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Oral administration of epidermal growth factor in suckling rats stimulates cell DNA synthesis in fundic and antral gastric mucosae as well as in intestinal mucosa and pancreas
Regulatory Peptides, 20 (1988) 53-64
53
Elsevier RPT 00652
Oral administration of epidermal growth factor in suckling rats stimulates cell D N A synthesis in fundic and antral gastric mucosae as well as in intestinal mucosa and pancreas F r a n q o i s P u c c i o a n d Th6r+se L e h y Unitk de Recherche de Gastroentkrologie, INSERM U.IO H6pital Bichat, Paris (France)
(Received 8 April 1987; revised version received21 July 1987; accepted 31 August 1987)
Summary The effect of orogastrically given epidermal growth factor (EGF) on the development of the digestive system was examined in suckling rats. In particular, D N A synthesis in progenitor cells of the fundic, antral and ileal mucosae and of the exocrine pancreas was analyzed through tritiated thymidine injection and histoautoradiographic study. E G F (10 or 100 #g/kg, 3 times daily) was instilled in pups between the 1lth and the 13th day of life. Controls received distilled water in a similar manner. All rats were killed 14h after the last orogastric instillation and 45 min after one pulse injection of tritiated thymidine. The highest dose of E G F increased the antral mucosal height (P < 0.005), the mean number of epithelial cells per crypt column in ileal mucosa, as well as the cell labeling indices of fundic, antral, ileal mucosae and of pancreatic acinar tissue (P < 0.001) as compared with controls. The lowest dose of E G F increased the cell labeling indices of antral and ileal mucosae and of the exocrine pancreas (P < 0.001) but did not modify that of fundic mucosa as compared with controls. It is concluded that (a) orally given E G F stimulates cell proliferation in the digestive system of suckling rats, (b) antral mucosa is more sensitive to E G F than fundic mucosa, (c) it is likely that E G F is absorbed and acts systemically on the pancreas. It remains to be determined whether E G F acts systemically or by activation of luminal receptors, on fundic, antral and ileal mucosae. Histoautoradiography; Mucosal growth; Progenitor cell D N A synthesis; Trophic effect; Development Correspondence. T. Lehy, INSERM U.10, 170 BoulevardNey, 75877 Paris Cedex 18, France.
0167-0115/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
54 Introduction
Epidermal growth factor (EGF), a polypeptide isolated from the mouse submandibular salivary gland [1]; is known to stimulate epithelial cell proliferation in a variety of tissues both in vitro and in vivo [2]. However, on the gastrointestinal tract of adult animals, the effects of E G F are somewhat controversial. Thus, it has been reported that it increases DNA synthesis in intestinal mucosa of fasted [3-5] but not of fed rodents [5] or, on the contrary, in intestinal mucosa of fed [6,7] but not of fasted rodents [8]. Discrepancies also exist concerning EGF's action on the stomach. It has been demonstrated that short intraperitoneal (i.p.) administration of E G F stimulates DNA synthesis in the gastric mucosa of fed mice, (depending on the time of administration) [9], and in the oxyntic gland area and pancreas of fasted rats [4]. After a longer period of administration, 5 days i.p., EGF was found to increase the DNA, RNA and protein content in the fundic gland of fasted rats and was considered to be atrophic factor for this mucosa [8]. However, this was not confirmed by others who used exactly the same protocol [3]. The latter authors did not find any trophic effect in the gastric mucosa of fasted rats and mice after estimation of the crypt cell production rate by histological methods, although such an effect was proved in intestinal mucosa [3]. Moreover, EGF, one of the nutrient factors detected in the maternal milk [10-13], is a likely candidate to play a role in the pre- and postnatal development of the gastrointestinal tract. Indeed, whether injected i.p. or subcutaneously (s.c.), it has been shown to influence maturation and/or proliferation in intestinal mucosa of fetal [14], and suckling animals [15,16]. Recent studies with EGF given i.p. or orogastrically to suckling rabbits have shown that both ways of administration result in different findings in intestinal mucosa and pancreas and that the orogastric route is less efficient than the i.p. route [17]. Some results concerning the stomach have also been obtained. In neonatal mouse stomach, one single s.c. injection of EGF promoted ornithine decarboxylase activity [18]. Recently, Dembinski and Johnson [19] using biochemical methods found, like in adult rats, that EGF, given i.p. for 5 days, stimulated oxyntic mucosal growth in suckling rats. On the whole, it appears that the trophic influence of E G F in vivo is not clearly established, especially on the gastric mucosa. The route of administration as well as the animals' condition, fed or fasted, could lead to conflicting results. In addition, the exact role that orally given EGF may play on gastric mucosal cell development has not yet been studied in newborn animals. It seemed interesting to elucidate this point. Thus, the present work was conducted in order to investigate in suckling rats whether a 3-day orogastric administration of EGF would actually stimulate progenitor epithelial cell proliferation in the fundic and antral mucosae. Cell proliferation in ileal mucosa and exocrine pancreas were examined as well. This was achieved through in vivo [3H]thymidine pulse labeling and subsequent histoautoradiographic analysis of isotope incorporation into the DNA of cell nucleus. Two doses of E G F were studied.
55 Materials and Methods
Animals Pregnant Sprague-Dawley rats, weighing about 280 g, were used in this study. They arrived in our animal quarters on approximately the 15th day of gestation. They were housed in individual cages and were kept in a light-controlled room. The light was on from 07.00 to 19.00 h. The rats were checked for delivery several times a day. The day of birth was referred to as day zero. On the day after birth, each litter was restricted to 10 pups. During lactation, females were fed ad iibitum with a diet supplemented in calcium, and the pups' body weight curves were surveyed. At the beginning of the experiment, each litter was divided into two halves with strictly equal average body weights. Pups of one group received treatment while pups of the other group served as controls. From day 11 to day 13 after birth, pups received EGF dissolved in sterile distilled water 3 times a day at 07.00, 15.00 and 23.00 h. Mouse EGF (receptor grade, from submaxillary gland, certified to be 95% pure) was purchased from Biomedical Technology Inc. (Cambridge, U.S.A.). It was given by orogastric instillation at a dosage of 100 #g/kg or 10/~g/kg b.wt. Control pups received sterile distilled water in the same manner. The volume of injection varied from 0.26 to 0.36 ml according to the pups' changing weight. Instillation was performed as quickly and quietly as possible. During the dark phase, all instillations were done in dim light, and the instilled rats were immediately returned to the dark environment. Forty-five minutes before sacrifice, each rat was injected i.p. with 1/~Ci/g b.wt. of methyl-[3H]thymidine (spec. act. 5 Ci/mmol; Amersham, U.K.). Rats were killed 14-16 h after the last orogastric instillation, i.e. between 13.00 and 15.00 h. At the time of killing, pups were anesthetized with ether. The pancreas in its totality, stomach and intestine (small and large bowel) were carefully removed and weighed.
Morphological procedures All stomachs were opened along the greater curvature, pinned flat on a paraffin wax block (the mucosal side up), and fixed in a freshly prepared cold Carnoy's solution for 30 min. After fixation, stomachs were dehydrated and parallel strips were resected perpendicularly to the cardia-pylorus axis, embedded in paraplast and cut into 3-#m-thick sections, rigorously perpendicular to the mucosal surface. The pancreas and 2 cm of ileum excised at 95% of the length of the small intestine from the pylorus (i.e. 3 cm from the ileocecal junction) were also processed for histological embedding. The following parameters were studied. Mucosal height. For the fundic, antral and ileal mucosae, measurements were performed along the whole length of strip sections, at l-ram intervals, using a calibrated ocular grid (magnification, × 252). Autoradiographic studies. Three-/~m-thick paraffin sections of fundic, antral and ileal mucosae were cleared in toluol, dehydrated and stained with the periodic acid-Schiff sequence. Slides were dipped in Ilford K5 photographic emulsion diluted 1:2 with
56
0 b
Fig,
I, Autoradiographs;
pancreas Arrows
fundic
of an EGF-treated indicate
the highest glands
mucosa
of a control
rat (c). Note
and the lowest epithelial (arrows)
exhibit
rat (a), antral
the wide distribution labeled
mucosa
of an EGF-treated
of labeled
cells in the fundic
cells. In the antral
several labeled nuckated
cells.
mucosa,
rat (b), mucosa.
well-oriented
x 560.
distilled water, and stored in light-proof boxes at 4°C for 3 weeks. Autoradiographs were developed with Kodak D19, fixed, then stained with hematoxilineosin for stomach and ileum or with hematoxil-acid fuchsin for pancreas. Cells were considered labeled when five or more silver grains overlaid the nucleus. Care was taken not to count nuclei of conjunctive cells. All counts were made under oil immersion by one observer (magnification x 630).
57 In the rat fundic mucosa, at this stage of development, proliferating cells are not restricted to the normal proliferative zone (pit-gland junction) as in adult mucosa but are widely distributed towards the bottom of the glands (Fig. la). At this stage, also, fundic glands are very close together, rarely cut along their entire length. It was therefore difficult to count labeled cells in well-individualized glands. Consequently, as previously described in the adult rat [20], the proliferative zone was defined as the mucosal portion comprised between the highest and the lowest labeled cells in a well-oriented section. Using an ocular grid, we counted labeled and unlabeled epithelial nucleated cells in rectangular 65-/~m wide areas covering the height of the proliferative zone. In each strip section, 4 rectangular areas in the greater curvature and 4 rectangular areas in the lower curvature were selected. Two sections from two different strips of fundic mucosa were studied per animal, leading to the examination of 2000-2400 nucleated epithelial cells. For antral and ileal mucosae, only well-oriented glands or crypts were examined [21]. In the antrum, the well-oriented gland with its pit extended from the base of the mucosa to the luminal surface. We selected 30 well-oriented antral glands with a single layer of cells along the pit-gland column. In these columns, we counted the number of labeled and unlabeled nucleated cells. A total of about 850-1100 cells were counted per animal (Fig. lb). In the ileum, 30 longitudinally sectioned crypts whose lumen was visible from the bottom to the crypt-villus junction were also selected, leading to the examination of about 600 cells per rat. In addition the number of cells in each crypt column, from the middle of the crypt bottom to the edge of the villus, was counted. In exocrine pancreas, 12-15 different tissues areas were studied at random, using a calibrated ocular grid. Labeled and unlabeled nucleated acinar cells were counted in each area, leading to the examination of more than 1000 cells per animal (Fig. lc). From these measurements we calculated the labeling index (i.e. the ratio in percentage of the labeled nuclei to the total number of nuclei in the proliferative zone) for each rat and for each organ. Results are expressed as mean 4- 1 S.E.M. Statistical comparisons between groups for independent populations were made using the Student's t-test. The level of significance was set at P < 0.05.
Results
Animals and organ weights Whatever the dose of intragastrically instilled EGF, the mean body weights did not vary between treated and control groups of rats after 3 days of treatment (Fig. 2). The weights of pancreas, stomach and intestine were the same for the two groups at the end of treatment with the two doses used (data not shown).
58 EGF (1OOIJg/kg)
EGF (1OtJg/kg) g
g
26 30_
24
26
TROL
22_
22
o GF
N=IO 2O ,
,
11
12
1'3
14
p
,
,
,
days
11
12
13
IlL
1~,
days
Fig. 2. Body weight curves of control and EGF-treated suckling rats from the 11th to the 14th day of life in two experimental series (I0 or 100 #g/kg per dose of EGF 3 times daily).
Morphological results Mucosal heights. EGF, given at the 100/~g/kg dose 3 times a day, enhanced the height of antral mucosa in the EGF-treated group by 12% (P < 0.005) over control values. Mucosal heights of fundic and ileal mucosae were not modified (Fig. 3). Nevertheless, in the ileal mucosa, there was a slight increase (+ 8%, P < 0.001), in the mean length of crypt column, expressed as the number of epithelial cell nuclei, in the EGF-treated rats as compared with controls. EGF
pm
100 pg/kg
~Jm
EGF
10 pg/kg
pm IJm
IJm
I.im~
17(
90.
230
140
80
185.
150
80.
210
130
70
175.
0:
O
O~ Fundic
Antral
Mucosa
Mucosa
CONTROL n=lO
Ileal Mucosa
m
Findie Mucosa EGF
n=lO
~
Antral Mucosa **
Ileal Mucosa
p < 0.005
Fig. 3. Height of fundic, antral and ileal mucosae in control and EGF-treated suckling rats on the 14th day of life, 14-16 h after the last orogastric instillation. Values are mean + 1 S.E.M. in two experimental series (10 or 100/lg/kg per dose of EGF, 3 times daily).
59
With the 10 #g/kg dose of EGF 3 times a day, the fundic, antral and ileal mucosal heights did not vary as compared with controls (Fig. 3).
Proliferative parameters. In 14-day-old rats, the highest dose of intragastrically instilled EGF (100 #g/kg), significantly increased (P < 0.001) the cell labeling indices in the exocrine pancreas (+ 85%), fundic (+ 19%), antral (+ 35%) and ileal mucosae (+ 33%) as compared with control values (Fig. 4). The lowest dose of EGF (10 #g/kg), although less potent than the highest dose, significantly increased the cell labeling indices (P < 0.001) in the exocrine pancreas ( + 35 %), antral ( + 19 %) and ileal mucosae ( + 19%) as compared with control values, whereas it was without effect on the cell labeling index in the fundic mucosa
(Fig. 5).
Discussion
Our current findings clearly indicate that EGF, orally given to suckling rats, is resistant enough to acid and proteolytic digestion to be capable of exerting a stimulatory effect on epithelial cell proliferation of gastric mucosa as well as on that of EGF
100 pg/kg
No. labeled cells (*/,)
No. labeled cells (%)
1
I o
13 25 11
o
I 1
1
f
I 8
20.
I
1 15
10
i 5
5
Rm
oT
0
Fundic
Pancreas
Mucosa
•
CONTROL
n:lO
Ileal
Antral Mucosa
o
EGF
n:lO
Mucosa
***
p
Fig. 4. Labeling indices of acinar cells in the exocrine pancreatic tissue and of epithelial cells in the fundic, antral and ileal mucosae of control and EGF-treated rats on the 14th day of life, 14-16 h after the last orogastric instillation. The dose of E G F was 100/~g/kg, 3 times daily for 3 days. Values are m e a n ± 1 S.E.M.
60
ileal mucosa and exocrine pancreas. These data support the assumption recently made that EGF plays a role in the development of gastric mucosa [19]. In this last experiment, EGF was given by the i.p. route. Original points of our study were the combination of orogastric administration of EGF and histoautoradiographic approach to demonstrate its growth-promoting action on the gastrointestinal tract and pancreas of the suckling rats. The stimulating effect of EGF on epithelial/parenchymal progenitor cell DNA synthesis was greater with the highest dose but was also present with the lowest dose. The latter (10 #g/kg dose, 3 times daily) is very close to the physiological dose of EGF that a pup could ingest in 24 h, considering (a) the quantity of milk ingested per pup during that time [22] and (b) the concentration of EGF in the milk of lactating Sprague-Dawley rats (38.85 ng/ml) [13]. However, other authors have estimated the daily EGF intake from milk per pup to be about 8 ktg/kg/day [23]. Our lowest dose would correspond to 3.5 times that dose. It should be noted that we used mouse EGF which is a more potent stimulant of DNA synthesis than rat EGF [24]. In this study, whatever the doses of EGF used, organ weights were not significantly increased as compared with controls. With longer i.p. administration in suckling animals, i.e. 5 days in rats [19] and 15 days in rabbits [17], an increase of organ weights was observed in EGF-treated animals. Three days of treatment may be too short to obtain a detectable increase of organ weight even with the highest dose. However, with that dose, the height of antral mucosa and the mean crypt length in the ileal mucosa, expressed as the number of epithelial cell nuclei per crypt column, were significantly increased in the EGF-treated rats as compared with controls. This suggests that EGF did exert a real trophic influence on the gastrointestinal mucosa. EG F
10 lug//kg
No. labeled cells (~o)
No. labeled (:ells (~o)
I
l
I
$
t
7.
•
1
1
g
l &
20
t
15
I
El
10
5.
I i
3.
or
5
r--l
r-~
Pancreas
•
CONTROL
r'-1
r~ Antral
Mucosa
Mucosa
n=lO
I
o
Fundic
o
EGF
n=10
Ileal Mucosa
***
p
Fig. 5. Same legend as for Fig. 4, except that the dose of E G F was 10/~g/kg, 3 times daily for 3 days.
61 In agreement with our data, Pollack et al. [23] did not show any difference in colon weight between EGF-treated and control suckling rats after 4 days of E G F enteral treatment, while DNA content was significantly augmented in EGF-treated rats. Previous findings in unweaned rats [19] and in undernourished suckling rats [25] showed that E G F (i.p. or intramuscularly) significantly increased DNA, RNA and protein content of the fundic gland but had no effect on antral and serum gastrin levels [19,25] as well as on gastric acid and pepsin secretion [19]. From these studies, it has been concluded that E G F does not affect the maturation of gastric function. In our study, fundic and antral mucosae have been examined separately. We showed here that antral mucosa is more sensitive to E G F than fundic mucosa. Indeed, as compared with control rats, study of progenitor cell proliferation under E G F treatment indicates that mean labeling index was more greatly increased in antral mucosa (+ 35%) than in fundic mucosa ( + 19%) with the highest dose. This parameter was augmented by 19% in antral mucosa while not modified in fundic mucosa with the lowest dose. Moreover, only the antral mucosal height was enhanced under EGF. We cannot exclude the possibility that the effect of E G F on the fundic mucosa would have been more important, particularly in the case of the lowest dose, if the animals had been sacrified earlier after the last orogastric instillation. We chose to kill animals 14 h after the last instillation because maximal stimulation of DNA synthesis seemed to occur about 16 h postinjection in the intestine of the adult rat [3]. However, in the adult mouse, for most of the digestive tissues, this maximal stimulation became evident between 8 and 12 h after injection [6,9]. Of the organs studied, the pancreas is the most sensitive to E G F action, and this with both doses tested. Previously, a stimulating effect on pancreatic DNA synthesis was observed in adult rats when EGF was administered i.p. but not orogastrically [4]. By contrast, other preliminary results showed that E G F would inhibit the proliferation of pancreatic tissue and significantly reduce the total pancreatic DNA content in adult rats [26]. To our knowledge there is only one report studying the effect of EGF on the developing pancreas. In this last work, EGF, when given i.p. for 15 days in suckling rabbits increased pancreatic weight, protein content and amylase activity, but when orogastrically given, it affected only pancreatic weight [17]. In the authors' opinion, cellular hypertrophy rather than hyperplasia was obtained. However, our data demonstrate that EGF affects the structural development of the pancreas by enhancing cell multiplication. Besides, it was proved that E G F is able to directly increase in vitro DNA synthesis in mouse pancreatic acinar cell culture [27]. The antral and ileal mucosae showed quite comparable responses to the two EGF doses. A growth-promoting effect of E G F on the intestinal mucosa of suckling animals, i.e. on ileal and duodenal mucosae [15-17] and on the colon [15-17, 23] was found by other authors. However, when EGF was given by the orogastric route, it was found to stimulate DNA content only in the distal part of small intestine [17] and in the colon [23]. It appears that in the newborn animals, as in adults, the route of E G F administration is important in obtaining a trophic effect. It has been mentioned above that i.p. and orogastric administration give different results on the gastrointestinal tract and pancreas of suckling rabbits [17], the second being less efficient. Several theories
62
can be put forward to explain how a hormone/peptide, luminally given, may induce a proliferative response in the gastrointestinal tract. First, it could be absorbed and act systemically. Second, it could be absorbed and act locally without appearing in the general circulation. Third, it could activate luminal receptors without being absorbed [28]. Indeed , in the neonatal period, kidneys and salivary glands begin to produce EGF m R N A by two weeks postpartum or after weaning, respectively and milk appears to be the physiological source of E G F [29]. 125I-EGF ingested by neonates was proven to be absorbed intact into the circulation and to reach many internal organs [13,29]. In our study, despite some likely hydrolysis, E G F given by orogastric instillation retained enough activity to stimulate digestive cells. Concerning its effect on the pancreas, it appears certain that it was absorbed by digestive mucosae and acted through the circulation. It has been proven that E G F receptors are present in pancreatic acini [30]. Concerning EGF action on the gastric and ileal mucosae, it is not possible at present to make any clear-cut statement about the possible mechanisms at work. Specific binding sites for E G F have been demonstrated in small intestinal cells of suckling [31] and adult rats [32]. This suggests a mechanism for intraluminal regulation of enterocyte proliferation. In addition, in suckling rodents, intestinal crypt epithelial cells seem to have a greater affinity for EGF than do villus epithelial cells [33,34]. In conclusion, nutrient factors present in breast milk like bombesin [35] and E G F appear to regulate the developmental growth of the digestive system in suckling rats. In this study, the stimulating effect of orogastric E G F instillation on the epithelial/ parenchymal cell proliferation of the gastrointestinal mucosa and exocrine pancreas was clearly established.
Acknowledgements We express our thanks to Mrs. L6one Gr~s and Danielle Labeille for expert technical assistance.
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63 mucosal proliferation in the small intestine of adult rats, Gastroenterology, 91 (1986) 1134-1140. 8 Johnson, L.R. and Guthrie, P.D., Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor, Am. J. Physiol., 238 (1980) G4~G49. 9 Scheving, L.A., Yeh, Y.C., Tsai, T.H. and Scheving, L.E., Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the tongue, esophagus and stomach of the adult male mouse, Endocrinology, 105 (1979) 1475-1480. 10 Beardmore, J.M. and Richards, R.C., Concentrations of epidermal growth factor in mouse milk throughout lactation. J. Endocrinol., 96 (1983) 287-292. 11 Hazum, E., Hormones and neurotransmitters in milk, Trends Pharmacol. Sci., 4 (1983) 454-456. 12 Moran, J.R., Courtney, M.E., Orth, D.N., Vaughan, R., Coy, S., Mount, C.D., Sherrell, B.J. and Greene, H.L., Epidermal growth factor in human milk: daily production and diurnal variation during early lactation in mothers delivering at term and at premature gestation, J. Pediatr., 103 (1983) 402 405. 13 Thornburg, W., Matrisian, L., Magun, B. and Koldovsky, O., Gastrointestinal absorption of epidermal growth factor in suckling rats, Am. J. Physiol., 246 (1984) G80-G85. 14 Calvert, R., Beaulieu, J.F. and Mrnard, D., Epidermal growth factor (EGF) accelerates the maturation of fetal mouse intestinal mucosa in utero, Experientia, 38 (1982) 1096-1097. 15 Malo, C. and Mrnard, D., Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa, Gastroenterology, 83 (1982) 28-35. 16 Oka, Y., Ghishan, F.K., Greene, H.L. and Orth, D.N., Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine, Endocrinology, 112 (1983) 940-944. 17 O'Loughlin, E.V., Chung, M., Hollenberg, M., Hayden, J., Zahavi, I. and Gall, D.G., Effect of epidermal growth factor on ontogeny of the gastrointestinal tract, Am. J. Physiol., 249 (1985) G674G678. 18 Feldman, E.J., Aures, D. and Grossman, M.I., Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of mouse, Proc. Soc. Exp. Biol. Med., 159 (1978) 40(0402. 19 Dembinski, A.B. and Johnson, L.R., Effect of epidermal growth factor on the development of rat gastric mucosa, Endocrinology, 116 (1985) 90-94. 20 Eastwood, G.L. and Quimby, G.F., Effect of chronic cimetidine ingestion on fundic and antral epithelial proliferation in the rat, Dig. Dis. Sci., 28 (1983) 61-64. 21 Lehy, T., Accary, J.P., Dubrasquet, M. and Lewin, M.J.M., Growth hormone-releasing factor (Somatocrinin) stimulates epithelial cell proliferation in the rat digestive tract, Gastroenterology, 90 (1986) 646-653. 22 Yagil, R., Etzion, Z. and Berlyne, M., Changes in rat milk quantity and quality due to variations in litter size and high ambient temperature, Lab. Anim. Sci., 26 (1976) 33-37. 23 Pollack, P.F., Goda, T., Colony, P.C., Edmonds, J., Thornburg, W., Korc, M. and Koldovsky, O., Effects on enterally fed epidermal growth factor on the small and large intestine of the suckling rat, Regul. Peptides, 17 (1987) 121-132. 24 Schaudies, R.P. and Savage Jr., C.R., Isolation of rat epidermal growth factor (r-EGF): chemical, biological and immunological comparisons with mouse and human EGF, Comp. Biochem. Physiol., 84B (1986) 497-505. 25 Majumdar, A.P.N., Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats, J. Pediatr. Gastroenterol. Nutr., 3 (1984) 618-625. 26 Morisset, J., Korc, M. and Larose, L., Antagonistic and additive trophic effects of caerulein and EGF in rat pancreas, Gastroenterology, 90 (1986) 1557 (abstract). 27 Logsdon, C.D., Stimulation of pancreatic acinar cell growth by CCK, epidermal growth factor, and insulin in vitro, Am. J. Physiol., 251 (1986) G487-G494. 28 Johnson, L.R., Guthrie, P.D. and Dudrick, S.J., Effects of luminal gastrin on the growth of rat intestinal mucosa, Gastroenterology, 81 (1981) 71-77. 29 Popliker, M., Shatz, A., Avini, A., Ullrich, A., Schlessinger, J. and Webb, C.G., Onset of endogenous synthesis of epidermal growth factor in neonatal mice, Dev. Biol., 119 (1987) 38-44. 30 Korc, M., Matrisian, L., Planck, S.R. and Magun, B.E., Binding of epidermal growth factor in rat pancreatic acini. Biochem. Biophys. Res. Commun., 111 (1983) 1066-1073.
64 31 Thompson, J.F., Evidence for specific epidermal growth factor (EGF) receptors on fetal, suckling and adult rat intestinal microvillus membranes, Gastroenterology, 90 (1986) 1664 (abstract). 32 Chabot, J.G., Walker, P. and Pelletier, G., Demonstration epidermal growth factor binding sites in the adult rat small intestine by autoradiography, Can. J. Physiol, Pharmacol., 65 (1987) 109-112. 33 Rao, R.K., Thornburg, W., Korc, M., Matrisian, L,M., Magun, B.E. and Koldovsky, O., Processing of epidermal growth factor by suckling and adult rat intestinal cells, Am. J. Physiol., 250 (1986) G850-G855. 34 Gallo-Payet, N. and Hugon, J.S., Epidermal growth factor receptors in isolated adult mouse intestinal cells: studies in vivo and in organ culture, Endocrinology, 116 (1985) 194-201. 35 Lehy, T., Puccio, F., Chariot, J. and Labeille, D., Stimulating effect of bombesin on the growth of gastrointestinal tract and pancreas in suckling rats, Gastroenterology, 90 (1986) 1942-1949.
53
Elsevier RPT 00652
Oral administration of epidermal growth factor in suckling rats stimulates cell D N A synthesis in fundic and antral gastric mucosae as well as in intestinal mucosa and pancreas F r a n q o i s P u c c i o a n d Th6r+se L e h y Unitk de Recherche de Gastroentkrologie, INSERM U.IO H6pital Bichat, Paris (France)
(Received 8 April 1987; revised version received21 July 1987; accepted 31 August 1987)
Summary The effect of orogastrically given epidermal growth factor (EGF) on the development of the digestive system was examined in suckling rats. In particular, D N A synthesis in progenitor cells of the fundic, antral and ileal mucosae and of the exocrine pancreas was analyzed through tritiated thymidine injection and histoautoradiographic study. E G F (10 or 100 #g/kg, 3 times daily) was instilled in pups between the 1lth and the 13th day of life. Controls received distilled water in a similar manner. All rats were killed 14h after the last orogastric instillation and 45 min after one pulse injection of tritiated thymidine. The highest dose of E G F increased the antral mucosal height (P < 0.005), the mean number of epithelial cells per crypt column in ileal mucosa, as well as the cell labeling indices of fundic, antral, ileal mucosae and of pancreatic acinar tissue (P < 0.001) as compared with controls. The lowest dose of E G F increased the cell labeling indices of antral and ileal mucosae and of the exocrine pancreas (P < 0.001) but did not modify that of fundic mucosa as compared with controls. It is concluded that (a) orally given E G F stimulates cell proliferation in the digestive system of suckling rats, (b) antral mucosa is more sensitive to E G F than fundic mucosa, (c) it is likely that E G F is absorbed and acts systemically on the pancreas. It remains to be determined whether E G F acts systemically or by activation of luminal receptors, on fundic, antral and ileal mucosae. Histoautoradiography; Mucosal growth; Progenitor cell D N A synthesis; Trophic effect; Development Correspondence. T. Lehy, INSERM U.10, 170 BoulevardNey, 75877 Paris Cedex 18, France.
0167-0115/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
54 Introduction
Epidermal growth factor (EGF), a polypeptide isolated from the mouse submandibular salivary gland [1]; is known to stimulate epithelial cell proliferation in a variety of tissues both in vitro and in vivo [2]. However, on the gastrointestinal tract of adult animals, the effects of E G F are somewhat controversial. Thus, it has been reported that it increases DNA synthesis in intestinal mucosa of fasted [3-5] but not of fed rodents [5] or, on the contrary, in intestinal mucosa of fed [6,7] but not of fasted rodents [8]. Discrepancies also exist concerning EGF's action on the stomach. It has been demonstrated that short intraperitoneal (i.p.) administration of E G F stimulates DNA synthesis in the gastric mucosa of fed mice, (depending on the time of administration) [9], and in the oxyntic gland area and pancreas of fasted rats [4]. After a longer period of administration, 5 days i.p., EGF was found to increase the DNA, RNA and protein content in the fundic gland of fasted rats and was considered to be atrophic factor for this mucosa [8]. However, this was not confirmed by others who used exactly the same protocol [3]. The latter authors did not find any trophic effect in the gastric mucosa of fasted rats and mice after estimation of the crypt cell production rate by histological methods, although such an effect was proved in intestinal mucosa [3]. Moreover, EGF, one of the nutrient factors detected in the maternal milk [10-13], is a likely candidate to play a role in the pre- and postnatal development of the gastrointestinal tract. Indeed, whether injected i.p. or subcutaneously (s.c.), it has been shown to influence maturation and/or proliferation in intestinal mucosa of fetal [14], and suckling animals [15,16]. Recent studies with EGF given i.p. or orogastrically to suckling rabbits have shown that both ways of administration result in different findings in intestinal mucosa and pancreas and that the orogastric route is less efficient than the i.p. route [17]. Some results concerning the stomach have also been obtained. In neonatal mouse stomach, one single s.c. injection of EGF promoted ornithine decarboxylase activity [18]. Recently, Dembinski and Johnson [19] using biochemical methods found, like in adult rats, that EGF, given i.p. for 5 days, stimulated oxyntic mucosal growth in suckling rats. On the whole, it appears that the trophic influence of E G F in vivo is not clearly established, especially on the gastric mucosa. The route of administration as well as the animals' condition, fed or fasted, could lead to conflicting results. In addition, the exact role that orally given EGF may play on gastric mucosal cell development has not yet been studied in newborn animals. It seemed interesting to elucidate this point. Thus, the present work was conducted in order to investigate in suckling rats whether a 3-day orogastric administration of EGF would actually stimulate progenitor epithelial cell proliferation in the fundic and antral mucosae. Cell proliferation in ileal mucosa and exocrine pancreas were examined as well. This was achieved through in vivo [3H]thymidine pulse labeling and subsequent histoautoradiographic analysis of isotope incorporation into the DNA of cell nucleus. Two doses of E G F were studied.
55 Materials and Methods
Animals Pregnant Sprague-Dawley rats, weighing about 280 g, were used in this study. They arrived in our animal quarters on approximately the 15th day of gestation. They were housed in individual cages and were kept in a light-controlled room. The light was on from 07.00 to 19.00 h. The rats were checked for delivery several times a day. The day of birth was referred to as day zero. On the day after birth, each litter was restricted to 10 pups. During lactation, females were fed ad iibitum with a diet supplemented in calcium, and the pups' body weight curves were surveyed. At the beginning of the experiment, each litter was divided into two halves with strictly equal average body weights. Pups of one group received treatment while pups of the other group served as controls. From day 11 to day 13 after birth, pups received EGF dissolved in sterile distilled water 3 times a day at 07.00, 15.00 and 23.00 h. Mouse EGF (receptor grade, from submaxillary gland, certified to be 95% pure) was purchased from Biomedical Technology Inc. (Cambridge, U.S.A.). It was given by orogastric instillation at a dosage of 100 #g/kg or 10/~g/kg b.wt. Control pups received sterile distilled water in the same manner. The volume of injection varied from 0.26 to 0.36 ml according to the pups' changing weight. Instillation was performed as quickly and quietly as possible. During the dark phase, all instillations were done in dim light, and the instilled rats were immediately returned to the dark environment. Forty-five minutes before sacrifice, each rat was injected i.p. with 1/~Ci/g b.wt. of methyl-[3H]thymidine (spec. act. 5 Ci/mmol; Amersham, U.K.). Rats were killed 14-16 h after the last orogastric instillation, i.e. between 13.00 and 15.00 h. At the time of killing, pups were anesthetized with ether. The pancreas in its totality, stomach and intestine (small and large bowel) were carefully removed and weighed.
Morphological procedures All stomachs were opened along the greater curvature, pinned flat on a paraffin wax block (the mucosal side up), and fixed in a freshly prepared cold Carnoy's solution for 30 min. After fixation, stomachs were dehydrated and parallel strips were resected perpendicularly to the cardia-pylorus axis, embedded in paraplast and cut into 3-#m-thick sections, rigorously perpendicular to the mucosal surface. The pancreas and 2 cm of ileum excised at 95% of the length of the small intestine from the pylorus (i.e. 3 cm from the ileocecal junction) were also processed for histological embedding. The following parameters were studied. Mucosal height. For the fundic, antral and ileal mucosae, measurements were performed along the whole length of strip sections, at l-ram intervals, using a calibrated ocular grid (magnification, × 252). Autoradiographic studies. Three-/~m-thick paraffin sections of fundic, antral and ileal mucosae were cleared in toluol, dehydrated and stained with the periodic acid-Schiff sequence. Slides were dipped in Ilford K5 photographic emulsion diluted 1:2 with
56
0 b
Fig,
I, Autoradiographs;
pancreas Arrows
fundic
of an EGF-treated indicate
the highest glands
mucosa
of a control
rat (c). Note
and the lowest epithelial (arrows)
exhibit
rat (a), antral
the wide distribution labeled
mucosa
of an EGF-treated
of labeled
cells in the fundic
cells. In the antral
several labeled nuckated
cells.
mucosa,
rat (b), mucosa.
well-oriented
x 560.
distilled water, and stored in light-proof boxes at 4°C for 3 weeks. Autoradiographs were developed with Kodak D19, fixed, then stained with hematoxilineosin for stomach and ileum or with hematoxil-acid fuchsin for pancreas. Cells were considered labeled when five or more silver grains overlaid the nucleus. Care was taken not to count nuclei of conjunctive cells. All counts were made under oil immersion by one observer (magnification x 630).
57 In the rat fundic mucosa, at this stage of development, proliferating cells are not restricted to the normal proliferative zone (pit-gland junction) as in adult mucosa but are widely distributed towards the bottom of the glands (Fig. la). At this stage, also, fundic glands are very close together, rarely cut along their entire length. It was therefore difficult to count labeled cells in well-individualized glands. Consequently, as previously described in the adult rat [20], the proliferative zone was defined as the mucosal portion comprised between the highest and the lowest labeled cells in a well-oriented section. Using an ocular grid, we counted labeled and unlabeled epithelial nucleated cells in rectangular 65-/~m wide areas covering the height of the proliferative zone. In each strip section, 4 rectangular areas in the greater curvature and 4 rectangular areas in the lower curvature were selected. Two sections from two different strips of fundic mucosa were studied per animal, leading to the examination of 2000-2400 nucleated epithelial cells. For antral and ileal mucosae, only well-oriented glands or crypts were examined [21]. In the antrum, the well-oriented gland with its pit extended from the base of the mucosa to the luminal surface. We selected 30 well-oriented antral glands with a single layer of cells along the pit-gland column. In these columns, we counted the number of labeled and unlabeled nucleated cells. A total of about 850-1100 cells were counted per animal (Fig. lb). In the ileum, 30 longitudinally sectioned crypts whose lumen was visible from the bottom to the crypt-villus junction were also selected, leading to the examination of about 600 cells per rat. In addition the number of cells in each crypt column, from the middle of the crypt bottom to the edge of the villus, was counted. In exocrine pancreas, 12-15 different tissues areas were studied at random, using a calibrated ocular grid. Labeled and unlabeled nucleated acinar cells were counted in each area, leading to the examination of more than 1000 cells per animal (Fig. lc). From these measurements we calculated the labeling index (i.e. the ratio in percentage of the labeled nuclei to the total number of nuclei in the proliferative zone) for each rat and for each organ. Results are expressed as mean 4- 1 S.E.M. Statistical comparisons between groups for independent populations were made using the Student's t-test. The level of significance was set at P < 0.05.
Results
Animals and organ weights Whatever the dose of intragastrically instilled EGF, the mean body weights did not vary between treated and control groups of rats after 3 days of treatment (Fig. 2). The weights of pancreas, stomach and intestine were the same for the two groups at the end of treatment with the two doses used (data not shown).
58 EGF (1OOIJg/kg)
EGF (1OtJg/kg) g
g
26 30_
24
26
TROL
22_
22
o GF
N=IO 2O ,
,
11
12
1'3
14
p
,
,
,
days
11
12
13
IlL
1~,
days
Fig. 2. Body weight curves of control and EGF-treated suckling rats from the 11th to the 14th day of life in two experimental series (I0 or 100 #g/kg per dose of EGF 3 times daily).
Morphological results Mucosal heights. EGF, given at the 100/~g/kg dose 3 times a day, enhanced the height of antral mucosa in the EGF-treated group by 12% (P < 0.005) over control values. Mucosal heights of fundic and ileal mucosae were not modified (Fig. 3). Nevertheless, in the ileal mucosa, there was a slight increase (+ 8%, P < 0.001), in the mean length of crypt column, expressed as the number of epithelial cell nuclei, in the EGF-treated rats as compared with controls. EGF
pm
100 pg/kg
~Jm
EGF
10 pg/kg
pm IJm
IJm
I.im~
17(
90.
230
140
80
185.
150
80.
210
130
70
175.
0:
O
O~ Fundic
Antral
Mucosa
Mucosa
CONTROL n=lO
Ileal Mucosa
m
Findie Mucosa EGF
n=lO
~
Antral Mucosa **
Ileal Mucosa
p < 0.005
Fig. 3. Height of fundic, antral and ileal mucosae in control and EGF-treated suckling rats on the 14th day of life, 14-16 h after the last orogastric instillation. Values are mean + 1 S.E.M. in two experimental series (10 or 100/lg/kg per dose of EGF, 3 times daily).
59
With the 10 #g/kg dose of EGF 3 times a day, the fundic, antral and ileal mucosal heights did not vary as compared with controls (Fig. 3).
Proliferative parameters. In 14-day-old rats, the highest dose of intragastrically instilled EGF (100 #g/kg), significantly increased (P < 0.001) the cell labeling indices in the exocrine pancreas (+ 85%), fundic (+ 19%), antral (+ 35%) and ileal mucosae (+ 33%) as compared with control values (Fig. 4). The lowest dose of EGF (10 #g/kg), although less potent than the highest dose, significantly increased the cell labeling indices (P < 0.001) in the exocrine pancreas ( + 35 %), antral ( + 19 %) and ileal mucosae ( + 19%) as compared with control values, whereas it was without effect on the cell labeling index in the fundic mucosa
(Fig. 5).
Discussion
Our current findings clearly indicate that EGF, orally given to suckling rats, is resistant enough to acid and proteolytic digestion to be capable of exerting a stimulatory effect on epithelial cell proliferation of gastric mucosa as well as on that of EGF
100 pg/kg
No. labeled cells (*/,)
No. labeled cells (%)
1
I o
13 25 11
o
I 1
1
f
I 8
20.
I
1 15
10
i 5
5
Rm
oT
0
Fundic
Pancreas
Mucosa
•
CONTROL
n:lO
Ileal
Antral Mucosa
o
EGF
n:lO
Mucosa
***
p
Fig. 4. Labeling indices of acinar cells in the exocrine pancreatic tissue and of epithelial cells in the fundic, antral and ileal mucosae of control and EGF-treated rats on the 14th day of life, 14-16 h after the last orogastric instillation. The dose of E G F was 100/~g/kg, 3 times daily for 3 days. Values are m e a n ± 1 S.E.M.
60
ileal mucosa and exocrine pancreas. These data support the assumption recently made that EGF plays a role in the development of gastric mucosa [19]. In this last experiment, EGF was given by the i.p. route. Original points of our study were the combination of orogastric administration of EGF and histoautoradiographic approach to demonstrate its growth-promoting action on the gastrointestinal tract and pancreas of the suckling rats. The stimulating effect of EGF on epithelial/parenchymal progenitor cell DNA synthesis was greater with the highest dose but was also present with the lowest dose. The latter (10 #g/kg dose, 3 times daily) is very close to the physiological dose of EGF that a pup could ingest in 24 h, considering (a) the quantity of milk ingested per pup during that time [22] and (b) the concentration of EGF in the milk of lactating Sprague-Dawley rats (38.85 ng/ml) [13]. However, other authors have estimated the daily EGF intake from milk per pup to be about 8 ktg/kg/day [23]. Our lowest dose would correspond to 3.5 times that dose. It should be noted that we used mouse EGF which is a more potent stimulant of DNA synthesis than rat EGF [24]. In this study, whatever the doses of EGF used, organ weights were not significantly increased as compared with controls. With longer i.p. administration in suckling animals, i.e. 5 days in rats [19] and 15 days in rabbits [17], an increase of organ weights was observed in EGF-treated animals. Three days of treatment may be too short to obtain a detectable increase of organ weight even with the highest dose. However, with that dose, the height of antral mucosa and the mean crypt length in the ileal mucosa, expressed as the number of epithelial cell nuclei per crypt column, were significantly increased in the EGF-treated rats as compared with controls. This suggests that EGF did exert a real trophic influence on the gastrointestinal mucosa. EG F
10 lug//kg
No. labeled cells (~o)
No. labeled (:ells (~o)
I
l
I
$
t
7.
•
1
1
g
l &
20
t
15
I
El
10
5.
I i
3.
or
5
r--l
r-~
Pancreas
•
CONTROL
r'-1
r~ Antral
Mucosa
Mucosa
n=lO
I
o
Fundic
o
EGF
n=10
Ileal Mucosa
***
p
Fig. 5. Same legend as for Fig. 4, except that the dose of E G F was 10/~g/kg, 3 times daily for 3 days.
61 In agreement with our data, Pollack et al. [23] did not show any difference in colon weight between EGF-treated and control suckling rats after 4 days of E G F enteral treatment, while DNA content was significantly augmented in EGF-treated rats. Previous findings in unweaned rats [19] and in undernourished suckling rats [25] showed that E G F (i.p. or intramuscularly) significantly increased DNA, RNA and protein content of the fundic gland but had no effect on antral and serum gastrin levels [19,25] as well as on gastric acid and pepsin secretion [19]. From these studies, it has been concluded that E G F does not affect the maturation of gastric function. In our study, fundic and antral mucosae have been examined separately. We showed here that antral mucosa is more sensitive to E G F than fundic mucosa. Indeed, as compared with control rats, study of progenitor cell proliferation under E G F treatment indicates that mean labeling index was more greatly increased in antral mucosa (+ 35%) than in fundic mucosa ( + 19%) with the highest dose. This parameter was augmented by 19% in antral mucosa while not modified in fundic mucosa with the lowest dose. Moreover, only the antral mucosal height was enhanced under EGF. We cannot exclude the possibility that the effect of E G F on the fundic mucosa would have been more important, particularly in the case of the lowest dose, if the animals had been sacrified earlier after the last orogastric instillation. We chose to kill animals 14 h after the last instillation because maximal stimulation of DNA synthesis seemed to occur about 16 h postinjection in the intestine of the adult rat [3]. However, in the adult mouse, for most of the digestive tissues, this maximal stimulation became evident between 8 and 12 h after injection [6,9]. Of the organs studied, the pancreas is the most sensitive to E G F action, and this with both doses tested. Previously, a stimulating effect on pancreatic DNA synthesis was observed in adult rats when EGF was administered i.p. but not orogastrically [4]. By contrast, other preliminary results showed that E G F would inhibit the proliferation of pancreatic tissue and significantly reduce the total pancreatic DNA content in adult rats [26]. To our knowledge there is only one report studying the effect of EGF on the developing pancreas. In this last work, EGF, when given i.p. for 15 days in suckling rabbits increased pancreatic weight, protein content and amylase activity, but when orogastrically given, it affected only pancreatic weight [17]. In the authors' opinion, cellular hypertrophy rather than hyperplasia was obtained. However, our data demonstrate that EGF affects the structural development of the pancreas by enhancing cell multiplication. Besides, it was proved that E G F is able to directly increase in vitro DNA synthesis in mouse pancreatic acinar cell culture [27]. The antral and ileal mucosae showed quite comparable responses to the two EGF doses. A growth-promoting effect of E G F on the intestinal mucosa of suckling animals, i.e. on ileal and duodenal mucosae [15-17] and on the colon [15-17, 23] was found by other authors. However, when EGF was given by the orogastric route, it was found to stimulate DNA content only in the distal part of small intestine [17] and in the colon [23]. It appears that in the newborn animals, as in adults, the route of E G F administration is important in obtaining a trophic effect. It has been mentioned above that i.p. and orogastric administration give different results on the gastrointestinal tract and pancreas of suckling rabbits [17], the second being less efficient. Several theories
62
can be put forward to explain how a hormone/peptide, luminally given, may induce a proliferative response in the gastrointestinal tract. First, it could be absorbed and act systemically. Second, it could be absorbed and act locally without appearing in the general circulation. Third, it could activate luminal receptors without being absorbed [28]. Indeed , in the neonatal period, kidneys and salivary glands begin to produce EGF m R N A by two weeks postpartum or after weaning, respectively and milk appears to be the physiological source of E G F [29]. 125I-EGF ingested by neonates was proven to be absorbed intact into the circulation and to reach many internal organs [13,29]. In our study, despite some likely hydrolysis, E G F given by orogastric instillation retained enough activity to stimulate digestive cells. Concerning its effect on the pancreas, it appears certain that it was absorbed by digestive mucosae and acted through the circulation. It has been proven that E G F receptors are present in pancreatic acini [30]. Concerning EGF action on the gastric and ileal mucosae, it is not possible at present to make any clear-cut statement about the possible mechanisms at work. Specific binding sites for E G F have been demonstrated in small intestinal cells of suckling [31] and adult rats [32]. This suggests a mechanism for intraluminal regulation of enterocyte proliferation. In addition, in suckling rodents, intestinal crypt epithelial cells seem to have a greater affinity for EGF than do villus epithelial cells [33,34]. In conclusion, nutrient factors present in breast milk like bombesin [35] and E G F appear to regulate the developmental growth of the digestive system in suckling rats. In this study, the stimulating effect of orogastric E G F instillation on the epithelial/ parenchymal cell proliferation of the gastrointestinal mucosa and exocrine pancreas was clearly established.
Acknowledgements We express our thanks to Mrs. L6one Gr~s and Danielle Labeille for expert technical assistance.
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63 mucosal proliferation in the small intestine of adult rats, Gastroenterology, 91 (1986) 1134-1140. 8 Johnson, L.R. and Guthrie, P.D., Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor, Am. J. Physiol., 238 (1980) G4~G49. 9 Scheving, L.A., Yeh, Y.C., Tsai, T.H. and Scheving, L.E., Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the tongue, esophagus and stomach of the adult male mouse, Endocrinology, 105 (1979) 1475-1480. 10 Beardmore, J.M. and Richards, R.C., Concentrations of epidermal growth factor in mouse milk throughout lactation. J. Endocrinol., 96 (1983) 287-292. 11 Hazum, E., Hormones and neurotransmitters in milk, Trends Pharmacol. Sci., 4 (1983) 454-456. 12 Moran, J.R., Courtney, M.E., Orth, D.N., Vaughan, R., Coy, S., Mount, C.D., Sherrell, B.J. and Greene, H.L., Epidermal growth factor in human milk: daily production and diurnal variation during early lactation in mothers delivering at term and at premature gestation, J. Pediatr., 103 (1983) 402 405. 13 Thornburg, W., Matrisian, L., Magun, B. and Koldovsky, O., Gastrointestinal absorption of epidermal growth factor in suckling rats, Am. J. Physiol., 246 (1984) G80-G85. 14 Calvert, R., Beaulieu, J.F. and Mrnard, D., Epidermal growth factor (EGF) accelerates the maturation of fetal mouse intestinal mucosa in utero, Experientia, 38 (1982) 1096-1097. 15 Malo, C. and Mrnard, D., Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa, Gastroenterology, 83 (1982) 28-35. 16 Oka, Y., Ghishan, F.K., Greene, H.L. and Orth, D.N., Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine, Endocrinology, 112 (1983) 940-944. 17 O'Loughlin, E.V., Chung, M., Hollenberg, M., Hayden, J., Zahavi, I. and Gall, D.G., Effect of epidermal growth factor on ontogeny of the gastrointestinal tract, Am. J. Physiol., 249 (1985) G674G678. 18 Feldman, E.J., Aures, D. and Grossman, M.I., Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of mouse, Proc. Soc. Exp. Biol. Med., 159 (1978) 40(0402. 19 Dembinski, A.B. and Johnson, L.R., Effect of epidermal growth factor on the development of rat gastric mucosa, Endocrinology, 116 (1985) 90-94. 20 Eastwood, G.L. and Quimby, G.F., Effect of chronic cimetidine ingestion on fundic and antral epithelial proliferation in the rat, Dig. Dis. Sci., 28 (1983) 61-64. 21 Lehy, T., Accary, J.P., Dubrasquet, M. and Lewin, M.J.M., Growth hormone-releasing factor (Somatocrinin) stimulates epithelial cell proliferation in the rat digestive tract, Gastroenterology, 90 (1986) 646-653. 22 Yagil, R., Etzion, Z. and Berlyne, M., Changes in rat milk quantity and quality due to variations in litter size and high ambient temperature, Lab. Anim. Sci., 26 (1976) 33-37. 23 Pollack, P.F., Goda, T., Colony, P.C., Edmonds, J., Thornburg, W., Korc, M. and Koldovsky, O., Effects on enterally fed epidermal growth factor on the small and large intestine of the suckling rat, Regul. Peptides, 17 (1987) 121-132. 24 Schaudies, R.P. and Savage Jr., C.R., Isolation of rat epidermal growth factor (r-EGF): chemical, biological and immunological comparisons with mouse and human EGF, Comp. Biochem. Physiol., 84B (1986) 497-505. 25 Majumdar, A.P.N., Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats, J. Pediatr. Gastroenterol. Nutr., 3 (1984) 618-625. 26 Morisset, J., Korc, M. and Larose, L., Antagonistic and additive trophic effects of caerulein and EGF in rat pancreas, Gastroenterology, 90 (1986) 1557 (abstract). 27 Logsdon, C.D., Stimulation of pancreatic acinar cell growth by CCK, epidermal growth factor, and insulin in vitro, Am. J. Physiol., 251 (1986) G487-G494. 28 Johnson, L.R., Guthrie, P.D. and Dudrick, S.J., Effects of luminal gastrin on the growth of rat intestinal mucosa, Gastroenterology, 81 (1981) 71-77. 29 Popliker, M., Shatz, A., Avini, A., Ullrich, A., Schlessinger, J. and Webb, C.G., Onset of endogenous synthesis of epidermal growth factor in neonatal mice, Dev. Biol., 119 (1987) 38-44. 30 Korc, M., Matrisian, L., Planck, S.R. and Magun, B.E., Binding of epidermal growth factor in rat pancreatic acini. Biochem. Biophys. Res. Commun., 111 (1983) 1066-1073.
64 31 Thompson, J.F., Evidence for specific epidermal growth factor (EGF) receptors on fetal, suckling and adult rat intestinal microvillus membranes, Gastroenterology, 90 (1986) 1664 (abstract). 32 Chabot, J.G., Walker, P. and Pelletier, G., Demonstration epidermal growth factor binding sites in the adult rat small intestine by autoradiography, Can. J. Physiol, Pharmacol., 65 (1987) 109-112. 33 Rao, R.K., Thornburg, W., Korc, M., Matrisian, L,M., Magun, B.E. and Koldovsky, O., Processing of epidermal growth factor by suckling and adult rat intestinal cells, Am. J. Physiol., 250 (1986) G850-G855. 34 Gallo-Payet, N. and Hugon, J.S., Epidermal growth factor receptors in isolated adult mouse intestinal cells: studies in vivo and in organ culture, Endocrinology, 116 (1985) 194-201. 35 Lehy, T., Puccio, F., Chariot, J. and Labeille, D., Stimulating effect of bombesin on the growth of gastrointestinal tract and pancreas in suckling rats, Gastroenterology, 90 (1986) 1942-1949.