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Influences of dexamethasone on the maturation of fetal mouse intestinal mucosa in organ culture
Camp. Eiochem. Physiol. Vol. 82A, No. I, pp. 91-95, 1985 Printed in Great Britain
0
0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd
INFLUENCES OF DEXAMETHASONE ON THE MATURATION OF FETAL MOUSE INTESTINAL MUCOSA IN ORGAN CULTURE* Dkpartement
d’anatomie
J.-F. BEAULIEU? and R. CALVERT~ et de biologie cellulaire, FacultC de mtdecine, Sherbrooke, Qutbec, Canada JIH 5N4 (Received 12 December
Universitk de Sherbrooke,
1984)
Abstract-l. The effects of glucocorticoids on the maturation of the fetal small been studied using duodenal explants resected at 17 days of gestation and cultured in the presence or absence of dexamethasone (30-300 ng/ml). 2. Dexamethasone (a) increases specifically alkaline phosphatase, maltase, activities and (b) allows an accumulation of goblet cells along the villi at a faster
intestinal mucosa have in a serum-free medium trehalase and sucrase rate than that occuring
in utero. 3. These results indicate that glucocorticoids influence directly the differentiation of absorptive cells and
goblet cells in the small intestine during the fetal period.
INTRODUCTION
MATERIALS
AND METHODS
Culture method
The modulatory influences of hormones on the maturation of the small intestine of rodents during the perinatal period are well established by means of in view studies (for review see Moog, 1981; Koldovsky, 1981). Nevertheless, for most hormones we still do not know if they act directly on the intestinal mucosa or not. During the past few years the introduction of organ culture methods has permitted the study of morphogenesis and maturation of the small intestine of rodents with completely synthetic media (DeRitis et al., 1975; Calvert and Micheletti, 1981; Beaulieu and Calvert, 1981, 1984b). When RPM1 medium is supplemented with 10% heat inactivated fetal calf serum, dexamethasone (a glucocorticoid analog) can induce some brush border enzymic activities (SimonAssmann et al., 1982). In the absence of serum, the same authors observed that L-triiodothyronine has no direct effect on several brush border enzymes (Simon-Assmann et al., 1984b). We have shown recently that the fetal mouse small intestine at 17 days of gestation can be cultured for at least 48 hr with Leibovitz L-15 medium without serum (Beaulieu and Calvert, 1984a). With this method preliminary observations revealed that dexamethasone had a direct effect on the maturation of the fetal intestinal mucosa. The aim of the present study was to complement and further analyse the influences of dexamethasone on the maturation of the fetal small intestine in organ culture. Effects on goblet cells and on the fine structural and biochemical differentiation of absorptive cells are reported.
Timed pregnant Swiss ICR mice at 17, 18 and 19 days of gestation were anesthetized with ether under sterile conditions. Fetuses were removed by laparotomy and decapitated. The proximal 2 cm of their small intestine was resected, cut-in two sections and each of them was placed into a Petri dish containing Leibovitz L- 15 medium without serum. Pancreatic tissue was carefully removed. The tubular explants were cut into large rings (5 mm), opened lengthwise and cultured as previously described (Beaulieu and Calvert, 1984a) under a carboxygen atmosphere (95% 0,/5x CO,) at 37°C. Dexamethasone (Dx) (Sigma) was added to the culture medium at concentrations of 3, 30 and 300ng/ml (7.6 x 10-9-7.6 x lo-‘M). The optimal effect on the induction of brush border enzymes was comparable with 30 and 300 ng/ml; the morphological preservation of the tissues was greatly improved with the higher dose. Consequently, the study was done with 300ng/ml. Morphological studies
For base line studies, duodenal segments from 17-19-days mouse fetuses were fixed. Furthermore, duodenal explants from 17-day mouse fetuses cultured for 6 hr and 48 hr with or without Dx were also fixed for morphological observations. The tissues were fixed in ice-cold 2.8% glutaraldehvde buffered with 0.1 M cacodvlate for 1 hr. washed in cacodylate buffer 0.2 M for 20 min, post-fixed in 2”/, osmium tetroxide for 1 hr, dehydrated at room temperature and embedded in Epon. Thick sections were stained with aldehyde fuchsin (Calvert and Pusterla, 1974) and thin sections were contrasted with uranyl acetate and lead citrate. Biochemical determinations
The behavior of brush border enzymic activities was evaluated in duodenal explants of 17-day mouse fetuses cultured for 48 hr with or without Dx. The results were compared to enzymic activities in duodenal explants of 17and 19-day mouse fetus cultured for 6 hr without Dx. The explants were carefully washed with the culture medium, pooled (for each assay, the proximal 2 cm of two small intestines were divided into four culture dishes), weighed, and homogenized into ice-cold redistilled water
*This work was supported by a research grant from the Medical Research Council of Canada, No. MT6069. tJ.-F. Beaulieu is the recipient of a studentship from the “Conseil de la Recherche en Santi: du Q&be?. $To whom request for reprints should be sent. 91
J.-F. BEAULIEUand R. CALVERT
92
with a Polytron PT-7 (Brickman Instruments Inc.. Rexdale, Ontario) during 30 set at half speed. Lactase, maltase, sucrase and trehalase activities were assayed according to a modification bv, Llovd . , of Dahlavist’s . and Whelan (1969) method (1964) and alkaline phosphatase activity was measured according to Eichholz (1967) in homogenates and in corresponding media. Determinations were done over a blank homogenate when enzyme activities were low and over a blank medium for disaccharidase activities in the media since glucose can be released during the culture. Protein content was measured by the method of Lowry ef a/. (1951).The results are reported as mean + SEM and the statistical significance of the difference of the means was determined by the Student’s f-test. RESULTS
parable to that observed at 19 days of gestation in utero (Fig. lc). With the light microscope, the number of goblet cells per 100 epithelial cells was recorded at 17-19 days of gestation in the duodenum in utero and in duodenal explants cultured for 48 hr with or without Dx (Fig. 2). In viva, the percentage of goblet cells increases during the last two days of gestation (P < 0.0005). In explants cultured for 48 hr without Dx, the percentage of goblet cells is not significantly different from that observed at 17 days of gestation. However, in the presence of Dx, the percentage of goblet cells in the duodenal explants increases significantly, at a level largely exceeding that observed at 19 days of gestation (P < 0.0005).
Morphological observations
Biochemical determinations
At the fine structural level, the addition of Dx to the culture medium does not seem to induce important morphological modifications to the intestinal epithelial cells; in fact only the striated border is slightly affected. After 48 hr of culture microvilli on absorptive cells of explants cultured without Dx are shorter (Fig. la) than those of explants cultured with Dx (Fig. lb). However, even the presence of Dx is not sufficient to induce a development of microvilli com-
Protein contents and specific activities of alkaline phosphatase (ALP), maltase (M), lactase (L), trehalase (T) and sucrase (S) were assayed in 17-day explants cultured for 48 hr with and without Dx and the results were compared to those measured in duodenal segments resected at 17 and 19 days of gestation and cultured for 6 hr (Fig. 3). During the 4%hr culture period, in explants cultured without Dx, the specific activities of M and S remain stable while ALP and T activities decreasing slightly (P < 0.025) and that of L falls significantly (P < 0.0005). The specific activities of all five enzymes measured in explants cultured in the presence of Dx are significantly higher (P < 0.0005) than those observed in explants cultured without Dx after 48 hr; on the other hand when compared to the levels of enzymic activity measured after 6 hr of culture, only ALP, M, T and S specific activities have increased significantly during the 4%hr treatment with Dx (increases of 1.7, 3.1,2.7 and 2.1 respectively). Finally, when compared to the values obtained in duodenal segments at 19 days of gestation, the specific activities of ALP, M and S are significantly higher in duodenal explants cultured during 48 hr in the presence of Dx (P < 0.025). If the enzymic activities are expressed as mU/culture (Table 1) instead of as IU/g protein (Fig. 3) similar observations are made; ALP, M, T and S activities are significantly higher in explants cultured with Dx when compared to values measured at 17
GOSLET
CEFLS
atl
% 4.0
17
Fig. 1. Fine structure of the apical portion of absorptive cells located on the villi of duodenal explants resected at 17 days of gestation cultured during 48 hr with L-15 medium in absence (A) or in presence of Dx (B) and at 19 days of gestation in utero (C). (x 12,092).
18
Fig. 2. Percentages of goblet cells determined in utero between 17 and 19 days of gestation (-) and in 17-day explants cultured during 48 hr in absence (C) or in presence of Dx (Dx). Level of significance between Dx and C. 17. 18 and 19: P < 0.0005, iV > 6.
Effect of Dx on fetal intestine Table I. Protein contents and enzymic activities in explants? ______~..._ Protein Alkaline phosphatase Maltase Lactase Trehalase Sucrase
C
C, 1.63 + 0.07 346.0 k 29.01 48.52 k 3.16 57.23 f 5.62 0.67 & 0.06 0.20 * 0.04
216.1 rt 14.2 624.4 + 60.8. 37.92 k4.18 153.27 t 14.59” 18.50 + 1.63 45.26 + 4.41 0.35 2 0.04 1.92+0.21* 0. II it 0.02 0.33 i 0.06’
*Significantly increased from C,. P <: 0.05. iProtein contents (mg/culture) and enzymic activities (mU/culture) m 17-day explants cultured during 6 hr (C,) or during 48 hr in the absence (C) or presence of Dx (Dx). $Mean + SEM, N z 10.
days of gestation (P < 0.05). In the same conditions, however, L activity remains stable. Finally, to ensure that the increases of specific enzymic activities caused by the treatment are not artificially produced by a reduction of the cell mass, mean protein contents measured in explants are also reported in Table 1. After 48 hr of culture, protein contents in control explants are significantly reduced (P < 0.0005) while in explants cultured with Dx, they remain comparable to those measured at the onset of the culture period. Table 2 shows the percentages of enzymic activities remaining in the tissues after 48 hr of culture. In the control group cultured without Dx, 5&65x of the enzymic activities are found in the explants while in the Dx-treated group for all the enzymes, except for S, over 80% of the activities remained in the explants. DISCUSSION
It has already been reported that the addition of Dx to a completely synthetic medium influences positively the morphological preservation of duodenal villi in organ culture (Beaulieu and Calvert, 1984a). This improvement in the preservation of the integrity of the intestinal explants by glucocorticoids
A
0 iO0
ALKALINE
MALTA%
Tabie 2. Enzymic activities remaining in explantst
DX
I .08 2 0.07 __--1.55 f 0.09
PHOSPHATASE
93
Alkaline phosphataseg Malta@ Lactase$ Trehataseji Sucrase//
C
Dx
57. I I + 2.542 58.26 t 2.51 51.48k5.39 45.39 * I. 10 64.40 If: 14.87
85.25 i I .32* 85.61 + 1.49’ 79.32+ 1.81* 88.14+2.31* 69.20 _t 7.98
*P -co.01
between C and Dx. tpercentages of activities measured in tissue (Tissue x IOO/Tissue + medium) cultured 48 hr in absence (C) or in presence of Dx (Dx). IMean i SEM. &V2 IO for each group. j/N 3 5.
has also been observed by others (Black, 1978; Kedinger et a/., 1980; Scott and Peters, 1983). Herein we have shown that this protective effect on the morphology is supported by biochemical evidence also: in the Dx-treated group less than 20% of the enzymic activities are released in the media during the 48 hr culture period whereas over 35”/, are released by the non-treated explants during the same period. These releases could result (1) from the cell lost by the villi since these structures are longer in the treated group (Beaulieu and Caivert, 1984a), (2) from the fo~ation of microvesicles produced by a partiai fra~entation of microvilli (Black et a(., IPSO), and/or (3) from the inherent solubility of the brush border membrane enzymes, already present at this stage (Beaulieu et al., 1984). Another important direct influence of Dx on the fetal duodenal mucosa in organ culture is observed at the level of brush border enzymic activities. Dx induces increases in ALP, M, T and S activities, expressed either as specific activity or as total activities. This effect is specific since L activity does not increase in the same experimental conditions. The modulatory effect of glucocorticoids on the maturation of maltase and sucrase during the prenatal development has been established by means of in uiuo
’
1 LACTASE
’
) TREHALASE
“i,
Fig. 3. Specific activities (W/g protein k SEM) of alkaline phosphatase (A), maltase (B), lactase (C), trehalase (D) and sucrase (E) assayed in 17-day duodenal explants cultured during 48 hr in absence (C) or in presence of Dx (Dx). Control explants were taken at 17 and 19 days of gestation and cultured only during 6 hr, N > 10.
94
J.-F. BEAULIEUand R.
studies (Jumawan et al., 1977; Celano et al., 1977) and also in organ culture using a medium supplemented with fetal calf serum (Simon-Assmann et al., 1982). Here we show that this effect of Dx is direct and specific since the presence of other factor(s) found in utero or in fetal calf serum is not required. It is interesting to note that although trehalase activity is very low in the fetal mouse small intestine (Calvert et al., 1982) this enzymic activity can be increased by glucocorticoids. It is already known that glucocorticoids have a similar effect during the suckling period in the mouse (Arsenault and Menard, 1984). During the suckling period in the rat a single injection of hydrocortisone does not affect the cells located on the villi (Henning et al., 1975; Menard and Malo, 1979); nevertheless an increase in sucrase activity was noticed 48 hr after the injection. These authors concluded that the action of glucocorticoids on enterocytes occurs only when the cells are in the proliferative phase. We have shown by autoradiography that the proliferative area in mouse fetal duodenum is restricted to the intervillous area and to the lower part of the villi (Beaulieu and Calvert, 1984a). Hence, although a 20% decrease in the length of the villi is observed in the Dx-treated group during the culture period (Beaulieu and Calvert, 1984a) it is not unexpected that Dx can still act on the explants because the proliferative area is well maintained in culture. The effect of Dx on the level of lactase and alkaline phosphatase of the fetal mouse small intestine in culture reported above is at variance with the observations made with the same hormone in the fetal rat (Simon-Assmann et al., 1982): in the latter when Dx is added to the culture medium, lactase activity increases to a level comparable to that measured in utero while alkaline phosphatase activity remains stable. The absence of fetal calf serum in our culture medium, the use of Leiboritz L- 15 medium instead of RPM1 medium and the fact that we have measured the enzymic activities in total homogenates instead of in brush border fractions might explain these discrepancies. Moreover, there could be some species differences between mouse and rat. It has been recently suggested that in the fetal rat small intestine the mechanism by which Dx induces lactase activity takes place in part at the transcriptional level while the absence of direct effect of Dx on alkaline phosphate could be explained by the existence of an actinomycin D-sensitive repressor (Simon-Assmann et ul., 1984a). In the fetal mouse, the lack of induction of lactase activity by Dx remains to be elucidated but it is obvious that the mechanism which regulates the expression of alkaline phosphatase activity is distinct from that postulated for the rat. Finally, the influences of glucocorticoids on the maturation of the fetal intestinal mucosa are not restricted to absorptive cells since with Dx in the medium, goblet cells accumulate on the duodenal villi at a faster rate than in utero during the same period of time. A stimulation of goblet cell proliferation by Dx is doubtful since this hormone reduces intestinal epithelial cells proliferation in organ culture (Beaulieu and Calvert, 1984a). Possible explanations could be that Dx acts by stimulating the maturation of
CALVERT
undifferentiated or poorly differentiated precursors or that it reduces the secretion of mature goblet cells. It is pertinent to note here that in chick embryo duodenal explants, hydrocortisone prevents an increase in goblet cell number which is normally observed in non-treated explants (Black and Moog, 1977). We have shown herein that glucocorticoids have specific and direct influences on the maturation of fetal intestinal absorptive cells. Furthermore, these influences are not confined to absorptive cells only since the maturation of goblet cells is also positively influenced. Acknowledgements-We are grateful to MS Anne Vezina for her expert technical assistance and to Mr Robert Dumont for the photomicrography.
REFERENCES Arsenault P. and Menard D. (1984) Comparative study oi the effect of hydrocortisone and thyroxine on suckling mouse small intestine in organ culture. Comp. Biochem. Physiol. 77A, 121-125. Beaulieu J.-F. and Calvert R. (198 1) The effect of epidermal growth factor (EGF) on the differentiation of the rough endoplasmic reticulum in fetal mouse small intestine in organ culture. J. Histochem. Cytochem. 29, 765-770. Beaulieu J.-F. and Calvert R. (1984a) Effect of dexamethasone on the fetal mouse small intestine in organ culture. Anat. Rec. 210, 61-71. Beaulieu J.-F. and Calvert R. (1984b) Maturation of the fetal small intestinal mucosa. Self-differentiation and modulation by dexamethasone in organ culture. J. Steroid Biochem. 20, 1424. Beaulieu J.-F., Minard D. and Calvert R. (1984) Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse. Camp. Biochem. Physiol. IIA, 261-265. Black B. L. (1978) Morphological development of the epithelium of the embryonic chick intestine in culture: Influence of thyroxine and hydrocortisone. Am. J. Anat. 153, 573-600.
Black B. L. and Moog F. (1977) Goblet cells in embryonic intestine: accelerated differentiation in culture. Science 197, 368-370. Black B. L., Yoneyama Y. and Moog F. (1980) Microvillous membrane vesicle accumulation in media during culture of intestine of chick embryo. Biochim. biophys. Acfa 601, 343-348.
Calvert R., Beaulieu J.-F. and Menard D. (1982) Epidermal growth factor (EGF) accelerates the maturation of fetal mouse intestinal mucosa in utero. Experientia 38, 1096-1097. Calvert R. and Micheletti P. (198 1) Selection of a chemically defined medium for culturing fetal mouse small intestine. In Vitro 17, 331-344. Calvert R. and Pusterla A. (1974) Epon-embedded tissue stained with aldehyde-fuchsin for autoradiography. Stain Technol. 49, 323-327.
Celano P., Jumawan J., Horowitz C., Law H. and Koldovsky 0. (1977) Prenatal induction of sucrase activity in rat jejunum. Biochem. J. 162, 469-472. Dahlqvist A. (1964) Method for assay of intestinal disaccharidases. Anal. Biochem. 7, 8-25. DeRitis G., Falchuk Z. M. and Trier J. S. (1975) Differentiation and maturation of cultured fetal rat jejunum. Dev. Biol. 45, 304-317. Eichholz A. (1967) Structural and functional organization
Effect of Dx on fetal intestine of the brush border of intestinal epithelial cells. III. Enzyme activities and chemical composition of various fractions of tris-disrupted brush borders. Eiochim. biophys. Acta 135, 415482. Henning S. J., Helman T. A. and Kretchmer N. (1975) Studies on normal and precocious appearance of jejunal sucrase in suckling rats. Biol. Neonate 26, 249-262. Jumawan J., Celano P., Horowitz C., Law H. and Koldovsky 0. (1977) Effect of cortisone or L-triiodothyronine administration to pregnant rat on the activity of fetal intestinal disaccharidases and lysosomal acid /?-galactosidase. Biol. Neonate 32, 21 I-217. Kedineer M.. Simon P. M.. Raul F.. Grenier J. F. and Ha&n K. ‘(1980) The effect of dexamethasone on the development of rat intestinal brush border enzymes in organ culture. Dev. Biol. 74, 9-21. Koldovsky 0. (1981) Developmental, dietary and hormonal control of intestinal disaccharidases in mammals (including man). In Carbohydrate Metabolism and its Disorders (Edited bv Randle P. J.. Steiner D. F. and Whelan W. J.). ” vol. 3, pp. 481-522. Academic Press, London. Lloyd S. and Whelan W. (1969) An improved method for enzymatic determination of glucose in the presence of maltose. Anal. Biochem. 30, 467-470. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall
95
R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Mtnard D. and Malo C. (1979) Insulin-evoked precocious appearance of intestinal sucrase activity in suckling mice. Dev. Biol. 69, 661-665.
Moog F. (198 1) Perinatal development of the enzymes of the brush border membrane. In Textbook ofGastroenterology and Nutrition in Infancy (Edited by Lebenthal, E.), pp. 139-147. Raven Press, New York. Scott J. and Peters T. J. (1983) Protection of epithelial function in human jejunum cultured with hydrocortisone. Am. J. Physiol. 244, G532-G540.
Simon-Assmann P. M., Kedinger M., Grenier J. F. and Haffen K. (1982) Control of brush border enzymes by dexamethasone in the fetal rat intestine cultured in vitro. J. Pediatr. Gastroenterol. Nutr. 1, 257-265.
Simon-Assmann P., Kedinger M., Grenier J. F. and Haffen K. (1984a) Organ culture of fetal rat intestine. Effects on brush borders enzyme activities of the combined administration of dexamethasone and cycloheximide or actinomycin D. Enzyme 31, 65-72. Simon-Assmann P., Kedinger M. and Haffen K. (1984b) Ability of L-triidothyronine to modulate glucocorticoid evoked brush border enzyme activities in cultured fetal rat intestine. Cell Biol. Int. Rep 8, 41-45.
0
0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd
INFLUENCES OF DEXAMETHASONE ON THE MATURATION OF FETAL MOUSE INTESTINAL MUCOSA IN ORGAN CULTURE* Dkpartement
d’anatomie
J.-F. BEAULIEU? and R. CALVERT~ et de biologie cellulaire, FacultC de mtdecine, Sherbrooke, Qutbec, Canada JIH 5N4 (Received 12 December
Universitk de Sherbrooke,
1984)
Abstract-l. The effects of glucocorticoids on the maturation of the fetal small been studied using duodenal explants resected at 17 days of gestation and cultured in the presence or absence of dexamethasone (30-300 ng/ml). 2. Dexamethasone (a) increases specifically alkaline phosphatase, maltase, activities and (b) allows an accumulation of goblet cells along the villi at a faster
intestinal mucosa have in a serum-free medium trehalase and sucrase rate than that occuring
in utero. 3. These results indicate that glucocorticoids influence directly the differentiation of absorptive cells and
goblet cells in the small intestine during the fetal period.
INTRODUCTION
MATERIALS
AND METHODS
Culture method
The modulatory influences of hormones on the maturation of the small intestine of rodents during the perinatal period are well established by means of in view studies (for review see Moog, 1981; Koldovsky, 1981). Nevertheless, for most hormones we still do not know if they act directly on the intestinal mucosa or not. During the past few years the introduction of organ culture methods has permitted the study of morphogenesis and maturation of the small intestine of rodents with completely synthetic media (DeRitis et al., 1975; Calvert and Micheletti, 1981; Beaulieu and Calvert, 1981, 1984b). When RPM1 medium is supplemented with 10% heat inactivated fetal calf serum, dexamethasone (a glucocorticoid analog) can induce some brush border enzymic activities (SimonAssmann et al., 1982). In the absence of serum, the same authors observed that L-triiodothyronine has no direct effect on several brush border enzymes (Simon-Assmann et al., 1984b). We have shown recently that the fetal mouse small intestine at 17 days of gestation can be cultured for at least 48 hr with Leibovitz L-15 medium without serum (Beaulieu and Calvert, 1984a). With this method preliminary observations revealed that dexamethasone had a direct effect on the maturation of the fetal intestinal mucosa. The aim of the present study was to complement and further analyse the influences of dexamethasone on the maturation of the fetal small intestine in organ culture. Effects on goblet cells and on the fine structural and biochemical differentiation of absorptive cells are reported.
Timed pregnant Swiss ICR mice at 17, 18 and 19 days of gestation were anesthetized with ether under sterile conditions. Fetuses were removed by laparotomy and decapitated. The proximal 2 cm of their small intestine was resected, cut-in two sections and each of them was placed into a Petri dish containing Leibovitz L- 15 medium without serum. Pancreatic tissue was carefully removed. The tubular explants were cut into large rings (5 mm), opened lengthwise and cultured as previously described (Beaulieu and Calvert, 1984a) under a carboxygen atmosphere (95% 0,/5x CO,) at 37°C. Dexamethasone (Dx) (Sigma) was added to the culture medium at concentrations of 3, 30 and 300ng/ml (7.6 x 10-9-7.6 x lo-‘M). The optimal effect on the induction of brush border enzymes was comparable with 30 and 300 ng/ml; the morphological preservation of the tissues was greatly improved with the higher dose. Consequently, the study was done with 300ng/ml. Morphological studies
For base line studies, duodenal segments from 17-19-days mouse fetuses were fixed. Furthermore, duodenal explants from 17-day mouse fetuses cultured for 6 hr and 48 hr with or without Dx were also fixed for morphological observations. The tissues were fixed in ice-cold 2.8% glutaraldehvde buffered with 0.1 M cacodvlate for 1 hr. washed in cacodylate buffer 0.2 M for 20 min, post-fixed in 2”/, osmium tetroxide for 1 hr, dehydrated at room temperature and embedded in Epon. Thick sections were stained with aldehyde fuchsin (Calvert and Pusterla, 1974) and thin sections were contrasted with uranyl acetate and lead citrate. Biochemical determinations
The behavior of brush border enzymic activities was evaluated in duodenal explants of 17-day mouse fetuses cultured for 48 hr with or without Dx. The results were compared to enzymic activities in duodenal explants of 17and 19-day mouse fetus cultured for 6 hr without Dx. The explants were carefully washed with the culture medium, pooled (for each assay, the proximal 2 cm of two small intestines were divided into four culture dishes), weighed, and homogenized into ice-cold redistilled water
*This work was supported by a research grant from the Medical Research Council of Canada, No. MT6069. tJ.-F. Beaulieu is the recipient of a studentship from the “Conseil de la Recherche en Santi: du Q&be?. $To whom request for reprints should be sent. 91
J.-F. BEAULIEUand R. CALVERT
92
with a Polytron PT-7 (Brickman Instruments Inc.. Rexdale, Ontario) during 30 set at half speed. Lactase, maltase, sucrase and trehalase activities were assayed according to a modification bv, Llovd . , of Dahlavist’s . and Whelan (1969) method (1964) and alkaline phosphatase activity was measured according to Eichholz (1967) in homogenates and in corresponding media. Determinations were done over a blank homogenate when enzyme activities were low and over a blank medium for disaccharidase activities in the media since glucose can be released during the culture. Protein content was measured by the method of Lowry ef a/. (1951).The results are reported as mean + SEM and the statistical significance of the difference of the means was determined by the Student’s f-test. RESULTS
parable to that observed at 19 days of gestation in utero (Fig. lc). With the light microscope, the number of goblet cells per 100 epithelial cells was recorded at 17-19 days of gestation in the duodenum in utero and in duodenal explants cultured for 48 hr with or without Dx (Fig. 2). In viva, the percentage of goblet cells increases during the last two days of gestation (P < 0.0005). In explants cultured for 48 hr without Dx, the percentage of goblet cells is not significantly different from that observed at 17 days of gestation. However, in the presence of Dx, the percentage of goblet cells in the duodenal explants increases significantly, at a level largely exceeding that observed at 19 days of gestation (P < 0.0005).
Morphological observations
Biochemical determinations
At the fine structural level, the addition of Dx to the culture medium does not seem to induce important morphological modifications to the intestinal epithelial cells; in fact only the striated border is slightly affected. After 48 hr of culture microvilli on absorptive cells of explants cultured without Dx are shorter (Fig. la) than those of explants cultured with Dx (Fig. lb). However, even the presence of Dx is not sufficient to induce a development of microvilli com-
Protein contents and specific activities of alkaline phosphatase (ALP), maltase (M), lactase (L), trehalase (T) and sucrase (S) were assayed in 17-day explants cultured for 48 hr with and without Dx and the results were compared to those measured in duodenal segments resected at 17 and 19 days of gestation and cultured for 6 hr (Fig. 3). During the 4%hr culture period, in explants cultured without Dx, the specific activities of M and S remain stable while ALP and T activities decreasing slightly (P < 0.025) and that of L falls significantly (P < 0.0005). The specific activities of all five enzymes measured in explants cultured in the presence of Dx are significantly higher (P < 0.0005) than those observed in explants cultured without Dx after 48 hr; on the other hand when compared to the levels of enzymic activity measured after 6 hr of culture, only ALP, M, T and S specific activities have increased significantly during the 4%hr treatment with Dx (increases of 1.7, 3.1,2.7 and 2.1 respectively). Finally, when compared to the values obtained in duodenal segments at 19 days of gestation, the specific activities of ALP, M and S are significantly higher in duodenal explants cultured during 48 hr in the presence of Dx (P < 0.025). If the enzymic activities are expressed as mU/culture (Table 1) instead of as IU/g protein (Fig. 3) similar observations are made; ALP, M, T and S activities are significantly higher in explants cultured with Dx when compared to values measured at 17
GOSLET
CEFLS
atl
% 4.0
17
Fig. 1. Fine structure of the apical portion of absorptive cells located on the villi of duodenal explants resected at 17 days of gestation cultured during 48 hr with L-15 medium in absence (A) or in presence of Dx (B) and at 19 days of gestation in utero (C). (x 12,092).
18
Fig. 2. Percentages of goblet cells determined in utero between 17 and 19 days of gestation (-) and in 17-day explants cultured during 48 hr in absence (C) or in presence of Dx (Dx). Level of significance between Dx and C. 17. 18 and 19: P < 0.0005, iV > 6.
Effect of Dx on fetal intestine Table I. Protein contents and enzymic activities in explants? ______~..._ Protein Alkaline phosphatase Maltase Lactase Trehalase Sucrase
C
C, 1.63 + 0.07 346.0 k 29.01 48.52 k 3.16 57.23 f 5.62 0.67 & 0.06 0.20 * 0.04
216.1 rt 14.2 624.4 + 60.8. 37.92 k4.18 153.27 t 14.59” 18.50 + 1.63 45.26 + 4.41 0.35 2 0.04 1.92+0.21* 0. II it 0.02 0.33 i 0.06’
*Significantly increased from C,. P <: 0.05. iProtein contents (mg/culture) and enzymic activities (mU/culture) m 17-day explants cultured during 6 hr (C,) or during 48 hr in the absence (C) or presence of Dx (Dx). $Mean + SEM, N z 10.
days of gestation (P < 0.05). In the same conditions, however, L activity remains stable. Finally, to ensure that the increases of specific enzymic activities caused by the treatment are not artificially produced by a reduction of the cell mass, mean protein contents measured in explants are also reported in Table 1. After 48 hr of culture, protein contents in control explants are significantly reduced (P < 0.0005) while in explants cultured with Dx, they remain comparable to those measured at the onset of the culture period. Table 2 shows the percentages of enzymic activities remaining in the tissues after 48 hr of culture. In the control group cultured without Dx, 5&65x of the enzymic activities are found in the explants while in the Dx-treated group for all the enzymes, except for S, over 80% of the activities remained in the explants. DISCUSSION
It has already been reported that the addition of Dx to a completely synthetic medium influences positively the morphological preservation of duodenal villi in organ culture (Beaulieu and Calvert, 1984a). This improvement in the preservation of the integrity of the intestinal explants by glucocorticoids
A
0 iO0
ALKALINE
MALTA%
Tabie 2. Enzymic activities remaining in explantst
DX
I .08 2 0.07 __--1.55 f 0.09
PHOSPHATASE
93
Alkaline phosphataseg Malta@ Lactase$ Trehataseji Sucrase//
C
Dx
57. I I + 2.542 58.26 t 2.51 51.48k5.39 45.39 * I. 10 64.40 If: 14.87
85.25 i I .32* 85.61 + 1.49’ 79.32+ 1.81* 88.14+2.31* 69.20 _t 7.98
*P -co.01
between C and Dx. tpercentages of activities measured in tissue (Tissue x IOO/Tissue + medium) cultured 48 hr in absence (C) or in presence of Dx (Dx). IMean i SEM. &V2 IO for each group. j/N 3 5.
has also been observed by others (Black, 1978; Kedinger et a/., 1980; Scott and Peters, 1983). Herein we have shown that this protective effect on the morphology is supported by biochemical evidence also: in the Dx-treated group less than 20% of the enzymic activities are released in the media during the 48 hr culture period whereas over 35”/, are released by the non-treated explants during the same period. These releases could result (1) from the cell lost by the villi since these structures are longer in the treated group (Beaulieu and Caivert, 1984a), (2) from the fo~ation of microvesicles produced by a partiai fra~entation of microvilli (Black et a(., IPSO), and/or (3) from the inherent solubility of the brush border membrane enzymes, already present at this stage (Beaulieu et al., 1984). Another important direct influence of Dx on the fetal duodenal mucosa in organ culture is observed at the level of brush border enzymic activities. Dx induces increases in ALP, M, T and S activities, expressed either as specific activity or as total activities. This effect is specific since L activity does not increase in the same experimental conditions. The modulatory effect of glucocorticoids on the maturation of maltase and sucrase during the prenatal development has been established by means of in uiuo
’
1 LACTASE
’
) TREHALASE
“i,
Fig. 3. Specific activities (W/g protein k SEM) of alkaline phosphatase (A), maltase (B), lactase (C), trehalase (D) and sucrase (E) assayed in 17-day duodenal explants cultured during 48 hr in absence (C) or in presence of Dx (Dx). Control explants were taken at 17 and 19 days of gestation and cultured only during 6 hr, N > 10.
94
J.-F. BEAULIEUand R.
studies (Jumawan et al., 1977; Celano et al., 1977) and also in organ culture using a medium supplemented with fetal calf serum (Simon-Assmann et al., 1982). Here we show that this effect of Dx is direct and specific since the presence of other factor(s) found in utero or in fetal calf serum is not required. It is interesting to note that although trehalase activity is very low in the fetal mouse small intestine (Calvert et al., 1982) this enzymic activity can be increased by glucocorticoids. It is already known that glucocorticoids have a similar effect during the suckling period in the mouse (Arsenault and Menard, 1984). During the suckling period in the rat a single injection of hydrocortisone does not affect the cells located on the villi (Henning et al., 1975; Menard and Malo, 1979); nevertheless an increase in sucrase activity was noticed 48 hr after the injection. These authors concluded that the action of glucocorticoids on enterocytes occurs only when the cells are in the proliferative phase. We have shown by autoradiography that the proliferative area in mouse fetal duodenum is restricted to the intervillous area and to the lower part of the villi (Beaulieu and Calvert, 1984a). Hence, although a 20% decrease in the length of the villi is observed in the Dx-treated group during the culture period (Beaulieu and Calvert, 1984a) it is not unexpected that Dx can still act on the explants because the proliferative area is well maintained in culture. The effect of Dx on the level of lactase and alkaline phosphatase of the fetal mouse small intestine in culture reported above is at variance with the observations made with the same hormone in the fetal rat (Simon-Assmann et al., 1982): in the latter when Dx is added to the culture medium, lactase activity increases to a level comparable to that measured in utero while alkaline phosphatase activity remains stable. The absence of fetal calf serum in our culture medium, the use of Leiboritz L- 15 medium instead of RPM1 medium and the fact that we have measured the enzymic activities in total homogenates instead of in brush border fractions might explain these discrepancies. Moreover, there could be some species differences between mouse and rat. It has been recently suggested that in the fetal rat small intestine the mechanism by which Dx induces lactase activity takes place in part at the transcriptional level while the absence of direct effect of Dx on alkaline phosphate could be explained by the existence of an actinomycin D-sensitive repressor (Simon-Assmann et ul., 1984a). In the fetal mouse, the lack of induction of lactase activity by Dx remains to be elucidated but it is obvious that the mechanism which regulates the expression of alkaline phosphatase activity is distinct from that postulated for the rat. Finally, the influences of glucocorticoids on the maturation of the fetal intestinal mucosa are not restricted to absorptive cells since with Dx in the medium, goblet cells accumulate on the duodenal villi at a faster rate than in utero during the same period of time. A stimulation of goblet cell proliferation by Dx is doubtful since this hormone reduces intestinal epithelial cells proliferation in organ culture (Beaulieu and Calvert, 1984a). Possible explanations could be that Dx acts by stimulating the maturation of
CALVERT
undifferentiated or poorly differentiated precursors or that it reduces the secretion of mature goblet cells. It is pertinent to note here that in chick embryo duodenal explants, hydrocortisone prevents an increase in goblet cell number which is normally observed in non-treated explants (Black and Moog, 1977). We have shown herein that glucocorticoids have specific and direct influences on the maturation of fetal intestinal absorptive cells. Furthermore, these influences are not confined to absorptive cells only since the maturation of goblet cells is also positively influenced. Acknowledgements-We are grateful to MS Anne Vezina for her expert technical assistance and to Mr Robert Dumont for the photomicrography.
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Simon-Assmann P. M., Kedinger M., Grenier J. F. and Haffen K. (1982) Control of brush border enzymes by dexamethasone in the fetal rat intestine cultured in vitro. J. Pediatr. Gastroenterol. Nutr. 1, 257-265.
Simon-Assmann P., Kedinger M., Grenier J. F. and Haffen K. (1984a) Organ culture of fetal rat intestine. Effects on brush borders enzyme activities of the combined administration of dexamethasone and cycloheximide or actinomycin D. Enzyme 31, 65-72. Simon-Assmann P., Kedinger M. and Haffen K. (1984b) Ability of L-triidothyronine to modulate glucocorticoid evoked brush border enzyme activities in cultured fetal rat intestine. Cell Biol. Int. Rep 8, 41-45.