some blunt processes are seen projecting from the surface which is covered by a fine fibrillar material. At some points continuity of the fibrils with the outer lamina of the plasma membrane is seen (arrows). x 62,000. FtG. 13. Portions of two superficial horny cells. The intercellular space is clearly shown, x 30,000. Fro. 14. In the intercellular space of the horny layer of ruminal epithelium are seen cell processes sectioned through various planes. The bilaminar horny cell membrane is shown (arrow). A sharp junction is seen between fibrils associated with the cell membrane and material found in the intercellular space, x 58,000.
i!
iiiii~i ¸ ii~!iiii!!i ~
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FI~. 15. Microorganisms observed at the surface of the epithelium. Portions of two horny cells are included in the lower part of the field. The denser rim of cytoplasm (arrows) and lighter vacuoles in the center of each cell are shown, x 8000. nuclei (cf. 30, 36). In general, precipitates are less numerous around basal cells but are noted in the plasma of subepithelial blood vessels.
DISCUSSION Farquhar and Palade (15) have proposed a model for sodium transport across amphibian epidermis which is based on the electrophysiological evidence of KoefoedJohnsen and Ussing (20) and their own ultrastructural findings. One notable feature of this model is the proposal that sodium is transported through the intercellular spaces. They suggest that sodium diffuses across the stratum corneum and moves intracellularly across the epithelium by way of the maculae and zonulae occludentes. Sodium is then pumped into the intercellular space by a mechanism involving an adenosine triphosphatase located in the plasma membranes of epithelial cells. Sodium pumped into the intercellular space, at approximately the mid level of the epithelium, then diffuses along the space to the dermis. Further insight into the problem of sodium transport across other epithelia has been provided by recent studies on gall bladder epithelium by Diamond and Tormey (8), Kaye et al. (19), and Tormey and Diamond (32) and on renal tubular epithelium by Thorburn and Molyneux (31) and Schmidt-Neilsen and Davis (26). In the present study of ruminal epithelium the site of high sodium concentration, as judged by precipitates of sodium pyroantimonate, was in the widened intercellular spaces of the mid-epithelial layers. The concentration of sodium decreased in the more basal layers of the epithelium. This distribution of sodium might be anticipated if the Standing-Gradient Osmotic Flow Theory of Diamond and Bossert (9) were applicable to ruminal epithelium. The model proposes that sodium is actively trans-
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Fins. 16-18. Ruminal epithelium exposed to potassium pyroantimonate during fixation by osmium tetroxide. Fro. 16. Particles of sodium pyroantimonate are seen on the luminal surface, at the top of the field, and smaller particles are noted in the large vacuole which occupies much of the horny cell. Very little sodium pyroantimonate is observed in the peripheral rim of denser cytoplasm (arrows). Unstained. x 25,000. Fins. 17 and 18. The intercellular space (*) in the mid-epithelium. The space is relatively wide and electron-opaque deposits of sodium pyroantimonate line the cell membrane (arrows). Fig. 17 stained; Fig. 18 unstained. ×25,000.
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ported into the intercellular spaces, thereby producing a localized region of high solute concentration. The hyperosmotic region draws water into the space and as the fluid passes between the cells toward the basal lamina, more water is drawn in and the fluid leaves the space isosmotic with the intracellular fluid. Essential to these models is the presence of a barrier in the intercellular space to prevent or minimize the back diffusion of sodium from the site of high sodium concentration to the lumen. A significant back diffusion would decrease the net sodium flux and short-circuit the transepithelial potential. Such a barrier would also prevent the free movement of other solutes along the intercellular spaces from the lumen to the blood. There is evidence to suggest that the permeability of ruminal epithelium is low (34); for example, the water permeability may be calculated to be in the range of that found for frog skin. Therefore, there does appear to be an effective barrier preventing the free intercellular movement of water-soluble compounds across ruminal epithelium. Hyd~n and Sperber (17) proposed that passage of materials across ruminal epithelium is largely impeded by the microvillous processes on basal cells. Schnorr and Vollmerhaus (28) concluded that the barrier in ruminal epithelium is at the level of proximal horny cells and distal granular cells, and also that the barrier is related to the narrowness of the intercellular space at this level in the epithelium. The present study tends to support the observations of Schnorr and Vollmerhaus. However, two additional fine structural features have been described which may be of importance in limiting membrane permeability: the points of fusion between plasma membranes and the polysaccharide coating of cornified cells. The intercellular space between cells in the stratum corneum is narrow and the surfaces of these cells may be deeply infolded, although dilatations of the intercellular space are sometimes seen. Desmosomes and maculae occludentes lie along the surface. It might be expected that the flow of materials through the intercellular space would be influenced both by the maculae occludentes and the complex nature of the intercellular channel. The surface coating on keratinized cells of ruminal epithelium is morphologically similar to coatings found on a variety of other types of cells and might be considered an example of a "glycocalyx" (3). The mucopolysaccharide nature of the coating on intestinal microvilli has been demonstrated histochemically and the continuity of the fine fibrils forming the surface coat with the outer lamina of the unit membrane may be seen by electron microscopy (18). In studying the surface coat on various cells from the rat Rambourg and co-workers (24, 25) came to the conclusion that the surface coating is not part of the plasma membrane and is composed of glycoproteins and acidic residues. In the present study, the PAS and Alcian blue reactivity at the surface of keratinized cells probably is related to the surface coating or "fuzz,"
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some of which is seen by electron microscopy to be continuous with the outer lamina of the thickened cell membrane. It is likely that this surface coating of mucopolysaccharide would influence the movement of substances through the intercellular space. The possibility of a barrier existing more proximally in ruminal epithelium must be considered. Recent experiments performed in this laboratory (Henrikson and Stacy, unpublished) have shown that horseradish peroxidase, infused into a small ruminal artery, penetrates the epithelium but stops at the level of proximal cornified cells. While the tortuous intercellular space, cellular processes, desmosomes, and maculae occludentes in the deeper levels of the epithelium do not function as an absolute barrier, they probably retard the transepithelial movement of solutes and fluids. In the present study of ruminal epithelium some cell-to-cell contacts are formed by fusion of the outer leaflets of the plasma membranes. The extracellular space is not seen at the point of fusion. Similar examples of this have been described by many investigators and have been called "maculae occludentes" and "zonulae occludentes" (13) and "nexuses" (7). Points of membrane fusion or maculae occludentes are found throughout ruminal epithelium from the basal layer to the superficial cornified cells. The length of the zone of fusion varies from about 300 ~ to approximately 1 #. In general, the maculae occludentes between cornified cells are smaller and less numerous than the maculae occludentes found deeper in the epithelium. The points of membrane fusion are less numerous than desmosomes. The maculae occludentes appear not to be oriented in any particular plane in the epithelium. The zones of membrane fusion between ruminal epithelial cells are punctate; there is little evidence that the maculae occludentes are extended to form "belts" or "zonulae" which are described by Farquhar and Palade (14) in the superficial layers of amphibian epidermis. This type of membrane fusion has been termed a "zonula occludens" and is thought to seal off the intercellular space of the epithelium from the external environment. It is evident that the passage of metabolites across ruminal epithelium is not dependent on the intercellular space being completely blocked by zonulae occludentes. The possibility does exist, however, that in ruminal epithelium the punctate maculae occludentes are partial barriers to diffusion through the intercellular space as well as sites of cell-to-cell diffusion of solutes. Despite indications from earlier work (17) there appears to be no need to invoke new mechanisms of sodium transport across ruminal epithelium. The basic essentials of the model discussed by Farquhar and Palade (15) appear to be present, with the exception of zonulae occludentes, the function of which is probably served by other structures. The present results with ruminal epithelium may therefore be adequately explained on the combined basis of the structural features enunciated by Farquhar 2 6 - 691822 J . Ultras~ruc~ure Research
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and Palade (15) and the theory of transport function developed by Diamond and Bossert (9). The author thanks R. T. Gemmell for assistance. REFERENCES 1. ANNISON, E. F., in DOUGHERTY, R. W. et al. (Eds.), Physiology of Digestion in the Ruminant, p. 185. Butterworths, Washington, D.C., 1965. 2. ARMSTRONG,O. G., ibid. p. 272. 3. BENNETT,H. S., J. Histochem. Cytochem. 11, 14 (1963). 4. BLAXTER,K. L., The Energy Metabolism of Ruminants. Hutchinson, London, 1962. 5. BRODY, I., J. Ultrastruct. Res. 2, 482 (1959). 6. CHEETrIAM,S. E. and STEVEN, D. H., J. Physiol. (London) 186, 56P (1966). 7. DEWEY, M. M. and BARR, L., J. Cell Biol. 23, 553 (1964). 8. DIAMOND,J. M. and TORMEY,J. McD., Nature. 210, 817 (1966). 9. DIAMOND,J. M. and BOSSERT,W. H., J. Gen. Physiol. 50, 2061 (1967). 10. DOBSON,A., J. Physiol. (London) 146, 235 (1959). 11. DOBSON,A. and PIMLLIPSON,A. T., in CODE, C. E. (Ed.), Handbook of Physiology, Sect. 6, Vol. 5, p. 2761. Am. Physiol. Soc., Washington, D.C., 1968. 12. DOBSON, M. J., BROWN, W. C. B., DOBSON,A. and PHILLIPSON,A. T., Quart. J. Exptl. Physiol. 41, 247 (1956). 13. EARQUHAR,M. G. and PALADE, G. E., J. Cell Biol. 17, 375 (1963). 14. - ibid. 26, 263 (1965). 15. - - - - ibid. 30, 359 (1966). 16. HYD~N, S., Lantbrukshogskol. Ann. 27, 273 (1961). 17. HYD£N, S. and SVERBER,I., in DOUGHBRTY,R. W. et al. (Eds.), Physiology of Digestion in the Ruminant, p. 51. Butterworths, Washington, D.C., 1965. 18. ITO, S., J. CellBiol. 27, 475 (1965). 19. KAYE, G. I., WHEELER,H. O., WHITLOCK,R. T. and LANE, N., J. Cell Biol. 30, 237 (1966). 20. KOEFOED--JoHNSEN,V. and USSING, H. H., Acta Physiol. Scand. 42, 298 (1958). 21. KOMNICK,H., Protoplasma 55, 414 (1962). 22. LINDH~.,B. and SVERBER,I., Lantbrukshogskol. Ann. 25, 321 (1959). 23. MATOLTSY,A. G. and PARAKKAL,P. F., in ZELICKSON,A. S. (Ed.), Ultrastructure of Normal and Abnormal Skin, p. 76. Lea & Febiger, Philadelphia, Pennsylvania, 1967. 24. RAMBOURG,A., NEUTRA, M., and LEBLONO,C. P., Anat. Record 154, 41 (1966). 25. RAMBOURG,A., and LEBLOND,C. P., Y. Cell Biol. 32, 27 (1967). 26. SCHMIDT--NIELSEN,B. and DAVIS, L. E., Science 159, 1105 (1968). 27. SCHNORR,B. and VOLLMERHAUS,B., Zentr. Veterinaermed. A 14, 93 (1967). 28. - - - - ibid. 14, 789 (1967). 29. - ibid. 15, 799 (1968). 30. SVlCER, S. S., HARDIN, J. H. and GREENE, W. B., J. Cell Biol. 39, 216 (1968). 31. THORBURN,G. D. and MOL'zZqEUX,G. S., Bull. Post Grad. Comm. Med. Univ. Sydney, 23, 199 (1967).
RUMINAL EPITHELIUM 32. 33. 34. 35. 36.
401
TORMEY,J. McD. and DIAMOND,J. M., J. Gen. Physiol. 50, 2031 (1967). VOLLMERHAUS,B. and SCHNORR,B., Zentr. Veterinaermed. A 14, 761 (1967). WARNER, A. C. I. and STACY, B. D., Brit. J. Nutr. 22, 389 (1968). ZADUNAISKY,J. A., J. Cell Biol. 31, Cll (1966). ZADUNAISKY,J. A., GENNARO, J. F., JR., BASHIRELAHI,N. and HILTON, M., Y. Gen. Physiol. 51, 290s (1968).