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/ . Embryol. exp. Morph. Vol. 25, 3, pp. 365-376, 1971
Printed in Great Britain
365
Accelerating effect of
hydrocortisone on the keratinization of chick
embryonic skin growing in a
chemically defined medium
By MASANOBU SUGIMOTO1 AND HIROYOSHI ENDO1
From the Department of Physiological Chemistry,
Faculty of Pharmaceutical Sciences, University of Tokyo
SUMMARY
In an attempt to examine histologically, chemically and biochemically the effect of hydrocortisone in a minimal concentration on keratinization of 13-day chick embryonic shank skin,
a simple replicate culture method ('Millipore' filter-roller-tube method) was devised to
cultivate rather large pieces of the skin in a chemically defined medium, BGJb supplemented
with ascorbate.
Hydrocortisone added in a minimal concentration of 0001 /tg/ml produced a heavily
cornified eosinophilic layer over the epidermis after 4 days' cultivation, whereas in the absence
of the steroid no sign of cornification could be found during culture.
Determination of total protein and analysis of amino acid composition of whole protein
of the epidermis indicated that hydrocortisone accelerated epidermal cornification as compared with in ovo development.
Pregnenolone and progesterone showed no effect on the in vitro keratinization of the
epidermis and deoxycorticosterone gave a slight effect: thus the cornification-accelerating
effect of hydrocortisone seems to be attributable to its glucocorticoid activity.
INTRODUCTION
One of the most important functions of the skin is the production and deposition of keratin by epidermal cells for protecting the living organism from the
external environment. Keratinization consists of a series of finely organized
processes of cell maturation in which synthesis of specific proteins within the
cells is accompanied by degradation of the cellular constituents. The process has
long been one of the most attractive subjects in tissue culture studies, since
Strangeways & Fell (1926) cultured chick embryonic skin by the watch-glass
method using the natural medium of a plasma clot. Later, Fell (1962) and
Weissmann& Fell (1962) observed the accelerating effect of hydrocortisone on
the keratinization of chick and rat embryonic skin cultured on a plasma clot.
Recently, on the other hand, chemically denned media have been successfully
1
Authors' address: Department of Physiological Chemistry, Faculty of Pharmaceutical
Sciences, University of Tokyo, Tokyo 113, Japan.
366
M. SUGIMOTO AND H. ENDO
used for cultivating rat (Trowell, 1959), chick (Wessells, 1961) and human foetal
skin (Pullar, 1964). Wessells (1961) observed that thyroxine initiated the keratinization of the epidermis of chick embryonic skin growing in such a medium.
The histological findings suggest that hormones may play an important role in
the embryonic development of the skin.
In an attempt to examine both histologically and biochemically the effects of
hydrocortisone in a minimal concentration on keratinization the authors devised
a simple replicate culture method for growing rather large pieces of chick
embryonic skin in a chemically denned medium. Our study revealed that hydrocortisone in very low concentrations dramatically accelerated cornification as
judged by the morphological and biochemical results. A preliminary report of
this work has been published (Sugimoto & Endo, 1969).
MATERIALS AND METHODS
The shank of a 13-day-old chick embryo from which the distal and proximal
portions had been removed (ca. 7 mm in length) was slit open with fine scissors.
The skin (ca. 7 x 7 mm2) was spread out in a drop of physiological saline solution
on a Petri dish by carefully removing the bones, muscle and loose connective
tissue under a dissecting microscope. The' Millipore' filter (type HA) was placed
on a sheet of the skin. The skin was then ready for culture, the dermis being
tightly in contact with the filter. Thefilterwith a stretched sheet of the skin was
put into a roller-tube containing 1 ml of the medium. These procedures are
diagrammatically illustrated in Fig. 1. The tubes were incubated at 37 °C while
rotating 10 times/h in a roller-drum which held them at an angle of approximately 5°. The medium was renewed every other day.
The medium used was the chemically defined one, BGJb (Biggers, Gwatkin
& Heyner, 1961), supplemented with 50/tg/ml of sodium ascorbate, unless
otherwise mentioned. Hydrocortisone hemisuccinate (Upjohn Co.) was added
to the medium at concentrations of 10-0-0001 /*g/ml. Before adding to the
medium, hydrocortisone and other natural steroids, pregnenolone, progesterone
and 11-deoxycorticosterone, were dissolved in ethanol, the concentration of
ethanol always being adjusted to 0-2% of the final medium.
For histological examination the explant on the filter wasfixedfor 2 h with
Bouin's solution, then put in 80 % ethanol and dehydrated. It was transferred
to toluene, embedded in paraffin wax and sectioned at a thickness of 5 or 6 fi.
Sections were stained with Mayer's haematoxylin and eosin. For chemical and
biochemical analyses the epidermis of cultured skin or of 13- and 17-day
embryonic skin was separated from the dermis by incubating the skin at 37 °C
for 1-5-2 h in Ca2+- and Mg2+-free Tyrode's solution containing 0-4% EDTA
(Mordoh & Lustig, 1966). The cornified epidermis of 21-day chick embryonic
shank skin was obtained by incubating the skin for 30 min at 37 °C in the solution
containing both 0-02 % EDTA and 0-25 % trypsin (Difco 1:300) and thereafter
Hydro cortisone on keratinization in vitro
367
by removing the digested cell layers with forceps. The epidermis thus obtained
was homogenized with a Potter-Elvehjem glass homogenizer, and the whole
homogenate was used for determining the amount of protein or its amino acid
composition. Protein was assayed after Lowry's method (Lowry, Rosebrough,
Farr & Randall, 1951) by using bovine serum albumin as the standard. Amino
acid composition was determined by a Hitachi KLA-3B automatic amino acid
analyser on triplicate samples of the homogenate hydrolysed in 6 N-HC1 at
110°Cfor24h.
Fig. 1. Diagrammatic illustration of procedure of the 'Millipore' filter-roller-tube
culture method for chick embryonic shank skin. See text for details of the procedures.
RESULTS
The epidermis of the 13-day chick embryonic shank skin at the start of culture
consisted of a single layer of basal columnar cells and on this two layers of intermediate and flattened cells (Fig. 2 A). When the skin was cultured in the chemically
defined medium the explant grew well as a whole (Fig. 3 A) and the epidermis
grew as thick as six to eight cell layers after 4 days' cultivation (Fig. 3B).
During growth in vitro, however, no sign of cornification could be observed over
the epidermis, though eosinophilic granules appeared in subpericytes as well as
in pericytes and sometimes the swollen cells just below the subpericytes became
diffusely eosinophilic (Figs. 3B, 4 A). Even after 6 days' cultivation the cornified
layer could not be discerned effectively all over the explants except in a few
368
M. SUGIMOTO AND H. ENDO
Hydrocortisone on keratinization in vitro
369
limited areas at the edges, despite the fact that the number of eosinophilic
swollen cells in the epidermis increased.
When hydrocortisone was added to the culture medium at a concentration of
0-01 /tg/ml, however, a heavily eosinophilic, compact layer devoid of cellular
structures was deposited in the uppermost stratum of the epidermis after
4 days' cultivation (Figs. 3C, 4B). As compared with the epidermis of control
explants, the cells under the eosinophilic layer seemed to be rather larger and
the basal cells shorter and wider. Moreover, the basal cell layer of the epidermis
was rather even in control explants (Fig. 3B), whereas it was fairly undulating
in treated explants (Fig. 3C). These histological features observed in the epidemis of the hydrocortisone-treated skin were very similar to physiological
cornification in the course of normal development of the chick (Fig. 2C). These
findings strongly indicated that hydrocortisone added in vitro vigorously
accelerated the cornification of the epidermis.
On separating the epidermis from the dermis with use of EDTA solution, the
junction between them was tighter in treated skin than in control skin. When
the separated epidermis was seen under the dissecting microscope, the control
was semitransparent and soft while the treated was opaque and hard. Chemical
analysis of the epidermis in all the cultures revealed that after 4 days' cultivation
hydrocortisone at a concentration of 001 /*g/ml always gave statistically significant acceleration of the progressive increase of total protein of the explant
during culture. Representative results are shown in Fig. 5. As shown in Table 1
moreover, the amino acid composition of the epidermal protein demonstrated
several qualitative differences between tissue cultivated in control medium and
that grown in medium containing the steroid at a concentration of 0-01 /tg/ml:
the content of serine, glycine and methionine was increased by the steroid while
that of lysine, proline and alanine was diminished. The most striking effect was
found for glycine, the content of which was 11-7 % in control skin and 16-7 % in
treated skin. Moreover, the changes in the content of each amino acid produced
by the treatment generally paralleled those in development in ovo of the chick
embryo, although this was not the case with proline and methionine (Table 1).
Even when cultured in medium containing hydrocortisone at a much higher
concentration of 1-0 /*g/ml, the epidermis showed all the above-mentioned
changes in the amino acid composition, though the rate of protein increase
during cultivation was not significantly affected (Sugimoto & Endo, 1969).
Considered together with the histological findings mentioned above, these data
from chemical determinations strongly suggest that hydrocortisone accelerated
FIGURE 2
(A) Thirteen-day chick embryonic shank skin, the starting material for the culture.
x400.
(B) Seventeen-day chick embryonic shank skin, x 400.
(C) Two-day chick shank, x 400.
370
M. SUGIMOTO AND H. ENDO
A
Hydrocortisone on keratinization in vitro
371
or induced in vitro the synthesis of some of the specific proteins involved in the
processes of physiological cornification of chick embryonic skin.
To determine the minimal effective dose of hydrocortisone for such a cornification-accelerating effect, skin explants were cultured in medium containing
the steroid at concentrations ranging from 10 to 0-0001 /*g/ml and examined
histologically. All the concentrations from 0-001 /*g/ml upwards produced
clearly recognizable keratinization and even the lowest concentration of
0-0001 /tg/ml showed a very slight but discernible effect after 4 days' cultivation.
When skin was cultured in a semi-defined medium (the chemically defined
medium plus 5% (v/v) bovine serum) a heavily cornified, eosinophilic layer
appeared even in the absence of hydrocortisone (Fig. 4C). Therefore added
steroid even at a concentration as high as 10/*g/ml, could not be shown to
accelerate the cornification of the epidermis throughout 6 days of cultivation.
To investigate the specificity of the effect of hydrocortisone, three natural
steroids biosynthetically related to this glucocorticoid were examined for the
ability to accelerate histologically visible cornification. At a concentration of
1-0 /Ag/ml the precursors, pregnenolone and progesterone, showed no effect at all
while a mineralocorticoid, deoxycorticosterone, produced a slight effect comparable with that of 0-0001 ^g/ml of hydrocortisone. Therefore it seems probable
that the cornification-accelerating effect of hydrocortisone can be attributed to
its glucocorticoid activity.
DISCUSSION
Hormonal factors controlling the keratinization of the epidermis have been
studied in tissue cultures of chick and/or rat embryonic skin (Wessells, 1961;
Fell, 1962; Weissmann & Fell, 1962). Although these histological studies strongly
suggested that hormones may play an important role in keratinization of the
skin in embryonic development, further chemical and biochemical elucidation
remained to be carried out. Thus the main aims of this work were first to devise
a simple replicate culture method for cultivating rather large pieces of the skin
in order to obtain a large enough amount of the explants for chemical and biochemical analyses and secondly to make the cultures successful in a chemically
defined medium in order to exclude the participation of any unknown factors
contained in biological materials such as serum or embryo extract. These objects
were fully achieved on chick embryonic shank skin by using the ' Millipore'
FIGURE 3
(A) Thirteen-day chick embryonic shank skin cultured for 4 days in the chemically
defined medium, x 40.
(B) Thirteen-day chick embryonic shank skin cultured for 4 days in the chemically
defined medium, x 400.
(C) Thirteen-day chick embryonic shank skin cultured in the chemically defined
medium containing hydrocortisone hemisuccinate at a concentration of 001
x400.
372
M. SUGIMOTO AND H. ENDO
B
Hydrocortisone on keratinization in vitro
373
filter-roller-tube method diagrammatically illustrated in Fig. 1 and a chemically
denned medium, BGJb supplemented with ascorbate.
The present paper reports that the accelerating effect of hydrocortisone on
keratinization of the skin growing in such a medium is far more dramatic than
the effect demonstrated by Weissmann & Fell (1962). They cultured rat embryonic skin on a plasma clot by use of the watch-glass method and observed a
stimulatory effect of the steroid at a concentration of 7-5 /*g/ml. In striking
contrast with their finding, in embryonic chicken skin the authors find that the
300
x 200
Control
100
•"—"• Hydrocortisone
Days in culture
Fig. 5. Effect of hydrocortisone on the increase of total protein of 13-day chick
embryonic shank skin growing in chemically defined medium. Hydrocortisone
hemisuccinate was added to the culture medium at a concentration of 001 /*g/ml at
the start of culture and when the medium was renewed after 2 days' cultivation.
Each determination was made on three to four explants. The vertical line at each
point represents ± standard error of the mean.
enhancing effect of hydrocortisone on keratinization occurs at a concentration
of 0-001 /tg/ml and is discernible even at a concentration of 0-0001 /^g/ml.
Considering that a natural liquid medium - BGJb supplemented with ascorbate
plus 5% bovine serum - permitted marked keratinization in the absence of
hydrocortisone (Fig. 4C), the wide difference in the effective concentrations of
FIGURE 4
(A) Thirteen-day chick embryonic shank skin cultured for 4 days in the chemically
defined medium, x 1000.
(B) Thirteen-day chick embryonic shank skin cultured for 4 days in the chemically
defined medium containing hydrocortisone hemisuccinate at a concentration of
0-01/tg/ml. xlOOO.
(C) Thirteen-day chick embryonic shank skin cultured for 6 days in the semi-defined
medium containing 5% bovine serum, x 1000.
25
E M B 25
A minn
1
6-79f
1-94
6-74
8-94
4-47
6-79
12-42
6-27
11-68
7-69
605
0-95
4-59
8-47
2-95
3-26
c
3*
7-39
2-21
6-72
8-97
4-60
6-85
12-71
6-54
11-62
8-61
5-41
0-59
4-41
8-42
2-17
2-79
2
702
1-73
6-44
9-46
4-45
6-96
13 01
5-86
11-67
7-78
613
0-58
4-53
8-44
2-79
315
A
Control
7 07
1-96
6-63
912
4-51
6-87
12-71
6-22
11-66
803
5-86
0-71
4-51
8-44
2-64
307
Mean
5-96
1-95
6-52
810
4-14
8-43
11-88
4-79
16-90
5-96
5-40
1-59
4-71
7-72
2-97
2-97
1
5-89
1 95
6-36
7-89
411
903
1208
4-95
1615
611
5-43
1-52
4-80
7-71
301
301
2
5-77
1-95
616
8-20
4-34
9-24
12-10
3-64
17-17
5-94
5-12
1-43
4 91
7-81
3-12
308
3
5-87
1-95
6-35
806
4-20
8-90
1202
4-46
16-74
600
5-32
1-51
4-81
7-75
303
3 02
Mean
Hydrocortisone (001 /ig/ml)
8 07
2-20
6-25
1012
4-82
607
12-78
504
9-68
8-54
6-39
0-41
4-86
8-66
2-41
3-72
13-day
5-54
200
7-18
8-31
4-45
7-57
1205
5-60
13-94
711
6-34
0-35
4-55
809
3-65
3-26
17-day
A
2-65
1-42
5-38
5-93
3-93
10-58
811
7-21
23-17
5-29
5-85
0-77
3-80
6-83
5-77
3-33
21-daj
In ovo development
* Triplicate samples.
f Each figure represents the number of amino acid residues per 100 amino acid residues of the whole tissue protein.
Lys
His
Arg
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
He
Leu
Tyr
Phe
acid
j \ 11111IU
In vitro culture
Table 1. Amino acid composition of the epidermal protein of chick embryo skin in the
normal course to hatching or cultured in vitro
w
H
O
o
C/3
U)
Hydrocortisone on keratinization in vitro
375
the steroid could result from the fact that the authors employed a chemically defined medium in which no sign of cornification occurred during the cultivation of
the control skin (Fig. 3B). From the histological findings of the present work it
might be said that hydrocortisone in physiological concentrations showed the
effect of inducing rather than accelerating the keratinization of chick embryonic
skin growing in vitro. As to the chemical development of the epidermis represented by the amino acid composition of its protein (Table 1), however, tissue
grown in the absence of hydrocortisone appears to continue to a lesser degree
the normal course of development. From a chemical standpoint, therefore, at
the present time hydrocortisone might be taken to accelerate rather than induce
keratinization of chick embryonic epidermis. But it should be pointed out here
that the amino acid composition of the whole protein of the epidermis (Table 1)
represents only mean values for a mixture of many kinds of protein in the tissue.
Thus, the possibility remains that hydrocortisone might induce a small amount
of specific protein(s) exclusively responsible for inducing histologically visible
keratinization while at the same time the steroid produced an increase of other
epidermal proteins not necessarily involved in this change in keratinization.
Concerning these points, the authors' recent studies gave some interesting
results: incorporation of [3H]glycine into epidermal protein was strongly
increased by hydrocortisone and the electrophoretic pattern of the chemically
modified epidermal protein showed a very sharp difference in a few bands
between the control and treated explants. These chemical and biochemical data
will soon be published elsewhere.
Lastly, as to embryological problems, keratinization of the epidermis of
shank skin could not be observed until 18 days of incubation (Mottett & Jensen,
1968), and it is said that adrenal cortex begins to secrete corticoids a few days
before this developmental stage (cf. Watterson, 1959; Piddington, 1970).
Considering all this together with the authors' findings that hydrocortisone in
very low concentrations strikingly accelerated keratinization of 13-day chick
embryonic shank skin and that the specificity of the effect of the steroid was
attributable to its glucocorticoid activity, it seems possible that glucocorticoids
participate in keratinization of the epidermis in the embryonic development of
the chick. Similarly, Piddington (1967) reported that glutamine synthetase
activity in the neural retina of the chick embryo increases sharply during the
final period of embryonic development and that hydrocortisone increases the
enzyme activity of the retina in culture at a minimal concentration of
0-003 /fcg/ml. There is a good correspondence between this finding and the
authors' own ones, both in the minimal effective dose of hydrocortisone and
in the developmental stage at which the biological reactions begin to occur.
This may be taken to indicate that glucocorticoids generally play an
important role in the embryonic development of the chick.
25-2
376
M. SUGIMOTO AND H. ENDO
The authors wish to express their sincere thanks to Dame Honor B. Fell of the Strangeways
Research Laboratory, Cambridge, England, for her continued encouragement and kind
revision of the manuscript. They are also grateful to the Central Research Laboratories,
Ajinomoto Co. Inc., Kawasaki, Japan, for analyses of the amino acid composition. This
investigation was supported in part by a Grant-in-Aid for Scientific Research from the
Ministry of Education of Japan.
REFERENCES
J. D., GWATKIN, R. B. L. & HEYNER, S. (1961). Growth of embryonic avian and
mammalian tibiae on a relatively simple chemically defined medium. Expl Cell Res. 25,
41-58.
FELL, H. B. (1962). The influence of hydrocortisone on the metaplastic action of vitamin A
on the epidermis of embryonic chicken skin in organ culture. /. Embryol. exp. Morph. 10,
389-409.
LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. C. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. /. biol. Chem. 193, 265-275.
MORDOH, P. R. & LUSTIG, E. A. (1966). Dermoepidermal interactions in tissue culture between heterologous species. Expl Cell Res. 42, 384-388.
MOTTET, N. K. & JENSEN, H. M. (1968). The differentiation of chick embryonic skin. Expl
Cell Res. 52, 261-283.
PIDDINGTON, R. (1967). Hormonal effects on the development of glutamine synthetase in the
embryonic chick retina. Devi Biol. 16, 168-188.
PIDDINGTON, R. (1970). Steroid control of the normal development of glutamine synthetase
in the embryonic chick retina. J. Embryol. exp. Morph. 23, 729-737.
PULLAR, P. (1964). Keratin formation in a chemically defined medium. /. Path. Bact. 88, 203212.
STRANGEWAYS, T. S. P. & FELL, H. B. (1926). Experimental studies on the differentiation of
embryonic tissues growing in vivo and in vitro. I. The development of the undifferentiated
limb-bud (a) when subcutaneously grafted into the post-embryonic chick and (b) when
cultivated in vitro. Proc. R. Soc. B 99, 340-360.
SUGFMOTO, M. & ENDO, H. (1969). Effect of hydrocortisone on the keratinization of chick
embryonic skin cultured in a chemically defined medium. Nature, Lond. 222, 1270-1272.
TROWELL, O. A. (1959). The culture of mature organs in a synthetic medium. Expl Cell Res.
16, 118-147.
WATTERSON, R. L. (1959). Ontogeny of selected endocrine glands: The adrenals. In Endocrines
in Development, ed. R. L. Watterson, pp. 13-28. Chicago: The University of Chicago
Press.
WEISSMANN, G. & FELL, H. B. (1962). The effect of hydrocortisone on the response of fetal rat
skin in culture to ultraviolet irradiation. /. exp. Med. 116, 365-380.
WESSELLS, N. K. (1961). Thyroxine initiation of epidermal differentiation as studied in vitro
in chemically defined medium. Expl Cell Res. 24, 131-142.
BIGGERS,
{Manuscript received 10 August 1970)