The Effects of Vitamin A and Citral on Epithelial ...epithelium is well ciliated and contains...

J. Embryol. exp. Morph., Vol. 11, Part 1, pp. 279-291, March 1963Printed in Great Britain

The Effects of Vitamin A and Citral on Epithelial

Differentiation in vitro 1. The Chick Tracheal

Epithelium

by MARGARET B. AYDELOTTE1

From the Physiological Laboratory, Cambridge

WITH FOUR PLATES

INTRODUCTION

I N many animals the tracheal epithelium is one of the first tissues to respond todeficiency of vitamin A. Mori (1922a, b) in a careful study of the histologicalchanges in vitamin A deficient rats, showed that the secretion of mucus fromthe tracheal epithelium and many other mucous membranes and glands wasreduced, and that the secretory epithelia were gradually replaced by thicker,drier, keratinized membranes. Similar changes have been demonstrated in manyother vitamin A deficient animals, including chickens (Beach, 1923; Seifried,1930; Jungherr, 1943).

Though vitamin A deficiency appears to have relatively little effect on skinand other epithelia that are normally keratinized, these epithelia change withhigh concentrations of vitamin A. When the vitamin was applied locally to theskin of rats (Sabella, Bern & Kahn, 1951) or administered orally in very largedoses (Studer & Frey, 1949), the skin failed to keratinize normally, while theimmature, non-keratinized cells proliferated rapidly and formed a thickepithelium. Similarly, keratinization of the epithelium of the hamster cheek-pouch was inhibited by local treatment with high concentrations of vitamin A,and in some regions the epithelium became thinner and mucus-secreting(Lawrence & Bern, 1960). High concentrations of vitamin A also influencedepidermal differentiation in vitro: when embryonic chick skin grown by theorgan culture method was treated with relatively high concentrations of vitaminA, normal keratinization was inhibited and a mucus-secreting, sometimesciliated epithelium, remarkably similar to that of the normal nasal mucosa,developed instead (Fell & Mellanby, 1953; Fell, 1957).

From these results it is clear that vitamin A exerts considerable influence overepithelial differentiation: excess inhibits keratinization of epithelia and some-

1 Author's address: Physiological Laboratory, Cambridge, U.K.

280 MARGARET B. AYDELOTTE

times induces mucous metaplasia, whereas deficiency in vivo inhibits mucus-secretion and causes keratinization of many mucous membranes.

The effects of citral have been studied much less than those of vitamin A, butLeach & Lloyd (1956) reported that this compound produced endothelialdamage in rabbits and monkeys. They showed that vitamin A could protectagainst and reverse the effects of citral, and they suggested that citral was acompetitive inhibitor of vitamin A aldehyde.

In the present experiments, the role of vitamin A in epithelial differentiationhas been investigated further by testing its effects in vitro over a fairly widerange of concentrations. Vitamin A alcohol was added to the culture mediumto produce hyper-vitamino sis, and citral was used to try to inhibit the vitaminnaturally present in the plasma of the medium, and so cause hypo-vitaminosis.The results of these experiments give further evidence that citral may be acompetitive inhibitor of vitamin A.

MATERIALS AND METHODS

Culture method

Explants, supported by pieces of cellulose-acetate net (Shaffer, 1956), weregrown on a semi-solid medium, in sealed embryological watch-glasses. Thebasic culture medium consisted of 50 per cent fresh cock plasma, 25 per centTyrode's solution and 25 per cent chick embryo extract (prepared from equalvolumes of minced 13-day embryo and Tyrode's solution). Antibiotics werenot used. For experimental media, vitamin A (synthetic crystalline vitamin Aalcohol, prepared by Eastman Kodak Co., U.S.A.) and/or citral (B.D.H.laboratory chemical) were dissolved in ethyl alcohol and added to the plasmabefore making up the media. An equal amount of alcohol was added to theplasma for the control cultures, but the final concentration of alcohol in themedium never exceeded 0-20 per cent.

The trachea was dissected from a 13-day chick embryo and placed in sterileTyrode's solution. Under a dissecting microscope, loose connective tissue wascleaned away and the trachea was cut transversely into three equal lengths.Each segment was then divided by two longitudinal incisions to give twotrough-shaped explants, each approximately 2 x 3 mm. in size. For someexplants the tracheal epithelium, with a little underlying connective tissue, wasdissected carefully away from the cartilage and placed over a thin sheet ofmuscle and connective tissue from the ventral body wall.

The explants were transferred to pieces of rayon net and orientated with theepithelium upwards and the connective tissue in contact with the rayon andculture medium. The cultures were incubated at 38°C. Every 2 days theexplants (still attached to the rayon) were washed in Tyrode's solution andtransferred to fresh medium.

In most experiments, the explants were divided into four groups and cultured

VITAMIN A AND EPITHELIAL DIFFERENTIATION IN VITRO 281

on the following media: 1. Normal medium (72 explants); 2. + Vitamin Amedium (48 explants); 3. +Citral medium (48 explants); 4. + Vitamin A + cit-ral medium (36 explants). In a few preliminary experiments, geraniol, £-iononeand crotonaldehyde were tested on fifty-three tracheal cultures. Explantswere usually fixed after 4 and 6 days, but were sometimes cultured for 8 or10 days. The cultures were examined histologically and compared with eachother and with material from a chick embryo of corresponding age.

Histology

At the end of the culture period, the explants were fixed in Zenker-formol or3 per cent acetic-Zenker and embedded in paraffin wax. Serial sections 5 /x inthickness were stained with Mayer's acid haemalum and alcian blue, and withperiodic acid Schiff and haemalum. Sheets of tracheal epithelium, which hadbeen dissected away from the cartilage of the explant after fixing in formol-alcohol (Moe, 1952), were stained with alcian blue to show the distributionand numbers of mucous cells. Material for comparison with the cultures wasremoved from chick embryos or young chicks, washed briefly in Tyrode'ssolution, and fixed, embedded, sectioned and stained in the same way as theexplants. Altogether, 257 explants of trachea and forty specimens from controlchicks were examined histologically.

RESULTS

Normal development of the tracheal epithelium in vivo

In a 13-day embryo, the tracheal epithelium contained no differentiatedciliated or mucous elements, but consisted of two layers of simple, rounded,actively dividing cells (Plate 1, Fig. A). The first signs of differentiation wereseen in a 16-day embryo, where a few of the cells were ciliated, and otherscontained a mucopolysaccharide which stained with alcian blue. At 17 days,fully differentiated ciliated and mucous cells could be found, and these graduallybecame more numerous until, at the time of hatching, almost all except thebasal cells were differentiated.

Tracheal development in normal medium

In culture, differentiation proceeded at about the same rate as in vivo forthe first 6 days. When the living cultures were examined, ciliation (indicated bymovement of mucus and cell-debris over the surface of the explants) couldfirst be detected after 3 days, and from 4 days onwards it became stronger andmore widespread.

Histological examination showed that the epithelium in these explants wasusually shghtly thinner than normal (probably because of migration from thecut edges of the explants), but otherwise it resembled that of the normal tracheaof corresponding age quite closely. After 4 days in culture, a few well-filled


goblet cells, and many others just beginning to synthesize mucus, were inter-spersed between groups of ciliated cells. After 6 days in vitro (Plate 1, Fig. B),most of the columnar cells were either ciliated or secretory, and the epitheliumresembled that from a 19-day embryo (Plate 1, Fig. C). Mucous cells developedin largest numbers near the edges of the explants above the cut ends of thecartilage bars, but in the rest of the epithelium they were rarely as numerousas in the intact animal.

Explants of tracheal epithelium on connective tissue (epithelial cultures)often differentiated better than those containing cartilage: there was lessmigration and, because of the absence of cartilage, the epithelium came intomuch closer contact with the nutrient medium. The epithelium became pseudo-stratified after 6 days, with groups of ciliated and mucous cells, and a fewapparently undifferentiated cells (Plate 2, Fig. D). When small ridges andfolds appeared in the mucosa, the mucous cells were usually in the hollowsforming intra-epithelial glands, whilst the ridges were ciliated; this patternof differentiation was strikingly similar to that of the normal chick trachealepithelium.

Usually, fewer mucous cells differentiated in culture than in vivo, but thenumber was very variable, and there is some evidence that this was due, at leastin part, to variations in the level of natural vitamin A in the medium. Apartfrom this the tracheal epithelium in both types of explant differentiated in afairly normal manner, so it was possible to use these cultures for testing theeffects of vitamin A and citral.

EXPLANATION OF PLATES

The figures are photomicrographs of tissue stained with Mayer's acid haemalum andalcian blue; mucus, and other alcian blue-positive material appears dark.

PLATE 1

FIG. A. Transverse section of the trachea of a 13-day chick embryo, showing the epitheliumat the time of explantation.

FIG. B. Section of a tracheal explant, cultured for 6 days on a normal medium. Theepithelium is well ciliated and contains several mucous cells.

FIG. C. Section of the trachea of a 19-day embryo, i.e. at an age corresponding to a 6-dayculture. The epithelium is folded and well ciliated and a few mucous cells are developing.

PLATE 2

FIG. D. Section of an explant of tracheal epithelium, grown for 6 days on a normal medium.Mucous cells are beginning to form small, intra-epithelial glands.

FIG. E. Sheet of epithelium dissected from a tracheal explant which had been grown for4 days on a normal medium. The mucous cells, stained with alcian blue, appear as small,dark spots.

FIG. F. Sheet of epithelium dissected from a tracheal explant which had been grown for4 days on a medium containing 7 • 5 i.u. of vitamin A/ml. Note how few mucous cells havedeveloped in comparison with the control culture shown in Fig. E.

FIG. G. Section of tracheal epithelium grown for 6 days on a medium containing 10 i.u.of vitamin A/ml. The epithelium is well ciliated but devoid of mucous cells.

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Tracheal development in + vitamin A medium

Tracheal explants grown in media containing additional vitamin A showedgood ciliation and usually secreted less mucus than the control cultures. Whenthe cultures were washed and changed to fresh medium, the + A explants hadlittle or no detectable secretion, whilst the control cultures were sticky withmucus.

The inhibitory effect of vitamin A on mucus-secretion was also observed infixed explants: whole mounts of epithelium stained with alcian blue for mucouscells, showed that the + A explants (Plate 2, Fig. F) contained far fewer mucouscells than the controls (Plate 2, Fig. E). Development of mucous cells was oftenmost strongly inhibited in the migrating epithelium over the outgrowth, butthis region could not be preserved in whole mounts.

Vitamin A favoured the differentiation of ciliated cells, so that a greaterproportion of the surface became ciliated in the + A cultures than in thecontrols. The effect of the vitamin depended upon the dose used: with a lowconcentration, e.g. 2-5 i.u./ml. of medium, ciliation was noticeably better thanin the controls, but differentiation of mucous cells was only slightly inhibited.In higher concentrations of vitamin A, e.g. 12-5 i.u./ml. of medium, the trachealepithelium became columnar and very well ciliated, and mucus synthesis wasalmost completely inhibited during the first 4 days in vitro. After 6 or 8 days,however, some cells had begun to synthesize mucus; vitamin A apparentlydelayed synthesis in these cells but did not suppress it indefinitely.

In response to high concentrations of vitamin A, cultures of tracheal epithe-lium with connective tissue but no cartilage showed changes similar to those

PLATE 3

FIG. H. Section of a tracheal explant grown for 4 days on a medium containing 2 0 mMcitral. Many of the superficial cells contain mucus but no ciliated cells have developed.

FIG. I. Section of a tracheal explant cultured for 6 days on a medium containing 3-0 mMcitral. The basal cells have multiplied and formed a stratified epithelium. The superficialcells contain very little mucus, although much has already been secreted and can be seenabove the epithelium.

FIG. J. Section of the central part of an explant of tracheal epithelium, grown for 6 dayson a medium containing 2-0 mM citral. Some of the superficial cells are ciliated (c), but theyare being pushed away from the basement membrane by the hyperplastic basal cells.

PLATE 4

FIG. K. Section of the edge of the explant shown in Fig. J, cultured for 6 days on a mediumcontaining 2 • 0 mM citral. The mucous cells are vacuolated and being sloughed, as a stratified,keratinizing epithelium develops underneath.

FIG. L. Section of tracheal epithelium cultured for 6 days on a medium containing 10 i.u.of vitamin A/ml and 2 • 5 mM citral. This culture shows a very slight citral effect: the epithe-lium is well ciliated and contains rather more mucous cells than that of the control shown inFig. D. Many of the cells are vacuolated, which is quite common for cultures grown in thepresence of citral.


described above. Mucus synthesis was almost completely inhibited for the first6 days, and the majority of the superficial cells became ciliated (Plate 2, Fig. G).Small groups of apparently undifferentiated cells, which neither containedmucus nor bore cilia, but resembled the basal cells in size and staining properties,were found between the large groups of ciliated cells. Probably under normalcircumstances these cells would have differentiated into goblet cells, but theirnormal synthetic activity was inhibited or delayed by the high concentration ofvitamin A.

Trachea! development in + citral medium

Tracheal explants were cultured in concentrations of citral ranging from0 • 2-5 • 0 mM for periods up to 8 days. In the lowest concentration, no differencescould be detected during cultivation, between the control explants and thosegrown with citral, but with concentrations of 1 • 0-3 • 0 mM the followingobservations were made on the living cultures. Citral inhibited the outgrowthof fibroblasts. Ciliation could not be detected after 4 days in vitro, and onlyvery slight activity was seen after 6 days. Citral stimulated the secretion ofmucus by the tracheal epithelium, and when explants were washed and changedto fresh medium, large quantities of viscous material covered each explant.Epithelial cultures grown on the + citral medium appeared thicker and moreopaque than the controls. Citral was toxic at concentrations above 3-0 mM,and the explants were usually dead after 2-3 days.

Histological examination showed that the lowest dose of citral (0-2 mM)stimulated the differentiation of mucous cells, especially in the migratingepithelium. Higher concentrations activated the differentiation of mucous cellsat the expense of ciliated cells, so that after 4 days on a medium containing1 • 0 mM citral, the epithelium contained only a few small groups of columnarciliated cells but many mucous elements. After 6 days on this medium, theepithelium was better ciliated than at 4 days, but many non-ciliated patchespersisted. Mucus synthesis and secretion had increased between 4 and 6 days,and secreted mucus lay thickly over the epithelium. Citral also stimulatedmitosis, with the result that the epithelium gradually became stratified. Thiswas often noticeable in the epithelium over the outgrowth, which instead ofbeing thin and composed of one or two layers of flattened cells as in the controlcultures, was much thicker with about four layers of very flattened cells beneaththe superficial mucous cells.

Higher concentrations of citral produced more drastic changes: after 4 dayson a medium containing 2-0 mM citral, there were practically no ciliated cellsin the epithelium, and a high proportion of the superficial columnar cellscontained mucus (Plate 3, Fig. H). The basal cells were dividing rapidly andhad formed a complete layer below the outer cells. During the next 2 days,.synthesis and secretion of mucus continued and the epithelium became thicker.

Differentiation of ciliated cells was inhibited almost completely by a con-


centration of 3 • 0 mM citral, and after 4 days the majority of the superficialcells contained mucus. After 6 days, the epithelium was stratified, and althoughthe explant was covered by a thick layer of mucus, the superficial cells hadalmost exhausted their supplies of secretion (Plate 3, Fig. I).

The effects of citral, however, were seen more clearly in explants of epitheliumwithout cartilage. Concentrations of 2 • 0 or 2 • 5 mM gave the best results, asthese were the highest doses that could be used consistently without killing thecultures. The response to citral varied from one region of the explant to another.In the central part (Plate 3, Fig. J), approximately half of the outermost cellswere ciliated after 6 days: some of these ciliated cells were still columnar, butothers had lost contact with the basement membrane and become spherical.Narrower, columnar mucous cells were found between the groups of ciliatedelements, and many small cells on the basement membrane were also syn-thesizing mucus. Near the edge of the explant, no cilia developed, but theepithelium was composed of cuboidal mucous cells above two or three layersof low cuboidal cells. At the extreme edge of the culture, the superficial layerof cuboidal mucous cells was becoming vacuolated and being sloughed, leavinga thick, stratified, squamous and keratinizing epithelium underneath (Plate 4,Fig. K).

The most marked citral effect in these cultures, therefore, was at the extremeedge where the connective tissue was thinnest. Probably the epithelium of thecentral region began to differentiate almost normally at the beginning of theculture period, before the citral penetrated the connective tissue. At the edge,however, citral at first stimulated the development of mucous cells, but thesewere not long maintained because later, the rapid division of the basal cellsgave rise to a keratinizing epithelium. Variation in thickness of the connectivetissue, therefore, may account for the regional differences in the epithelium ofthese explants.

Tracheal development in + vitamin A + citral medium

Cultures grown in media containing vitamin A and citral were comparedwith those grown on normal medium and on media containing the same con-centrations of vitamin A or citral alone. The results varied according to theproportions of vitamin A and citral: some explants, examined while living,could not be distinguished from those on normal medium, whilst others showedchanges similar to those in cultures grown on media containing low concentra-tions of citral.

Cultures grown on a medium containing 10 i.u. of vitamin A/ml, and 2-0 mMcitral showed a slight citral effect: the epithelium contained more mucous cellsand fewer ciliated cells than the control explants, and the basal layer was slightlyhyperplastic. This was a mild citral effect, since 2-0 mM citral alone causedmarked hyperplasia of the basal cells, and almost completely suppressedciliation in favour of the differentiation of mucous cells. In this experiment,


therefore, vitamin A lowered the effective concentration of citral but did notovercome its eifects completely.

In another experiment, in which the concentrations of vitamin A and citralwere 2-5 i.u./ml. and 3-0 mM respectively, the cultures showed a greater citraleffect than those of the previous experiment: in the central parts of the explants,the epithelium was thick, composed of a hyperplastic basal layer, a high pro-portion of mucous cells and no ciliated cells. The edges of the cultures were notvery healthy but they survived better than those grown on a medium containing3-0 mM citral alone. Thus vitamin A reduced the toxicity of citral.

Cultures of tracheal epithelium, grown on a medium containing 10 i.u. ofvitamin A/ml, and 2 • 5 mM citral, showed a very slight citral effect: the epitheliumhad rather more mucous cells, and was not quite as well ciliated as that of thecontrols, but the basal cells were not hyperplastic (Plate 4, Fig. L).

These results indicate that the effects of vitamin A were inhibited by addingcitral to the medium. The combined effect of vitamin A and citral dependedupon their relative concentrations, and varied from almost zero to an effectsimilar to that produced by a lower concentration of citral alone.

The effects ofgeraniol, fi-ionone and crotonaldehyde

In some preliminary experiments a few compounds chemically related tocitral were also tested on cultures of chick trachea. Geraniol, the alcoholcorresponding to citral, had a very slight inhibitory effect on the ciliated cells,and stimulated mucus-secretion slightly at a concentration of 2 0 mM, but itdid not cause hyperplasia of the basal cells of the tracheal epithelium. Theseeffects, therefore, were much less marked than those of citral. ]8-ionone ap-peared to have no specific effect on the differentiation of the tracheal epithelium,but it was toxic at about the same concentration as citral (i.e. 3-0-4-0 mM).The unsaturated aldehyde, crotonaldehyde, was much more toxic to thetracheal epithelium than citral, but it did not specifically affect differentiation.

DISCUSSION

Although vitamin A is essential for the normal maintenance of mucus-secretory epithelia, and in high concentrations it can promote mucous meta-plasia of some keratinizing epithelia, in the experiments just described highconcentrations of vitamin A actually inhibited synthesis and secretion of mucusby the chick tracheal epithelium. Previous experiments, however, show thatthe concentration of vitamin A need not be abnormally high to inhibit mucus-secretion by the tracheal epithelium; indeed, in young chicks, secretion ispartly inhibited by the normal concentration of vitamin A in the body (Aydelotte,unpublished). In the early stages of vitamin A deficiency, secretion in-creased as the mucous cells and glands became larger, whilst the ciliated cellswere gradually reduced in number. With more severe deficiency, the basal


cells divided rapidly and formed a stratified, keratinizing epithelium, whicheventually replaced the secretory epithelium.

Experiments on chick skin in culture (Fell & Mellanby, 1953; Fell, 1957)also indicate that high concentrations of vitamin A are inhibitory to mucouscells: when skin cultures which had undergone mucous metaplasia in responseto excess of vitamin A, were transferred to normal medium, mucus-secretionincreased for the first few days, and then the secretory cells atrophied as theywere replaced by a stratified, squamous, keratinizing epithelium. These resultsshow that relatively high concentrations of vitamin A are inhibitory to mucus-secretion, and they suggest that secretion is maximal at a particular concentra-tion of vitamin A, which varies with different epithelia.

Since the mucous cells of the chick tracheal epithelium are so sensitive tothe environmental concentration of vitamin A, variation in the level of thevitamin in the plasma of the medium may account for the variation in thenumber of mucous cells that differentiated in the control cultures. Normally,fewer mucous cells developed in culture than in vivo, which suggests that thenormal culture medium was slightly hypervitaminotic to the tracheal epithelium.

Citral added to the medium in fairly low concentrations, produced effectsdirectly opposite to those of vitamin A, and inhibited the development ofciliated cells whilst stimulating the differentiation of mucous cells and synthesisof mucus. Higher concentrations stimulated division of the basal cells, so thateventually the secretory cells were replaced by a stratified epithelium whichoccasionally keratinized. These changes in the tracheal epithelium in responseto citral were remarkably similar to those in vitamin A deficiency in vivodescribed above. This suggests that in the present experiments citral inhibitedthe natural vitamin A in the plasma of the culture medium, thereby producinghypovitaminosis A in vitro.

This hypothesis was tested further by culturing the chick trachea in a mediumto which both vitamin A and citral had been added, to see if citral could alsoinhibit synthetic vitamin A added to the plasma. In these experiments, citralovercame the effects of vitamin A, and the combined action of the two com-pounds resembled a citral effect. The final result, however, was similar to thatproduced by a lower concentration of citral than was actually present, so thatthe vitamin reduced the effectiveness of the citral, and epithelial differentiationvaried according to the ratio of the two compounds.

Variations in the level of natural vitamin A in the plasma also influenced theresponse to citral: when the plasma was rich in natural vitamin A, as indicatedby the differentiation of the control explants, the citral effect was corre-spondingly mild. Although a vitamin A effect was not seen in tracheal culturesgrown with both vitamin A and citral, this has been observed in cultures ofother tissues (Aydelotte, 1963, in preparation), and it could almost certainly beachieved in the trachea by using less citral with more vitamin A. The concentra-tions of vitamin A and citral that most nearly balanced each other were 10


i.u./ml. and 2-0 mM respectively; in this medium, the molecular ratio of vitaminA to citral was approximately 1:200.

There seems good evidence, therefore, that in this culture system vitamin Ais antagonized by citral. The mechanism of inhibition, however, is not under-stood. Leach & Lloyd (1956) pointed out that since citral is chemically similarto part of the vitamin A molecule, it might act as a competitive inhibitor ofvitamin A. In addition, they reported that other unsaturated aldehydes similarto citral, with the group —C:CH.CHO (e.g. crotonaldehyde, furfural, cin-namic aldehyde and crocetin aldehyde), and their corresponding alcohols andacids, also produced changes similar to those of citral poisoning. The struc-tures of vitamin A, citral and other compounds tested on tracheal cultures are

TABLE 1

Structures of compounds which were tested on organ cultures of chick trachea

Vitamin Ax

H 3 C\ /CH3 CH3 CH3C I I

C ^C—CH=CH—C=CH—CH=CH—C=CH—CH,0H

CH2 CH3

Citral (2 molecules)

C H C ? H s

HO* CH—CHO 3 ^C=CH—CH2—CH2—C=CH—CHO

1

CH2

Geraniol (2 molecules)

3 H C I 3

CH—CH2OH 3 XO=CH—CH2—CH2—O-CH—CH2OH

B-ionone/CH3 CH3

' VC—CH^CH—C=0

2 CH3

Crotonaldehyde

CH3

HC=CH—CHO

shown in Table 1. It can be seen that the citral molecule is approximately halfthe size of that of vitamin A and, moreover, citral resembles not only theside-chain, but also the jS-ionone ring of vitamin A aldehyde.


In an attempt to find whether citral acts as an inhibitor of vitamin A becauseof its similarity either to the £-ionone ring or to the unsaturated side-chain ofvitamin A, several other related compounds were tested on chick trachealcultures. The results of these preliminary experiments, however, give no clearindication of how citral inhibits vitamin A, and it has not yet been possible totest vitamin A and citral together over a very wide range of concentrations, aswould be necessary to demonstrate competitive inhibition.

So far, relatively little is known of the way in which vitamin A acts onepithelia, but possibly citral may help to elucidate this problem. Recentexperiments indicate that vitamin A may function as a coenzyme in mucopoly-saccharide synthesis (Wolf, Varandani & Johnson, 1961; Moretti & Wolf, 1961).It is unlikely, however, that the only influence of vitamin A in epithelia is itsdirect effect on mucus synthesis; it also seems to affect the rate of proliferationof the basal cells. Since the tracheal basal cells normally have a low mitoticrate, the epithelium is replaced slowly, but in vitamin A deficiency and in citraltreatment, the basal cells divide much more frequently and form a stratifiedepithelium. Possibly the normal concentration of vitamin A in the bodypartially inhibits mitosis in this tissue, but when the animal becomes vitamin Adeficient or when the trachea is treated with citral, the cells are released fromthis inhibition. When the basal cells divide very actively, their daughter-cellsare soon pushed into the higher layers of the epithelium and become separatedfrom the basement membrane. Since the nutrients necessary for mucussynthesis may not be readily available to cells in the superficial layers of astratified epithelium, such cells may keratinize instead of becoming secretory.In this way the effect of vitamin A on the mitotic rate of an epithelium may wellbe important in influencing differentiation.

SUMMARY

1. Differentiation of the tracheal epithelium of embryonic chicks was studiedin organ cultures in a normal medium and in media containing added vitamin Aand citral.

2. In normal medium, the tracheal epithelium differentiated well, but usuallyfewer mucous cells developed than in the intact chick.

3. High concentrations of vitamin A alcohol inhibited the differentiation ofmucous cells and favoured that of ciliated cells.

4. Citral, in fairly low concentrations, had opposite effects: it stimulatedmucus-secretion and inhibited the differentiation of ciliated cells. In higherconcentrations it stimulated division of the basal cells, and the epitheliumbecame stratified and sometimes keratinized.

5. Vitamin A and citral added together to the culture medium produced acitral effect, which varied in intensity according to the proportions of the twocompounds, but was always milder than that resulting from treatment with thesame concentration of citral alone.

19


6. These results suggest that citral inhibits both the natural vitamin A in theplasma and the added synthetic vitamin A, thus producing changes in thetracheal epithelium characteristic of vitamin A deficiency.

7. The results indicate that secretion of mucus by the tracheal epithelium ismaximal at a lower concentration of vitamin A than that to which it is normallyexposed in the body. It is suggested that vitamin A may influence epithelialdifferentiation partly by affecting the mitotic rate.

RESUME

Les effets de la vitamine A et du citral sur la differentiation epitheliale in vitro./. Vepithelium tracheen du poulet

1. La differenciation de l'epithelium tracheen de Pembryon de poulet a eteetudiee en cultures d'organes dans un milieu normal et dans des milieuxadditionnes de vitamine A et de citral.

2. En milieu normal, l'epithelium tracheen se differencie bien, mais generale-ment il se developpe moins de cellules a mucus que dans Pembryon entier.

3. De fortes concentrations d'alcool-vitamine A inhibent la differenciationdes cellules a mucus et favorisent celle des cellules ciliees.

4. Le citral, a des concentrations tres faibles, a des effets opposes: il stimulela secretion de mucus et inhibe la differenciation de cellules ciliees. A de plusfortes concentrations, il stimule la division des cellules basales, l'epitheliumdevient stratifie et parfois se keratinise.

5. La vitamine A et le citral ajoutes ensemble au milieu de culture ont lememe effet que le citral; Pintensite ce cette action varie avec les proportionsdes deux composants. Elle est toujours plue faible qu'avec la meme concentra-tion de citral seul.

6. Ces resultats suggerent que le citral inhibe aussi bien la vitamine A duplasma que la vitamine A synthetique ajoutee au milieu; il determine ainsi desmodifications de l'epithelium tracheen caracteristiques d'une carence envitamine A.

7. Ces resultats indiquent que la secretion de mucus par l'epithelium tracheenatteint son maximum a une concentration de vitamine A plus faible que celle alaquelle il est soumis normalement dans Pembryon. On suppose que la vitamineA agit sur la differenciation epitheliale, en particulier en modifiant le taux desmitoses.

ACKNOWLEDGEMENTS

The author wishes to thank Dr E. N. Willmer for his helpful guidance in this research,and Dr H. B. Fell for her kind interest and advice during the progress of the work, and helpfulcriticism during the preparation of this paper. Financial support from the Medical ResearchCouncil (Scholarship for training in research methods) is gratefully acknowledged.


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high vitamin A. / . Physiol. 119, 470-88.JUNGHERR, E. (1943). Nasal histopathology and liver storage in subtotal vitamin A deficiency

of chickens. Bull. Storrs. agric. Exp. Sta. 250, 1-36.LAWRENCE, D. J. & BERN, H. A. (1960). Mucous metaplasia and mucous gland formation

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LEACH, E. H. & LLOYD, J. P. F. (1956). Citral poisoning. Proc. Nutr. Soc. 15, xv.MOE, H. (1952). Mapping goblet cells in mucous membranes. Stain Tech. 27, 141-6.MORETTI, A. & WOLF, G. (1961). Decrease in mucopolysaccharide-bound hexosamine of

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(Manuscript received 5th October 1962)

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The Effects of Vitamin A and Citral on Epithelial ...epithelium is well ciliated and contains...

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