The Ultrastructure of the Neonatal Pig Colon F. B. P. WOODING, M. W. SMITH AND H. CRAIG ARCZnstitute ofAnirna1 Physiologv. Babraharn, Cambridge CB2 4AT, England
ABSTRACT The neonatal pig colon has several unique structural and developmental features. A t birth it has a variable population of epithelial cells which in their arrangement on villus-like protrusions and in their capability for protein uptake into large preformed supranuclear vacuoles closely resemble neonatal ileal cells. Such villus-like protrusions and vacuolated cells are not present in the 2-day-old piglet. On the first day after birth absorptive epithelial cells which lack supranuclear vacuoles transiently accumulate a large number of lipid droplets, each separated from the cytoplasm only by a proteolipid interface. None of the much smaller lipid droplets bounded by a unit membrane of the smooth endoplasmic reticulum and characteristic of normal small intestinal fat uptake were ever seen in these cells. Very few of the large lipid drops remain on the second day after birth. This initial capacity of the colon for protein and lipid uptake never reappears. The pattern of colonic amino acid transport also changes markedly in the first four days of independent life and this may be correlated with the observation that the absorptive cells a t birth have microvilli which are twice the length of those on similar cells a t and after two days old. These morphological results are discussed in terms of implied functional changes in the neonatal period. Changes in the structure and function of the digestive system may be of great importance to the survival of neonatal animals. Several developmental studies have been carried out on the ultrastructure and physiology of the stomach and small intestines of various neonatal animals, e.g., opossum (Krause e t al., '761, pig (Staley e t al., '68; Hardy et al., '71; Moon, '72) and rat (Helander and Olivecrona, '701, but little attention has been given so far to the large intestine. Two recent studies describe the ultrastructure of the postnatal rat colon (Helander, '73, '75; Ono, '76) but this study of the pig colon is the first known to the authors describing the ultrastructure of neonatal development in the colon of any domesticated animal of agricultural importance. One of the most notable ultrastructural features of the colonic surface epithelium of the pig is the presence a t birth of cells with large supranuclear vacuoles equivalent to those found in the terminal ileum. Unlike the latter, colonic cells never accumulate protein after suckling has started. In addition one day after birth in cells without supranuclear vacuoles there is a transient accumulation of lipid by a route which differs from that found a t the norAM. J. ANAT. (1978)152: 269-286.
mal site of lipid uptake, the proximal intestinal cells. The neonatal pig colon has many physiological similarities with adult small intestines. For example, it can actively transport amino acids such as methionine and this transport stimulates the absorption of Na' (James and Smith, '76). These transport capabilities are lost rapidly and the adult pattern is established two or three days after birth. The present work details the rapid changes that take place in the structure of the pig colon immediately after birth. These results, with a study of cell kinetics, have been used to define the changes in transport function after birth in the neonatal pig colon (Jarvis e t al., '77). MATERIALS AND METHODS
Pigs were taken from a herd of Large Whites bred a t Babraham. Parturition was induced routinely by the intramuscular injection of prostaglandin analogues on days 109111 of gestation. Birth occurred some 24 hours later (Ash and Heap, '73). Piglets were used newborn before suckling, or a t days 1, 2, 4 or Accepted Oct. 1, '77.
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10. The animals were killed by cervical dislocation and portions of the proximal colon taken for microscopy. Samples were also taken after mounting the colonic mucosa flat in an Ussing chamber for transport experiments (James and Smith, '76). The solutions bathing either side of the mucosa were replaced simultaneously with fixative. Fixation was carried out a t a time when the mucosal preparation had achieved a steady state in the physiological experiment. Samples of fresh mucosa were fixed by opening the colon longitudinally and cutting the tissue into pieces in the fixative, or fixed intact after stretching the tissue flat with pins on cork. This latter procedure produced a much flatter, thinner preparation, easier to orient and section than the contorted pieces resulting from unrestrained slicing of the mucosa into small cubes. No significant ultrastructural differences were observed between samples prepared by the two methods. Tissue was fixed for one to two hours in 4% glutaraldehyde in 0.1 M NaH2/Na2HPO, buffer, pH 7.2, containing 2% sucrose. Tissue was then osmium tetroxide treated fcr one hour with 1% in 0.1 M veronal-acetate buffer, pH 7.2, sometimes with 1.5%potassium ferrocyanide added (see figure legends for details), followed in some cases by treatment for half an hour with 2%aqueous uranyl acetate. Dehydration was in alcohol and, finally, propylene oxide. All the processing was a t room temperature. Embedding was carried out in Araldite. Sections were stained with uranyl acetate (saturated solution in 50%ethanol) and lead citrate and examined in an AE1 EM 6B or a JEOL lOOC microscope.
shown a flat surface epithelium with crypt openings and goblet cells as the only surface features. In the newborn piglet colon, however (within 10-15 cm of the ileocecal junction), the results in this paper show a significant but very variable number of villus-like structures very similar to those found in the ileum. Such villus-like structures were not found in the colon of piglets one day after birth or older. This paper deals with cells in the surface epithelium and the apical eighth of the crypts, only - cells which are mature and do not alter their fine-structural characters prior to cell death and elimination a t the surface of the epithelium.
Unsuckled, newborn Three main cell types were found a t this stage: goblet (fig. 4), absorptive (fig. 1) and vacuolated (fig. 4). The goblet cell fine-structure was identical to goblet cells found elsewhere in the intestine and in other species. Absorptive cells had a microvillar brushborder, under which lay a zone of cytoplasm, devoid of organelles save for the microfibrillar roots of the microvillar cores. Then came a region containing mitochondria, the occasional cisterna of rough endoplasmic reticulum (RER) and many glycogen granules. Just above or beside the nucleus there was a small Golgi body and, below the nucleus, more mitochondria, RER and glycogen, this last frequently in massive amounts. The lateral cell membrane was considerably folded, and sealed at the apex by a fairly wide tight junction. These absorptive cells formed the total cell population (fig. 1) (excluding goblet cells) at the luminal surface of the colon, except where Intestinal bypass studies vacuolated cells occurred. Two newborn piglets were anesthetised Vacuolated cells were found in significant with Fluothane (ICI, Macclesfield, Cheshire) number only in the colons of newborn piglets and a cecal cannula inserted after opening the and, even a t this stage, their occurrence was abdomen. Five ml of pig colostrum was in- very unpredictable. They were usually the jected directly into the proximal colon, after major cell type for 2-3 cm beyond the ileocecal the ileum had been sutured a t the ileocecal junction (figs. 4, 61, although they were presjunction, and the cannula sealed. The animal ent in only a small percentage of any one cowas allowed to recover and five to six hours lonic diameter. They occurred on protuberlater was killed and the colon sampled for elec- ances in the epithelium which projected above the general level of the flat colonic epithelium tron microscopy as detailed above. and resembled miniature ileal villi (figs. 6,7). RESULTS After the first 2 cm their occurrence was very As a general rule the colonic mucosal epi- sporadic, but groups of protuberances bearing thelium was distinguished easily from that in vacuolated cells have been found 15 cm from the small intestine, by the absence of villi. In the ileocecal junction and a group of two or the 6-day-old pig Newman et al. ('76) have three vacuolated cells were often to be found
271
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in the normal colonic surface epithelium up to 25 cm from the ileocecal junction (fig. 7). Usually the tissue used for incubations in the Ussing chamber was taken 3 or 4 cm beyond the ileocecal junction, so did not contain a significant number of vacuolated cells. However, one example was found where most of the surface cells of the experimental piece of colon were vacuolated. The transport capacity for methionine was in no way different from that found in newborn colons containing virtually no vacuolated cells. The microvilli (fig. 8) were shorter than those on absorptive cells and there were numerous invaginations of the plasmalemma a t the base of the microvilli down into the cell (figs. 11,121. The plasmalemma of the bases of the microvilli and the invaginations bore a regular array of particles on the luminal surface (figs. 10, 11).In the apical cytoplasm between and beneath the invaginations lay numerous vesicles and tubules of fairly constant diameter which could occasionally be seen to be continuous with larger vacuoles (figs. 9, 12). The vesicles and tubules had an asymmetrically stained plasmalemma, with the luminal leaflet much wider than that on the cytoplasmic side (figs. 9,12). Beneath this zone of vesicles and tubules the “supranuclear vacuoles” were found. These contained occasional dense areas inside their bounding membrane, and a loose flocculent material filled the vacuole itself (figs. 4,8). They varied in size from 550 p m and were bounded by a symmetrical unit membrane. Mitochondria, RER and glycogen were found between the vacuoles. There was a large Golgi body just above the nucleus and, if there were only a few small supranuclear vacuoles, they seemed to be associated with the Golgi membranes. Very rarely a vacuole was found below the nucleus, but usually this area had only mitochondria, RER and glycogen (fig. 4). In the unsuckled newborn animals used in this study the supranuclear vacuoles were present in both ileum and proximal colon. The colon was normally filled with meconium, and the vacuoles showed a dispersed, flocculent content, but after colostrum was injected through a cecal cannula the supranuclear vacuoles became filled with protein (fig. 5) and lipid droplets appeared in all the absorptive cells. Such lipid droplets were never seen in untreated unsuckled newborn piglet colonic epithelium, but started to appear about five to six hours after suckling had begun.
Suckled, one day old
The most obvious differences from the newborn were the almost complete absence of vacuolated cells showing the tubulovesicular system and/or supranuclear vacuoles and the appearance of numerous lipid droplets in the cytoplasm of the absorptive cells (figs. 13, 14). Lipid droplets, not bound by membranes (fig. 151, varied in size from 0.1-20 pm, and were most frequent in the supranuclear cytoplasm, but did occur throughout the cell (figs. 13,14). There was no indication of membrane-bound lipid except, rarely, in the immediate vicinity of the Golgi body. Here a uniform population of small lipid droplets could sometimes be found within what were probably vesicles arising from the Golgi body (fig. 16). Lipid droplets of a similar size were also found occasionally in the intercellular space between cells below the level of the Golgi body (fig. 17). Other organelles in the absorptive cell were as found in the newborn. Very rarely a cell with some of the tubulovesicular system of the vacuolated cells could be found a t the surface away from the crypt mouth, but there were no large supranuclear vacuoles. Suckled, two days old
At this stage most of the large lipid droplets characteristic of the 1-day-old had disappeared, although most of the absorptive cells showed a scattering of small lipid droplets. No lipid droplets were seen within Golgi vesicles or intercellular spaces. The characteristic feature of the apical cytoplasm was the presence of several phagolysosome-type bodies containing vesicles and dense material of various sorts. There was less glycogen but more mitochondria and RER than in the newborn. The Golgi body was slightly larger and so was the basal region below the nucleus which had more mitochondria and less glycogen. Four days and ten days old
There were only two types of cells, goblet and absorptive, both of which showed the characteristic structure found in such cells wherever they occur in large or small intestine. The absorptive cells had large aggregations of mitochondria interspersed with RER apically and basally. The supranuclear Golgi body was well developed, and multivesicular bodies were present (fig. 2). There was virtually no glycogen or lipid.
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Epithelium after incubation in physiological medium The most striking difference seen after incubation of any epithelium was the vast increase in the volume of the intercellular spaces (compare figs. 2 and 3). This was greatest a t the base of the epithelium and narrowed to the apical junctional complex, whose structure was completely unaffected by the incubation. The tight junction showed no sign of internal widening and there was no evidence of any difference in the extent or structure of the tight junction from the newborn to the 10-day-oldpiglet. The epithelium in figure 2 was twice the height of figure 3 at the same magnification, although they were both from the same colon. The difference in cell heights was due to the fact that the tissue in figure 2 was fixed without prior stretching, whereas in figure 3 the tissue was stretched, incubated and then fixed in the incubation chamber. Stretching an unincubated colon never produced any significant widening of the intercellular spaces. The tubulovesicular system of the newborn vacuolated cells and the lipid droplets in the 1-day-old suckled piglets appeared largely unchanged after incubation for up to two hours, although there were probably fewer lipid droplets in the cells.
Length of microvilli and presence of a glycocalyx There was a marked decrease in the average length of colonic microvilli on the absorptive cells of the newborn piglet, when compared to the microvilli on the 2-day-old piglet absorptive cells, after which there was little change. The microvilli on the newborn vacuolated cells were much shorter than on absorptive cells a t the same age (compare figs. 18 and 8) (table 1).There was a well developed zone of delicate extracellular microfibrils attached to the outer leaflet of the microvillar plasmalemTABLE 1
Change of length of microvilli with age No. of pigs
51 4 3
4 5
Age
h n @ h in pM Width in p M
Newborn absorptive Newborn vacuolated 1 day 2 day 4 day 10 day
1.220.2(25) 0.120.03 0.520.02(9) 0.120.02 1.020.1(24) 0.120.02 0.620.2(24) 0.0820.02 0.6-+0.1(24) O.l-tO.01 0.520.1(15) 0.120.02
ma a t all stages examined (figs. 18, 19). This presumably corresponds to the “glycocalyx” observed by other workers on intestinal brush borders. There was no apparent difference in the extent of this layer at any age examined, although i t was usually wider a t the bottom of the descending spiral of the colon than close to the ileocecal junction. DISCUSSION
The sporadic occurrence of vacuolated cells in the unsuckled newborn piglet colon suggests that the ileocecal junction does not provide a clear cut demarcation line for the “ileal” type of intestinal differentiation. This contention is supported by the observations that vacuolated cells were found most frequently within two or three centimetres of the junction, but rarely below this, and that the vacuolated cells appeared in prolongations of the normally flat surface epithelium of the colon which, although much shorter, were typical of the ileal villi. After one day the piglet colon showed no structures equivalent to villi; when stretched the epithelium was flat with frequent deep crypts, and very few vacuolated cells were found. This indicated a remarkably rapid loss of the small protuberances or villi bearing these vacuolated cells, a cell elimination which is apparently much faster than that established for the absorptive and goblet cell populations (Jarvis et al., ‘77). The unpredictable occurrence of the small villi would make any accurate estimation of turnover rate very difficult if not impossible to determine. The presence of regular arrays of particles on the surface of the plasmalemma invaginations and at the bases of the microvilli of the vacuolated cells clearly demonstrates the similarity of these cells in the piglet ileum (Hardy e t al., ’71) and the colon. Particles similar in location, size and organisation into arrays have been investigated extensively in rat neonatal ileal cells (Graney, ’64; Knutton e t al., ’74). The vacuolated cells in the colon were present prior to suckling, as has also been reported for the ileal vacuolated cells (Hardy e t al., ’71; Moon, ’72). The colonic vacuolated cells normally contained only a loose flocculent material, but if colostrum was injected directly into the newborn colon they were as capable as ileal cells of taking it up from the lumen. Presumably, in normal development these cells are lost before protein reaches the colon in any
COLONIC ULTRASTRUCTURE
quantity. They apparently have no direct function in postnatal life. Thus the presence of these vacuolated cells in the upper part of the newborn proximal colon is probably the result of a fortuitous and unpredictable longitudinal spread of the factors which cause the ileal type of differentiation. The brief presence of vacuolated cells in the colon seems unlikely to be connected to the transient amino acid transport-capability of the proximal colon (James and Smith, ’76). This transport is uniform in its distribution down the proximal colon, and the occurrence of vacuolated cells is far too sporadic to account for this capacity. The short-lived occurrence of vacuolated cells in the piglet colon is in sharp contrast to the considerable proliferation of such cells in the rat neonatal colon. Ono (‘76) has shown that in the rat the proportion of vacuolated cells increases from less than 1%a t birth to 75% of the total non-goblet surface epithelial cells at day 5. This high percentage was measured at the midpoint of the proximal colon, whereas in the piglet, even in the newborn, we have never observed more than a n individual vacuolated cell a t this level. Lipid uptake has not previously been reported from the neonatal colon. The uptake differs from the “classical” process found in the jejunum (Dobbins, ’69, for a review) in that the lipid droplets which first appeared in the apical cytoplasm as early as six hours after the initial suckling of the newborn were never bounded by a unit membrane. They were also much larger than the chylomicron-sized drops bounded by smooth endoplasmic reticulum found in studies of the apical cytoplasm of the small intestine during “classical” lipid absorption. However, chylomicron-sized droplets of lipid were found in vesicles closely associated with the Golgi body and also in the intercellular spaces in the 1-day-old piglet colonic surface epithelium. Thus, although the initial stages of lipid uptake are “non classical” and equivalent to those found in opossum stomachs (Krause e t al., ’76) and r a t (Helander e t al., ’70), the Golgi body does seem to play the role classically assigned to i t of packaging and export of the absorbed lipid. The process in the neonatal colon seems to be a combination of features of the two previously described systems for intestinal uptake of lipid, equivalent to the uptake in the terminal ileum described by Jersild and Clayton (’71).
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In the normal piglet colon, the transience of the occurrence of lipid droplets in the absorptive cells is probably merely an indication of the rapid increase in efficiency of lipid absorption by the upper digestive tract. If lipid is presented directly as colostrum then the piglet colon can absorb it readily a t birth and at 10 days old. The swelling of the intercellular spaces of transporting epithelia, in vitro, is a common observation (Smulders e t al., ’72). There is no indication that it affects the permeability of the epithelium to any significant degree, since the tight junctions are normally completely unaffected. A recent report (Fredericksen and Rostgaard, ’741, t h a t the swollen intercellular space is purely an artifact of electron microscopic preparation, may well be true for tissue fixed initially in osmium tetroxide, which is a poor primary fixative. There is no evidence that this swelling occurs with the glutaraldehyde-fixed material used in this or previous studies in which we found that the swelling of the intercellular space was quite independent of any of the processing procedures used (France e t al., ’76). The final notable developmental change in the piglet colon, the decrease in length of the microvilli, does appear to correlate with the short-lived amino-acid-transport capability. The change was found uniformly along the length of the proximal colon, and could well be a n expression of the loss of a population of cells having very different properties to those which replace them. Changes in the microvillar length of the most apical absorptive cells on the villus during neonatal development have been reported for guinea pig ileum (Merrill e t al., ’67) and mouse jejunum (Overton, ’65). In both these cases a n increase in length with age was observed, whereas the piglet colonic microvilli decreased in length. The small intestine and colon differ considerably in function in the adult, and this difference in developmental progression is, therefore, not surprising. The observed change in the length of the colonic microvilli reinforces the idea t h a t the newborn absorptive cells and the vacuolated cells are transient types, and that the cells which replace them have different functional and structural characteristics. The hypothesis of a change in functional cell population between birth and day 2 accounts satisfactorily for the two main structural changes observed: the loss of all the vacuolated cells and the change in the apical ab-
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F. B. P. WOODING, M. W. SMITH AND H. CRAIG
sorptive cell population characterised by the length of their microvilli. This latter population of cells would be responsible for the initial amino-acid-transport capability. Evidence from cell-kinetic studies supports the hypothesis of a loss of cell population correlating with the loss of certain structural and physiological parameters (Jarvis e t al., '77). Whether this initial population is a remnant of fetal life or a necessary bridge between the fetal and an independent existence remains to be determined. LITERATURE CITED Ash, R. W., and R. B. Heap 1973 The induction and synchronisation of parturition in sows treated with ICI 79939 an analogue of prostaglandin F2=. J. Agric. Sci. Camb., 81: 365-368. Dobbins, W. 0. 1969 Morphological aspects of lipid absorption. Amer. J. Clin. Nutrit., 22: 257-265. France, V. M.,M. W. Stanier and F. B. P. Wooding 1976 The effect of hormones and of an osmotic gradient on the structure and properties of mammalian foetal urinary bladder in uitro. J. Physiol. (London), 258: 393-408. Frederiksen, O., and J. Rostgaard 1974 Absence of dilated lateral intercellular spaces in fluid transporting frog gall bladder epithelium. J. Cell Biol., 61: 830-834. Graney, D. 0. 1964 Ultrastructure of the apical plasma membrane of intestinal lining cells. Anat. Rec., 148:
373-374.
the r a t colon during development. Acta Anat., 91:
330-349. Helander, H., and T. Olivecrona 1970 Lipolysis and lipid absorption in the stomach of the suckling rat. Gastroent., 59: 22-35. James, P.S., and M. W. Smith 1976 Methionine transport by pig colonic mucosa measured during early postnatal development. J. Physiol. (London), 262: 151-168. Jarvis, L.G., G. Morgan, M. W. Smith and F. B. P. Wooding 1977 Cell replacement and changing transport function in the neonatal pig colon. J. Physiol. (London), 273:
717-731. Jersild, R. A,, and R. T. Clayton 1971 A comparison of lipid absorption in the jejunum and ileum of the adult rat. Am. J. Anat., 131: 481-504. Knutton, S., A. R. Limbrick and J. D. Robertson 1974 Regular structures in membranes 1. Membranes in the endocytotic complex of ileal cells. J. Cell Biol., 62:
679-694. Krause, W. J., J. H. Cutts and C. R. Leeson 1976 The postn a t a l development of t h e alimentary canal i n the opossum. J. Anat. (London), 122: 499-519. Merrill, T. G., H. Sprinz and A. J. Tousimis 1967 Changes in intestinal absorptive cells during maturation. An electron microscope study of prenatal, postnatal and adult guinea pig ileum. J. Ult. Res., 19: 304-326. Moon, H.W. 1972 Vacuolated villous epithelium of the small intestine of young pigs. Vet. Path., 9: 3-21. Ono, K. 1976 Ultrastructure of the surface principal cells of the large intestine in postnatal developing rats. Anat. Embryol., 149: 155-171. Overton, J. 1965 Fine structure of t h e free cell surface in developing mouse intestinal mucosa. J. Exp. Zwl., 159:
195-202.
Hardy, R. N., A. R. Hockday and R. L. Tapp 1971 Observations on the structure of the small intestine in foetal, neonatal and suckling pigs. Phil. Trans. Roy. SOC. Lond. B., 259: 517-531. Helander, H. 1973 Morphological studies on the development of the r a t colonic mucosa. Acta Anat., 85: 153-176. 1975 Enzyme patterns and protein absorption in
-
Smulders, A. P.,J. McD. Tormey andE. M. Wright 1972The effect of osmotically induced water flows on the permeability and ultrastructure of the rabbit gall bladder. J. Memb. Biol., 7: 164-197. Staley, T. E., E. Wynn-Jones and A. E. Marshall 1968 The jejunal absorptive cell of the newborn pig: An electron microscopic study. Anat. Rec., 161: 497-516.
PLATE I EXPLANATION OF FIGURES
1 Newborn piglet. Absorptive cells from the surface epithelium. Note t h e small Golgi bodies (arrowheads) and the glycogen masses (open arrows) below the nuclei. Glutaraldehyde, Osmium + Ferrocyanide. X 4,500.
2 Ten-day-old piglet. Absorptive cells from the surface epithelium. There are, perhaps, more mitochondria both apically and basally (asterisks) when compared with figure 1, the Golgi bodies are much larger (arrowheads), multivesicular bodies (double arrowheads) are often present and there is no glycogen. The other fine-structure is very similar to t h a t found in the newborn. Glutaraldehyde, Osmium, Uranyl Acetate. X
4,500.
3 Ten-day-old piglet. Tissue from the same colon as figure 2 after incubation in a n Ussing chamber. Note the dilated intercellular spaces (asterisks). The cell length is much less in figure 3 (after allowing for the difference in magnification) b c a u s e this tissue was stretched (for incubation) prior to fixation. The tissue of figure 2 was not stretched before fixation. Stretching by itself has no effect on t h e intercellular spaces. x 2,000.
COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
4 Newborn piglet, unsuckled. Vacuolated cells from the surface epithelium. The vacu-
oles contain a loose flocculent material and a n occasional dense body. Note the extensive glycogen areas in the bases of the cells (asterisks) and the goblet cell (arrow). Glutaraldehyde, Osmium. X 2,400. 5 Newborn piglet, colon injected with colostrum via the cecum. The vacuoles are full of a dense material which can be shown to be protein by histochemical procedures. Ferrocyanide. X 3,500. Glutaraldehyde, Osmium
+
6, 7 Newborn piglets, unsuckled. Light micrographs of semi-thin Araldite sections stained with 1%Toluidine blue in 1%sodium borate. The vacuolated cells are present on villus-like extensions which occur sporadically along the length of the colon, protruding above the usual surface epithelial level. Figure 6 : 1 cm, figure 7: 5 cm from the ileocecal junction. Figure 6 X 130,figure 7 X 50.
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COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
PLATE 2
277
PLATE 3 EXPLANATION OF FIGURES
Figs. 8, 9 Newborn piglets, unsuckled. Details of the apical tubulovesicular system and vacuoles.
8 The tubulovesicular system can be seen to be continuous (open arrow) with a small vacuole (1) with less content than those below i t (2).Glutaraldehyde, Osmium. x 22,000. 9 Traces of regular beading (which can be seen better on the plasmalemma in figs. 10. 11) are present in t h e tubulovesicular system (double open arrow) and small apical vacuole (single open arrows). The apical vacuole (equivalent to 1 in fig. 8 ) is continuous with elements of the tubulovesicular system (asterisks). Glutaraldehyde, Osmium, Uranyl Acetate. X 68,000.
278
COLONIC ULTRASTRUCTURE F B P Wooding, M W Smlth and H Cralg
PLATE 3
PLATE 4 EXPLANATION OF FIGURES
Figs. 10-12 Newborn piglets, unsuckled. Details of the apical tubulovesicular system and vacuoles. 1 0 , l l Regular beading on the outer leaflet of the plasmalemma of apical invaginations (fig. 11) and the bases of the microvilli (open arrows) (fig. 10). Glutaraldehyde, Osmium, Uranyl Acetate. Figure 10 X 62,000, fig. 11 X 76,000. 12 Traces of the regular beading seen on the plasmalemma in figures 10 and 11 are present on elements of the tubulovesicular system (open arrows). This system is continuous (single asterisk) with the small apical vacuole a t the base of the micrograph. Neither t h e vacuole (equivalent to l in fig. 8)nor the apical plasmalemma1 invagination (double asterisk) clearly show the beading, because the plane of section is unsuitable. Glutaraldehyde, Osmium, Uranyl Acetate. X 90,000.
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COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
PLATE 4
281
PLATE 5 EXPLANATION OF FIGURES
Figs. 13-17 One-day-old piglets, suckled. 13 Surface epithelial cells showing lipid droplets (round white areas) throughout the cytoplasm. Glutaraldehyde, Osmium, Uranyl Acetate. X 2,100. 14 Light micrograph of a semi-thin Araldite section stained with toluidine blue and subsequently heated. Lipid
droplets are the only structures retaining a significant amount of stain after this procedure. The restriction of the lipid to the surface cells of the epithelium is clearly demonstrated. X 250. 15 Apical cytoplasm of a n absorptive cell. Compare the single dense line bounding the lipid droplets (asterisks) with the unit membrane of the plasmalemma (black arrow). The lipid droplets are thus available to cytoplasmic enzymes, in contrast to the smaller lipid drops in figure 16, which are segregated within the unit membranes of the Golgi vesicles. I n normal fat absorption (e.g., in the jejunum) the majority of the small lipid droplets are produced within the unit membranes of t h e smooth endoplasmic reticulum. Glutaraldehyde, Osmium, Uranyl Acetate. X 65,000. 16 Lipid droplets in the chylomicron size-range, packed into vesicles (asterisks) in close proximity to the Golgi body (black arrow). This micrograph was from a specimen incubated in an Ussing chamber; the intercellular space is widely dilated (double asterisk). Glutaraldehyde, Osmium, Uranyl Acetate. x 45,000. 17 Basal area of a n epithelial cell. Note t h e accumulation of chylomicron-sized particles in the intercellular spaces (asterisks), and the larger lipid droplets, not bound by membranes, in the cytoplasm (black arrows). Glutaraldehyde, Osmium, Uranyl Acetate. x 31,000.
282
PLATE 5 COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
283
PLATE 6 EXPLANATION OF FIGURES
18 Newborn piglet, unsuckled. Apical cytoplasm of absorptive cells, microvillar brush border and glycocalyx (open arrow). Glutaraldehyde, Osmium + Ferrocyanide. X 34,000. 19 Ten-day-old piglet. Apical cytoplasm of absorptive cells, microvillar brush border and glycocalyx (open arrow). Glutaraldehyde, Osmium, Uranyl Acetate. X 34,000.
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COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W Smith and H. Cralg
PLATE 6
285
ABSTRACT The neonatal pig colon has several unique structural and developmental features. A t birth it has a variable population of epithelial cells which in their arrangement on villus-like protrusions and in their capability for protein uptake into large preformed supranuclear vacuoles closely resemble neonatal ileal cells. Such villus-like protrusions and vacuolated cells are not present in the 2-day-old piglet. On the first day after birth absorptive epithelial cells which lack supranuclear vacuoles transiently accumulate a large number of lipid droplets, each separated from the cytoplasm only by a proteolipid interface. None of the much smaller lipid droplets bounded by a unit membrane of the smooth endoplasmic reticulum and characteristic of normal small intestinal fat uptake were ever seen in these cells. Very few of the large lipid drops remain on the second day after birth. This initial capacity of the colon for protein and lipid uptake never reappears. The pattern of colonic amino acid transport also changes markedly in the first four days of independent life and this may be correlated with the observation that the absorptive cells a t birth have microvilli which are twice the length of those on similar cells a t and after two days old. These morphological results are discussed in terms of implied functional changes in the neonatal period. Changes in the structure and function of the digestive system may be of great importance to the survival of neonatal animals. Several developmental studies have been carried out on the ultrastructure and physiology of the stomach and small intestines of various neonatal animals, e.g., opossum (Krause e t al., '761, pig (Staley e t al., '68; Hardy et al., '71; Moon, '72) and rat (Helander and Olivecrona, '701, but little attention has been given so far to the large intestine. Two recent studies describe the ultrastructure of the postnatal rat colon (Helander, '73, '75; Ono, '76) but this study of the pig colon is the first known to the authors describing the ultrastructure of neonatal development in the colon of any domesticated animal of agricultural importance. One of the most notable ultrastructural features of the colonic surface epithelium of the pig is the presence a t birth of cells with large supranuclear vacuoles equivalent to those found in the terminal ileum. Unlike the latter, colonic cells never accumulate protein after suckling has started. In addition one day after birth in cells without supranuclear vacuoles there is a transient accumulation of lipid by a route which differs from that found a t the norAM. J. ANAT. (1978)152: 269-286.
mal site of lipid uptake, the proximal intestinal cells. The neonatal pig colon has many physiological similarities with adult small intestines. For example, it can actively transport amino acids such as methionine and this transport stimulates the absorption of Na' (James and Smith, '76). These transport capabilities are lost rapidly and the adult pattern is established two or three days after birth. The present work details the rapid changes that take place in the structure of the pig colon immediately after birth. These results, with a study of cell kinetics, have been used to define the changes in transport function after birth in the neonatal pig colon (Jarvis e t al., '77). MATERIALS AND METHODS
Pigs were taken from a herd of Large Whites bred a t Babraham. Parturition was induced routinely by the intramuscular injection of prostaglandin analogues on days 109111 of gestation. Birth occurred some 24 hours later (Ash and Heap, '73). Piglets were used newborn before suckling, or a t days 1, 2, 4 or Accepted Oct. 1, '77.
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10. The animals were killed by cervical dislocation and portions of the proximal colon taken for microscopy. Samples were also taken after mounting the colonic mucosa flat in an Ussing chamber for transport experiments (James and Smith, '76). The solutions bathing either side of the mucosa were replaced simultaneously with fixative. Fixation was carried out a t a time when the mucosal preparation had achieved a steady state in the physiological experiment. Samples of fresh mucosa were fixed by opening the colon longitudinally and cutting the tissue into pieces in the fixative, or fixed intact after stretching the tissue flat with pins on cork. This latter procedure produced a much flatter, thinner preparation, easier to orient and section than the contorted pieces resulting from unrestrained slicing of the mucosa into small cubes. No significant ultrastructural differences were observed between samples prepared by the two methods. Tissue was fixed for one to two hours in 4% glutaraldehyde in 0.1 M NaH2/Na2HPO, buffer, pH 7.2, containing 2% sucrose. Tissue was then osmium tetroxide treated fcr one hour with 1% in 0.1 M veronal-acetate buffer, pH 7.2, sometimes with 1.5%potassium ferrocyanide added (see figure legends for details), followed in some cases by treatment for half an hour with 2%aqueous uranyl acetate. Dehydration was in alcohol and, finally, propylene oxide. All the processing was a t room temperature. Embedding was carried out in Araldite. Sections were stained with uranyl acetate (saturated solution in 50%ethanol) and lead citrate and examined in an AE1 EM 6B or a JEOL lOOC microscope.
shown a flat surface epithelium with crypt openings and goblet cells as the only surface features. In the newborn piglet colon, however (within 10-15 cm of the ileocecal junction), the results in this paper show a significant but very variable number of villus-like structures very similar to those found in the ileum. Such villus-like structures were not found in the colon of piglets one day after birth or older. This paper deals with cells in the surface epithelium and the apical eighth of the crypts, only - cells which are mature and do not alter their fine-structural characters prior to cell death and elimination a t the surface of the epithelium.
Unsuckled, newborn Three main cell types were found a t this stage: goblet (fig. 4), absorptive (fig. 1) and vacuolated (fig. 4). The goblet cell fine-structure was identical to goblet cells found elsewhere in the intestine and in other species. Absorptive cells had a microvillar brushborder, under which lay a zone of cytoplasm, devoid of organelles save for the microfibrillar roots of the microvillar cores. Then came a region containing mitochondria, the occasional cisterna of rough endoplasmic reticulum (RER) and many glycogen granules. Just above or beside the nucleus there was a small Golgi body and, below the nucleus, more mitochondria, RER and glycogen, this last frequently in massive amounts. The lateral cell membrane was considerably folded, and sealed at the apex by a fairly wide tight junction. These absorptive cells formed the total cell population (fig. 1) (excluding goblet cells) at the luminal surface of the colon, except where Intestinal bypass studies vacuolated cells occurred. Two newborn piglets were anesthetised Vacuolated cells were found in significant with Fluothane (ICI, Macclesfield, Cheshire) number only in the colons of newborn piglets and a cecal cannula inserted after opening the and, even a t this stage, their occurrence was abdomen. Five ml of pig colostrum was in- very unpredictable. They were usually the jected directly into the proximal colon, after major cell type for 2-3 cm beyond the ileocecal the ileum had been sutured a t the ileocecal junction (figs. 4, 61, although they were presjunction, and the cannula sealed. The animal ent in only a small percentage of any one cowas allowed to recover and five to six hours lonic diameter. They occurred on protuberlater was killed and the colon sampled for elec- ances in the epithelium which projected above the general level of the flat colonic epithelium tron microscopy as detailed above. and resembled miniature ileal villi (figs. 6,7). RESULTS After the first 2 cm their occurrence was very As a general rule the colonic mucosal epi- sporadic, but groups of protuberances bearing thelium was distinguished easily from that in vacuolated cells have been found 15 cm from the small intestine, by the absence of villi. In the ileocecal junction and a group of two or the 6-day-old pig Newman et al. ('76) have three vacuolated cells were often to be found
271
COLONIC ULTRASTRUCTURE
in the normal colonic surface epithelium up to 25 cm from the ileocecal junction (fig. 7). Usually the tissue used for incubations in the Ussing chamber was taken 3 or 4 cm beyond the ileocecal junction, so did not contain a significant number of vacuolated cells. However, one example was found where most of the surface cells of the experimental piece of colon were vacuolated. The transport capacity for methionine was in no way different from that found in newborn colons containing virtually no vacuolated cells. The microvilli (fig. 8) were shorter than those on absorptive cells and there were numerous invaginations of the plasmalemma a t the base of the microvilli down into the cell (figs. 11,121. The plasmalemma of the bases of the microvilli and the invaginations bore a regular array of particles on the luminal surface (figs. 10, 11).In the apical cytoplasm between and beneath the invaginations lay numerous vesicles and tubules of fairly constant diameter which could occasionally be seen to be continuous with larger vacuoles (figs. 9, 12). The vesicles and tubules had an asymmetrically stained plasmalemma, with the luminal leaflet much wider than that on the cytoplasmic side (figs. 9,12). Beneath this zone of vesicles and tubules the “supranuclear vacuoles” were found. These contained occasional dense areas inside their bounding membrane, and a loose flocculent material filled the vacuole itself (figs. 4,8). They varied in size from 550 p m and were bounded by a symmetrical unit membrane. Mitochondria, RER and glycogen were found between the vacuoles. There was a large Golgi body just above the nucleus and, if there were only a few small supranuclear vacuoles, they seemed to be associated with the Golgi membranes. Very rarely a vacuole was found below the nucleus, but usually this area had only mitochondria, RER and glycogen (fig. 4). In the unsuckled newborn animals used in this study the supranuclear vacuoles were present in both ileum and proximal colon. The colon was normally filled with meconium, and the vacuoles showed a dispersed, flocculent content, but after colostrum was injected through a cecal cannula the supranuclear vacuoles became filled with protein (fig. 5) and lipid droplets appeared in all the absorptive cells. Such lipid droplets were never seen in untreated unsuckled newborn piglet colonic epithelium, but started to appear about five to six hours after suckling had begun.
Suckled, one day old
The most obvious differences from the newborn were the almost complete absence of vacuolated cells showing the tubulovesicular system and/or supranuclear vacuoles and the appearance of numerous lipid droplets in the cytoplasm of the absorptive cells (figs. 13, 14). Lipid droplets, not bound by membranes (fig. 151, varied in size from 0.1-20 pm, and were most frequent in the supranuclear cytoplasm, but did occur throughout the cell (figs. 13,14). There was no indication of membrane-bound lipid except, rarely, in the immediate vicinity of the Golgi body. Here a uniform population of small lipid droplets could sometimes be found within what were probably vesicles arising from the Golgi body (fig. 16). Lipid droplets of a similar size were also found occasionally in the intercellular space between cells below the level of the Golgi body (fig. 17). Other organelles in the absorptive cell were as found in the newborn. Very rarely a cell with some of the tubulovesicular system of the vacuolated cells could be found a t the surface away from the crypt mouth, but there were no large supranuclear vacuoles. Suckled, two days old
At this stage most of the large lipid droplets characteristic of the 1-day-old had disappeared, although most of the absorptive cells showed a scattering of small lipid droplets. No lipid droplets were seen within Golgi vesicles or intercellular spaces. The characteristic feature of the apical cytoplasm was the presence of several phagolysosome-type bodies containing vesicles and dense material of various sorts. There was less glycogen but more mitochondria and RER than in the newborn. The Golgi body was slightly larger and so was the basal region below the nucleus which had more mitochondria and less glycogen. Four days and ten days old
There were only two types of cells, goblet and absorptive, both of which showed the characteristic structure found in such cells wherever they occur in large or small intestine. The absorptive cells had large aggregations of mitochondria interspersed with RER apically and basally. The supranuclear Golgi body was well developed, and multivesicular bodies were present (fig. 2). There was virtually no glycogen or lipid.
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Epithelium after incubation in physiological medium The most striking difference seen after incubation of any epithelium was the vast increase in the volume of the intercellular spaces (compare figs. 2 and 3). This was greatest a t the base of the epithelium and narrowed to the apical junctional complex, whose structure was completely unaffected by the incubation. The tight junction showed no sign of internal widening and there was no evidence of any difference in the extent or structure of the tight junction from the newborn to the 10-day-oldpiglet. The epithelium in figure 2 was twice the height of figure 3 at the same magnification, although they were both from the same colon. The difference in cell heights was due to the fact that the tissue in figure 2 was fixed without prior stretching, whereas in figure 3 the tissue was stretched, incubated and then fixed in the incubation chamber. Stretching an unincubated colon never produced any significant widening of the intercellular spaces. The tubulovesicular system of the newborn vacuolated cells and the lipid droplets in the 1-day-old suckled piglets appeared largely unchanged after incubation for up to two hours, although there were probably fewer lipid droplets in the cells.
Length of microvilli and presence of a glycocalyx There was a marked decrease in the average length of colonic microvilli on the absorptive cells of the newborn piglet, when compared to the microvilli on the 2-day-old piglet absorptive cells, after which there was little change. The microvilli on the newborn vacuolated cells were much shorter than on absorptive cells a t the same age (compare figs. 18 and 8) (table 1).There was a well developed zone of delicate extracellular microfibrils attached to the outer leaflet of the microvillar plasmalemTABLE 1
Change of length of microvilli with age No. of pigs
51 4 3
4 5
Age
h n @ h in pM Width in p M
Newborn absorptive Newborn vacuolated 1 day 2 day 4 day 10 day
1.220.2(25) 0.120.03 0.520.02(9) 0.120.02 1.020.1(24) 0.120.02 0.620.2(24) 0.0820.02 0.6-+0.1(24) O.l-tO.01 0.520.1(15) 0.120.02
ma a t all stages examined (figs. 18, 19). This presumably corresponds to the “glycocalyx” observed by other workers on intestinal brush borders. There was no apparent difference in the extent of this layer at any age examined, although i t was usually wider a t the bottom of the descending spiral of the colon than close to the ileocecal junction. DISCUSSION
The sporadic occurrence of vacuolated cells in the unsuckled newborn piglet colon suggests that the ileocecal junction does not provide a clear cut demarcation line for the “ileal” type of intestinal differentiation. This contention is supported by the observations that vacuolated cells were found most frequently within two or three centimetres of the junction, but rarely below this, and that the vacuolated cells appeared in prolongations of the normally flat surface epithelium of the colon which, although much shorter, were typical of the ileal villi. After one day the piglet colon showed no structures equivalent to villi; when stretched the epithelium was flat with frequent deep crypts, and very few vacuolated cells were found. This indicated a remarkably rapid loss of the small protuberances or villi bearing these vacuolated cells, a cell elimination which is apparently much faster than that established for the absorptive and goblet cell populations (Jarvis et al., ‘77). The unpredictable occurrence of the small villi would make any accurate estimation of turnover rate very difficult if not impossible to determine. The presence of regular arrays of particles on the surface of the plasmalemma invaginations and at the bases of the microvilli of the vacuolated cells clearly demonstrates the similarity of these cells in the piglet ileum (Hardy e t al., ’71) and the colon. Particles similar in location, size and organisation into arrays have been investigated extensively in rat neonatal ileal cells (Graney, ’64; Knutton e t al., ’74). The vacuolated cells in the colon were present prior to suckling, as has also been reported for the ileal vacuolated cells (Hardy e t al., ’71; Moon, ’72). The colonic vacuolated cells normally contained only a loose flocculent material, but if colostrum was injected directly into the newborn colon they were as capable as ileal cells of taking it up from the lumen. Presumably, in normal development these cells are lost before protein reaches the colon in any
COLONIC ULTRASTRUCTURE
quantity. They apparently have no direct function in postnatal life. Thus the presence of these vacuolated cells in the upper part of the newborn proximal colon is probably the result of a fortuitous and unpredictable longitudinal spread of the factors which cause the ileal type of differentiation. The brief presence of vacuolated cells in the colon seems unlikely to be connected to the transient amino acid transport-capability of the proximal colon (James and Smith, ’76). This transport is uniform in its distribution down the proximal colon, and the occurrence of vacuolated cells is far too sporadic to account for this capacity. The short-lived occurrence of vacuolated cells in the piglet colon is in sharp contrast to the considerable proliferation of such cells in the rat neonatal colon. Ono (‘76) has shown that in the rat the proportion of vacuolated cells increases from less than 1%a t birth to 75% of the total non-goblet surface epithelial cells at day 5. This high percentage was measured at the midpoint of the proximal colon, whereas in the piglet, even in the newborn, we have never observed more than a n individual vacuolated cell a t this level. Lipid uptake has not previously been reported from the neonatal colon. The uptake differs from the “classical” process found in the jejunum (Dobbins, ’69, for a review) in that the lipid droplets which first appeared in the apical cytoplasm as early as six hours after the initial suckling of the newborn were never bounded by a unit membrane. They were also much larger than the chylomicron-sized drops bounded by smooth endoplasmic reticulum found in studies of the apical cytoplasm of the small intestine during “classical” lipid absorption. However, chylomicron-sized droplets of lipid were found in vesicles closely associated with the Golgi body and also in the intercellular spaces in the 1-day-old piglet colonic surface epithelium. Thus, although the initial stages of lipid uptake are “non classical” and equivalent to those found in opossum stomachs (Krause e t al., ’76) and r a t (Helander e t al., ’70), the Golgi body does seem to play the role classically assigned to i t of packaging and export of the absorbed lipid. The process in the neonatal colon seems to be a combination of features of the two previously described systems for intestinal uptake of lipid, equivalent to the uptake in the terminal ileum described by Jersild and Clayton (’71).
273
In the normal piglet colon, the transience of the occurrence of lipid droplets in the absorptive cells is probably merely an indication of the rapid increase in efficiency of lipid absorption by the upper digestive tract. If lipid is presented directly as colostrum then the piglet colon can absorb it readily a t birth and at 10 days old. The swelling of the intercellular spaces of transporting epithelia, in vitro, is a common observation (Smulders e t al., ’72). There is no indication that it affects the permeability of the epithelium to any significant degree, since the tight junctions are normally completely unaffected. A recent report (Fredericksen and Rostgaard, ’741, t h a t the swollen intercellular space is purely an artifact of electron microscopic preparation, may well be true for tissue fixed initially in osmium tetroxide, which is a poor primary fixative. There is no evidence that this swelling occurs with the glutaraldehyde-fixed material used in this or previous studies in which we found that the swelling of the intercellular space was quite independent of any of the processing procedures used (France e t al., ’76). The final notable developmental change in the piglet colon, the decrease in length of the microvilli, does appear to correlate with the short-lived amino-acid-transport capability. The change was found uniformly along the length of the proximal colon, and could well be a n expression of the loss of a population of cells having very different properties to those which replace them. Changes in the microvillar length of the most apical absorptive cells on the villus during neonatal development have been reported for guinea pig ileum (Merrill e t al., ’67) and mouse jejunum (Overton, ’65). In both these cases a n increase in length with age was observed, whereas the piglet colonic microvilli decreased in length. The small intestine and colon differ considerably in function in the adult, and this difference in developmental progression is, therefore, not surprising. The observed change in the length of the colonic microvilli reinforces the idea t h a t the newborn absorptive cells and the vacuolated cells are transient types, and that the cells which replace them have different functional and structural characteristics. The hypothesis of a change in functional cell population between birth and day 2 accounts satisfactorily for the two main structural changes observed: the loss of all the vacuolated cells and the change in the apical ab-
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F. B. P. WOODING, M. W. SMITH AND H. CRAIG
sorptive cell population characterised by the length of their microvilli. This latter population of cells would be responsible for the initial amino-acid-transport capability. Evidence from cell-kinetic studies supports the hypothesis of a loss of cell population correlating with the loss of certain structural and physiological parameters (Jarvis e t al., '77). Whether this initial population is a remnant of fetal life or a necessary bridge between the fetal and an independent existence remains to be determined. LITERATURE CITED Ash, R. W., and R. B. Heap 1973 The induction and synchronisation of parturition in sows treated with ICI 79939 an analogue of prostaglandin F2=. J. Agric. Sci. Camb., 81: 365-368. Dobbins, W. 0. 1969 Morphological aspects of lipid absorption. Amer. J. Clin. Nutrit., 22: 257-265. France, V. M.,M. W. Stanier and F. B. P. Wooding 1976 The effect of hormones and of an osmotic gradient on the structure and properties of mammalian foetal urinary bladder in uitro. J. Physiol. (London), 258: 393-408. Frederiksen, O., and J. Rostgaard 1974 Absence of dilated lateral intercellular spaces in fluid transporting frog gall bladder epithelium. J. Cell Biol., 61: 830-834. Graney, D. 0. 1964 Ultrastructure of the apical plasma membrane of intestinal lining cells. Anat. Rec., 148:
373-374.
the r a t colon during development. Acta Anat., 91:
330-349. Helander, H., and T. Olivecrona 1970 Lipolysis and lipid absorption in the stomach of the suckling rat. Gastroent., 59: 22-35. James, P.S., and M. W. Smith 1976 Methionine transport by pig colonic mucosa measured during early postnatal development. J. Physiol. (London), 262: 151-168. Jarvis, L.G., G. Morgan, M. W. Smith and F. B. P. Wooding 1977 Cell replacement and changing transport function in the neonatal pig colon. J. Physiol. (London), 273:
717-731. Jersild, R. A,, and R. T. Clayton 1971 A comparison of lipid absorption in the jejunum and ileum of the adult rat. Am. J. Anat., 131: 481-504. Knutton, S., A. R. Limbrick and J. D. Robertson 1974 Regular structures in membranes 1. Membranes in the endocytotic complex of ileal cells. J. Cell Biol., 62:
679-694. Krause, W. J., J. H. Cutts and C. R. Leeson 1976 The postn a t a l development of t h e alimentary canal i n the opossum. J. Anat. (London), 122: 499-519. Merrill, T. G., H. Sprinz and A. J. Tousimis 1967 Changes in intestinal absorptive cells during maturation. An electron microscope study of prenatal, postnatal and adult guinea pig ileum. J. Ult. Res., 19: 304-326. Moon, H.W. 1972 Vacuolated villous epithelium of the small intestine of young pigs. Vet. Path., 9: 3-21. Ono, K. 1976 Ultrastructure of the surface principal cells of the large intestine in postnatal developing rats. Anat. Embryol., 149: 155-171. Overton, J. 1965 Fine structure of t h e free cell surface in developing mouse intestinal mucosa. J. Exp. Zwl., 159:
195-202.
Hardy, R. N., A. R. Hockday and R. L. Tapp 1971 Observations on the structure of the small intestine in foetal, neonatal and suckling pigs. Phil. Trans. Roy. SOC. Lond. B., 259: 517-531. Helander, H. 1973 Morphological studies on the development of the r a t colonic mucosa. Acta Anat., 85: 153-176. 1975 Enzyme patterns and protein absorption in
-
Smulders, A. P.,J. McD. Tormey andE. M. Wright 1972The effect of osmotically induced water flows on the permeability and ultrastructure of the rabbit gall bladder. J. Memb. Biol., 7: 164-197. Staley, T. E., E. Wynn-Jones and A. E. Marshall 1968 The jejunal absorptive cell of the newborn pig: An electron microscopic study. Anat. Rec., 161: 497-516.
PLATE I EXPLANATION OF FIGURES
1 Newborn piglet. Absorptive cells from the surface epithelium. Note t h e small Golgi bodies (arrowheads) and the glycogen masses (open arrows) below the nuclei. Glutaraldehyde, Osmium + Ferrocyanide. X 4,500.
2 Ten-day-old piglet. Absorptive cells from the surface epithelium. There are, perhaps, more mitochondria both apically and basally (asterisks) when compared with figure 1, the Golgi bodies are much larger (arrowheads), multivesicular bodies (double arrowheads) are often present and there is no glycogen. The other fine-structure is very similar to t h a t found in the newborn. Glutaraldehyde, Osmium, Uranyl Acetate. X
4,500.
3 Ten-day-old piglet. Tissue from the same colon as figure 2 after incubation in a n Ussing chamber. Note the dilated intercellular spaces (asterisks). The cell length is much less in figure 3 (after allowing for the difference in magnification) b c a u s e this tissue was stretched (for incubation) prior to fixation. The tissue of figure 2 was not stretched before fixation. Stretching by itself has no effect on t h e intercellular spaces. x 2,000.
COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
4 Newborn piglet, unsuckled. Vacuolated cells from the surface epithelium. The vacu-
oles contain a loose flocculent material and a n occasional dense body. Note the extensive glycogen areas in the bases of the cells (asterisks) and the goblet cell (arrow). Glutaraldehyde, Osmium. X 2,400. 5 Newborn piglet, colon injected with colostrum via the cecum. The vacuoles are full of a dense material which can be shown to be protein by histochemical procedures. Ferrocyanide. X 3,500. Glutaraldehyde, Osmium
+
6, 7 Newborn piglets, unsuckled. Light micrographs of semi-thin Araldite sections stained with 1%Toluidine blue in 1%sodium borate. The vacuolated cells are present on villus-like extensions which occur sporadically along the length of the colon, protruding above the usual surface epithelial level. Figure 6 : 1 cm, figure 7: 5 cm from the ileocecal junction. Figure 6 X 130,figure 7 X 50.
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COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
PLATE 2
277
PLATE 3 EXPLANATION OF FIGURES
Figs. 8, 9 Newborn piglets, unsuckled. Details of the apical tubulovesicular system and vacuoles.
8 The tubulovesicular system can be seen to be continuous (open arrow) with a small vacuole (1) with less content than those below i t (2).Glutaraldehyde, Osmium. x 22,000. 9 Traces of regular beading (which can be seen better on the plasmalemma in figs. 10. 11) are present in t h e tubulovesicular system (double open arrow) and small apical vacuole (single open arrows). The apical vacuole (equivalent to 1 in fig. 8 ) is continuous with elements of the tubulovesicular system (asterisks). Glutaraldehyde, Osmium, Uranyl Acetate. X 68,000.
278
COLONIC ULTRASTRUCTURE F B P Wooding, M W Smlth and H Cralg
PLATE 3
PLATE 4 EXPLANATION OF FIGURES
Figs. 10-12 Newborn piglets, unsuckled. Details of the apical tubulovesicular system and vacuoles. 1 0 , l l Regular beading on the outer leaflet of the plasmalemma of apical invaginations (fig. 11) and the bases of the microvilli (open arrows) (fig. 10). Glutaraldehyde, Osmium, Uranyl Acetate. Figure 10 X 62,000, fig. 11 X 76,000. 12 Traces of the regular beading seen on the plasmalemma in figures 10 and 11 are present on elements of the tubulovesicular system (open arrows). This system is continuous (single asterisk) with the small apical vacuole a t the base of the micrograph. Neither t h e vacuole (equivalent to l in fig. 8)nor the apical plasmalemma1 invagination (double asterisk) clearly show the beading, because the plane of section is unsuitable. Glutaraldehyde, Osmium, Uranyl Acetate. X 90,000.
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COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
PLATE 4
281
PLATE 5 EXPLANATION OF FIGURES
Figs. 13-17 One-day-old piglets, suckled. 13 Surface epithelial cells showing lipid droplets (round white areas) throughout the cytoplasm. Glutaraldehyde, Osmium, Uranyl Acetate. X 2,100. 14 Light micrograph of a semi-thin Araldite section stained with toluidine blue and subsequently heated. Lipid
droplets are the only structures retaining a significant amount of stain after this procedure. The restriction of the lipid to the surface cells of the epithelium is clearly demonstrated. X 250. 15 Apical cytoplasm of a n absorptive cell. Compare the single dense line bounding the lipid droplets (asterisks) with the unit membrane of the plasmalemma (black arrow). The lipid droplets are thus available to cytoplasmic enzymes, in contrast to the smaller lipid drops in figure 16, which are segregated within the unit membranes of the Golgi vesicles. I n normal fat absorption (e.g., in the jejunum) the majority of the small lipid droplets are produced within the unit membranes of t h e smooth endoplasmic reticulum. Glutaraldehyde, Osmium, Uranyl Acetate. X 65,000. 16 Lipid droplets in the chylomicron size-range, packed into vesicles (asterisks) in close proximity to the Golgi body (black arrow). This micrograph was from a specimen incubated in an Ussing chamber; the intercellular space is widely dilated (double asterisk). Glutaraldehyde, Osmium, Uranyl Acetate. x 45,000. 17 Basal area of a n epithelial cell. Note t h e accumulation of chylomicron-sized particles in the intercellular spaces (asterisks), and the larger lipid droplets, not bound by membranes, in the cytoplasm (black arrows). Glutaraldehyde, Osmium, Uranyl Acetate. x 31,000.
282
PLATE 5 COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W. Smith and H. Craig
283
PLATE 6 EXPLANATION OF FIGURES
18 Newborn piglet, unsuckled. Apical cytoplasm of absorptive cells, microvillar brush border and glycocalyx (open arrow). Glutaraldehyde, Osmium + Ferrocyanide. X 34,000. 19 Ten-day-old piglet. Apical cytoplasm of absorptive cells, microvillar brush border and glycocalyx (open arrow). Glutaraldehyde, Osmium, Uranyl Acetate. X 34,000.
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COLONIC ULTRASTRUCTURE F. B. P. Wooding, M. W Smith and H. Cralg
PLATE 6
285
