Changes in small intestinal mucosa morphology and cell renewal in suckling, prolonged-suckling, and weaned lambs DIDIER
ATTAIX
AND
JEAN-CLAUDE
MESLIN
Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Ceyrat; and Laboratoire de Nutrition et Skuritk Alimentaire, 78352 Jouy-en-Josas Cedex, France
ATTAIX, DIDIER, AND JEAN-CLAUDE MESLIN. Changes in small intestinal mucosa morphology and cell renewal in suckling, prolonged-suckling, and weaned lambs. Am. J. Physiol. 261
(Regulatory Integrative Comp. Physiol. 30): R811-R818, 1991.-No information concerning the effect of weaning on intestinal cell proliferation is currently available in largespecies with early intestinal morphogenesis,a group including most domesticanimalsand humans. Changesin intestinal morphology and epithelial cell renewal were investigated in l-, 5, and 8wk-old suckling and 8-wk-old weaned lambs after injection of [“Hlthymidine. In suckling lambsa gradual increasein crypt depth occurred with age, especially in the proximal intestine, whereasvillus height was significantly reduced in the distal regions. At 8 wk of age weanedand prolonged-suckling lambs exhibited no significant differences in crypt depth throughout the intestine and in villus height proximally. However, weaned lambs had shorter villi in the jejunum and ileum. The highest enterocyte migration rates (4.4-9.7 pm/h) were observed in lwk-old lambs. In suckling animals, migration rates decreased with age by 60, 51, and 11% in the duodenum, jejunum, and ileum, respectively. Weaned and prolonged-suckling 8-wk-old lambshad a similar rate of enterocyte migration in the ileum. Furthermore, ruminating animalsexhibited only slightly higher migration ratesin the duodenumand the jejunum (53 and 15%, respectively). In suckling lambs,epithelial cell renewalrequired 2.1-4.0, 4.5-6.3, and 4.0-5.3 days at 1, 5, and 8 wk of age, respectively, whereaslabeled cells reachedthe tips of the villi within 3.0-3.1 days in weanedanimals. These data suggestthat the suckling period correspondsto a gradual and important phaseof postnatal intestinal adaptation in the sheep,a species with early patterns of intestinal cell replacement. By contrast, and although weaning is the major event in gastrointestinal adaptation in ruminants, the transition from milk to solid feeding, or temporally related events, is not associatedwith abrupt changesin intestinal epithelial morphology and cell kinetics as reported in altricial species.
intestinal crypt-villus; cell proliferation; development; weaning
THE EPITHELIUM OF THE vertebrate small intestine, cells are produced in the crypts and continuously shed from the villi tips (19). Between birth and weaning, two critical periods in gastrointestinal adaptation, this dynamic and essential process for intestinal growth is under the control of intrinsic factors (genetic endowment, developmental clock; see Ref. 17 for definitions) and is influenced by extrinsic or environmental factors (hormonal, neural, dietary, luminal, and growth factors; Refs. 14, 15). Changes that occur perinatally have been extenIN
0363-6119/91
$1.50 Copyright
sively studied in suckling and weaned rodents (13). However, in species such as the rat and the mouse cryptvillus differentiation occurs during the latest stages of fetal growth (ZO), and weaning represents a major event in gastrointestinal maturation (13). By contrast, small intestinal development in the sheep and the pig, two species with a long gestational period, occurs earlier in utero and may be more representative of human development in the fetal and neonatal periods (13). Unlike the pig, the sheep exhibits patterns of intestinal cell migration and replacement at an early stage of fetal development (38, 39), as do humans (1). Immediately after birth, we (5) and others (23, 31, 42) have described rapid epithelium renewal times (2-4 days) in contrast to observations made in rodents (13). Only Moon and Joel (23) have briefly reported the postnatal evolution of small intestinal morphology and cell migration in lambs. Unfortunately these data are very difficult to interpret because no indications relative to animal breeding and diets were given. Furthermore, to our knowledge, no information concerning the effect of weaning on intestinal cell proliferation is available in large species with early intestinal morphogenesis, a group including most domestic animals and humans. Based on in vivo protein synthesis measurements performed in lambs, we have recently provided evidence that the 5- to 8-wk-period of age corresponds in the ovine species to an intrinsic developmentally controlled period for both pancreatic (4) and intestinal maturation (3), similar to the 3rd wk of life in rodents (10). Therefore, to establish whether developmental factors or weaning exerts a major influence on intestinal maturation in the sheep, we have measured changes in mucosal morphology and cell renewal in l- and 5-wk-old suckling lambs and in either prolonged suckling or weaned 8-wk-old animals. Although weaning is a critical phase of gastrointestinal adaptation in ruminants, the data suggest that the suckling period, and not weaning, corresponds in sheep to a major phase of progressive intestinal maturation, in terms of altered mucosal morphology and cell migration. MATERIALS
AND
METHODS
Animals and diets. Thirty-four male lambs (Ile de France x Romanov-Limousin) were used from the Sheep Production Laboratory Flock (Institut National de la Recherche Agronomique, Theix, France). Animals were separated from their dams 12-24 h postpartum and ran-
0 1991 the American
Physiological
Society
R811
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RN2
INTESTINAL
CELL
RENEWAL
IN
LAMBS
the proximal duodenum (presence of Brunner’s glands), domly divided into four groups. At the time of the experiments, suckling lambs were 1, 5, and 8 wk old, whereas the jejunum (30% of the intestinal length), and the ileum weaned animals were 8 wk old (Table 1). Suckling lambs (immediately above the terminal aggregate of Peyer’s had no access to any solid food. These animals were patches that covers -1-2.8 m above the ileocecal junction housed in individual metallic cages with mesh floor in a in l- and 8-wk-old animals, respectively). In some lambs, clean and controlled environment for temperature (21 “C) specimens were also collected from the distal duodenum and humidity (50-60%). They were given a commercial (no Brunner’s glands but above the ligament of Treitz). milk substitute (Agnodor, Union Univor, Paris) ad libiSamples were fixed according to Pout (29) with minor turn, containing [in %dry matter (DM)] 69 spray-dried modifications (21). The dehydrated specimens were emskim milk, 18 tallow fat, 9 coconut fat, trace starch, 3 bedded in paraffin, sectioned at 5 ,um, and then coated mineral and vitamin mixture (27). Protein content was with K2 autoradiographic emulsion (Ilford, Ilford, UK). 24.3% DM, and gross and metabolizable energy was 23.3 The autoradiographs were exposed for at least 6 wk, and 22.1 MJ/kg, respectively. Milk was prepared twice developed in Refinal (Agfa-Gevaert, Leverkusen, Beldaily and placed in nipple feeders (4). Weaned lambs gium), fixed in sodium hyposulfite, and then stained with were housed in a separate room in standard pens. During nuclear fast red picroindigo carmine. weaning, performed between 25 and 31 days of age, Except in the distal duodenum where only crypt-villus decreasing amounts of milk substitute were given, so that dimensions were measured, the height of the crypt-villus 32day-old lambs received only water, hay (protein con- columns, the depth of crypts, and the distance migrated tent, 9.3% DM; gross and metabolizable energy, 17.7 and by epithelial cells from the bottom of the crypt to the 7.8 MJ/kg, respectively) and a concentrate feed (A 8302, leading edge of the labeled zone on the villus were reUcabec, Lapeyrouse, France; protein content, 18.1% DM; corded manually using an ocular micrometer. Measuregross and metabolizable energy, 17.8 and 11.5 MJ/kg, ments were made in at least 10 well-oriented crypt-villus respectively) ad libitum. The concentrate composition systems for each intestinal sampling site. Values were was (in %DM) 10.0 wheat, 56.75 barley, 12.5 alfalfa, 2.25 then averaged to give an estimate per site and per lamb. peanut meal, 13.0 rapeseed meal, 2 molasses, 0.85 calcium To minimize variations observed within lambs as well as carbonate, 0.3 sodium chloride, 1.0 ammonium chloride, between animals and previously reported in the fetal (39) and 1.35 mineral and vitamin mix (4). Live weight and or neonatal (5,23) sheep small intestine, each percentage food intake were recorded daily in the 1st wk of life and of villus height migrated was expressed using the indiafter catheterization (see below) or weekly between these vidual corresponding villus height, before an average two periods (Table 1). estimate was obtained for a given lamb. The relative migration rate of enterocytes was calcuExperimental procedure. Catheters were inserted into external jugular veins 1 day (1-wk-old lambs) or 2 days lated as the slope of the linear regression of the percentage of villus height migrated by labeled cells against time (5- and 8-wk-old animals) before isotope administration. after thymidine injection. An estimate of the absolute Lambs were injected intravenously with 0.6-0.7 mCi/kg migration rate (in pm/h) was calculated by multiplying body weight [ methyl-3H] thymidine in sterile aqueous the relative rate by the related mean villus height. The solution (1 Ci/mmol, 1 mCi/ml; Commissariat a 1’Energie epithelium renewal time, i.e., the time taken for labeled Atomique, Saclay, France) at 1000 h to minimize diurnal variation. In each group, pairs of lambs were given the cells to reach 100% of villus height, was predicted from label at various times before slaughter (i.e., 24, 36, 48, the regression equations relating the percentage of villus and 60 h; 24,36,48, and 96 h; 24,36,48,60, and 72 h for height migrated by labeled cells and the time period after [ methyl-“H] thymidine injection. I-, 5- and 8-wk-old suckling lambs, respectively; 24, 36, Statistical procedures. Values were expressed as means 60, and 72 h for 8-wk-old weaned lambs). Animals were t SE. When appropriate, the significance of differences killed under pentobarbital sodium anesthesia to prevent between means was assessed by the Newman-Keuls mulcell sloughing in the small intestine (5). tiple range test (35). Barlett’s test revealed heterogeneity Samples (lo-20 mm long) were quickly excised from of variances for some data, and logarithmic transformations failed to stabilize variances. These peculiar data TABLE 1. Body weight of lambs and their weight gain, were subjected to the Kruskal-Wallis test and mean DM, crude protein (nitrogen x 6.25), or gross energy values were compared with the Mann-Whitney U test intake at time of experiment (35) Intake The standard errors for slopes and intercepts of regresBody Wt, Wt Gain, Age, Group wk n sion lines and the estimates of epithelium renewal times Protein, Gross energy, g/day DM, kg MJ/day g/day g/day with their 95% confidence limits were computed as described by Sokal and Rohlf (35). The significance of Suckling 1 8 4.9kO.l 295&12 225k12 55&3 5.2t0.3 differences between the slopes of best fit was assessed 5 8 13.220.5 267tlO 320t21 78k5 7.5t0.5 8 10 19.0k0.2 283t4 435k18 106k4 10. MO.4 according to Snedecor and Cochran (33). Weaned
8
8 15.6k0.5
213t8
610&26*
lOlt6*
10.8kO.5”
Values are means t SE for n lambs. Wt gain measured from birth (or 2 days of age, l-wk-old group) to slaughter. Intake measured between catheterization and slaughter. Lambs were weaned between 25 and 31 days of age: * 496 t 48 and 114 k 32 g of dry matter (DM)/ day, 90 & 9 and 11 * 3 g of protein/day, and 8.8 * 0.8 and 2.0 t 0.6 MJ/day was provided by concentrate and hay, respectively.
RESULTS
MorphologicaL observations. In suckling groups, the villi were significantly taller at the jejunal site than in the duodenum and ileum, whereas the deepest crypts were
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INTESTINAL
CELL RENEWAL
observed in either the proximal or distal duodenum (Fig. 1). However, there were no significant differences between intestinal sites for crypt depth in 1-wk-old lambs. With increasing age, no significant variation for villus height occurred in the proximal and distal duodenum of suckling animals (Table 2). By contrast, villus height decreased significantly in the jejunum (33%, Fig. 2) and the ileum (28%). Crypt depth increased progressively and significantly in the proximal and distal duodenum, by 70 and 142%, respectively. In the more distal regions, crypt depth was either reduced (jejunum) or not modified (ileum) between 1 and 5 wk of age. However, prolonged suckling 8wk-old lambs exhibited a marked increase in crypt depth in these regions (61%), compared with 5-wkold animals. Variations in villus height or crypt depth, or both, resulted in a significant reduction (39-65%) of the ratio, villus height-crypt depth, in all intestinal sites. The comparison of suckling and weaned Swk-old 0
Proximal
n
Jejunum
Duodenum
q
DistalDuodenum
q
Ileum
.3
E .-ul a .c ul a = .> -0 r 7i 4s
600
b
400 200
-200 4.
"A
u
Group
. . . . 1s
a
aa
8s
5s
1 a 8W
FIG. 1. Villus height (above 0) and crypt depth (below 0) in small intestine of l-, 5, and 8-wk-old suckling (IS, 5S, and 8S, respectively) and 8-wk-old weaned (8W) lambs. Values are means f SE (vertical bars) for 8-10 animals, except in distal duodenum when n = 3-5. Same superscript letter for villus height or crypt depth within group indicates no significant difference (P > 0.05) between intestinal sites (a-c, Newman-Keuls multiple-range test; A-C, Mann-Whitney U test).
R813
IN LAMBS
groups indicated that weaning by itself resulted in no significant variation (P > 0.05) for crypt depth throughout the small intestine and for villus height in the proximal and distal duodenum (Table 2). However, weaned animals had smaller villi in the jejunum and ileum than their prolonged suckling 8-wk-old counterparts. As a result of these changes, villus height profiles along the intestine that were very similar in suckling groups, showed a different pattern in weaned lambs, so that the normal proximodistal gradient was established from the distal duodenum toward the cecum (Fig. 1). Epithelial cell migration and renewal. The relationship between the percentage of villus height migrated by labeled cells and the time period after [methyl-3H]thymidine injection was significant for all intestinal sites and groups of lambs (Table 3). In suckling animals, relative migration rates (i.e., b, the slope of best fit in the model described in Table 3) decreased significantly with age in the duodenum but did not change in the jejunum and ileum (P > 0.05). Weaning resulted in an increased migration rate at all intestinal sites that was significant only in the jejunum. The relative migration rates at different intestinal sites were not significantly different (P > 0.05), except in the youngest group of lambs. Calculated absolute migration rates (in pm/h) are given in Table 4. Although not analyzed statistically, these data show that there is a clear reduction in migration rate between 1 and 5 wk of age, by 71, 57, and 41% in the duodenum, jejunum, and ileum, respectively. Table 4 also shows that weaning did not affect absolute migration rate in the ileum and only increased cell migration in the duodenum and the jejunum by 53 and 15%, respectively. The epithelium renewal times ranged between 2.1 and 6.3 days in suckling groups (Table 4). Differences between intestinal sites were especially noticeable in the youngest animals and were completely abolished in weaned lambs. Epithelium renewal times were the longest at 5 wk of age (4.5-6.3 days). In weaned animals, labeled enterocytes reached the villi tips within 3.0-3.1
TABLE 2. Small intestinal mucosa morphology in suckling and weaned lambs Site Proximal duodenum
Distal duodenum
Jejunum
Ileum
Group Suckling Weaned Suckling Weaned Suckling Weaned Suckling
Age, wk
n
crypt Depth, pm
Villus Height, pm
1
8 8
161+8A 212f6' 274fLtiC
268+15" 221rt14"
5 8 8
1 5 8 8
1 5 8 8
1
10 8 4 4 5 3 8 8
10 8 8 8
290f17C 13OH2" 228+13' 314+23b 315+56b 142+7* 99*3B
159f8* ’ 202+23' 137f7" 122+17'
268219” 294&19” 394k40" 336tll" 348543" 386+43" 722~k42~ 511+45b 487k33b
311+21’ 418t38*
Crypt-Villus Height, wrn 429f208 433215" 542f30h 584+28b 524+4gA 564+4*
662f5gA 701+92A 865k48" 610+43b 646+37" 513+38b 555k43" 352f23b 497529" 399+32b
Villus Height: Crypt Depth 1.7kO.09 l.lf0.09b l.O~k0.06~ l.lt0.07b 3.EzO.23" 1.5+0.13b
1.1t0.12b 1.3+0.13b 5.11?I0.17* 5.4f0.61A 3.1t0.17B 1.7ko.17c
3.1*0.19* 2.1+o.22B 10 197+11b 300f19C 1.6&0.06C Weaned 8 175+14b 224k20' 1.3+0.07n Values are means + SE for n lambs. Values within column in each site sharing common superscript letter were not significantly different (P > 0.05): a-c, Newman-Keuls multiple-range test; A-D, Mann-Whitney U test. 5 8 8
230z!~8~
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R814
INTESTINAL
CELL RENEWAL
IN LAMBS
FIG. 2. Autoradiograms of jejunum sections in l-, 5-, and 8wk-old suckling (A, B, and C, respectively) and 8-wk-old weaned (D) lambs. Animals were killed 48 (A and B) or 60 (C and D) h after [3H]thymidine injection. A-C illustrate villus height reduction in suckling lambs. C and D illustrate lack of major differences between suckling and weaned 8wk-old animals in both intestinal morphology and cell migration. Calibration bars indicate 50 pm. Arrows denote position of labeled enterocytes up villi.
days compared with 4.0-5.2 days in prolonged lambs of the same age.
suckling
DISCUSSION
Effect of age on mucosal morphology. In suckling rodents, although Yeh (46) observed a significant increase in both crypt depth and villus height in the duodenum of 6- to 16-day-old rats, it is generally admitted that no evident variations in villus height-to-crypt depth ratios can be detected before weaning, because both dimensions remain rather unchanged in the jejunum and ileum (11,
13). However, a recent study by Trahair (37) suggests that a dramatic shortening of villi occurred at -15 days after birth in the distal regions of the rat small intestine. By contrast, the rapid reduction in villus height and the deepening of crypts reported here during the suckling period are the continuation of a trend occurring by 6 days after birth in mother-fed lambs (42). A similar pattern has been observed in pigs (9, 12, 22) and in the upper intestine of the growing guinea pig, a rodent species with early intestinal maturation compared with the rat and mouse (45). However, these alterations in crypt depth and villus height did not occur before the 2nd to
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INTESTINAL
CELL
3. Regression equations relating percentage of villus height migrated by labeled cells (%; y) to time after thymidine injection (h; x) in intestine of suckling and weaned lambs: y = a + bx TABLE
Linear Site
Proximal duodenum
Group
Suckling
Age, wk
1
5 8
Jejunum
Weaned Suckling
8
Weaned Suckling
8
Ileum
Weaned
8
1 5 8 1 5 8
Values are means t SE for [“Hlthymidine injection, 100% 6. Values within column in each were not significantly different
n
a, % Villus height
6* -59.5t17.9 8 -22.8klO.l 10 -19.9t8.3 8 -32.8tll.l 8 -20.7215.0 8 -23.5k4.7 10 -22.9k7.3 8 -24.2kll.5 8 -0.8t16.7 8 -31.9k6.1 10 -3l.Ot6.6 8 -28.1t8.9
Coefficient b, % Villus height/h
3.15-1-0.48" l.12f0.17b 1.25f0.14"" 1.78kO.21" 1.35k0.34"l' 0.82&0.08b 0.96+0.12b 1.73t0.22" 1.05kO.38"" 1.14kO.10" l.31f0.11ab l.72+0.17b
’
co.005 ~0.001 cO.001 CO.001 CO.01 CO.001 ~0.001 CO.001 CO.05 0.05).
4. Epithelial cell migration rate and renewal time in suckling and weaned lambs
TABLE
Site
Group
Age, ---l-
n
Migration Rate,
WK
Proximal duodenum
Suckling
1
6*
5 8
8 10
Weaned Suckling
8
8
Jejunum
1 5 8
8 8 10
Ileum
Weaned Suckling
8 1
8 8
5 8
8 10
8
8
Weaned
8.6 2.5 3.4 5.2 9.7 4.2 4.7 5.4 4.4 2.6 3.9 3.9
Epithelium Renewal Time, h
50.7 109.3 95.9 74.6 89.6 150.9 128.2 71.7 96.0 116.0 99.9 74.6
(42.4-65.3) (86.3-151.6) (82.5-114.7) (63.5-90.8) (67.8-169.2) (128.7-185.1) (108.2-161.2) (60.2-88.3) (65.9-494.1) (100.3-138.7) (89.0-114.1) (65.1-87.4)
Migration rate is percentage of villus height migrated by labeled cells/h multiplied by villus height. Note that no attempt of error was made for this calculation. Epithelium renewal time predicted from equations given in Table 3; values in parentheses are 95% confidence limits. * Villus height was 273 & 19 pm for these 6 lambs.
3rd wk of postnatal life in piglets (9, 12, 22). The precise mechanisms responsible for villus atrophy are not well understood. In pigs (12, 24) and lambs (34) it is clear that part of this process results from intestinal pathogens. An alternative explanation is that the cessation of endocytosis of luminal macromolecules (closure) in the distal regions results in a reduction of nutrient utilization, which in turn may result in involution of the villi (44). In sheep (31) and guinea pig (45), closure occurs within a few days after birth, and villus involution is immediately apparent (42, 45). By contrast, in rodents (37) and piglets (12) reduction in villus height seemed to be delayed until closure occurs, either during the weaning period or at the end of the 2nd wk of age, respectively. Effect of weaning on mucosal morphology. In the rat and mouse, there is a marked elongation of both crypts and villi around weaning, between ‘14 and 28 days after birth (11, 13).
RENEWAL
IN
LAMBS
R815
The only significant effect of weaning on small intestinal morphology in lambs was a reduction in villus height in the jejunum and ileum sites (Table 2), also reported in piglets (9, 22). However, villus height was more reduced proximally, whereas distal crypts elongated the most, in pigs (9) but not in lambs. Therefore, although there are similarities between species with early intestinal morphogenesis, different mechanisms are probably responsible for small intestinal longitudinal specialization. A possible effect of short-chain fatty acids (SCFA) on intestinal structure in ruminants can be ruled out because prolonged suckling lambs exhibited the same pattern for crypts and for proximal villi as weaned animals (Table 2). The alterations in mucosal architecture reported in altricial rodent species, although temporarily related to the cessation of breast feeding, are essentially mediated by intrinsic factors and modulated by extrinsic or environmental factors (14, 15). Our data suggest that an intrinsic developmental clock could also explain the deepening of crypts observed in ruminating lambs. First, the alterations in crypt size occurring between 1 and 8 wk of age were remarkably similar in prolonged-suckling and weaned animals. Second, the 5- to 8-wk period was characterized by a sharp increase in crypt depth (61%) in the jejunum and ileum of suckling animals. In other experiments performed in four groups of lambs raised in identical conditions to those of the present study, we have recently reported a significant increase in pancreatic RNA content between 5 and 8 wk of age (4). At the intestinal level, both prolonged-suckling and weaned 8-wk-old lambs exhibited in vivo a pronounced negative gradient of protein synthesis from the proximal to the distal small intestine (3) that did not prevail in either l(2) or 5-wk-old suckling animals (3). Villus shorthening presumably results in a reduction of the area of nutrients absorption in weaned lambs, because there was no significant alteration for villus density in the jejunum, the intestinal region where most of the absorption occurs (Table 5). However, this was partially cancelled by a clear increase (P < 0.005) in intestinal length expressed per kilogram empty body weight compared with prolonged suckling 8-wk-old animals (Table 5), probably resulting from the trophic effect of intraluminal nutrients during the weaning period (7). Validity of epithelial cell migration and renewal estimates. The model used assumes a steady state during the
period of measurements that was not fullfilled because the mucosa was subject to rapid changes. The maximal observed reduction in villus height occurred in the ileum between 1 and 5 wk of age (45%). Assuming a constant and progressive villus involution during this period, this corresponded to a 0.06% villus height reduction per hour. This computed value represents the apparent progression of labeled cells up the villus in the absence of any actual movement. Relative migration rates were in considerable excess (17- to 19fold) of this estimate in l- and 5-wkold animals, respectively. Consequently, changes in intestinal structure had probably little effect on our estimations. Smith and Peacock (32) reported differential migration of enterocytes and vacuolated cells in neonate piglets
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R816 TABLE
INTESTINAL
CELL
RENEWAL
IN LAMBS
5. Small intestinal mass or length and jejunal villus density in suckling and weaned lambs Intestinal Group
Suckling
Age, wk 1 5
n
g
8
142klO”
Mass
Intestinal
g/k empty body wt
3O.Ok1.6”
m
11.6*0.5”
Length m/k empty body wt
2.45&O. lO*
Villus Density, no. of villi/mm 11.1+0.5*
7.8+0.3B 8 1.oo+o.04c 7.5&0.3B c Weaned 8 8 416&13” 30.8t1.2” 16.6t0.9” 1.23+0.06B 6.7&O. 1’ Values are means t SE for n lambs. Values within column sharing common superscript letter were not significantly different (P > 0.05): a-c, Newman-Keuls multiple-range test; A-C, Mann-Whitney U test. 8 10
296+17b 396t16”
23.9*0.gb 22.5+0.gb
and suggested that renewal patterns might be uneven in immature animals. By contrast, we found that the relationship between the distance along the villus that the labeled cells had migrated and the time period after [3H] thymidine injection was highly significant (P < 0.001) for all intestinal sites, except in the youngest lambs. In this l-wk-old group, the relationship (P < 0.05-0.005) still supported a constant progression of cells up the villus in agreement with observations reported in fetal (39) and neonate lambs (5) or in suckling (46) and weanling rats (11). However, the presence of vacuolated cells in the distal intestine, which in the sheep are detectable until 5 days after birth (43), may explain the greater variability of our measurements in the ileum (Table 3). Effect of age on epithelial cell migration and renewal.
Enterocyte migration rates are very low in suckling rodents until 15-19 days after birth (11, 13, 16), so that cell transit time along the villus is -7 days in 6-day-old animals (46). Lambs differ fundamentally from altricial rodent species in that the highest absolute migration rates were observed in the youngest suckling animals (Table 4). Accordingly, cell replacement was achieved within 2-4 days. Rapid rates of cell renewal previously reported in neonatal lambs (23,31) resulted from a dramatic increase in the mitotic index (31) or in the proportion of crypt cells labeled (42) at birth. These data support the concept that in the sheep [as in the rat, mouse, and pig, but not in the rabbit, hamster, or guinea pig (13)], there is a causal relationship between cell turnover and closure. The cessation of absorption of immunoglobulins occurs normally 2 days after birth in lambs (31). However, we used artificially fed animals. The extent to which this may affect closure is unfortunately unknown. The major effect of age reported in this study is a marked reduction in absolute cell migration rate, between 1 and 5 wk after birth. Moon and Joel (23) previously reported similar observations in lambs between 1 day and 3 wk of age. In addition, the migration rate was more reduced in the duodenum than in the jejunum and ileum in both studies. The gradual bacterial colonization of the small intestine could explain these regional variations. Intestinal microflora, which increases in number in the distal small intestine, is known to accelerate cell migration in the ileum of various species, and has been recently reported to stimulate crypt cell division in the sheep (28). As to the question of why rates of cell migration and renewal are so dramatically reduced with aging, we can
14.3+0.5b 17.6kO.7”
1. 16+0.04B
only speculate. Adrenocortical hormones have been hypothesized to play a major role in the regulation of cell proliferation and migration in the neonatal rodent intestine (14, 15). The sheep fetal intestine appears very sensitive to cortisol, the major glucocorticoid hormone in this species. Cortisol infusion resulted in an increase in both migration rates and proportion of labeled crypt cells (41), whereas adrenalectomy reduced only enterocyte migration (40). In addition, the acceleration in cell turnover observed at birth in the ovine species may result from the increased plasma cortisol concentration that characterizes the latest days of fetal life and peaks at term (42). After birth, circulating cortisol levels sharply decline during the 1st mo of life (26), including in artificially fed lambs (6), and may contribute to the decrease in cellular migration that we reported. Effect of weaning on epithelial cell migration and renewal. In rats, where weaned animals have longer villi,
the increased rate of cell loss results essentially from a fourfold increase in migration rates (11, 13, 16). Alterations in cell renewal observed in the sheep at weaning were of very low amplitude and in striking contrast with observations reported in rodents (Fig. 2). First, a slight or no increase in absolute migration rates (53, 15, and 0% in the duodenum, jejunum, and ileum, respectively) contributed to a shorter cell transit time in weaned lambs at 8 wk of age. Indeed, only the increase for the relative migration rate of labeled enterocytes in the jejunum was significantly higher in ruminating animals than in their prolonged-suckling counterparts (Table 3). Second, the acceleration in cell replacement observed in weaned lambs accounted for both an increase in relative migration rates and a reduction in villus height in the distal regions. It is clear, however, that these differences did not totally result from weaning in itself, because in 8wk-old suckling animals migration rates increased slightly but not significantly (Table 3), at a time when they were significant deepenings of crypts at all intestinal sites (Table 2). These observations suggest that part of the increased cellular proliferation observed at weaning in lambs resulted from intrinsic factors, as previously demonstrated in rodents (18). These findings also do not support any effect of SCFA on intestinal cell renewal in the sheep of physiological importance, at least in our experimental conditions, in contrast with recent data obtained in rats (30). However, SCFA concentrations are extremely low in the sheep small intestine, even in the distal segments (25), and SCFA clearly affected intestinal cell proliferation in a
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INTESTINAL
CELL RENEWAL
dose-dependent manner (30). More likely, weaning-related effects on cell renewal in the sheep may be explained by the interactions between intrinsic (see above) and extrinsic factors. For example, dietary factors such as a higher solid food (7) or fiber content (8, 36) in the diet of ruminating animals are more likely candidates to explain an increased cell turnover rate in the sheep, via direct or indirect effects. Conclusion. The present experiments give the first comprehensive description of the changes in both mucosal morphology and cell renewal occurring between birth and weaning in species with early intestinal morphogenesis similar to most domestic animals and humans. Although obtai .ned i.n a rumi .nant species, our findings suggest that the suckling period, and not weaning, is a major phase of gradual postnatal intestinal adaptation. These data may be representative of human development during the suckling period. However, suckling lambs and piglets, two species with early intestinal differentiation, exhibit major differences in intestinal architecture and cell proliferation patterns. We suggest that the timing of closure may explain most of the differences observed. Because closure is not well defined in humans (l3), more information must be gained before extrapolation of animal data to humans is possible. The authors thank M. Sallas and M. Selle for animal care, M. Serezat for expert technical assistance, and G. Bayle and E. Aurousseau for computer data processing. This study was supported by a grant from the French Ministry of Research and Technology. Address for reprint requests: D. Attaix, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-FerrandTheix, Laboratoire d’Etude du Metabolisme Azote, 63122 Ceyrat, France.
J. J., AND P. SUNSHINE. Postnatal development of the small intestine of the rat. Changes in mucosal morphology at weaning. Pediatr. Res. 3: 27-33, 1969. KENWORTHY, R. Observations on the effects of weaning in the young pig. Clinical and histopathological studies of intestinal function and morphology. Res. Vet. Sci. 21: 69-75, 1976. KLEIN, R. M. Small intestinal proliferation during development. In: Human Gastrointestinal Development, edited by E. Lebenthal. New York: Raven, 1989, p. 367-392. KLEIN, R. M. Cell proliferative regulation in developing small intestine. In: Human Gastrointestinal Development, edited by E. Lebenthal. New York: Raven, 1989, p. 393-436. KLEIN, R. M., AND J. C. MCKENZIE. The role of cell renewal in the ontogeny of the intestine. II. Regulation of cell proliferation in adult, fetal, and neonatal intestine. J. Pediatr. GustroenteroZ. Nutr.
11. HERBST,
12.
13.
14.
15.
2: 204-228,1983. 16. KOLDOVSKY,
O., P. SUNSHINE, AND N. KRETCHMER. Cellular of intestinal epithelia in suckling and weaned rats.
migration
Nature Lond. 212: 1389-1390,1966. 17. LEBENTHAL, E. Concepts in gastrointestinal man Gastrointestinal Development, edited
development. In: Huby E. Lebenthal. New
York: Raven, 1989, p. 3-18. E., P. SUNSHINE, AND N. KRETCHMER. Effect of prolonged nursing on the activity of intestinal lactase in rats.
18. LEBENTHAL,
Gustroenterology 19. LIPKIN, M.
64: 1136-1141,
1973.
Proliferation and differentiation of gastrointestinal cells in normal and disease states. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson, M. I. Grossman, E. D. Jacobson, and S. G. Schultz. New York: Raven, 1987, p. 145168. 20. MATHAN, M., P. C. MOXEY, AND J. S. TRIER. Morphogenesis of fetal rat duodenal villi. Am. J. Anat. 146: 73-92, 1976. 21. MESLIN, J. C., AND F. DABURON. Duree du renouvellement de l’epithelium intestinal chez le port adulte selon deux modes d’administration de la thymidine tritiee. Ann. BioZ. Anim. Biochim. Biophys. 22. MILLER,
17: 33-41,
1977.
B. G., P. S. JAMES, M. W. SMITH, AND F. J. BOURNE. Effect of weaning on the capacity of pig intestinal villi to digest and absorb nutrients. J. Agric. Sci. Cambridge 107: 579-589, 1986. 23. MOON, H. W., AND D. D. JOEL. Epithelial cell migration in the small intestine of sheep and calves. Am. J. Vet. Res. 36: 187-189, 1975. 24. MOON, H. D. BOOTH.
Received 30 August 1990; accepted in final form 26 April 1991. REFERENCES P., AND D. MENARD. Cell proliferation in developing human jejunum. Biol. Neonate 51: 297-304,1987. 2. ATTAIX, D., AND M. ARNAL. Protein synthesis and growth in the gastrointestinal tract of the young preruminant lamb. Br. J. Nutr.
W., L. J. KEMENY, G. LAMBERT, S. L. STARK, AND G. Age dependent resistance to transmissible gastroenteritis of swine. III. Effects of epithelial cell kinetics on coronavirus production and on atrophy of intestinal villi. Vet. PathoZ. 12: 434-
1. ARSENAULT,
58: 159-169, 1987. 3. ATTAIX, D., E. AUROUSSEAU, G. BAYLE, A. MANGHEBATI, ARNAL. Protein synthesis and degradation in growing European Association for Animal Production Symposium,
R817
IN LAMBS
M. lambs. In: AND
Publ.
35.
Restock, GDR: Eur. Assoc. Anim. Prod., 1987, p. 24-25. 4. ATTAIX, D., E. AUROUSSEAU, G. BAYLE, D. ROSOLOWSKA-HUSZCZ, A MANGHEBATI, AND M. ARNAL. Influences of age and weaning on in vivo pancreatic protein synthesis in the lamb. J. Nutr. 119: 463-470,1989. 5. ATTAIX, D.,
J. C. MESLIN, AND E. COMBE. Intestinal epithelium renewal time in the young lamb. Nutr. Rep. Int. 29: 689-697, 1984. 6. CABELLO, G., AND D. LEVIEUX. Hormonal status in the newborn lamb (cortisol, T3, T4). Relationships to the birth weight and the length of gestation: effect of the litter size. Biol. Neonate 39: 208216, 1981. 7. CASTILLO, R. O., J. J. FENG, D. K. STEVENSON, J. A. KERNER, AND L. K. KWONG. Regulation of intestinal ontogeny by intraluminal nutrients. J. Pediatr. Gustroenterol. Nutr. 10: 199-205, 1990. 8. ECKNAUER, R., B. SIRCAR, AND L. R. JOHNSON. Effect of dietary
bulk on small intestinal morphology and cell renewal in the rat. Gastroenterology 81: 781-786, 1981. 9. HAMPSON, D. J. Alterations in piglet small intestinal structure at weaning. Res. Vet. Sci. 40: 32-40, 1986. 10. HENNING, S. J. Postnatal development: coordination of feeding, digestion, and metabolism. Am. J. Physiol. 241 (Gastrointest. Liver Physiol. 4): G199-G214, 1981.
445,1975. 25.
OH, J. H., I. D. HUME, AND D. T. TORRELL. Development of microbial activity in the alimentary tract of lambs. J. Anim. Sci.
35: 450-459,1972. 26. PAISEY, R. B., AND
P. W. NATHANIELSZ. Plasma cortisol levels in the newborn lamb from birth to 30 days. J. Endocrinol 50: 7Ol-
702,197l. 27. PAPET,
I., D. BREUILLE, F. GLOMOT, AND M. ARNAL. Nutritional and metabolic effects of dietary leucine excess in preruminant lamb. J. Nutr. 118: 450-455, 1988. 28. PARKER, D. S. Manipulation of the functional activity of the gut by dietary and other means (antibiotics/probiotics) in ruminants. J. Nutr.
120: 639-648,
1990.
29. POUT, D. D. The mucosal surface patterns of the small intestine of grazing lambs. Br. Vet. J. 126: 357-363, 1970. 30. SAKATA, T. Stimulatory effect of short-chain fatty acids on epithelial cell proliferation in the rat intestine: a possible explanation for trophic effects of fermentable fibre, gut microbes and luminal trophic factors. Br. J. Nutr. 58: 95-103, 1987. 31. SMEATON, T. C., AND M. W. SIMPSON-MORGAN. Epithelial cell renewal and antibody transfer in the small intestine of the foetal and neonatal lamb. Aust. J. Exp. Biol. Med. Sci. 63: 41-51, 1985. 32. SMITH, M. W., AND M. A. PEACOCK. Anomalous replacement of foetal enterocytes in the neonatal pig. Proc. R. Sot. Lond. B Biol. Sci. 206: 411-420,198O. 33. SNEDECOR, G. W., AND
W. G. COCHRAN. Methodes Statistiques (6th ed.). Paris: Association de Coordination Agricole, 1971. 34. SNODGRASS, D. R., A. FERGUSON, F. ALLAN, K. W. ANGUS, AND B. MITCHELL. Small intestinal morphology and epithelial cell
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R818 kinetics 1979.
INTESTINAL in lamb
rotavirus
infections.
Gastroenterology
CELL
RENEWAL
76: 477-481,
35. SOKAL, R. R., AND F. J. ROHLF. Biometry. San Francisco: Freeman, 1969. 36. SOUTHON, S., G. LIVESEY, J. M. GEE, AND I. T. JOHNSON. Differences in intestinal protein synthesis and cellular proliferation in well-nourished rats consuming conventional laboratory diets. Br. J. Nutr. 53: 87-95, 1985. 37. TRAHAIR, J. F. Remodeling of the rat small intestinal mucosa during the suckling period. J. Pediatr. Gastroenterol. Nutr. 9: 232237, 1989. 38. TRAHAIR, J. F., R. A. PERRY, M. SILVER, AND P. M. ROBINSON. The autoradiographic localization of [3H] thymidine incorporation in the fetal sheep small intestine. J. Pediatr. Gastroenterol. Nutr. 5: 648-654, 1986. 39. TRAHAIR, J. F., R. A. PERRY, M. SILVER, AND P. M. ROBINSON. Enterocyte migration in the fetal sheep small intestine. Biol. Neonate 50: 214-220, 1986. 40. TRAHAIR, J. F., R. A. PERRY, M. SILVER, AND P. M. ROBINSON. Studies on the maturation of the small intestine of the fetal sheep.
41.
42.
43.
44.
45.
46.
IN
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I. The effects of bilateral adrenalectomy. Q. J. Exp. Physiol. 72: 61-69,1987. TRAHAIR, J. F., R. A. PERRY, M. SILVER, AND P. M. ROBINSON. Studies on the maturation of the small intestine of the fetal sheep. II. The effects of exogenous cortisol. Q. J. Exp. Physiol. 72: 71-79, 1987. TRAHAIR, J. F., AND P. M. ROBINSON. Perinatal development of the small intestine of the sheep. Reprod. Nutr. Deu. 26: 1255-1263, 1986. TRAHAIR, J. F., AND P. M. ROBINSON. Enterocyte ultrastructure and uptake of immunoglobulins in the small intestine of the neonatal lamb. J. Anat. 166: 103-111, 1989. TSUBOI, K. K., AND R. 0. CASTILLO. Development of jejunoileal differences in rat intestine. J. Pediatr. Gastroenterol. Nutr. 9: l40143,1989. WEAVER, L. T., AND B. M. CARRICK. Changes in upper intestinal epithelial morphology and kinetics in the growing guinea pig. Pediatr. Res. 26: 31-33, 1989. YEH, K. Y. Cell kinetics in the small intestine of suckling rats. I. Influence of hypophysectomy. Anat. Rec. 188: 69-76, 1977.
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ATTAIX
AND
JEAN-CLAUDE
MESLIN
Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Ceyrat; and Laboratoire de Nutrition et Skuritk Alimentaire, 78352 Jouy-en-Josas Cedex, France
ATTAIX, DIDIER, AND JEAN-CLAUDE MESLIN. Changes in small intestinal mucosa morphology and cell renewal in suckling, prolonged-suckling, and weaned lambs. Am. J. Physiol. 261
(Regulatory Integrative Comp. Physiol. 30): R811-R818, 1991.-No information concerning the effect of weaning on intestinal cell proliferation is currently available in largespecies with early intestinal morphogenesis,a group including most domesticanimalsand humans. Changesin intestinal morphology and epithelial cell renewal were investigated in l-, 5, and 8wk-old suckling and 8-wk-old weaned lambs after injection of [“Hlthymidine. In suckling lambsa gradual increasein crypt depth occurred with age, especially in the proximal intestine, whereasvillus height was significantly reduced in the distal regions. At 8 wk of age weanedand prolonged-suckling lambs exhibited no significant differences in crypt depth throughout the intestine and in villus height proximally. However, weaned lambs had shorter villi in the jejunum and ileum. The highest enterocyte migration rates (4.4-9.7 pm/h) were observed in lwk-old lambs. In suckling animals, migration rates decreased with age by 60, 51, and 11% in the duodenum, jejunum, and ileum, respectively. Weaned and prolonged-suckling 8-wk-old lambshad a similar rate of enterocyte migration in the ileum. Furthermore, ruminating animalsexhibited only slightly higher migration ratesin the duodenumand the jejunum (53 and 15%, respectively). In suckling lambs,epithelial cell renewalrequired 2.1-4.0, 4.5-6.3, and 4.0-5.3 days at 1, 5, and 8 wk of age, respectively, whereaslabeled cells reachedthe tips of the villi within 3.0-3.1 days in weanedanimals. These data suggestthat the suckling period correspondsto a gradual and important phaseof postnatal intestinal adaptation in the sheep,a species with early patterns of intestinal cell replacement. By contrast, and although weaning is the major event in gastrointestinal adaptation in ruminants, the transition from milk to solid feeding, or temporally related events, is not associatedwith abrupt changesin intestinal epithelial morphology and cell kinetics as reported in altricial species.
intestinal crypt-villus; cell proliferation; development; weaning
THE EPITHELIUM OF THE vertebrate small intestine, cells are produced in the crypts and continuously shed from the villi tips (19). Between birth and weaning, two critical periods in gastrointestinal adaptation, this dynamic and essential process for intestinal growth is under the control of intrinsic factors (genetic endowment, developmental clock; see Ref. 17 for definitions) and is influenced by extrinsic or environmental factors (hormonal, neural, dietary, luminal, and growth factors; Refs. 14, 15). Changes that occur perinatally have been extenIN
0363-6119/91
$1.50 Copyright
sively studied in suckling and weaned rodents (13). However, in species such as the rat and the mouse cryptvillus differentiation occurs during the latest stages of fetal growth (ZO), and weaning represents a major event in gastrointestinal maturation (13). By contrast, small intestinal development in the sheep and the pig, two species with a long gestational period, occurs earlier in utero and may be more representative of human development in the fetal and neonatal periods (13). Unlike the pig, the sheep exhibits patterns of intestinal cell migration and replacement at an early stage of fetal development (38, 39), as do humans (1). Immediately after birth, we (5) and others (23, 31, 42) have described rapid epithelium renewal times (2-4 days) in contrast to observations made in rodents (13). Only Moon and Joel (23) have briefly reported the postnatal evolution of small intestinal morphology and cell migration in lambs. Unfortunately these data are very difficult to interpret because no indications relative to animal breeding and diets were given. Furthermore, to our knowledge, no information concerning the effect of weaning on intestinal cell proliferation is available in large species with early intestinal morphogenesis, a group including most domestic animals and humans. Based on in vivo protein synthesis measurements performed in lambs, we have recently provided evidence that the 5- to 8-wk-period of age corresponds in the ovine species to an intrinsic developmentally controlled period for both pancreatic (4) and intestinal maturation (3), similar to the 3rd wk of life in rodents (10). Therefore, to establish whether developmental factors or weaning exerts a major influence on intestinal maturation in the sheep, we have measured changes in mucosal morphology and cell renewal in l- and 5-wk-old suckling lambs and in either prolonged suckling or weaned 8-wk-old animals. Although weaning is a critical phase of gastrointestinal adaptation in ruminants, the data suggest that the suckling period, and not weaning, corresponds in sheep to a major phase of progressive intestinal maturation, in terms of altered mucosal morphology and cell migration. MATERIALS
AND
METHODS
Animals and diets. Thirty-four male lambs (Ile de France x Romanov-Limousin) were used from the Sheep Production Laboratory Flock (Institut National de la Recherche Agronomique, Theix, France). Animals were separated from their dams 12-24 h postpartum and ran-
0 1991 the American
Physiological
Society
R811
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (139.184.014.150) on September 27, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
RN2
INTESTINAL
CELL
RENEWAL
IN
LAMBS
the proximal duodenum (presence of Brunner’s glands), domly divided into four groups. At the time of the experiments, suckling lambs were 1, 5, and 8 wk old, whereas the jejunum (30% of the intestinal length), and the ileum weaned animals were 8 wk old (Table 1). Suckling lambs (immediately above the terminal aggregate of Peyer’s had no access to any solid food. These animals were patches that covers -1-2.8 m above the ileocecal junction housed in individual metallic cages with mesh floor in a in l- and 8-wk-old animals, respectively). In some lambs, clean and controlled environment for temperature (21 “C) specimens were also collected from the distal duodenum and humidity (50-60%). They were given a commercial (no Brunner’s glands but above the ligament of Treitz). milk substitute (Agnodor, Union Univor, Paris) ad libiSamples were fixed according to Pout (29) with minor turn, containing [in %dry matter (DM)] 69 spray-dried modifications (21). The dehydrated specimens were emskim milk, 18 tallow fat, 9 coconut fat, trace starch, 3 bedded in paraffin, sectioned at 5 ,um, and then coated mineral and vitamin mixture (27). Protein content was with K2 autoradiographic emulsion (Ilford, Ilford, UK). 24.3% DM, and gross and metabolizable energy was 23.3 The autoradiographs were exposed for at least 6 wk, and 22.1 MJ/kg, respectively. Milk was prepared twice developed in Refinal (Agfa-Gevaert, Leverkusen, Beldaily and placed in nipple feeders (4). Weaned lambs gium), fixed in sodium hyposulfite, and then stained with were housed in a separate room in standard pens. During nuclear fast red picroindigo carmine. weaning, performed between 25 and 31 days of age, Except in the distal duodenum where only crypt-villus decreasing amounts of milk substitute were given, so that dimensions were measured, the height of the crypt-villus 32day-old lambs received only water, hay (protein con- columns, the depth of crypts, and the distance migrated tent, 9.3% DM; gross and metabolizable energy, 17.7 and by epithelial cells from the bottom of the crypt to the 7.8 MJ/kg, respectively) and a concentrate feed (A 8302, leading edge of the labeled zone on the villus were reUcabec, Lapeyrouse, France; protein content, 18.1% DM; corded manually using an ocular micrometer. Measuregross and metabolizable energy, 17.8 and 11.5 MJ/kg, ments were made in at least 10 well-oriented crypt-villus respectively) ad libitum. The concentrate composition systems for each intestinal sampling site. Values were was (in %DM) 10.0 wheat, 56.75 barley, 12.5 alfalfa, 2.25 then averaged to give an estimate per site and per lamb. peanut meal, 13.0 rapeseed meal, 2 molasses, 0.85 calcium To minimize variations observed within lambs as well as carbonate, 0.3 sodium chloride, 1.0 ammonium chloride, between animals and previously reported in the fetal (39) and 1.35 mineral and vitamin mix (4). Live weight and or neonatal (5,23) sheep small intestine, each percentage food intake were recorded daily in the 1st wk of life and of villus height migrated was expressed using the indiafter catheterization (see below) or weekly between these vidual corresponding villus height, before an average two periods (Table 1). estimate was obtained for a given lamb. The relative migration rate of enterocytes was calcuExperimental procedure. Catheters were inserted into external jugular veins 1 day (1-wk-old lambs) or 2 days lated as the slope of the linear regression of the percentage of villus height migrated by labeled cells against time (5- and 8-wk-old animals) before isotope administration. after thymidine injection. An estimate of the absolute Lambs were injected intravenously with 0.6-0.7 mCi/kg migration rate (in pm/h) was calculated by multiplying body weight [ methyl-3H] thymidine in sterile aqueous the relative rate by the related mean villus height. The solution (1 Ci/mmol, 1 mCi/ml; Commissariat a 1’Energie epithelium renewal time, i.e., the time taken for labeled Atomique, Saclay, France) at 1000 h to minimize diurnal variation. In each group, pairs of lambs were given the cells to reach 100% of villus height, was predicted from label at various times before slaughter (i.e., 24, 36, 48, the regression equations relating the percentage of villus and 60 h; 24,36,48, and 96 h; 24,36,48,60, and 72 h for height migrated by labeled cells and the time period after [ methyl-“H] thymidine injection. I-, 5- and 8-wk-old suckling lambs, respectively; 24, 36, Statistical procedures. Values were expressed as means 60, and 72 h for 8-wk-old weaned lambs). Animals were t SE. When appropriate, the significance of differences killed under pentobarbital sodium anesthesia to prevent between means was assessed by the Newman-Keuls mulcell sloughing in the small intestine (5). tiple range test (35). Barlett’s test revealed heterogeneity Samples (lo-20 mm long) were quickly excised from of variances for some data, and logarithmic transformations failed to stabilize variances. These peculiar data TABLE 1. Body weight of lambs and their weight gain, were subjected to the Kruskal-Wallis test and mean DM, crude protein (nitrogen x 6.25), or gross energy values were compared with the Mann-Whitney U test intake at time of experiment (35) Intake The standard errors for slopes and intercepts of regresBody Wt, Wt Gain, Age, Group wk n sion lines and the estimates of epithelium renewal times Protein, Gross energy, g/day DM, kg MJ/day g/day g/day with their 95% confidence limits were computed as described by Sokal and Rohlf (35). The significance of Suckling 1 8 4.9kO.l 295&12 225k12 55&3 5.2t0.3 differences between the slopes of best fit was assessed 5 8 13.220.5 267tlO 320t21 78k5 7.5t0.5 8 10 19.0k0.2 283t4 435k18 106k4 10. MO.4 according to Snedecor and Cochran (33). Weaned
8
8 15.6k0.5
213t8
610&26*
lOlt6*
10.8kO.5”
Values are means t SE for n lambs. Wt gain measured from birth (or 2 days of age, l-wk-old group) to slaughter. Intake measured between catheterization and slaughter. Lambs were weaned between 25 and 31 days of age: * 496 t 48 and 114 k 32 g of dry matter (DM)/ day, 90 & 9 and 11 * 3 g of protein/day, and 8.8 * 0.8 and 2.0 t 0.6 MJ/day was provided by concentrate and hay, respectively.
RESULTS
MorphologicaL observations. In suckling groups, the villi were significantly taller at the jejunal site than in the duodenum and ileum, whereas the deepest crypts were
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INTESTINAL
CELL RENEWAL
observed in either the proximal or distal duodenum (Fig. 1). However, there were no significant differences between intestinal sites for crypt depth in 1-wk-old lambs. With increasing age, no significant variation for villus height occurred in the proximal and distal duodenum of suckling animals (Table 2). By contrast, villus height decreased significantly in the jejunum (33%, Fig. 2) and the ileum (28%). Crypt depth increased progressively and significantly in the proximal and distal duodenum, by 70 and 142%, respectively. In the more distal regions, crypt depth was either reduced (jejunum) or not modified (ileum) between 1 and 5 wk of age. However, prolonged suckling 8wk-old lambs exhibited a marked increase in crypt depth in these regions (61%), compared with 5-wkold animals. Variations in villus height or crypt depth, or both, resulted in a significant reduction (39-65%) of the ratio, villus height-crypt depth, in all intestinal sites. The comparison of suckling and weaned Swk-old 0
Proximal
n
Jejunum
Duodenum
q
DistalDuodenum
q
Ileum
.3
E .-ul a .c ul a = .> -0 r 7i 4s
600
b
400 200
-200 4.
"A
u
Group
. . . . 1s
a
aa
8s
5s
1 a 8W
FIG. 1. Villus height (above 0) and crypt depth (below 0) in small intestine of l-, 5, and 8-wk-old suckling (IS, 5S, and 8S, respectively) and 8-wk-old weaned (8W) lambs. Values are means f SE (vertical bars) for 8-10 animals, except in distal duodenum when n = 3-5. Same superscript letter for villus height or crypt depth within group indicates no significant difference (P > 0.05) between intestinal sites (a-c, Newman-Keuls multiple-range test; A-C, Mann-Whitney U test).
R813
IN LAMBS
groups indicated that weaning by itself resulted in no significant variation (P > 0.05) for crypt depth throughout the small intestine and for villus height in the proximal and distal duodenum (Table 2). However, weaned animals had smaller villi in the jejunum and ileum than their prolonged suckling 8-wk-old counterparts. As a result of these changes, villus height profiles along the intestine that were very similar in suckling groups, showed a different pattern in weaned lambs, so that the normal proximodistal gradient was established from the distal duodenum toward the cecum (Fig. 1). Epithelial cell migration and renewal. The relationship between the percentage of villus height migrated by labeled cells and the time period after [methyl-3H]thymidine injection was significant for all intestinal sites and groups of lambs (Table 3). In suckling animals, relative migration rates (i.e., b, the slope of best fit in the model described in Table 3) decreased significantly with age in the duodenum but did not change in the jejunum and ileum (P > 0.05). Weaning resulted in an increased migration rate at all intestinal sites that was significant only in the jejunum. The relative migration rates at different intestinal sites were not significantly different (P > 0.05), except in the youngest group of lambs. Calculated absolute migration rates (in pm/h) are given in Table 4. Although not analyzed statistically, these data show that there is a clear reduction in migration rate between 1 and 5 wk of age, by 71, 57, and 41% in the duodenum, jejunum, and ileum, respectively. Table 4 also shows that weaning did not affect absolute migration rate in the ileum and only increased cell migration in the duodenum and the jejunum by 53 and 15%, respectively. The epithelium renewal times ranged between 2.1 and 6.3 days in suckling groups (Table 4). Differences between intestinal sites were especially noticeable in the youngest animals and were completely abolished in weaned lambs. Epithelium renewal times were the longest at 5 wk of age (4.5-6.3 days). In weaned animals, labeled enterocytes reached the villi tips within 3.0-3.1
TABLE 2. Small intestinal mucosa morphology in suckling and weaned lambs Site Proximal duodenum
Distal duodenum
Jejunum
Ileum
Group Suckling Weaned Suckling Weaned Suckling Weaned Suckling
Age, wk
n
crypt Depth, pm
Villus Height, pm
1
8 8
161+8A 212f6' 274fLtiC
268+15" 221rt14"
5 8 8
1 5 8 8
1 5 8 8
1
10 8 4 4 5 3 8 8
10 8 8 8
290f17C 13OH2" 228+13' 314+23b 315+56b 142+7* 99*3B
159f8* ’ 202+23' 137f7" 122+17'
268219” 294&19” 394k40" 336tll" 348543" 386+43" 722~k42~ 511+45b 487k33b
311+21’ 418t38*
Crypt-Villus Height, wrn 429f208 433215" 542f30h 584+28b 524+4gA 564+4*
662f5gA 701+92A 865k48" 610+43b 646+37" 513+38b 555k43" 352f23b 497529" 399+32b
Villus Height: Crypt Depth 1.7kO.09 l.lf0.09b l.O~k0.06~ l.lt0.07b 3.EzO.23" 1.5+0.13b
1.1t0.12b 1.3+0.13b 5.11?I0.17* 5.4f0.61A 3.1t0.17B 1.7ko.17c
3.1*0.19* 2.1+o.22B 10 197+11b 300f19C 1.6&0.06C Weaned 8 175+14b 224k20' 1.3+0.07n Values are means + SE for n lambs. Values within column in each site sharing common superscript letter were not significantly different (P > 0.05): a-c, Newman-Keuls multiple-range test; A-D, Mann-Whitney U test. 5 8 8
230z!~8~
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R814
INTESTINAL
CELL RENEWAL
IN LAMBS
FIG. 2. Autoradiograms of jejunum sections in l-, 5-, and 8wk-old suckling (A, B, and C, respectively) and 8-wk-old weaned (D) lambs. Animals were killed 48 (A and B) or 60 (C and D) h after [3H]thymidine injection. A-C illustrate villus height reduction in suckling lambs. C and D illustrate lack of major differences between suckling and weaned 8wk-old animals in both intestinal morphology and cell migration. Calibration bars indicate 50 pm. Arrows denote position of labeled enterocytes up villi.
days compared with 4.0-5.2 days in prolonged lambs of the same age.
suckling
DISCUSSION
Effect of age on mucosal morphology. In suckling rodents, although Yeh (46) observed a significant increase in both crypt depth and villus height in the duodenum of 6- to 16-day-old rats, it is generally admitted that no evident variations in villus height-to-crypt depth ratios can be detected before weaning, because both dimensions remain rather unchanged in the jejunum and ileum (11,
13). However, a recent study by Trahair (37) suggests that a dramatic shortening of villi occurred at -15 days after birth in the distal regions of the rat small intestine. By contrast, the rapid reduction in villus height and the deepening of crypts reported here during the suckling period are the continuation of a trend occurring by 6 days after birth in mother-fed lambs (42). A similar pattern has been observed in pigs (9, 12, 22) and in the upper intestine of the growing guinea pig, a rodent species with early intestinal maturation compared with the rat and mouse (45). However, these alterations in crypt depth and villus height did not occur before the 2nd to
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INTESTINAL
CELL
3. Regression equations relating percentage of villus height migrated by labeled cells (%; y) to time after thymidine injection (h; x) in intestine of suckling and weaned lambs: y = a + bx TABLE
Linear Site
Proximal duodenum
Group
Suckling
Age, wk
1
5 8
Jejunum
Weaned Suckling
8
Weaned Suckling
8
Ileum
Weaned
8
1 5 8 1 5 8
Values are means t SE for [“Hlthymidine injection, 100% 6. Values within column in each were not significantly different
n
a, % Villus height
6* -59.5t17.9 8 -22.8klO.l 10 -19.9t8.3 8 -32.8tll.l 8 -20.7215.0 8 -23.5k4.7 10 -22.9k7.3 8 -24.2kll.5 8 -0.8t16.7 8 -31.9k6.1 10 -3l.Ot6.6 8 -28.1t8.9
Coefficient b, % Villus height/h
3.15-1-0.48" l.12f0.17b 1.25f0.14"" 1.78kO.21" 1.35k0.34"l' 0.82&0.08b 0.96+0.12b 1.73t0.22" 1.05kO.38"" 1.14kO.10" l.31f0.11ab l.72+0.17b
’
co.005 ~0.001 cO.001 CO.001 CO.01 CO.001 ~0.001 CO.001 CO.05 0.05).
4. Epithelial cell migration rate and renewal time in suckling and weaned lambs
TABLE
Site
Group
Age, ---l-
n
Migration Rate,
WK
Proximal duodenum
Suckling
1
6*
5 8
8 10
Weaned Suckling
8
8
Jejunum
1 5 8
8 8 10
Ileum
Weaned Suckling
8 1
8 8
5 8
8 10
8
8
Weaned
8.6 2.5 3.4 5.2 9.7 4.2 4.7 5.4 4.4 2.6 3.9 3.9
Epithelium Renewal Time, h
50.7 109.3 95.9 74.6 89.6 150.9 128.2 71.7 96.0 116.0 99.9 74.6
(42.4-65.3) (86.3-151.6) (82.5-114.7) (63.5-90.8) (67.8-169.2) (128.7-185.1) (108.2-161.2) (60.2-88.3) (65.9-494.1) (100.3-138.7) (89.0-114.1) (65.1-87.4)
Migration rate is percentage of villus height migrated by labeled cells/h multiplied by villus height. Note that no attempt of error was made for this calculation. Epithelium renewal time predicted from equations given in Table 3; values in parentheses are 95% confidence limits. * Villus height was 273 & 19 pm for these 6 lambs.
3rd wk of postnatal life in piglets (9, 12, 22). The precise mechanisms responsible for villus atrophy are not well understood. In pigs (12, 24) and lambs (34) it is clear that part of this process results from intestinal pathogens. An alternative explanation is that the cessation of endocytosis of luminal macromolecules (closure) in the distal regions results in a reduction of nutrient utilization, which in turn may result in involution of the villi (44). In sheep (31) and guinea pig (45), closure occurs within a few days after birth, and villus involution is immediately apparent (42, 45). By contrast, in rodents (37) and piglets (12) reduction in villus height seemed to be delayed until closure occurs, either during the weaning period or at the end of the 2nd wk of age, respectively. Effect of weaning on mucosal morphology. In the rat and mouse, there is a marked elongation of both crypts and villi around weaning, between ‘14 and 28 days after birth (11, 13).
RENEWAL
IN
LAMBS
R815
The only significant effect of weaning on small intestinal morphology in lambs was a reduction in villus height in the jejunum and ileum sites (Table 2), also reported in piglets (9, 22). However, villus height was more reduced proximally, whereas distal crypts elongated the most, in pigs (9) but not in lambs. Therefore, although there are similarities between species with early intestinal morphogenesis, different mechanisms are probably responsible for small intestinal longitudinal specialization. A possible effect of short-chain fatty acids (SCFA) on intestinal structure in ruminants can be ruled out because prolonged suckling lambs exhibited the same pattern for crypts and for proximal villi as weaned animals (Table 2). The alterations in mucosal architecture reported in altricial rodent species, although temporarily related to the cessation of breast feeding, are essentially mediated by intrinsic factors and modulated by extrinsic or environmental factors (14, 15). Our data suggest that an intrinsic developmental clock could also explain the deepening of crypts observed in ruminating lambs. First, the alterations in crypt size occurring between 1 and 8 wk of age were remarkably similar in prolonged-suckling and weaned animals. Second, the 5- to 8-wk period was characterized by a sharp increase in crypt depth (61%) in the jejunum and ileum of suckling animals. In other experiments performed in four groups of lambs raised in identical conditions to those of the present study, we have recently reported a significant increase in pancreatic RNA content between 5 and 8 wk of age (4). At the intestinal level, both prolonged-suckling and weaned 8-wk-old lambs exhibited in vivo a pronounced negative gradient of protein synthesis from the proximal to the distal small intestine (3) that did not prevail in either l(2) or 5-wk-old suckling animals (3). Villus shorthening presumably results in a reduction of the area of nutrients absorption in weaned lambs, because there was no significant alteration for villus density in the jejunum, the intestinal region where most of the absorption occurs (Table 5). However, this was partially cancelled by a clear increase (P < 0.005) in intestinal length expressed per kilogram empty body weight compared with prolonged suckling 8-wk-old animals (Table 5), probably resulting from the trophic effect of intraluminal nutrients during the weaning period (7). Validity of epithelial cell migration and renewal estimates. The model used assumes a steady state during the
period of measurements that was not fullfilled because the mucosa was subject to rapid changes. The maximal observed reduction in villus height occurred in the ileum between 1 and 5 wk of age (45%). Assuming a constant and progressive villus involution during this period, this corresponded to a 0.06% villus height reduction per hour. This computed value represents the apparent progression of labeled cells up the villus in the absence of any actual movement. Relative migration rates were in considerable excess (17- to 19fold) of this estimate in l- and 5-wkold animals, respectively. Consequently, changes in intestinal structure had probably little effect on our estimations. Smith and Peacock (32) reported differential migration of enterocytes and vacuolated cells in neonate piglets
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R816 TABLE
INTESTINAL
CELL
RENEWAL
IN LAMBS
5. Small intestinal mass or length and jejunal villus density in suckling and weaned lambs Intestinal Group
Suckling
Age, wk 1 5
n
g
8
142klO”
Mass
Intestinal
g/k empty body wt
3O.Ok1.6”
m
11.6*0.5”
Length m/k empty body wt
2.45&O. lO*
Villus Density, no. of villi/mm 11.1+0.5*
7.8+0.3B 8 1.oo+o.04c 7.5&0.3B c Weaned 8 8 416&13” 30.8t1.2” 16.6t0.9” 1.23+0.06B 6.7&O. 1’ Values are means t SE for n lambs. Values within column sharing common superscript letter were not significantly different (P > 0.05): a-c, Newman-Keuls multiple-range test; A-C, Mann-Whitney U test. 8 10
296+17b 396t16”
23.9*0.gb 22.5+0.gb
and suggested that renewal patterns might be uneven in immature animals. By contrast, we found that the relationship between the distance along the villus that the labeled cells had migrated and the time period after [3H] thymidine injection was highly significant (P < 0.001) for all intestinal sites, except in the youngest lambs. In this l-wk-old group, the relationship (P < 0.05-0.005) still supported a constant progression of cells up the villus in agreement with observations reported in fetal (39) and neonate lambs (5) or in suckling (46) and weanling rats (11). However, the presence of vacuolated cells in the distal intestine, which in the sheep are detectable until 5 days after birth (43), may explain the greater variability of our measurements in the ileum (Table 3). Effect of age on epithelial cell migration and renewal.
Enterocyte migration rates are very low in suckling rodents until 15-19 days after birth (11, 13, 16), so that cell transit time along the villus is -7 days in 6-day-old animals (46). Lambs differ fundamentally from altricial rodent species in that the highest absolute migration rates were observed in the youngest suckling animals (Table 4). Accordingly, cell replacement was achieved within 2-4 days. Rapid rates of cell renewal previously reported in neonatal lambs (23,31) resulted from a dramatic increase in the mitotic index (31) or in the proportion of crypt cells labeled (42) at birth. These data support the concept that in the sheep [as in the rat, mouse, and pig, but not in the rabbit, hamster, or guinea pig (13)], there is a causal relationship between cell turnover and closure. The cessation of absorption of immunoglobulins occurs normally 2 days after birth in lambs (31). However, we used artificially fed animals. The extent to which this may affect closure is unfortunately unknown. The major effect of age reported in this study is a marked reduction in absolute cell migration rate, between 1 and 5 wk after birth. Moon and Joel (23) previously reported similar observations in lambs between 1 day and 3 wk of age. In addition, the migration rate was more reduced in the duodenum than in the jejunum and ileum in both studies. The gradual bacterial colonization of the small intestine could explain these regional variations. Intestinal microflora, which increases in number in the distal small intestine, is known to accelerate cell migration in the ileum of various species, and has been recently reported to stimulate crypt cell division in the sheep (28). As to the question of why rates of cell migration and renewal are so dramatically reduced with aging, we can
14.3+0.5b 17.6kO.7”
1. 16+0.04B
only speculate. Adrenocortical hormones have been hypothesized to play a major role in the regulation of cell proliferation and migration in the neonatal rodent intestine (14, 15). The sheep fetal intestine appears very sensitive to cortisol, the major glucocorticoid hormone in this species. Cortisol infusion resulted in an increase in both migration rates and proportion of labeled crypt cells (41), whereas adrenalectomy reduced only enterocyte migration (40). In addition, the acceleration in cell turnover observed at birth in the ovine species may result from the increased plasma cortisol concentration that characterizes the latest days of fetal life and peaks at term (42). After birth, circulating cortisol levels sharply decline during the 1st mo of life (26), including in artificially fed lambs (6), and may contribute to the decrease in cellular migration that we reported. Effect of weaning on epithelial cell migration and renewal. In rats, where weaned animals have longer villi,
the increased rate of cell loss results essentially from a fourfold increase in migration rates (11, 13, 16). Alterations in cell renewal observed in the sheep at weaning were of very low amplitude and in striking contrast with observations reported in rodents (Fig. 2). First, a slight or no increase in absolute migration rates (53, 15, and 0% in the duodenum, jejunum, and ileum, respectively) contributed to a shorter cell transit time in weaned lambs at 8 wk of age. Indeed, only the increase for the relative migration rate of labeled enterocytes in the jejunum was significantly higher in ruminating animals than in their prolonged-suckling counterparts (Table 3). Second, the acceleration in cell replacement observed in weaned lambs accounted for both an increase in relative migration rates and a reduction in villus height in the distal regions. It is clear, however, that these differences did not totally result from weaning in itself, because in 8wk-old suckling animals migration rates increased slightly but not significantly (Table 3), at a time when they were significant deepenings of crypts at all intestinal sites (Table 2). These observations suggest that part of the increased cellular proliferation observed at weaning in lambs resulted from intrinsic factors, as previously demonstrated in rodents (18). These findings also do not support any effect of SCFA on intestinal cell renewal in the sheep of physiological importance, at least in our experimental conditions, in contrast with recent data obtained in rats (30). However, SCFA concentrations are extremely low in the sheep small intestine, even in the distal segments (25), and SCFA clearly affected intestinal cell proliferation in a
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INTESTINAL
CELL RENEWAL
dose-dependent manner (30). More likely, weaning-related effects on cell renewal in the sheep may be explained by the interactions between intrinsic (see above) and extrinsic factors. For example, dietary factors such as a higher solid food (7) or fiber content (8, 36) in the diet of ruminating animals are more likely candidates to explain an increased cell turnover rate in the sheep, via direct or indirect effects. Conclusion. The present experiments give the first comprehensive description of the changes in both mucosal morphology and cell renewal occurring between birth and weaning in species with early intestinal morphogenesis similar to most domestic animals and humans. Although obtai .ned i.n a rumi .nant species, our findings suggest that the suckling period, and not weaning, is a major phase of gradual postnatal intestinal adaptation. These data may be representative of human development during the suckling period. However, suckling lambs and piglets, two species with early intestinal differentiation, exhibit major differences in intestinal architecture and cell proliferation patterns. We suggest that the timing of closure may explain most of the differences observed. Because closure is not well defined in humans (l3), more information must be gained before extrapolation of animal data to humans is possible. The authors thank M. Sallas and M. Selle for animal care, M. Serezat for expert technical assistance, and G. Bayle and E. Aurousseau for computer data processing. This study was supported by a grant from the French Ministry of Research and Technology. Address for reprint requests: D. Attaix, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-FerrandTheix, Laboratoire d’Etude du Metabolisme Azote, 63122 Ceyrat, France.
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migration
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