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/ . Embryo!, exp. Morph. Vol. 27, 1, pp. 155-162,1972
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\ 55
Hydrocortisone-increased glycogen
deposition and its dependence on tissue
interactions in mouse embryonic lung
developing in vitro
By T. ALESCIO 1 AND A. M. DANI 1
From the Institute of Topographic Anatomy and II Chair of Human
Anatomy, University of Naples
SUMMARY
We have performed experiments showing that the epithelial tree of embryonic rudiments,
when cultured in vitro in the presence of hydrocortisone, sharply increases its glycogen content. The distribution of the polysaccharide remains very similar to that observed in the
absence of the hormone.
The increased glycogen deposition in response to the hormonal treatment requires the
presence of homologous (bronchial) mesenchyme, as shown by the complete inability of the
epithelium to accumulate glycogen when experimentally associated with metanephrogenic
mesenchyme.
The increased deposition of glycogen in the epithelial tree does not result in an enhanced
rate of budding activity.
INTRODUCTION
Recent investigations (Le Douarin, 1968; Morris & Moscona, 1970) have
demonstrated that the existence of the proper interrelations among cells and cell
groups is essential to the appearance of specific products in embryonic cells. In
this context we have shown (Alescio & Dani, 1971) that in the epithelium of
developing mouse lungs the presence of glycogen is dependent on the nature of
the surrounding mesenchyme, and strictly associated, both topographically and
chronologically, with the budding activity; hence a possible morphogenetic
significance of glycogen in the developing lung was indicated.
The purpose of the current paper is to show first that substances (glucocorticoids) which are especially potent in affecting carbohydrate metabolism
become almost totally ineffective when their target cells have lost the proper
associations. Second, the data also show that an increased amount of glycogen
in the embryonic lungs does not necessarily result in an enhanced rate of the
epithelial morphogenetic growth.
1
Authors' address: Istituto di Anatomia topografica, Via L. Armanni 5, 80138 Napoli,
Italy.
156
T. ALESCIO AND A. M. DANI
MATERIALS AND METHODS
The plan of the research has been as follows:
(1) Glycogen and protein determinations in 11-, 12- and 13-day lung rudiments developing in vivo.
(2) Glycogen and protein determinations in lung rudiments explanted at the
11th day of gestation and cultured in vitro for 24 and 48 h with and without
hydrocortisone (1 ^g/ml of culture medium).
(3) Effect of hydrocortisone-induced glycogen accumulation on the rate of
morphogenetic growth, measured as increase of the epithelial surface area.
(4) Effect of the heterologous association of the main left bronchus with
metanephrogenic mesenchyme on glycogen accumulation in the presence of
hydrocortisone.
Points (1) and (2) are intended to give a quantitative estimate of glycogen
content, relative to the total amount of protein, during the normal and in vitro
development, and of the modifications ensuing from hydrocortisone treatment;
point (3) should answer the question whether a modified glycogen content may
affect the rate of the epithelial morphogenetic growth, while point (4) is meant
to inform on the requirements for tissue interactions for the expression of the
steroid effect.
Finally, in a collateral experimental series the epithelial trees of 11-day rudiments have been completely separated from mesenchyme and cultured in
isolation in the presence of hydrocortisone, to show the direct effect of the
hormone on the epithelial glycogen.
First-generation mouse hybrids from ^C57BLx^BALB/c
have been used.
The discovery of the copulation plug, looked for each 12 h, was taken as the
beginning of the pregnancy. Lung rudiments have been cultured in a hanging
drop at the surface of a clot made up with equal volumes of chicken plasma and
9-day chick embryo extract. Hydrocortisone (hydrocortisone alcohol, Mann
Research Laboratory) was added to the culture medium, when required, at the
final concentration of 1 /tg/ml of culture medium. The grafting of the 11-day
metanephrogenic mesenchyme on the left main bronchus, and successive reassociation with freshly dissected 11-day bronchial mesenchyme was performed
as described previously (Alescio & Dani, 1971). Epithelial morphogenetic growth
was measured as relative increment of the epithelial surface area, as in earlier
experiments (Alescio & Colombo Piperno, 1967; Alescio & di Michele, 1968).
The complete separation of the epithelial tree from its mesenchymal mantle
has been obtained by mild trypsinization as follows: 5min in calcium- magnesium-free Tyrode solution (CMF), followed by a 5min treatment with 2 %
trypsin (Trypsin 1:250, Difco Laboratory) in CMF at 37 °C; the loosened mesenchyme has been then removed by cataract knives in 20 % horse serum containing
DNase, 20/tg/ml. Glycogen was determined by the anthrone reagent after
digesting the tissue in boiling 30 % KOH; protein was determined by the method
Glycogen deposition and tissue interactions in lungs
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Fig. 3
Fig. 1. Amount of protein, expressed as /tg per lung rudiment, as a function of
developmental age. HC = rudiments cultured in the presence of hydrocortisone.
Uncertainties are standard errors of the average of 3 to 8 determinations.
Fig. 2. Amount of glycogen, expressed as /tg per lung rudiment, as a function of
developmental age. HC = rudiments cultured in the presence of hydrocortisone.
Uncertainties are standard errors of the average of 3 to 8 determinations.
Fig. 3. Specific concentration of glycogen expressed as units of glycogen per unit of
protein, as a function of developmental age. HC = rudiments cultured in the
presence of hydrocortisone. Uncertainties are standard errors of the average of 3
to 8 ratios.
of Lowry et al. (1951), following ultrasound disintegration. The histochemical
localization of glycogen was obtained using the periodic acid-Schiff (PAS)
sequence after McManus (Pearse, 1968), with and without diastase digestion,
as previously indicated (Alescio & Dani, 1971).
158
T. ALESCIO AND A. M. DANI
RESULTS
The embryonic stage between the 11th and the 13th day of development in
vivo, morphogenetically characterized by intensive budding activity (Alescio,
1968), as expected, shows a marked increase in the amount of protein per rudiment (Fig. 1). This is representative of its intense growth rate, and is associated
with an even more marked rise in the amount of glycogen (Fig. 2). In consequence,
the specific concentration of glycogen (as expressed by the ratio: amount of
glycogen/amount of protein) is not kept constant but rather increases with age
(Fig. 3). The above sharp rise in protein and glycogen content is definitely but
evenly depressed under conditions of growth in culture (Figs. 1, 2), hence the
proportionality between the two is maintained and the specific concentration of
glycogen remains very close to that of normal development (Fig. 3).
The effect of hydrocortisone added to the culture medium at a final concentration of 1 /*g/ml consists in a moderate yet statistically significant (P < 0-05)
increase in the amount of protein per lung rudiment (Fig. 1) while the glycogen
content goes up strikingly to more than twice the value reached in culture in
the absence of the steroid (Fig. 2); consequently the specific concentration of
glycogen is clearly above the level of both the in vivo and in vitro development
(Fig. 3). The increased amount of glycogen follows closely the characteristic
distribution pattern of this polysaccharide in developing lungs, as shown in
Figs. 4 and 5, where it appears predominantly localized in the terminal
buds.
The rate of epithelial growth of the hydrocortisone-treated rudiments, as
determined by measurement of the epithelial surface area, is not significantly
higher than that of rudiments cultured in the absence of the steroid, as also
implied by the above mentioned divergent increase of protein and glycogen with
consequent rise of the polysaccharide specific concentration. Therefore the
increase of glycogen does not suffice, in itself, to bring about a clear-cut
enhancement of budding rate.
The next question to be examined is whether the above sharp increase in
glycogen concentration, resulting from hydrocortisone treatment, is dependent
on the existance of the proper cell associations. We have previously shown
(Alescio & Dani, 1971) that when the bronchial epithelium is associated with
metanephrogenic mesenchyme, budding activity ceases immediately and completely, and no PAS-positive, diastase-sensitive material can be traced in the
epithelial cells. When this experiment is performed in the presence of the steroid,
introduced in the culture medium at the final concentration of 1 /tg/ml, the
bronchial epithelium associated with metanephrogenic mesenchyme becomes
totally insensitive to the presence of the hormone. This is shown in Figs. 6 and 7,
where the main left bronchus surrounded by metanephrogenic mesenchyme
shows no progress of bronchial arborization and also complete absence of PASpositive material, in spite of the presence of the steroid; this is in sharp contrast
Glycogen deposition and tissue interactions in lungs
FIGURES 4-9
Fig. 4. Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone. Presence of large quantities of glycogen in the epithelial tree, with preferential localization in the terminal buds. PAS-haematoxylin, x 65.
Fig. 5. Higher magnification of the same rudiment as in Fig. 4. PAS-haematoxylin,
x240.
Fig. 6. Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone after association of the main left bronchus (arrows) with metanephrogenic
mesenchyme. Absence of epithelial glycogen as a result of the heterologous association. PAS-haematoxylin, x 70.
Fig. 7. Higher magnification of the same rudiment as in Fig. 6 to show the striking
difference between the main left bronchus (arrows) associated with metanephrogenic
mesenchyme and buds from the main right bronchus where bronchial mesenchyme
is present. PAS-haematoxylin, x 150.
Fig. 8. Eleven-day lung rudiment cultured for 2 days in the presence of hydrocortisone after association of the main left bronchus with metanephrogenic mesenchyme; the metanephrogenic mesenchyme has then been removed and bronchial
mesenchyme reassociated for two additional days in culture in the presence of the
steroid. Reappearance of the epithelial glycogen in the main left bronchus. PAShaematoxylin, x70.
Fig. 9. Higher magnification of the same rudiment as in Fig. 8. PAS-haematoxylin,
xl50.
159
160
T. ALESCIO AND A. M. DANI
with the controlateral bronchial branches from the main right bronchus, where
the normal tissue association was maintained (Fig. 7).
This is only a provisional effect of the heterologous association, as demonstrated by the subsequent reassociation of the morphogenetically quiescent main
left bronchus with bronchial mesenchyme (Figs. 8, 9): this immediately makes
the bronchial epithelium highly responsive to the steroid, as shown by the
intense deposition of glycogen in the epithelial cells and resumption of budding
activity.
The above results, suggesting a dependence of the epithelial responsiveness to
the steroid on the nature of the surrounding mesenchyme, give rise to the question
of whether the homologous mesenchyme is specifically required or whether the
heterologous mesenchyme may actively inhibit or somehow suppress the
epithelial response. Therefore the ability of the isolated epithelium to accumulate glycogen in the presence of the steroid was tested. The mesenchyme-deprived
epithelial tree cultured in hanging drop for 2 days shows progressive disorganization and spreading of the epithelial cells from tissue-like organization, and
absence of glycogen stores. The presence of hydrocortisone in the culture
medium does not modify the above situation.
DISCUSSION
We have shown in a previous paper (Alescio & Dani, 1971) that in the
epithelial tree of developing lungs the ability of the terminal buds to accumulate
glycogen is dependent on the nature of the mesenchymal environment, and that
the presence of glycogen is associated, with impressive constancy, to the budding
movement of the epithelial cells.
The main features of the present results are that (1) the responsiveness of the
epithelial cells to substances (corticosteroids) which are also specifically able to
interfere with glycogen metabolism in other embryonic organs (Greengard &
Dewey, 1970) is dependent on the surrounding mesenchyme; and (2) a remarkable increase in glycogen stores does not suffice,/w se, to determine an increased
rate of budding.
As far as point (1) is concerned, it is noteworthy first that the sharp increase
in the specific concentration of glycogen following hydrocortisone treatment
tends to conform with the normal pattern of glycogen distribution, so that the
final effect takes the appearance of some kind of magnification of the normal
behaviour of glycogen deposition.
The results of the experimental association with heterologous (metanephrogenic) mesenchyme show that the epithelial metabolic activities involved in
glycogen deposition, which are normally dependent on the availability of the
homologous mesenchyme (Le Douarin, 1968; Alescio & Dani, 1971), cannot be
elicited even by a specific and potent activator (hydrocortisone) in the absence
of external signals provided T)y tissue interactions. In this context Morris &
Glycogen deposition and tissue interactions in lungs
161
Moscona (1970) have recently shown that the existence of the proper organization at the cellular level is required for the hormonal induction of glutamine
synthetase in the embryonic neural retina. They have also shown that inducibility
is irreversibly lost after cell dissociation, as result of metabolism-dependent
processes following the persistent separation of the cells from tissue-like associations. Our findings stress the idea that, also at the level of tissue organization,
similar interacting phenomena are actively operating since an overall metabolic
process, namely glycogen metabolism, appears highly dependent on tissue interactions. Moreover, the degree of such dependence is so stringent as to suppress
completely the epithelial response to the hormone. This fact is noteworthy in
the evaluation of the developmental significance and specificity levels of tissue
interactions in developing lungs (Wessells, 1970), since it now seems likely that
mesenchymal 'information' is required for the maintenance of normal metabolic
patterns. This is also stressed by the fact that isolated pulmonary epithelium
appears insensitive to hydrocortisone, suggesting that the condition 'absence
of mesenchyme' is possibly equivalent, from this point of view, to the condition
of 'heterologous association'. Should this idea prove correct, not inhibition
from metanephrogenic mesenchyme but rather active involvement of bronchial
mesenchyme would explain the described results.
It would be of obvious biological interest to clarify the mechanism of the
mesenchymal dependence of the epithelial response to the steroid; at the moment
we cannot establish with the available data whether the site of the primary
hormonal effect resides in the mesenchymal or in the epithelial compartment
of the rudiment, and therefore the two hypotheses are in principle both acceptable. However, since increased glycogen stores expressed in the epithelium are
the final effect of the steroid, the evidence is that there must be, in any case, a
flow of influences from mesenchyme to epithelium. The binding of the steroid
to the epithelial cell receptors or any of the subsequent steps preceding the
expression of the steroid effects, as recently elucidated in rat thymus cells
(Mosher, Young & Munck, 1971), may represent the mesenchyme-dependent
steps in our experimental system.
Our second point indicates that an increase in glycogen stores does not suffice,
in itself, to enhance significantly the rate of morphogenetic growth. Clearly
this fails to prove that the presence of the polysaccharide is unrelated to budding
activity, and we must therefore conclude that we are still lacking the demonstration of a causal relationship between the two, beyond their coincidental
occurrence. An answer to the question of the possible morphogenetic significance
of glycogen in terminal buds might perhaps require an experimental system
where glycogen could be cleared from budding epithelium while preserving the
normal association with the morphogenetically active bronchial mesenchyme.
E M B 27
162
T. ALESCIO AND A. M. DANI
RIASSUNTO
Aumento da idrocortisone della deposizione di glicogeno e sua dipendenza dalle
interazioni tessutali nelpolmone embrionale di topo coltivato in vitro
Gli esperimenti eseguiti indicano che l'abbozzo epiteliale del polmone embrionale, coltivato
in vitro in presenza di idrocortisone, aumenta fortemente il proprio contenuto in glicogeno.
La sua distribuzione rimane simile a quella osservata in assenza deH'ormone.
L'aumento della deposizione di glicogeno in risposta al trattamento ormonale richiede la
presenza di mesenchima omologo (bronchiale), come e dimostrato dalla completa incapacita
deU'epitelio ad accumulare glicogeno quando sia sperimentalmente associato a mesenchima
metanefrogeno.
L'aumento della deposizione di glicogeno nell'albero epiteliale non provoca un aumento
del tasso di gemmazione.
This research has been performed with a contribution from the Italian C.N.R.
REFERENCES
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ALESCIO, T. & DANI, A. M. (1971). The influence of mesenchyme on the epithelial glycogen
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ALESCIO,
(Manuscript received 1 June 1971)