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The Ultrastructure of Normal Chick Intestinal Epithelium123
The infrastructure of Normal Chick Intestinal Epithelium1,2,3 C. D . HUMPHREY 4 AND D . E . T U R K 5
Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 (Received for publication August 20, 1973)
ABSTRACT Duodenal, jejunal and ileal mucosa of chicks, four to seven weeks old, were embedded in Epon-812 for examination by conventional electron microscopic procedures. A portion of the embedded tissues was also sectioned for examination by light microscopy. Principal cells, goblet cells, basal granular cells, intraepithelial lymphocytes, and globule leukocytes were observed in chick intestinal epithelium. The mucosal morphology of chick intestine varied little from that reported in the literature for mammals. The differences observed in chick intestinal mucosa included the following: a less dense glycocalyx (surface coat) covering the microvilli; smooth oval nuclei in the absorptive cells; an infrequency of mitochondrial dark granules; an absence of well developed lacteals; and the presence of nucleated erythrocytes in the lamina propria. Structural variations among similar types of cells were not observed in different parts of the small intestine. However, microvillar variations on the principal (absorptive) cells were seen. The microvilli on these cells were shorter near the villus tip and in the crypts than they were on cells in the central portion of the villi. There was also a tendency for the microvilli to be shorter, broader, and less numerous in distal areas of the intestine. POULTRY SCIENCE 53: 990-1000, 1974
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LTHOUGH there are several reports of the histology of normal avian small intestinal mucosa (Calhoun, 1954; Aitken, 1958; Farner, 1960), structural examinations with the electron microscope have been limited to studies of embryonic chick intestinal
1. This manuscript was taken in part from a dissertation submitted to the Graduate School of Clemson University by C. D. Humphrey in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2. Published with the approval of the Director of the South Carolina Agricultural Experimental Station as Technical Contribution No. 1094. 3. Presented in part at the 59th annual meeting of the Poultry Science Association and published as an abstract in Poultry Science 1970, 49: 1399 (abstr.). Other portions were presented at the 42nd annual meeting of the South Carolina Academy of Science and published as an abstract in the Bulletin of the South Carolina Academy of Science 1969, 31: 44. 4. Present address: Department of Medicine, Medical University of South Carolina, 80 Barre Street, Charleston, South Carolina 29401. 5. Address reprint requests to: D. E. Turk, Department of Poultry Science, Clemson University, Clemson, South Carolina 29631.
mucosa (Overton and Shoup, 1964), and one or two intestinal cell types in normal young chicks (Toner, 1964, 1965; Holman, 1968). There are no comprehensive ultrastructural studies of normal chick small intestinal mucosa. In particular, the ultrastructure of the principal or absorbing cell has been neglected. Since the chicken is an important, relatively inexpensive, nutritious food source and is a valuable nutritional study animal (Scott et al., 1969), a combined light and electron microscopic survey of its small intestinal mucosa, with emphasis upon the principal cell, was carried out. The findings are reported here. METHODS Sixteen, four to seven week old broiler type chicks (Gallus domesticus) fed a cornsoybean meal ration were killed after being fasted eight hours. The small intestine was removed immediately and specimens were obtained from the duodenal loop, midway between the duodenal loop and yolk stalk diverticulum, and midway between the yolk stalk diverticulum and the ceca. Specimens
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PLATE 1 FIG. 1. Low magnification light micrograph of duodenal mucosa from chick intestine. The mucosa is composed of finger-like villi (V) and glands of Lieberkiihn (GL) which open into the intestinal lumen (L) at the base of the villi. Methylene blue-azure II. (x 114).
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PLATE 2 FIG. 2. Light micrograph of chick intestinal villi. Differences between the brush border (BB) at the villus tip and its central portion may be compared. Absorptive cells (P), goblet cells (GC), and globule leukocytes (L) are present. Methylene blue-azure II.(x 682). FIG. 3. Light micrograph of the crypts of Lieberkuhn. The glands of Lieberkuhn (GL) are present and contain Paneth cells (PC) and basal granular cells (A). Nucleated erythrocytes (e) may be observed. Methylene blue-azure II. (x 586). Inset (x 1165).
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&£*!'.! PLATE 3 FIG. 4. Electron micrograph of intestinal epithelial cells. Globule leukocytes (L), intraepithelial lymphocytes (IL), goblet cells (GC), basal granular cells (A), and principal cells (P) are present. An erythrocyte (E) is in a capillary at the base of these cells. The cells contain mitochondria (m), lysosomes (ly), tight junctions (tj), Golgi (G), nuclei (n), and are lined with microvilli (mv). Desmosomes (d) are located along the cell membrane (cm). There is a basement membrane (bm) at the base of the cells, (x 5200).
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PLATE 4 FIG. 5. Electron micrograph of the apical cytoplasm of two absorptive cells. The microvilli (MV) are long finger-like structures covered by a web-like glycocalyx (GX). The plasma membrane (PM) occasionally indents to form pinocytotic vesicles (PV). A tight junction (TJ) is formed at the apical border of the cell (CM). Desmosomes (D) are observed on the cell membrane (CM).The round structures between the microvilli perhaps are degenerating microvilli, (x 25,480). Inset: The microvillar tips are covered by a glycocalyx (surface coat) (G) and contain filaments (F). A trilaminar plasma membrane (PM) is around the microvilli, (x 76,000). FIG. 6. Electron micrograph of the apical cytoplasm of two chick absorptive cells. Vesicles (V), microtubules (MT), and mitochondria (M) are present, (x 15,900). Inset: The mitochondria occasionally contain dense granules (arrows), (x 47,490).
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PLATE 5 FIG. 7. Electron micrograph of the apical portions of chick intestine principal cells. Granular (GER) and agranular endoplasmic reticulum (AER) are abundantly represented. Microtubules (MT) are also present. Free ribosomes and polysomes (PO) are scattered through this region. The microvilli (MV) have a trilaminar plasma membrane and contain filamentous structures which appear to support themselves in the terminal web (TW). The plasma membrane (PM) displays interdigitations and forms a tight junction (TJ) at the apical border. Mitochondria (M) are abundantly present in these cells, (x 37,500).
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PLATE 7 FIG. 9. Electron micrograph of the basal portion of a principal cell. Polysomes (PO), granular (GER) and agranular endoplasmic reticulum (AER), mitochondria (M) and lysosomes (LY) are present. A portion of the lateral plasma membrane (PM) is apparent. An erythrocyte (E) rests in a capillary adjacent to the basement membrane (BM). (x 32,500).
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were fixed in Karnovsky's paraformaldehyde-glutaraldehyde solution (Karnovsky, 1965), post-fixed in cold (5°) 2% Os0 4 , dehydrated in ethanol solutions of increasing concentrations, followed by propylene oxide, and infiltrated and embedded in Epon-812 (Luft, 1961). Thick sections (1000-2000 nm.) of Eponembedded tissue were cut, mounted on glass slides, and stained by a modification of a procedure originally described by Belanger (1961) for radioautographs. Sections were stained in a warm (60°) methylene blue-azure II solution for one hour and counterstained in a solution of basic f uchsin at room temperature for ten minutes. These sections were examined by light microscopy. Ultrathin sections (60-90 nm.) were cut with glass knives in a Porter-Blum MT-2 ultramicrotome, stained with 3% uranyl acetate for 20 minutes (Watson, 1958), followed by lead citrate for 20 seconds (Venable and Coggeshall, 1965), and examined in an Hitachi HS-8-1 electron microscope. Five to ten blocks from each level of the intestine in each chick were examined. RESULTS Normal chick small intestinal mucosa was composed of finger-like projections (villi) and simple tubular glands (crypts of Lieberkuhn) which opened into the intestinal lumen at the base of the villi (Fig. 1). Villi were most dense and longest in the duodenum. They became progressively less dense, shorter and broader in the distal small intestine. Branched villi were common throughout. The central core of the villi—the subepithelial lamina propria—contained capillaries, smooth muscle fibers, connective tissue, fibroblasts, lymphocytes and plasma cells. Lacteals were not identified. The major epithelial cell type was the principal cell (absorptive cells). Interspersed in the epithelium were goblet cells and basal granular cells (Figs. 1, 2, 3). Intraepithelial lymphocytes and globule leuko-
cytes were also observed in the epithelium (Fig. 2). Paneth cells were most often found in the crypt epithelium (Fig. 3). Brunner's glands were not found. By electron microscopy most of the cytoplasmic organelles of principal cells (e.g., microtubules, lysosomes, tight junctions, lateral plasma membranes, desmosomes, endoplasmic reticulum, Golgi, microvilli) were similar to those previously reported in mammals (Figs. 4-9) (Palay and Karlin, 1959a,b; Trier, 1963, 1967; Trier and Rubin, 1965; Toner, 1968; Moe, 1968). Microvilli averaged 1,000 nm. long and 100 nm. wide and consisted of a trilaminar plasma membrane. They enclosed a core of filamentous fibers (Figs. 5,7), and were shorter at the villous tip and in the crypts than they were on the central portion of the villi. Microvilli also were somewhat shorter, broader, and less numerous in the jejunum and ileum than they were in the duodenum. Some organelle differences were found between chick and mammalian principal cells. These included nuclei, mitochondria, and glycocalyx. The nuclei, which were basally located in principal and goblet cells, were smoother, more ovoid, and displaced a smaller portion of the cell interiors than did the nuclei of mammalian species (Figs. 2, 4, 8). The frequently observed dark granules in mitochondria of mammalian species were seen less often in chick principal cell mitochondria (Fig. 6). The surface coat (glycocalyx) covering the microvilli was not as extensive as that which has been reported in mammals (Fig. 5) (Ito, 1964, 1965). It was usually so diffuse as to be nearly invisible. DISCUSSION Calhoun (1954), Aitken (1958), and Farner (1960), have described the histology of the chick digestive tract. In general, our findings were similar to theirs; however, they described paraffin embedded tissue which does not offer the section thinness and resolution
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of plastic embedded tissue. Convincing evidence for the presence of lacteals in chick intestinal villi was not found. Calhoun (1954) observed lacteals in chick intestinal villi. However, Graney (1967) observed no system of lacteals, although he reported the presence of well defined blood capillaries. Our findings were similar. Thus, it is probable that the portal system is the major pathway for lipid absorption in the intestine by avians as postulated by Noyan et al. (1964). The ultrastructure of avian intestinal principal cells is similar to that previously reported in mammals. However, some differences between mammalian and chick glycocalyx and microvilli were noted. Yamada (1955) first described the surface coat on gallbladder epithelium and Bennett (1963) included intestinal surface coats in his broad description of the glycocalyx. Mukherjee and Williams (1967) and Mukherjee and Staehelin (1971) suggested that the surface coat may be an extension of microvillous filaments. Ito (1964,1965) described a surface coat of the intestinal absorbing cells in a number of mammals. He observed that the surface coat of dogs, rabbits, and rodents was less extensive than that of cats, bats and men. We also noted that the surface coat of chicks was not as extensive as reported in these latter animals. The surface coat may play a role in protection against harmful extraneous materials (Ito, 1964), selective binding of nutrients (Bennett, 1963), or the digestion of nutrients prior to their absorption (Fawcett, 1965). The microvilli were formerly identified by light microscopists as the striated, brush or cuticular border, but were later shown by Granger and Baker (1950) to be discrete finger-shaped structures. Although substantial variation in the dimensions of intestinal microvilli have been noted, an average dimension of 1000 nm. in length and 100 nm. in width is generally accepted as repre-
sentative for mammals. Brown (1962) suggested that one possible reason for the reported variations in the dimensions of microvilli might be due to different morphological characteristics on different portions of the villus. He also noted that the microvilli near the tips of villi were longer and thinner than those in the middle. However, in the growing chick it appeared that the microvilli were longer and thinner in the mid-portion of the villus than in the crypts or at the tip (Fig. 2). Perhaps the decrease in length of microvilli observed at the tips of villi was due to the degenerating condition of cells on this portion of the villus and/or to the greater friction of food particles in this region. Other ultrastructural differences between mammals and chicks noted in this study included smooth, ovoid nuclei, less numerous mitochondrial dark granules, and the presence of nucleated erythrocytes in chick intestinal mucosa. A previous study of chick intestinal argentaffin cells also suggested that only slight differences exist between avian and mammalian intestinal ultrastructure (Toner, 1964). ACKNOWLEDGEMENTS The authors wish to express their appreciation to Drs. J. F. Dickey, C. C. Fain, and R. M. Lavker for their assistance in the technical aspects of this project, and to Dr. F. E. Pittman and J. C. Pittman for their encouragement and critical review of the manuscript. REFERENCES Aitken, R. N. C , 1958. A biochemical study of the stomach of the chicken. J. Anat. 92: 453-566. Belanger, L. F., 1961. Staining processed radioautographs. Stain Techn. 36: 313-317. Bennett, H. S., 1963. Morphological aspects of extracellular polysaccharides. J. Histochem. Cytochem. 11: 14-23. Brown, A. F., 1962. Microvilli of the human jejunal ephithelial cell. J. Cell Biol. 12: 623-627. Calhoun, M. L., 1954. Microscopic Anatomy of the
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Digestive Tract. Iowa State College Press, Ames, Iowa. Farner, D. S., 1960. Digestion and the digestive system. In: Biology and Comparative Physiology of Birds. Ed., A. J. Marshall, Academic Press, Inc., New York, p. 411. Fawcett, D. W., 1965. Surface specializations of absorbing cells. J. Histochem. Cytochem. 13: 75-96. Graney, D. O., 1967. Electron microscopic observations on the morphology of intestinal capillaries in the chicken and the transcapillary passage of chylomicra during fat absorption. Anat. Rec. 157: 250. Granger, B., and R. F. Baker, 1950. Electron microscope investigation of the striated border of intestinal epithelium. Anat. Rec. 107: 423-441. Holman, J., 1968. Ultrastructure of the globule leucocytes in the lipids resorbing intestinal epithelium of the chick. Acta Universitat Agric. 37: 193-197. Ito, S., 1964. The surface coating of enteric microvilli. Anat. Rec. 148: 294. Ito, S., 1965. The enteric surface coating on cat intestinal microvilli. J. Cell Biol. 26: 475-491. Karnovsky, M. J., 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27: 137a. Luft, H. L., 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409-414. Moe, H., 1968. The goblet cells, paneth cells, and basal granular cells of the epithelium of the intestine. Int. Rev. Gen. Exp. Zool, 3: 241-287. Mukherjee, T. M., and A. W. Williams, 1967. A comparative study of the microvilli in the epithelium of the large and small intestine of mice. J. Cell Biol. 34: 447-461. Mukherjee, T. M., and L. A. Staehelin, 1971. The fine-structural organization of the brush border of intestinal epithelial cells. J. Cell Sci. 8: 573-599. Noyan, A., W. J. Lossow, N. Brot and I. F. Chaikoff, 1964. Pathway and form of absorption of palmitic acid in the chicken. J. Lipid Res. 5: 538-541.
Overton, J., and J. Shoup, 1964. Fine structure of cell surface specializations in the maturing duodenal mucosa of the chick. J. Cell Biol. 21: 75-85. Palay, S. F., and L. F. Karlin, 1959a. An electron microscopic study of the intestinal villus. I. The fasting animal. J. Biophys. Biochem. Cytol. 5: 363-372. Palay, S. F., and L. F. Karlin, 1959b. An electron microscopic study of the intestinal villus. II. The pathway of fat absorption. J. Biophys. Biochem. Cytol 5: 373-384. Scott, M. F., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, New York, p. 2. Toner, P. G., 1964. Fine structure of argophil and argentaffin cells in the gastrointestinal tract of the fowl. J. Zellforsch. 63: 830-839. Toner, P. G., 1965. The fine structure of the globule leucocyte in the fowl intestine. Acta Anat. (Basel) 61: 321-330. Toner, P. G., 1968. Cytology of intestinal epithelial cells. Int. Rev. Cytol. 24: 233-343. Trier, J. S., 1963. Studies on the small intestinal crypt epithelium. I. The fine structure of the crypt epithelium of the proximal small intestine of fasting humans. J. Cell Biol. 18: 599-620. Trier, J. S., and C. E. Rubin, 1965. Electron microscopy of the small intestine: A review. Gastroenterology, 49: 574-603. Trier, J. S., 1967. Structure of the small intestine as it relates to intestinal function. Fed. Proc. 26: 1391-1404. Venable, J. H., and R. Coggeshall, 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25: 407-408. Watson, M. L., 1958. Staining tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4: 475-478. Yamada, E., 1955. The fine structure of the gallbladder epithelium of the mouse. J. Biophys. Biochem. Cytol. 1:445-458.
JUNE 12-15. SPECIAL CONFERENCE ON THE ANALYSIS OF LIPIDS AND LIPOPROTEINS, AMERICAN OIL CHEMISTS' SOCIETY, SHERATON PARK HOTEL, WASHINGTON, D.C. JUNE 16-21.
25TH ANNUAL MEETING, AMERICAN INSTITUTE OF BIOLOGICAL SCIENCES, ARIZONA STATE UNIVERSITY, TEMPE.
JUNE 21-22. DELMARVA CHICKEN FESTIVAL AND DELMARVA CHICKEN COOKING CONTEST, SEAFORD, DELAWARE. JUNE 23-26. 67TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF AGRICULTURAL ENGINEERS, OKLAHOMA STATE UNIVERSITY, STILLWATER, OKLAHOMA.
Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 (Received for publication August 20, 1973)
ABSTRACT Duodenal, jejunal and ileal mucosa of chicks, four to seven weeks old, were embedded in Epon-812 for examination by conventional electron microscopic procedures. A portion of the embedded tissues was also sectioned for examination by light microscopy. Principal cells, goblet cells, basal granular cells, intraepithelial lymphocytes, and globule leukocytes were observed in chick intestinal epithelium. The mucosal morphology of chick intestine varied little from that reported in the literature for mammals. The differences observed in chick intestinal mucosa included the following: a less dense glycocalyx (surface coat) covering the microvilli; smooth oval nuclei in the absorptive cells; an infrequency of mitochondrial dark granules; an absence of well developed lacteals; and the presence of nucleated erythrocytes in the lamina propria. Structural variations among similar types of cells were not observed in different parts of the small intestine. However, microvillar variations on the principal (absorptive) cells were seen. The microvilli on these cells were shorter near the villus tip and in the crypts than they were on cells in the central portion of the villi. There was also a tendency for the microvilli to be shorter, broader, and less numerous in distal areas of the intestine. POULTRY SCIENCE 53: 990-1000, 1974
A
LTHOUGH there are several reports of the histology of normal avian small intestinal mucosa (Calhoun, 1954; Aitken, 1958; Farner, 1960), structural examinations with the electron microscope have been limited to studies of embryonic chick intestinal
1. This manuscript was taken in part from a dissertation submitted to the Graduate School of Clemson University by C. D. Humphrey in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2. Published with the approval of the Director of the South Carolina Agricultural Experimental Station as Technical Contribution No. 1094. 3. Presented in part at the 59th annual meeting of the Poultry Science Association and published as an abstract in Poultry Science 1970, 49: 1399 (abstr.). Other portions were presented at the 42nd annual meeting of the South Carolina Academy of Science and published as an abstract in the Bulletin of the South Carolina Academy of Science 1969, 31: 44. 4. Present address: Department of Medicine, Medical University of South Carolina, 80 Barre Street, Charleston, South Carolina 29401. 5. Address reprint requests to: D. E. Turk, Department of Poultry Science, Clemson University, Clemson, South Carolina 29631.
mucosa (Overton and Shoup, 1964), and one or two intestinal cell types in normal young chicks (Toner, 1964, 1965; Holman, 1968). There are no comprehensive ultrastructural studies of normal chick small intestinal mucosa. In particular, the ultrastructure of the principal or absorbing cell has been neglected. Since the chicken is an important, relatively inexpensive, nutritious food source and is a valuable nutritional study animal (Scott et al., 1969), a combined light and electron microscopic survey of its small intestinal mucosa, with emphasis upon the principal cell, was carried out. The findings are reported here. METHODS Sixteen, four to seven week old broiler type chicks (Gallus domesticus) fed a cornsoybean meal ration were killed after being fasted eight hours. The small intestine was removed immediately and specimens were obtained from the duodenal loop, midway between the duodenal loop and yolk stalk diverticulum, and midway between the yolk stalk diverticulum and the ceca. Specimens
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PLATE 1 FIG. 1. Low magnification light micrograph of duodenal mucosa from chick intestine. The mucosa is composed of finger-like villi (V) and glands of Lieberkiihn (GL) which open into the intestinal lumen (L) at the base of the villi. Methylene blue-azure II. (x 114).
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PLATE 2 FIG. 2. Light micrograph of chick intestinal villi. Differences between the brush border (BB) at the villus tip and its central portion may be compared. Absorptive cells (P), goblet cells (GC), and globule leukocytes (L) are present. Methylene blue-azure II.(x 682). FIG. 3. Light micrograph of the crypts of Lieberkuhn. The glands of Lieberkuhn (GL) are present and contain Paneth cells (PC) and basal granular cells (A). Nucleated erythrocytes (e) may be observed. Methylene blue-azure II. (x 586). Inset (x 1165).
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&£*!'.! PLATE 3 FIG. 4. Electron micrograph of intestinal epithelial cells. Globule leukocytes (L), intraepithelial lymphocytes (IL), goblet cells (GC), basal granular cells (A), and principal cells (P) are present. An erythrocyte (E) is in a capillary at the base of these cells. The cells contain mitochondria (m), lysosomes (ly), tight junctions (tj), Golgi (G), nuclei (n), and are lined with microvilli (mv). Desmosomes (d) are located along the cell membrane (cm). There is a basement membrane (bm) at the base of the cells, (x 5200).
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PLATE 4 FIG. 5. Electron micrograph of the apical cytoplasm of two absorptive cells. The microvilli (MV) are long finger-like structures covered by a web-like glycocalyx (GX). The plasma membrane (PM) occasionally indents to form pinocytotic vesicles (PV). A tight junction (TJ) is formed at the apical border of the cell (CM). Desmosomes (D) are observed on the cell membrane (CM).The round structures between the microvilli perhaps are degenerating microvilli, (x 25,480). Inset: The microvillar tips are covered by a glycocalyx (surface coat) (G) and contain filaments (F). A trilaminar plasma membrane (PM) is around the microvilli, (x 76,000). FIG. 6. Electron micrograph of the apical cytoplasm of two chick absorptive cells. Vesicles (V), microtubules (MT), and mitochondria (M) are present, (x 15,900). Inset: The mitochondria occasionally contain dense granules (arrows), (x 47,490).
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PLATE 5 FIG. 7. Electron micrograph of the apical portions of chick intestine principal cells. Granular (GER) and agranular endoplasmic reticulum (AER) are abundantly represented. Microtubules (MT) are also present. Free ribosomes and polysomes (PO) are scattered through this region. The microvilli (MV) have a trilaminar plasma membrane and contain filamentous structures which appear to support themselves in the terminal web (TW). The plasma membrane (PM) displays interdigitations and forms a tight junction (TJ) at the apical border. Mitochondria (M) are abundantly present in these cells, (x 37,500).
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PLATE 7 FIG. 9. Electron micrograph of the basal portion of a principal cell. Polysomes (PO), granular (GER) and agranular endoplasmic reticulum (AER), mitochondria (M) and lysosomes (LY) are present. A portion of the lateral plasma membrane (PM) is apparent. An erythrocyte (E) rests in a capillary adjacent to the basement membrane (BM). (x 32,500).
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were fixed in Karnovsky's paraformaldehyde-glutaraldehyde solution (Karnovsky, 1965), post-fixed in cold (5°) 2% Os0 4 , dehydrated in ethanol solutions of increasing concentrations, followed by propylene oxide, and infiltrated and embedded in Epon-812 (Luft, 1961). Thick sections (1000-2000 nm.) of Eponembedded tissue were cut, mounted on glass slides, and stained by a modification of a procedure originally described by Belanger (1961) for radioautographs. Sections were stained in a warm (60°) methylene blue-azure II solution for one hour and counterstained in a solution of basic f uchsin at room temperature for ten minutes. These sections were examined by light microscopy. Ultrathin sections (60-90 nm.) were cut with glass knives in a Porter-Blum MT-2 ultramicrotome, stained with 3% uranyl acetate for 20 minutes (Watson, 1958), followed by lead citrate for 20 seconds (Venable and Coggeshall, 1965), and examined in an Hitachi HS-8-1 electron microscope. Five to ten blocks from each level of the intestine in each chick were examined. RESULTS Normal chick small intestinal mucosa was composed of finger-like projections (villi) and simple tubular glands (crypts of Lieberkuhn) which opened into the intestinal lumen at the base of the villi (Fig. 1). Villi were most dense and longest in the duodenum. They became progressively less dense, shorter and broader in the distal small intestine. Branched villi were common throughout. The central core of the villi—the subepithelial lamina propria—contained capillaries, smooth muscle fibers, connective tissue, fibroblasts, lymphocytes and plasma cells. Lacteals were not identified. The major epithelial cell type was the principal cell (absorptive cells). Interspersed in the epithelium were goblet cells and basal granular cells (Figs. 1, 2, 3). Intraepithelial lymphocytes and globule leuko-
cytes were also observed in the epithelium (Fig. 2). Paneth cells were most often found in the crypt epithelium (Fig. 3). Brunner's glands were not found. By electron microscopy most of the cytoplasmic organelles of principal cells (e.g., microtubules, lysosomes, tight junctions, lateral plasma membranes, desmosomes, endoplasmic reticulum, Golgi, microvilli) were similar to those previously reported in mammals (Figs. 4-9) (Palay and Karlin, 1959a,b; Trier, 1963, 1967; Trier and Rubin, 1965; Toner, 1968; Moe, 1968). Microvilli averaged 1,000 nm. long and 100 nm. wide and consisted of a trilaminar plasma membrane. They enclosed a core of filamentous fibers (Figs. 5,7), and were shorter at the villous tip and in the crypts than they were on the central portion of the villi. Microvilli also were somewhat shorter, broader, and less numerous in the jejunum and ileum than they were in the duodenum. Some organelle differences were found between chick and mammalian principal cells. These included nuclei, mitochondria, and glycocalyx. The nuclei, which were basally located in principal and goblet cells, were smoother, more ovoid, and displaced a smaller portion of the cell interiors than did the nuclei of mammalian species (Figs. 2, 4, 8). The frequently observed dark granules in mitochondria of mammalian species were seen less often in chick principal cell mitochondria (Fig. 6). The surface coat (glycocalyx) covering the microvilli was not as extensive as that which has been reported in mammals (Fig. 5) (Ito, 1964, 1965). It was usually so diffuse as to be nearly invisible. DISCUSSION Calhoun (1954), Aitken (1958), and Farner (1960), have described the histology of the chick digestive tract. In general, our findings were similar to theirs; however, they described paraffin embedded tissue which does not offer the section thinness and resolution
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of plastic embedded tissue. Convincing evidence for the presence of lacteals in chick intestinal villi was not found. Calhoun (1954) observed lacteals in chick intestinal villi. However, Graney (1967) observed no system of lacteals, although he reported the presence of well defined blood capillaries. Our findings were similar. Thus, it is probable that the portal system is the major pathway for lipid absorption in the intestine by avians as postulated by Noyan et al. (1964). The ultrastructure of avian intestinal principal cells is similar to that previously reported in mammals. However, some differences between mammalian and chick glycocalyx and microvilli were noted. Yamada (1955) first described the surface coat on gallbladder epithelium and Bennett (1963) included intestinal surface coats in his broad description of the glycocalyx. Mukherjee and Williams (1967) and Mukherjee and Staehelin (1971) suggested that the surface coat may be an extension of microvillous filaments. Ito (1964,1965) described a surface coat of the intestinal absorbing cells in a number of mammals. He observed that the surface coat of dogs, rabbits, and rodents was less extensive than that of cats, bats and men. We also noted that the surface coat of chicks was not as extensive as reported in these latter animals. The surface coat may play a role in protection against harmful extraneous materials (Ito, 1964), selective binding of nutrients (Bennett, 1963), or the digestion of nutrients prior to their absorption (Fawcett, 1965). The microvilli were formerly identified by light microscopists as the striated, brush or cuticular border, but were later shown by Granger and Baker (1950) to be discrete finger-shaped structures. Although substantial variation in the dimensions of intestinal microvilli have been noted, an average dimension of 1000 nm. in length and 100 nm. in width is generally accepted as repre-
sentative for mammals. Brown (1962) suggested that one possible reason for the reported variations in the dimensions of microvilli might be due to different morphological characteristics on different portions of the villus. He also noted that the microvilli near the tips of villi were longer and thinner than those in the middle. However, in the growing chick it appeared that the microvilli were longer and thinner in the mid-portion of the villus than in the crypts or at the tip (Fig. 2). Perhaps the decrease in length of microvilli observed at the tips of villi was due to the degenerating condition of cells on this portion of the villus and/or to the greater friction of food particles in this region. Other ultrastructural differences between mammals and chicks noted in this study included smooth, ovoid nuclei, less numerous mitochondrial dark granules, and the presence of nucleated erythrocytes in chick intestinal mucosa. A previous study of chick intestinal argentaffin cells also suggested that only slight differences exist between avian and mammalian intestinal ultrastructure (Toner, 1964). ACKNOWLEDGEMENTS The authors wish to express their appreciation to Drs. J. F. Dickey, C. C. Fain, and R. M. Lavker for their assistance in the technical aspects of this project, and to Dr. F. E. Pittman and J. C. Pittman for their encouragement and critical review of the manuscript. REFERENCES Aitken, R. N. C , 1958. A biochemical study of the stomach of the chicken. J. Anat. 92: 453-566. Belanger, L. F., 1961. Staining processed radioautographs. Stain Techn. 36: 313-317. Bennett, H. S., 1963. Morphological aspects of extracellular polysaccharides. J. Histochem. Cytochem. 11: 14-23. Brown, A. F., 1962. Microvilli of the human jejunal ephithelial cell. J. Cell Biol. 12: 623-627. Calhoun, M. L., 1954. Microscopic Anatomy of the
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Digestive Tract. Iowa State College Press, Ames, Iowa. Farner, D. S., 1960. Digestion and the digestive system. In: Biology and Comparative Physiology of Birds. Ed., A. J. Marshall, Academic Press, Inc., New York, p. 411. Fawcett, D. W., 1965. Surface specializations of absorbing cells. J. Histochem. Cytochem. 13: 75-96. Graney, D. O., 1967. Electron microscopic observations on the morphology of intestinal capillaries in the chicken and the transcapillary passage of chylomicra during fat absorption. Anat. Rec. 157: 250. Granger, B., and R. F. Baker, 1950. Electron microscope investigation of the striated border of intestinal epithelium. Anat. Rec. 107: 423-441. Holman, J., 1968. Ultrastructure of the globule leucocytes in the lipids resorbing intestinal epithelium of the chick. Acta Universitat Agric. 37: 193-197. Ito, S., 1964. The surface coating of enteric microvilli. Anat. Rec. 148: 294. Ito, S., 1965. The enteric surface coating on cat intestinal microvilli. J. Cell Biol. 26: 475-491. Karnovsky, M. J., 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27: 137a. Luft, H. L., 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409-414. Moe, H., 1968. The goblet cells, paneth cells, and basal granular cells of the epithelium of the intestine. Int. Rev. Gen. Exp. Zool, 3: 241-287. Mukherjee, T. M., and A. W. Williams, 1967. A comparative study of the microvilli in the epithelium of the large and small intestine of mice. J. Cell Biol. 34: 447-461. Mukherjee, T. M., and L. A. Staehelin, 1971. The fine-structural organization of the brush border of intestinal epithelial cells. J. Cell Sci. 8: 573-599. Noyan, A., W. J. Lossow, N. Brot and I. F. Chaikoff, 1964. Pathway and form of absorption of palmitic acid in the chicken. J. Lipid Res. 5: 538-541.
Overton, J., and J. Shoup, 1964. Fine structure of cell surface specializations in the maturing duodenal mucosa of the chick. J. Cell Biol. 21: 75-85. Palay, S. F., and L. F. Karlin, 1959a. An electron microscopic study of the intestinal villus. I. The fasting animal. J. Biophys. Biochem. Cytol. 5: 363-372. Palay, S. F., and L. F. Karlin, 1959b. An electron microscopic study of the intestinal villus. II. The pathway of fat absorption. J. Biophys. Biochem. Cytol 5: 373-384. Scott, M. F., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. M. L. Scott and Associates, Ithaca, New York, p. 2. Toner, P. G., 1964. Fine structure of argophil and argentaffin cells in the gastrointestinal tract of the fowl. J. Zellforsch. 63: 830-839. Toner, P. G., 1965. The fine structure of the globule leucocyte in the fowl intestine. Acta Anat. (Basel) 61: 321-330. Toner, P. G., 1968. Cytology of intestinal epithelial cells. Int. Rev. Cytol. 24: 233-343. Trier, J. S., 1963. Studies on the small intestinal crypt epithelium. I. The fine structure of the crypt epithelium of the proximal small intestine of fasting humans. J. Cell Biol. 18: 599-620. Trier, J. S., and C. E. Rubin, 1965. Electron microscopy of the small intestine: A review. Gastroenterology, 49: 574-603. Trier, J. S., 1967. Structure of the small intestine as it relates to intestinal function. Fed. Proc. 26: 1391-1404. Venable, J. H., and R. Coggeshall, 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25: 407-408. Watson, M. L., 1958. Staining tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4: 475-478. Yamada, E., 1955. The fine structure of the gallbladder epithelium of the mouse. J. Biophys. Biochem. Cytol. 1:445-458.
JUNE 12-15. SPECIAL CONFERENCE ON THE ANALYSIS OF LIPIDS AND LIPOPROTEINS, AMERICAN OIL CHEMISTS' SOCIETY, SHERATON PARK HOTEL, WASHINGTON, D.C. JUNE 16-21.
25TH ANNUAL MEETING, AMERICAN INSTITUTE OF BIOLOGICAL SCIENCES, ARIZONA STATE UNIVERSITY, TEMPE.
JUNE 21-22. DELMARVA CHICKEN FESTIVAL AND DELMARVA CHICKEN COOKING CONTEST, SEAFORD, DELAWARE. JUNE 23-26. 67TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF AGRICULTURAL ENGINEERS, OKLAHOMA STATE UNIVERSITY, STILLWATER, OKLAHOMA.