J. Embryol. exp. Morph., Vol. 14, Part 2, pp. 169-179, October 1965
Printed in Great Britain
The maturation of cortisone-treated embryonic
duodenum in vitro. II. The striated border
by
R A Y M O N D L. H A Y E S J r . 1
From the Department of Anatomy, Harvard Medical School, Boston
WITH FOUR PLATES
of the morphological and physiological maturation of embryonic
duodenal mucosa by adrenocorticoid hormones has been demonstrated with a
variety of techniques. Single injections of cortisone acetate (Moog & Richardson,
1955; Moog & Thomas, 1957); multiple daily administrations of cortisone
(Moog & Ford, 1957; Moog, 1959); or in vitro maintenance of duodenal
fragments with adrenal steroids (Moog & Nehari, 1954; Moog & Kirsch, 1955)
all accelerate morphogenesis and the onset of alkaline phosphatase activity in
this tissue.
The differentiation of the duodenal mucosa is not only responsive to hormonal
application but is dependent on it. Hinni & Watterson (1963) have ablated
pituitary glands from chick embryos, hence interrupting the hypophysealadrenal axis and arresting adrenal function. Following this procedure, morphogenesis is retarded and alkaline phosphatase activity in the epithelium
diminished. Release from adrenocortical influence does not modify the course
of differentiation, but does affect the rate of cellular maturation.
• Implicit in the hormone-dependent quality of duodenal maturation is the
existence of a delicate balance between the rate of tissue differentiation and the
hormonal exposure. The present investigation has been conducted to examine
this relationship between the concentration of hormone applied to explants in
vitro and the responses induced in the epithelial striated border. This relationship
has been studied in embryos representing a series of incubation ages to observe
differential responsiveness of the tissue when explanted at various stages.
REGULATION
MATERIALS AND METHODS
Duodenal loops from White Leghorn chicken embryos of stages 37-45
(Hamburger & Hamilton, 1951) were removed aseptically and minced into
fragments 1-2 mm 3 in size. These explants were maintained as organ cultures
Author's address: Department of Anatomy and Cell Biology, University of Pittsburgh,
School of Medicine, Pittsburgh, Pennsylvania 15213, U.S.A.
170
R. L. HAYES, JR.
aboard a Millipore filter disc supported by a stainless steel grid (after Trowell,
1959). The nutritive medium was #199 (Morgan, Morton & Parker, 1950)
supplemented with 10 per cent, calf serum. Cortisone acetate was added in the
following concentrations: 0-01, 0-025, 0-05, 0-075, 0 1 , 0-25, 0-5, and 1-0
/xg./ml.
All explants were fixed after 48 hr. of incubation at 37° C. They, as well as
the unincubated control tissues, were fixed in 2 per cent, glutaraldehyde buffered
with sodium cacodylate (280-320 mOs; pH 7-2-7-6) for 1 hr. at 4° C. and subsequently washed overnight in an isotonic solution of sucrose in cacodylate buffer
(Gordon et al. 1963). For detection of alkaline phosphatase activity, the fixed
tissues were incubated in toto for 1 hr. at 37° C. in the presence of sodium
j8-glycerophosphate at pH 9 • 4. They then were reacted according to Gomori
(1941) to produce a visible precipitate of cobalt sulphide. Sample tissues were
incubated in media without substrate (glycerophosphate) to test for specificity
of the reaction.
Quantification of enzyme activity was achieved by a procedure reported by
Doyle (1950). After wet weights were obtained, controls and cultures were
reacted for alkaline phosphatase by a modified Gomori procedure to yield a
deposit of lead sulphide at sites of enzymatic activity. This deposit was extracted
and reacted with N,N-dimethyl-p-phenylenediamine sulphate to synthesize
methylene blue. After oxidation of this solution with ferric chloride, the content
of methylene blue was determined by spectrophotometric transmission at a wave
length of 670 m/x. Conversion of these optical readings to values per milligram
of tissue yielded a series of quantitative estimates of enzyme activity (Table).
Tissues were prepared for histological observation by post-fixation in 1 per
cent, osmium tetroxide in cacodylate buffer for 1 hr. at 4° C. Following alcoholic
dehydration, they were embedded in Epon 812 (Luft, 1961) and sectioned on a
Porter-Blum microtome. For light microscopic observation, 1 • 5 [x thick sections
were visualized by staining with 1 per cent, toluidine blue. Thin sections for
electron microscopy were cut from the same blocks and stained with lead citrate
(Venable & Coggeshall, 1965).
RESULTS
The localization of alkaline phosphatase activity
Inspection of thick sections of explants treated for alkaline phosphatase by
the Gomori technique reveals no reaction product in 11-, 13- and 15-day control
tissues. When cultivated without hormone, no change in activity is observed
in these tissues. A positive reaction, however, is observed in 17-day controls.
Also, the 19-day control is positive (Plate 2, Figs. E & F) and although this
activity diminishes during cultivation without hormone, the reaction is not lost
(Plate 2, Fig. G).
Addition of cortisone to the nutritive medium does not induce a positive
108
2-59
105
3-05
2-58
1-79
1-28
0-57
2-33
0
001
0-5
10
0-25
01
0 075
005
0 025
tissue
weight
(mg.)
Concentration
of cortisone
(ixg./ml.)
1
c
660
49 0
49 0
69 0
63 0
51-5
650
49-5
52-5
22-5
870
46-6
22-5
35-4
28-7
50-9
610
18-9
% transmission % trans 1
(670 mix)
mg.
13 Days
c
16
•50
•55
()-95
107
1-57
()-30
•22
1-54
920
69-5
95-5
660
90-5
75-5
93 0
67-5
950
tissue % transweight mission
(mg.) (670 mil)
15 Days
790
46-5
61 -5
69-5
850
480
3100
550
620
%transl
mg.
1 -63
1-22
0-38
0-39
0-90
018
0-49
0-45
0-76
tissue
weight
(mg.)
77-0
700
74-5
740
73-0
75-5
760
73-5
730
960
420-0
1550
1640
800
47-2
57-3
I960
1900
% transmission % trans1
(670 mil)
mg.
17 Days
Incubation Age of Embryonic Tissue
Results of Doyle technique for quantification of alkaline phosphatase activity
TABLE
0-77
1-59
1-26
100
108
0-91
1-22
0-89
1-61
tissue
weight
(mg.)
63 0
109-5
112-5
520
56-5
770
750
830
830
820
69 0
89-5
520
520
84-5
61 -5
89-5
51-5
% transmission % trans 1
mg.
(670 mix)
19 Days
I-
172
R. L. HAYES, JR.
reaction for alkaline phosphatase in the 11-day culture (Plate 1, Fig. A), but does
yield a reaction in the 13- and 15-day explants (Plate 1, Figs. B & C). The 17-day
tissues continue to demonstrate positive activity after hormone treatment
(Plate 1, Fig. D). Also, the 19-day tissue treated with hormone is positive for
enzymatic activity (Plate 2, Figs. H & I).
No precipitate is found on those tissues incubated in medium lacking j8-glycerophosphate but otherwise treated by the Gomori method. This is true even when
controls or cultures of the same age are positive for alkaline phosphatase.
Where a positive reaction is obtained, the precipitated sulphide is located
invariably on the luminal surface of the duodenal epithelium. This deposit is
scattered irregularly over the surface of villi on isolated cells as well as small
groups of cells. Specifically, the reaction product is localized on the microvillous
membrane and along the apical cell membrane separating microvilli (Plate 3,
Figs. J & K). Negatively-staining cells are found adjacent to positive ones, in
which case the reaction product ends abruptly at the intercellular junction (Plate
3, Fig. J). Whenever the microvilli are positive, the apical cell membrane is also.
Quantification of alkaline phosphatase activity in embryonic duodenal tissue
Results obtained from application of the Doyle procedure for quantifying
enzyme activity are presented in the Table. Spectrophotometric readings were
corrected for variation in luminal surface area of duodenal explants by conversion to values per milligram of tissue. These values are significant within any
one age group but are not comparable between age groups because of differences
in thickness of the duodenal wall.
Alkaline phosphatase activity reaches a maximum at a definite hormonal
concentration regardless of tissue age (Text-fig. 1). Increasing cortisone levels
beyond this optimal concentration reduces the enzymatic response of the tissue.
Moreover, the amount of cortisone necessary to induce maximal activity in
duodenal explants decreases with increasing incubation age of the tissue. In
other words, the older the tissue, the greater its sensitivity to the hormone
(Text-fig. 2).
Morphological alterations in the striated border
The only dramatic change in morphology of the striated border occurs in the
19-day tissue following hormonal exposure. Compared to that of the untreated
19-day culture (Plate 2, Fig. G), the striated border of the hormone-treated
epithelium is higher (Plate 2, Figs. H & I). Electron micrographs of this cortisonetreated epithelium reveal that the apparent increase in height of the border is
due to the apposition of a layer of vesicles onto the microvilli (Plate 3, Fig. M;
Plate 4). The thickness of the vesicular layer is 2-3 times the height of the
microvilli (Plate 4, Fig. N). The combined height of the vesicular and micro-
J. Embryo!, e.xp. Murph.
Vol. 14, Part 2
PLATE 1
Hormone-treated cultures reacted for alkaline phosphatase by the Gomori technique.
FIG. A. 11-day duodenum cultivated with 0-25 jixg./ml. cortisone. No visible enzymatic
activity is observed. Toluidine blue, x 1400.
FIG. B. 13-day duodenum cultivated with 0-5 /ng./ml. of cortisone. An irregular phosphatase
reaction is localized on the lateral sides of villi. x 1400.
FIG. C. 15-day duodenum cultivated with 0-5 /Ag./ml. of cortisone. Alkaline phosphatase
activity is shown on sides of villi. x 2000.
FIG. D. 17-day duodenum cultivated with 0-25 ju.g./ml. of cortisone. Individual cells and
isolated groups of cells stain positively for phosphatase. x 1400.
RAYMOND L. HAYES Jr.
{Facing page 172)
/ . Embryol. exp. Morph.
Vol. 14, Part 2
PLATE 2
19-day controls and cultures stained for alkaline phosphatase.
FIG. E. Control tissue. Striated border (sb) stains positively, x 1400.
FIG. F. Control tissue. Note irregular enzymatic activity, which changes abruptly at intercellular junctions, x1400.
FIG. G. 19-day duodenum cultivated 48 hr. with no hormone, x 1400.
FIG. H. 19-day duodenum cultivated 48 hr. with 0 01 /xg./ml. of cortisone. The striated
border is thicker and stains heavier than in (B) and more cells stain positively, x 1400.
FIG. I. 19-day duodenum cultivated 48 hr. with 0-5 ftg./ml. of cortisone. The striated border
is thicker than in G. x 2000.
RAYMOND L. HAYES Jr.
J. Embryo/, cxp. Morph.
Vol. 14, Part 2
mv
PLATE 3
FIG. J. Electron micrograph of 19-day tissue cultivated for 48 hr. with no hormone and stained
for alkaline phosphatase. Enzyme activity is localized on microvillous membrane and
apical cell membrane between microvilli. Reaction ends at intercellular junction (arrow).
x 24,100.
FIG. K. 19-day explant with 0 1 /xg./ml. of cortisone and stained for alkaline phosphatase.
Note phosphatase-positive vesicles and unstained hirsute coating (he) of membranes.
FIG. L. 19-day control tissue showing absence of vesicular layer on microvilli. x 7000.
FIG. M. 19-day culture exposed to 0-1 /xg./ml. of cortisone. Notice development of terminal
web (tw) and presence of vesicular layer (ves) on luminal surface of microvilli (mv). x 6300.
RAYMOND L. HAYES Jr.
Vol. 14, Part 2
/ . Embryol. exp. Morph.
PLATE 4
Electron micrographs of 19-day explants treated with 0.5 /xg./ml. of cortisone for 48 hr.
FIG. N. Vesicular layer (ves) is 2-3 times thicker than the height of the microvilli. x 12,100.
Insert shows lack of internal structure in vesicles.
FIG. O. Apical extensions or distortions on microvilli might represent developing vesicles
(arrows), x 17,800.
RAYMOND L. HAYES Jr.
{Facing page 173)
Cortisone-treated embryonic duodenum
173
Alkaline phosphatase activity in 48-hour cultures
of embryonic chick duodenum treated with cortisone
15 day
0
0-2
0-4
0-6
0-8
1-0
0
0-2
0-4 0-6
0-8
10
19 day
0
0-2
0-4
0-6
0-8 1-0
0
0-2
Cortisone acetate (y/ml.)
0-4
0-6
0-8
10
1. Alkaline phosphatase activity in the striated border of 48-hr, cultures of embryonic
chick duodenum treated with cortisone. Induction of enzyme activity reaches a maximum
at a definite hormonal concentration. Greatest response to the hormone is shown by 15and 17-day tissues.
TEXT-FIG.
Cortisone inducing maximal alkaline phosphatase activity
embryonic chick duodenum, 48-hour cultures
rtis
100-50-251)
018 0075005co 002500100-
13
15
17
Tissue age (days)
19
2. Cortisone inducing maximal alkaline phosphatase activity in embryonic chick
duodenum maintained in vitro. Tissue sensitivity to the hormone increases as tissue age
increases.
TEXT-FIG.
174
R. L. HAYES, JR.
villous layers is comparable to the height of the border as viewed in light micrographs. The spheroid units comprising the vesicular layer are slightly smaller in
diameter than the microvilli, are membrane-bound, and their external surfaces
are covered with a hirsute coating as characterizes the surface of enteric microvilli
(Ito, 1964). When stained for alkaline phosphatase, these extra-cellular spherules
demonstrate positive enzymatic activity (Plate 3, Fig. K). Both the hirtellous
coat and the phosphatase-positivity suggest that these vesicles are derived from
the cell membrane.
Further indication of the origin of these vesicles is provided in micrographs
showing apical distentions of the microvilli (Plate 4, Fig. O). These protrusions
of the apical membrane of the microvillus suggest that the gemmules are pinched
off from the tips of the microvilli. The surface filamentous coat retains these buds
in contact with one another and with the cell surface. Hence, a definite layering
of these vesicles on the luminal surface of the epithelium results. As hormone
concentration is augmented, the striated border increases in height through a
greater degree of vesiculation. During this process, the microvillus does not
decrease noticeably in height, suggesting a concurrent expansion of the
plasmalemma.
Although no internal structure is observed in the vesicles, there is a definite
increase in filamentous material within the microvillus proper. This change
resulting from hormone application is reflected in the development of the
terminal web region of the apical cell cytoplasm (cf. Plate 3, Figs. L & M).
DISCUSSION
Our data demonstrate a positive effect of cortisone, under the cultivation
conditions chosen, upon the maturation of the striated border of the embryonic
duodenal epithelium. The normal rate of differentiation of these cells in vivo
is approximated in vitro after hormonal administration. Untreated cultures
fail to attain comparable stages of maturation either morphologically or
physiologically.
The onset of functional activity, as evidenced by the accumulation of alkaline
phosphatase in the tissue, is stimulated by cortisone. As originally demonstrated
by Moog & Nehari (1954), this in vitro effect indicates a direct influence of the
hormone on the duodenal tissue. This response reaches maximal levels with
lesser concentrations of hormone as tissue age increases, provided the tissue is
competent to respond. In the present study, enzymatic activity is not visualized
in the 11-day tissue, but phosphatase activity is (1) induced in the 13- and 15-day
cultures and (2) intensified in the 17- and 19-day tissues.
Maximal enzymatic activity is reached with a definite hormonal concentration
such that submaximal levels are elicited by concentrations above or below this
value. That is, the duodenal mucosa is increasingly responsive to cortisone up
to a point where the induction of the phosphatase activity is optimal. This
Cortisone-treated embryonic duodenum
175
intensification of enzyme activity is attributed both to an increase in the number
of reactive cells and to an increase in reactivity per cell.
Once the optimal hormonal concentration is exceeded, maximal enzymatic
activity is lost. Hence, cortisone has a repressive effect upon the enzymatic
machinery of the cell when given in excess. Other studies indicate that overexposure to steroids is generally toxic to tissues (Karnofsky et al. 1951; Evans,
1953; Grossfeld, 1959; Moscona & Karnofsky, 1960). Moreover, the degree of
toxicity shown in vitro varies with the cell strain and the specific corticosteroid
utilized (Gillette & Buchsbaum, 1953; Holden et al. 1953; Grossfeld & Ragan,
1954; Holden & Adams, 1957; Perlman et al. 1962).
Lasfargues & Di Fine (1951) have reported the diminution of alkaline
phosphatase activity in 14-day tissue following administration of an adrenocortical extract, 'eschatine', but they did not vary the hormone concentration
to reveal the extent of this effect. Moog & Nehari (1954) have attributed these
negative results to the insensitivity of the tissue to hormone at that age as well as
to cultivation conditions favoring the monolayering of cells. Our data, however,
indicate that chick tissue is sensitive to corticoid influence as early as 13 days of
incubation. Consequently, Lasfargues & Di Fine's negative results might best
be attributed to exposure to excess hormone levels, which the authors have
expressed in 'dog units'.
In spite of the evidence for positive effects of cortisone on the duodenal
mucosa, other in vitro studies have suggested little or no dependence of the tissue
upon cortisone. Hancox and Hyslop (1953) report that 15-day tissue, which
exhibited positive phosphatase activity before cultivation, shows increased
activity after 2-3 days in vitro without hormone. Hancox (1954) has reported
that 12-day tissue, which is enzymatically negative at explantation, becomes
positive after 4 days of cultivation. Moog & Nehari (1954) report that 16-day
tissue responds similarly after 1-2 days in vitro. Such apparent indications of
steroid independence can be explained by a latency in the appearance of the
enzymatic activity. Adrenal function has been reported in the 12-day chick
embryo (Dawson, 1953; Moog, 1959), so the tissue used in the above studies
must have been exposed to the hormone. That this exposure, if sufficient, need
not be continuous is quite probable.
In addition to influencing enzymatic activity on the apical cell membrane,
cortisone induces morphological changes in the striated border. As hormone is
applied to 19-day explants, the microvilli appear to elongate. This increase in
length continues during exposure to hormone levels which coincidently repress
enzyme activity. Dissociation between the morphological and physiological
aspects of differentiation of duodenal epithelia has been reported previously by
Kato (1959) and by Moog & Nehari (1954). These workers based their deduction
on a change in enzymatic levels with no concurrent change in morphology, while
our data indicate structural changes with no corresponding increase in
phosphatase activity.
12
176
R. L. HAYES, JR.
The height of the striated border normally increases during maturation.
Microvilli elongate in vivo 6-fold between days 16 and 22, with the greatest
change occurring at day 21. This increase in surface area of the micro villi parallels
an increase in alkaline phosphatase activity along the apical cell surface (Overton
& Shoup, 1964).
Exposure to cortisone in vitro stimulates an increase in apical plasmalemmal
surface on the duodenal epithelium. This increase is complicated by vesiculation
of the tips of the microvilli. Although the microvillous membrane is expanded,
there is no significant increase in height of microvilli compared to 19-day controls.
Present attempts to explain vesiculation of microvilli are at best speculative.
Other work with corticosteroids, however, might be linked with the results
reported here. Fell & Thomas (1961) have demonstrated that hydrocortisone
retards the resorptive effects of Vitamin A on skeletal tissue in vitro. The vitamin
is thought to disrupt membrane integrity and to increase membrane permeability
(Lucy, 1964). These effects are exerted on both the plasmalemma (Dingle &
Lucy, 1962; Dingle et al. 1962; Glauert et al. 1963) and intracellular membranes
(Dingle, 1961; Fitton Jackson & Fell, 1963; Lucy et al. 1963). Hydrocortisone
is thought to counteract the influence of Vitamin A by stabilizing the membrane
systems of the cell.
Cortisone, likewise, might increase the integrity or stability of the apical cell
membrane and in so doing might inhibit increases in the surface area of this
portion of the cell membrane. If so, the hormone would be stimulating precocious
membrane-bound phosphatase activity, yet concomitantly restricting membrane
expansion which is linked developmentally to and which varies directly with
enzymatic activity.
Vesiculation of the cell membrane, under the influence of cortisone, might
represent the cell's attempt to maintain equilibrium between the physiological
and morphological phases of differentiation. The small portions of the membrane
and cytoplasm released as vesicles would no longer be identified as part of the
cell. Consequently, when the duodenal epithelium is exposed to concentrations
of cortisone greater than that yielding maximal enzyme activity, no disproportionate increase in apical surface area results.
SUMMARY
1. The effects of cortisone on the striated border of the embryonic chick
duodenal epithelium during in vitro maintenance are described.
2. The application of cortisone induces phosphatase activity in 13- and 15-day
tissue and intensifies this activity in 17- and 19-day explants.
3. The phosphatase activity obtained varies in quantity depending on the age
of the tissues and the concentration of hormone. The sensitivity of the tissue to
hormone increases with age; that is, a lower cortisone concentration is needed to
elicit a maximal enzymatic response in older tissues. As hormone concentration
Cortisone-treated embryonic duodenum
177
is increased for all explants from 13 to 19 days, phosphatase activity increases to
a maximum and thereafter decreases.
4. Cortisone induces a morphological change in the striated border of 19-day
tissue. The apical ends of microvilli are pinched off to form numerous spheroid
vesicles. A possible explanation for this phenomenon is discussed.
RESUME
La maturation in vitro du duodenum embryonnaire traite par la cortisone
II. La bordure stride
1. Les effets de la cortisone sur la bordure striee de l'epithelium duodenal de
l'embryon de Poulet cultive in vitro sont decrits.
2. L'application de cortisone induit l'activite phosphatasique dans le tissu
de 13etde 15 jours et intensifies cette activite dans les explants de 17etde 19jours.
3. L'activite phosphatasique obtenue varie quantitativement selon l'age du
tissu et la concentration en hormone. La sensibilite du tissu a l'hormone s'accroit
avec l'age; c'est-a-dire que plus les tissus sont ages plus la concentration de
cortisone necessaire pour produire la reponse enzymatique maximum est basse.
Lorsqu'on augmente la concentration d'hormone pour tous les explants de 13 a
19 jours, l'activite phosphatasique augmente jusqu'a un maximum puis decroit.
4. La cortisone induit un changement morphologique de la bordure striee
dans le tissu provenant d'embryons de 19 jours. Les terminaisons apicales de
microvillosites se fragmentent pour former de nombreuses vesicules plus ou
moins spheriques. Une explication possible de ce phenomene est discutee.
ACKNOWLEDGEMENT
This work was supported by U.S.P.H.S. Grant RG-6729.
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