Distribution of Ascorbic Acid (vitamin C) in cells and...

Distribution of Ascorbic Acid (vitamin C) incells and tissues of the Developing Chick.

By

S. A. Barnett and 6. Bourne*Department of Human Anatomy, Oxford.

With 35 Text-figures and Plates 14 and 15.

CONTENTS.PAGE

I. INTRODUCTION 259

II. MATERIAL AND METHODS 261

III. RESULTS 262

1. Skeletal Tissues, p. 262. 2. Other Mesenchyme Deriva-tives, p. 270. 3. Blood and Belated Tissues, p. 271.4. Liver, p. 273. 5. Thyroid, p. 276. 6. Epithelia andEpithelial Derivatives, p. 276. 7. Adrenal, p. 277.8. Gonads, p. 279. 9. Nephroi, p. 280. 10. NervousSystem, p. 284.

IV. GENERAL DISCUSSION 291

V. SUMMARY 294

I. INTRODUCTION.

THE distribution of ascorbic acid in chick embryos up to fourdays' incubation was described in a previous communication(Barnett and Bourne, 1941). Use of the acid silver nitratemethod for its histological demonstration indicated that ascorbicacid was present, up to this age, only in the extra-embryonictissues and the yolk underlying the embryo. In accordance withthe titrimetric results of Eay (1934) it was found that thevitamin appears in the embryonic tissues on the fourth day.The tissues which contain it are those of the central nervoussystem, certain mesoderm derivatives including the myomere,the liver and pancreas diverticula, and the epidermis. Theresults of continuing these investigations, on later stages in the

1 Formerly Beit Memorial Research Fellow.

NO. 331 T

260 S. A. BARNETT AND G. BOURNE

development of the chick, are described below. The periodcovered is from the fourth day of incubation to two days afterhatching. During this period the following were found to con-tain marked quantities of ascorbic acid: skeletal tissues;erythrocytes; liver; thyroid; adrenal; gonads; nephroi;nervoussystem. Some other tissues were found to contain ascorbic acid,and they are briefly considered in the sections on mesenchymederivatives and epithelia.

Previous studies of the distribution of ascorbic acid in tissueshave been carried out in three ways. In the first place, theamount present can be approximately established by biologicaltests on scorbutic animals. In the second place, its concentrationcan be more accurately determined by titration, for example bydichlorophenolindophenol (Harris and Ray, 1933); in additionfor small amounts a micrometric technique has been elabo-rated by Glick and Biskind (1935). None of these methodspermits the study of the differing contents of the cell types inmixed tissues, or of the relation of ascorbic acid to cell struc-tures. The third method permits both of these, but is notquantitative. The technique was adumbrated by Szent-Gyorgyi (1928) in his studies of a powerful reducing agent inthe adrenal gland; in these studies he showed that this agent,which was later shown to be vitamin C (Waugh and King, 1932),has a particularly strong reducing action on silver nitrate. Thisfact was used by Bourne (1933 a, b), Leblond (1934), and Giroud(1938 a) in elaborating a method for the specific demonstrationof ascorbic acid in microscopic preparations.

The acid silver nitrate technique, as developed by theseauthors, has been used in extensive studies of the cytology ofascorbic acid in adult tissues; surveys of this work have beenrecently published by Giroud (1938) and Tonutti (1939). Onthe other hand, little is known of the distribution of ascorbicacid during histogenesis, although the vitamin is believed toplay an important part in the laying-down of some intercellularsubstances (Wolbach and Howe, 1926; see also Dalldorf, 1939).The following survey is designed as a preliminary to the in-vestigation of the role of ascorbic acid in developmental pro-cesses.

ASCORBIC ACID IN CHICK 261

II. MATERIAL AND METHODS.

Owing to the solubility of ascorbic acid fixation and impregna-tion with silver are carried out together. The most satisfactorymixture for this purpose was found to be a 10 per cent, solutionof silver nitrate in 10 per cent, acetic acid. A similar solutioncontaining 50 per cent, alcohol was tried, but was found not togive even impregnation throughout relatively large pieces oftissue. Four-day embryos were fixed for 45 minutes, largerembryos for longer periods, up to 90 minutes for a 12-dayembryo. They were then thoroughly washed in distilled water,dehydrated, and embedded in paraffin. All the sections werecut at 5 /x, brought down to water, and toned in very dilute goldchloride for from 5 to 10 minutes. This was followed by a similarperiod in sodium thiosulphate, after which they were againwashed thoroughly in distilled water and dehydrated. Xylenewas used as a 'clearing' agent,since cedar-wood oil obscures thegranules of precipitated silver which indicate the presence ofascorbic acid. The sections were mounted in Canada balsam.Fixation and the subsequent treatment up to embedding werecarried out in the dark, and exposure to strong light wasavoided at all stages.

The evidence for the validity of this method has been con-sidered in detail elsewhere (see Barnett and Bourne, 1941). Itdepends in the first place on the relative difficulty with whichsilver nitrate is reduced, when in acid solution and in the dark.The use of silver nitrate in other histological procedures, suchas those of Cajal and da Fano, involves its reduction, by a furtherreagent, after it has been absorbed by the tissues. In the methodused here the silver nitrate is directly reduced. Leblond (1934)has shown that, of known reducing agents occurring in cells,only ascorbic acid reduces silver nitrate in the given conditions.The same author has demonstrated a parallelism between theascorbic acid contents of various plant and animal tissues, asestimated by titration, and the reaction shown with the silvernitrate reagent. Most important of all have been the observa-tions by various authors that the reaction disappears from thetissues of an animal in the course of the development of scurvy

262 S. A. BAENETT AND G. BOURNE

(Leblond, 1934; Bourne, 1935 a; Giroud and Leblond, 1936,1937). The present authors (1941), on the basis of this and otherevidence, have concluded that it is 'justifiable +o assume thatthe reactions observed are unlikely to be due to reducing sub-stances other than ascorbic acid'.

In Table 1 is set out the material used for the account whichfollows.

TABLE 1.—Material. 'Urogenitalia' includes adrenals. For the studyof ossification the femur and frontal bones were used, and in some oasesvertebrae and the clavicle. Eighteen- and 23-day bone was decalcified in5 per cent, trichloracetic acid for 24 hours. In all tissues adjacent sectionswere taken for a duplicate set, and stained with haematoxylin and eosin.

Days.

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III. EESULTS.

1. Ske le t a l T i ssues .E e s u l t s .

C a r t i 1 a g e.—Small amounts of ascorbic acid can be detectedin the sclerotome at the fourth day (see Barnett and Bourne,1941). At the fifth day ascorbic acid is present in larger quanti-ties in the mesenchyme which surrounds the notochord; thismesenchyme is destined to form cartilage. The first morpho-logical indication that mesenchyme cells are about to formchondroblasts is the withdrawal of their processes and theappearance of a 'mucinoid matrix' (Fell, 1925); the resultingtissue is called precartilage. Before this happens, and in regions

ASCOEBIC ACID IN CHICK 263

in which cartilage is about to develop, a deposit of ascorbic acidappears, mainly in the processes of the cells (Text-fig. 1). Asimilar process can be observed, not only in the early embryo,but later, when some cartilage is already formed and undergoingaccretionary growth: such cartilage shows a well defined outerzone of mesenchyme, within which is precartilage; both layerscontain ascorbic acid, and the amount diminishes with increas-ing distance from the surface (fig. 1, PL 14). The majority ofdifferentiated cartilage cells show little or no reaction, but small

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TEXT-BIG. 1.

Mesenchyme cells of 5-day embryo from region of notochord. Thejoining of the processes is an artefact due to bad fixation (Fell,1925). Ascorbic acid mainly in the processes.

groups of heavily impregnated cells occur. Sometimes thesegroups are wholly surrounded by ordinary cartilage cells, butmore often they are peripherally situated (figs. 1, 3, 4, PL 14).The deposit is typically in the form of granules which areapproximately spherical, and in the most heavily impregnatedcells the granules fill the cytoplasm; in almost all cases there issome concentration near the nucleus; this may take the formof granules evenly spread over the nuclear surface, or there maybe a group at one pole. There is much variation in the detailsof the arrangement. In some cases a series of cells can beobserved in the same cartilage, showing what appear to bedifferent stages in the accumulation of ascorbic acid; some havenone, some have a localized area of cytoplasm containing moreor less ascorbic acid, and in some the whole of the cytoplasm


is filled. The main types of distribution are illustrated in Text-figs. 2 and 3, and figs. 1, 3, 4, PL 14.

TEXT-FIG. 2.

Chondroblasts from 12-day vertebra, showing variations in ascorbicacid content.

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TEXT-FIG. 3.

Chondroblasts from 10-day vertebra, showing local impregnation.

Ascorbic acid is not confined to the cells in cartilage: it occursalso in the ground substance of precartilage and possibly of thefully developed tissue (fig. 2, PI. 14). In the latter it probably


occurs only in connexion with the development of replacingbone, which is described below.

Bone.—The mesenchyme which is destined to form mem-brane bone contains ascorbic acid distributed in the same wayas in precartilage. When the differentiation into osteoblasts

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TEXT-ITG. 4.

Twelve-day osteoblasts.

begins the cells lose their ascorbic acid; those which do notcomplete the differentiation, but remain on the surface of thedeveloping bone, continue to show no reaction; the earlyosteoblasts themselves generally contain a few granules; in welldeveloped bone, such as is seen in the femur two days afterhatching, a few cells are heavily impregnated (Text-figs. 4, 5).The deposit is always granular and diffuse. Klein (1938), usingAuCl3 instead of AgNO3 for the demonstration of ascorbic acid,has observed a similar impregnation in periosteal mesenchymein pig embryos; he found that the osteoblasts also give a strongreaction, either local or diffuse, both when on the surface andwhen embedded in the calcined matrix.

266 S. A. BAENBTT AND G. BOUBNE

In regions where calcification has not begun, but where thecollagenous 'osteogenic' fibres have appeared, extra-cellularascorbic acid is present; it occurs in granules which can oftenbe observed to be arranged in rows along the cohagen fibres(Text-fig. 6, and fig. 7, PI. 15). Very fine granules are also

1

TEXT-ITGS. 5-7.

Fig. 5. Twenty-three-day osteoblast with heavy deposit.Fig. 6. Collagenous, 'osteogenic' fibres between osteoid trabeculae

at 12 days; ascorbic acid is in association with the fibres in theground substance.

Fig. 7. Deposit visible in recently laid down bone after treatmentwith ammonia (see text). Twelve days.

present, rather unevenly distributed, in freshly calcified bonelaminae. These can only be observed after treatment withammonia (Text-fig. 7); when freshly developed bone is treatedwith acid silver nitrate a heavy black precipitate generallyappears in the bone laminae; this precipitate, which is presumedto be a silver salt, disappears if the tissue is left for five minutesin 5 per cent, ammonia (Barnett et al., 1941). Treatment ofother tissues, such as adrenal, with ammonia causes no alterationin the appearance of the preparation.

In the development of cartilage bone, whether endochondral

ASCOEBXC ACID IN CHICK 267

or perichondral, the osteoblasts do not differ from those ofmembrane bone. The special phenomenon associated with thisprocess is the appearance of a deposit in the cartilaginousmatrix around the hypertrophied cartilage cells; the latter donot contain ascorbic acid (fig. 2, Pi. 14). This deposit 'issimilar to that obtained by applying von Kossa's AgN03

technique for the detection of calcium to developing bone.Since the ammonia modification was not applied to thesepreparations, the precipitate may be due to the presence ofbone salts, and not to the action of ascorbic acid. (SeeBarnett et al., 1941.)' Before cartilage cells enlarge anddegenerate they have aligned themselves in a characteristicfashion which foreshadows the appearance of bone; at thisstage they may contain ascorbic acid in small amounts, generallylocalized at one pole of the nucleus.

Di scuss ion .It is well known that fragility of the bones is a characteristic

symptom of the terminal stages of acute human scurvy; thesame condition has been observed in cavies kept on a scorbuticdiet (Eddy and Dalldorf, 1937; Ham and Elliott, 1938). Asmight be expected, ascorbic acid is important in regenerationas well as in maintenance: Jessey and Korpassy (1934) andKlein (1938) have reported a failure of healing in the experi-mentally fractured bones of cavies on a vitamin C deficientdiet. According to Hanke (1935) even rabbits, which normallysynthesize all the ascorbic acid they require, produce a callusmore rapidly after a fracture if ascorbic acid is administered;Giangrasso (1939) reports a similar result.

It now seems well established that ascorbic acid plays anessential part in the laying-down of certain intercellular colloidalsubstances of mesenchymal origin. Wolbach and Howe (1926)studied these processes in the connective tissue of animalssuffering from experimental scurvy. They found that in vitaminC deficiency the formation of cartilage and bone matricesstops; the osteoblasts undergo a reversible change into a fibro-blast-like form, but continue to proliferate: the result of thesechanges is said to be an oedematous connective tissue (the

268 S. A. BARNBXT AND G. BOUBNB

Geriistmark) which can, on administration of vitamin C, beinduced to redifferentiate into skeletal tissue. Other worktends to confirm the importance of ascorbic acid in the laying-down of collagen: Mazoue (1937) found that the formation ofcollagen in granulomata in cavies is prevented by ascorbic acid;the formation of collagen in v i t ro has also been shown todepend on the presence of the vitamin (v. Jeney and Toro1936; Querido and Gaillard, 1939). With reference to main-tenance, Bayer (1931) has reported that in the weakened boneof scorbutic cavies the mineral content remains normal.

It is still not decided whether ascorbic acid affects the cells,or has a direct influence on the behaviour of the colloidalmaterial. Wolbach and Howe (1926) supposed that the latteris the case: the cells only degenerate in the final stages ofscurvy when the blood supply is seriously interrupted andextensive metabolic disturbances have occurred; these authorstherefore suggest that ascorbic acid is required for the gellingof the intercellular material. The observation, reported above,of the association of ascorbic acid with 'osteogenie' fibres,provides some support for this view, especially since the cellsthemselves contain very little of the vitamin. The gel theory has,however, been criticized by various authors (Fish and Harris,1934; Ham and Elliott, 1938; McClean et al., 1939). McCleanand his co-workers report that in young scorbutic cavies thedifferentiation of mesenchymal cells to osteoblasts, and theirfunctioning in bone formation, are impaired. The suggestionthat ascorbic acid is concerned in the differentiation of mesen-chymal cells to osteoblasts is concordant with the observation,made in this study, of its presence in cells undergoing thisdifferentiation. It is interesting in this connexion that in thecavy osteoblasts revert to a fibroblast structure when deprivedof ascorbic acid (Wolbach and Howe, 1926).

One further observation remains to be discussed: this is theoccurrence of the groups of heavily impregnated cartilage cellsdescribed above. The distribution of these cells, mainly at theedges and especially the ends of cartilages, suggests immediatelythat they occur in the regions of active cell-division. Thispossibility is confirmed by careful study of the most favourable


material, including the duplicate stained sections; in the latterthe heavily impregnated cells are easily picked out, and in bothstained and unstained sections they can b.e seen in some casesto be dividing (Text-fig. 8). The ascorbic acid is collected roundthe nucleus, •which is consequently obscured; when the spindle

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Dividing cartilage cells at 12 days, with concentration of ascorbicacid round nuclei; in b the spindle is visible, in c the daughternuclei are separate, but the cytoplasm has not yet begun to divide.Drawn from stained preparation.

elongates it, too, is surrounded by ascorbic acid; in what appearsto be telophase the reaction is again confined to the regions ofthe two daughter nuclei. Text-fig. 8 c may be compared withHam's (1932, p. 997) photograph of a cartilage cell in a latephase of mitosis. It is interesting that in the adjacent connec-tive tissue other dividing cells can be seen in the stained pre-parations ; and these do not contain the high concentrations of

270 S. A. BABNETT AND G. BOUKNB

ascorbic acid. Cartilage cells seem to be unique in accumulatingthe vitamin before division: as a rule, in animal tissues, thepresence of ascorbic acid is not a correlate of cellular prolifera-tion (see Barnett and Bourne, 1941).

2. Other Mesenchyme D e r i v a t i v e s .The main mesenchyme derivatives, with the exception of

muscle, are described in separate sections; here it is intendedbriefly to indicate the mesenchymal tissues in which ascorbicacid has been observed, but which have not been speciallystudied.

Barnett and Bourne (1941) described the distribution ofascorbic acid in the 4-day embryo: in the somites it was parti-cularly conspicuous in the myomere (myotome). Its presencehas been observed in other mesenchyme which is later to developinto muscle; for example, the mesenchyme surrounding theepithelium of the small intestine, which is destined to give riseto smooth muscle, shows a well marked reaction. Fullydeveloped muscle has not been found to contain detectablequantities of ascorbic acid. Other workers have reported thepresence of ascorbic acid associated with the myofibrils ofdeveloping muscle (see Tonutti, 1939), and these observationsconfirm their results. They are quite similar to those, reportedin the previous section, obtained in mesenchyme about to giverise to chondroblasts or osteoblasts.

The sub-epidermal mesenchyme shows a uniform reactionfrom the sixth day (fig. 1, PI. 14). Since smooth muscle andcollagen fibres are to develop in it, this is what would be ex-pected from the observations reported above.

At the fourth day the mesenchyme which invades the eye-cupin the formation of the vitreous humour can be seen to containascorbic acid. The surrounding mesenchyme with which it isin continuity contains little or none. The reaction remainswhile the cells are still visible as mesenehyme, but has almostdisappeared by the tenth day. The adult vitreous humour ofmammals is known from titrimetric studies to contain ascorbicacid (Miiller, 1935). These observations on the developing eyebring it into line with other mesenchvme derivatives in which


the cells lay down large amounts of intercellular groundsubstance.

3. Blood and R e l a t e d Tissues .

E ry t h r o c y t e s .

The silver nitrate method indicates the presence of ascorbicacid, certainly in the erythrocytes, probably in the plasma, notat all in the blood leucocytes. In a previous communication(Barnett and Bourne, 1941) it is suggested that even from thetwenty-fourth hour of incubation ascorbic acid may be presentin very small amounts in the plasma and tissue fluids. It isa normal constituent of adult mammalian plasma, and is knownto be present in the plasma of young chicks (Holmes et al.,1938). It does not occur in the erythrocytes until the fifth day,when a granular deposit is observed in some of them, evenlydistributed in the cytoplasm but varying in density from cell tocell. After this the blood of chick embryos of all ages examinedshows a similar reaction: from the tenth day the cells vary froma few which contain so much that under low power they appearas homogeneous black ovals, to the many which show noreaction at all (Text-fig. 9). Titrimetric investigations haveshown the presence of vitamin C in mammalian erythrocytes(Heinemann, 1936), and Giroud (1938 a, p. 62) refers to aninconstant reduction of silver nitrate in red blood-cells. Noneof these investigations accounts for the variation in quantitypresent in the different cells.

It is known that erythropoiesis is affected by ascorbic acid,and reduced or stopped in scurvy (Fleischhacker and Schiirer-Waldheim, 1938; Menshikov, 1938; Rohmer et al., 1938), andit might be supposed that it is the young red cells which containthe largest amounts of the vitamin. To investigate this possi-bility the yolk-sac, which is the blood-forming organ of thechick embryo (Dantschakoff, 1908), was examined at 16 and18 days; a reaction, in the form of a granular deposit, was foundin the walls of the blood-vessels, but the erythrocytes showed noreaction. This observation would appear to imply that it is theolder red cells which accumulate ascorbic acid. The observations


of various authors, that ascorbic acid injected into mam-malian blood gradually accumulates in the erythrocytes, are inaccordance with this view (Heinemann, 1938; Heinemann andHald, 1940; Butler and Cushaman, 1940). It seems likely thatascorbic acid is synthesized outside the embryo, or by the liver(q.v.), enters the plasma, and from there gradually passes intothe red cells.

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TEXT-FIGS. 9-10.Kg. 9. Erythrocytes from a 12-day embryo.Fig. 10. Histiocyte (?) from 10-day muscle.

B lood-vesse l s .One of the earliest symptoms of vitamin C deficiency is

fragility of the capillaries. Dalldorf (1939, p. 340) points outthat capillaries develop from mesenchyme, and that theirendothelium is believed to be fused together by a 'cementsubstance' which may, like other intercellular matrices, beaffected by the amount of ascorbic acid present. In the chickembryo there is usually a deposit in or on the walls of theblood-vessels, but the poor fixation obtained with acid silvernitrate does not permit the study of its exact distribution; thereis little doubt that a great part of the reaction is due to thepresence of the vitamin in the plasma or lymph.

H i s t i o c y t e s .

In Text-fig. 10 is illustrated a type of cell found in stripedmuscle. It is heavily impregnated, and resembles the histio-cytes figured by Tonutti (1939) and reported by him to accumu-


late ascorbic acid when stimulated to activity by bacterialinfection. It cannot be definitely asserted that the cells seen inchick embryos are histiocytes, but their appearance andscattered distribution make it probable.

4. L ive r .Eesu l t s .

A reaction appears in the liver diverticulum during the fourthday. At this stage the cells of the diverticulum are not histo-logically distinguishable from those of the gut-wall from which

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TEXT-FIG. 11.

Liver cells at 6 days, with diffuse impregnation.

they are derived. Nevertheless, while the cells of the definitivegut-wall contain only a few, isolated granules, those of thediverticulum contain them in fairly large numbers. Thegranules are spherical, and dispersed at random in the cyto-plasm. This type of distribution persists until the seventh dayof incubation (Text-fig. 11). By the eighth day most of the cellsshow little or no reaction; plenty of ascorbic acid is present inthe blood-vessels, and some in the cells immediately surroundingthem; the reaction fades with increasing distance from a blood-vessel.

274 S. A. BARNBTT AND .G. BOURNE

On the tenth day the granular deposit has wholly disappeared.A restricted region of the cytoplasm shows a heavy impregna-tion in some groups of cells; this region is usually at one poleof the nucleus, but there may be two regions at opposite poles(Text-fig. 12). The appearance is closely similar to that of the

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TEXT-FIG. 12.

Liver cells at 10 days, with localized impregnation, apparentlyin the Golgi substance.

figures of chick embryo liver Golgi substance published byDalton in 1934; in fact, the preparations are indistinguishablefrom those made by Kolatchev's method for the demonstrationof the Golgi material. This phenomenon is a transitory one:by the twelfth day, and at later ages, the liver.shows no reactionwhatever.

Discuss ion.

The liver occupies a special place in histological studies ofascorbic acid. It is known to contain large quantities of the


vitamin, from biological and titrometric investigations, but, atleast in the case of the adult mammalian organ, it does not givea reaction with acid silver nitrate (Harris and Eay, 1933;Giroud, 1938 a, pp. 32, 42). The ascorbic acid content ofembryo chick liver is comparable with that of a mammal (Eay,1934): in particular, it increases with age. The absence of areaction with silver nitrate in the chick Mver from the twelfthday is what would have been expected from previous work.

The failure to reduce silver nitrate has been supposed to bedue to the presence of an inhibiting substance (Harris and Eay,1933; Svirbely, .1935; Giroud et al., 1936); the authors havediscussed the evidence for this hypothesis elsewhere (Bamettand Bourne, 1941). What is interesting is that in the chickthe inhibiting action is not fully developed until after the tenthday; as we have seen, there is a reaction to silver nitrate beforethis, despite the fact that, according to Kay, the amount ofascorbic acid in the liver is least during the earlier period. Itis reasonable to suppose that until after the sixth day there is ~no inhibiting mechanism; the reactions observed are typicalfor an organ containing a relatively high concentration ofascorbic acid. It seems likely that the inhibiting substance, ifit is a substance, has begun to be synthesized during the seventhday, in view of the relatively slight reaction shown by most ofthe cells after this age. The tenth day is the last on which areaction has been observed, when it resembles that of an organcontaining comparatively small quantities of ascorbic acid con-fined to the Golgi substance (see Tonutti, 1939). It is, therefore,of interest that the chick liver becomes functional during theeleventh day of incubation; before this time glycogen synthesishas been carried out by the 'transitory liver' constituted bythe extra-embryonic tissues and the yolk (Needham, 1934).There is evidence that ascorbic acid is synthesized in the yolkfrom the earliest stages, and in the extra-embryonic tissues fromthe twenty-fourth hour of incubation (Bamett and Bourne,1941). The distribution of ascorbic acid in the liver at theeighth day in relation to the blood-vessels, already described,seenis to indicate that the vitamin is still synthesized elsewhereand carried to the liver in the blood.

NO. 331 u

276 S. A. BAENETT AND G. BOUENB

5. Thy ro id .Sufficient material was not obtained for a full survey of the

thyroid. At 10 days, when the vesicles have not developed, noascorbic acid can be detected in the thyroid rudiment. In onespecimen of 12 days' incubation quite large amounts werepresent in some vesicles, not only in the colloid but also insome of the cells of the glandular epithelium (fig. 5, PL 15).Other vesicles showed no reaction. In two thyroids from chicks2 days after hatching there was a negative reaction throughout.

6. B p i t h e l i a and E p i t h e l i a l D e r i v a t i v e s .This section, like the second, is devoted to various tissues,

containing ascorbic acid but not showing results of sufficientinterest to be dealt with at length.

The occurrence of a strong reaction in the ectoderm and endo-derm during the first 4 days has akeady been reported (Barnettand Bourne, 1941). When the skin differentiates, the cellsaccumulate melanin, and it is no longer possible to study thedistribution of ascorbic acid in them. For the same reason noobservations can be made on the developing feather. Certainepithelia are dealt with elsewhere in this paper: they includetwo important organs of passage for ascorbic acid, the kidneyand the choroid plexus. The epithelia of the organs of specialsense, and of the alimentary canal, have not been studied atevery stage, and will be described below as they appear in the12-day embryo. The state of affairs at 10 days is much the same.

A reaction is shown at 12 days in the olfactory epithelium,in the cells lining the utriculus and sacculus, in the cornea, andin the lens of the eye. In the first of these the reaction is slight:the deposit is granular, and most of it is close to the nucleus.In the sacculus of the inner ear the lining cells "show greatvariety in the reaction they present; in some there are heavyimpregnations, in others none at all; again, the deposits aregranular. The epithelium of the inner surface of the utriculusshows a reaction similar to that of the olfactory epithelium.

In the eye a granular reaction appears in the cornea; thecorneal epithelium is a continuation of the epidermis containing


no melanin, and as the melanin disappears the deposit of pre-cipitated silver becomes apparent. The lens is well known tocontain ascorbic acid in the adult mammal (Euler and Malmberg,1935; Glick and Biskind, 1936). In the chick a reaction is shownfrom the first appearance of the lens as a differentiated tissue ;at 12 days there is none in the lens epithelium; the most markedreaction is in the region of the ciliary processes.

In the alimentary canal the mouth and the small intestine

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TEXT-FIG. 13.

Cells of adrenal medulla at 16 days.

show positive reactions. In the former there is a fine deposit inthe superficial cells, and a much heavier one in the stratumgerminativum; at intervals this epithelium is interrupted bygroups of cells which appear to be developing into glands; thesecells show no reaction at all. In the epithelium of the smallintestine the cells show a granular deposit, mainly round thenucleus and wholly confined to the part of the cell farthest fromthe lumen.

7. A d r e n a l .A reaction appears hi both components of the adrenal at the

twelfth day, hi the form of a light granular deposit in most ofthe cells. At 14 days the two rudiments, though still topo-graphically separate, possess already the main histologicalcharacters of the adult tissues. They have also developeddifferent and specific distributions of ascorbic acid; after thisstage the amount present increases, but no other change isobserved. In the medulla at 16 days most of the cells contain

278 S. A. BARNBTT AND G. BOURNE

it distributed diffusely in the cytoplasm (Text-figs. 13, 15 a);the amounts vary greatly: a few cells are so full that they standout as conspicuous black objects under low powe*\ This typeof reaction also occurs in the mammalian adrenal (Bourne,19336). In the cortex the reaction is very much less markedthan in the medulla; Text-figs. 14 and 15& show two stages inthe development of this reaction.

Two days after hatching the adrenal has the adult arrange-

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Fig. 14. Adrenal cortical cell at 14 days; beginning of appearance ofascorbic acid.

Fig. 15. Adrenal cells at 16 days; a, medullary cell, 6, cortical cell;showing characteristic difference in strength of reaction.

i

ment of the component tissues, in which the two kinds of celloccur in small groups in a composite organ. The same type ofdistribution of ascorbic acid is observed as at 16 days, with themedullary cells showing, on the average, a much heavierdeposit than those of the cortex. (The terms 'medulla' and' cortex' are, of course, taken over from the mammalian adrenal,where they have a literal significance.) The deposit in both isgranular only. A few of the cells are again particularly heavilyimpregnated and can be picked out in the duplicate stainedpreparations. They can then be seen to be confined to the classof chromophil medullary cells described by Uotila (1939).

It is to be noted that there is a reversal of the conditions whichusually hold in the mammalian adrenal, regarding the reactionsshown by the two components of the gland. In the mammal the


cortex has an exceptional capacity for reducing silver nitrate,and although the medulla can be shown by other tests tocontain large quantities of ascorbic acid it resembles the liverin giving a disproportionately slight reaction (Bourne, 1983 b;Galvao and Cardoso, 1935). However, in the sheep the medullagives a stronger reaction than the cortex (Giroud, 19386), andin other mammals in certain physiological states the reaction isreversed (Bourne, 1935 c). For reasons indicated in the sectionon the liver, it would be valuable to have data on the sulphydrylcontent of the components of the embryo fowl adrenal.

8. Gonads .Ascorbic acid has not been observed in the testis. According

to Giroud and Leblond (1934), in the mammalian testis it is

? / [ / iA. K. \ / /

16a •^•••••^.....•••- / 16b

8(JU

TEXT-ira. 16.Cells from ovary at 16 days; localized deposits.

localized in the interstitial cells; the embryonic testes studiedwere not yet organized into tubules and separate interstitialtissue, although interstitial-type cells appear during the thir-teenth day (Willier, 1939, p. 84).

The ovary shows no reaction till the fourteenth day, and thenonly in certain large cells of the cortex; at this age the depositis confined to what is little more than a large granule, close tothe nucleus. At 16 days the impregnated area is larger (Text-fig. 16) and may be granular or reticular; a similar picture ispresented by the 23-day ovary (Text-fig. 17). Bourne (19356)reported the presence of ascorbic acid in the oocytes of the fox,although there was none in the surrounding follicular cells.


It is tempting to suppose that the cells which show a reactionin the embryonic fowl ovary are also germ-cells; their distribu-tion in the organ as a whole is compatible with 'Ms view.However, the deposits cannot be seen in the duplicate stainedpreparations, and it is consequently not possible to identifythe cells with certainty. It may be that the cells in questiondo not constitute all the germ cells, but only those which

TBST-HG. 17.

Cells from 23-day ovary.

have reached a certain stage in development. The primordialgerm-cells, according to Willier (1939, p. 85), have theirmitochondria generally distributed in the cytoplasm; themitochondria of the oogonia to which they give rise aregrouped on one side of the nucleus. Changes of this kindmay be paralleled by alterations in ascorbic acid content.

9. Nephro i .E e s u l t s .

Mesonephros.—The mesonephros shows a distinct re-action at the fifth day. Many tubule cells contain fine granulesin the cytoplasm, and slight reactions also appear in theglomeruli and the lumina of the tubules. The connective tissueassociated with the organ shows a stronger reaction than thatof the differentiated tissue: the granules are larger and morenumerous. This type of distribution persists until after thetenth day, but certain variations from it have been observedin some of the tubule cells. A few show intense impregnation ofthe cytoplasm located at a pole of the nucleus nearest the lumen;


these cells for the most part contain none of the ordinarygranular deposit (Text-fig. 18). This type of distribution hasnot been observed after the tenth day, though up to 16 daysgranules are observed in the tubule cells and glomemli; by thistime the mesonephros is rapidly degenerating, and a few daysafter hatching it is vestigial.

Metanephros.—The metanephros has not been observed

* .:

i

• 18a

18dTEXT-JIG. 18.

Tubule cells of 10-day mesonephros; various modes of localizationof ascorbic acid.

to show any reaction worth noting until the twelfth day ofincubation. At this time granules can be seen in the cells ofsome tubules, near the nucleus and usually on the side of itnearest the lumen. A deposit also appears in the region of theblood-vessels. At 14 and 16 days the deposit is more general(Text-figs. 19, 20); at these ages the ascorbic acid content ofthe metanephros is much greater than that of the mesonephros.

282 S. A. BAENETT AND G. BOUENE

Again there are cells with localized deposits at one or other poleof the tubule cells (Text-figs. 19,20 b, 21). At 23 days there is nogreat change from 16 days.

Discuss ion .The results are in general what would be expected, when it

is considered that a kidney is essentially an organ of passage

• . ' "• 1 "• / I f • / ••<•

TEXT-FIG. 19.

Metanephric tubule at 14 days; ascorbic acid on lumen side ofnucleus in each cell.

for ascorbic acid. The mesonephros is said to commence functionat the fifth day (Lillie, 1906, p. 382), and on that day it firstshows the presence of the vitamin. Similarly, the metanephrosbegins its secretory functions after the eleventh day (Lillie,1908, p. 390), and ascorbic acid appears in it on the twelfthday, but is absent on the tenth day and before. The detailsof the distribution of ascorbic acid, in particular the localizedintracellular deposits, must be supposed to be related todifferent functional states of the tubule cells. The most inter-esting observation on this point is that of the stages in a process,illustrated in Text-figs. 18 and 22, which appears either to bethe break-up of the Golgi material into dispersed granular

ASCORBIC ACID IN CHICK

20a

-5. - . . .

20 b

8|x

TEXT-ETG. 20.

Sixteen-day metatiephros: a, section through tubule and glomerulus,showing diffuse distribution of ascorbic acid; 6, section throughtubule, with adjacent mesenchyme and blood-vessel, showingascorbic acid mainly on far side of nuclei from lumen in thetubule cells, and deposits in the mesenehyme and blood-vessel.

bodies all bearing ascorbic acid, or the converse process. Inaddition, stages in the passage of ascorbic acid through the wallof the tubules can be observed (Text-fig. 18).

The only observation which could be of developmentalsignificance is the impregnation of the mesenchyme around


the mesonephros at the fifth day. This might be taken to suggestthat ascorbic acid is concerned in the process of the differentia-

•"*'•"'•-, I •*•••/' } /

i

\ J ^ 22.

TEXT-EIGS. 21-22.

Fig. 21. Cell of 16-day metanephric tubule, showing polarityreversed from condition illustrated in Text-fig. 19.

Fig. 22. Tubule cells of 23-day metanephros; schematic: variousphases in the distribution of ascorbic acid.

tion of mesenchymatous cells into kidney cells. Similar depositsoccur in the metanephric mesenchyme (Text-fig. 20b).

10. Nervous Sys tem.E e s u l t s .

The study of ascorbic acid in the nervous system presentsspecial difficulties. Nearly every one of the many cell-types isonly certainly identifiable by means of special staining methods,which could not be applied in material treated for the demon-stration of ascorbic acid; even the tissue organization, such asthe arrangement of cortical layers, can only be observed inspecial preparations. This account therefore begins with adescription of the various ways in which ascorbic acid has beenseen to be distributed in the cells of the nervous system; afterthis there follows a general review of the occurrence of the maintypes of distribution in certain parts of the brain, and in the'spinal cord and ganglia.

Cytology.—The distribution of ascorbic acid in nerve-cells


may be localized or diffuse. If localized, it may be in any ofthree types of situation. Very commonly the deposit is confinedto a relatively small area adjacent to the nucleus: this area isoften diametrically opposite the point of origin of the axon, andis generally in that half of the cell-body which does not includethe latter. This type of localized distribution does not become

{'

I = •:,.

M 24 £>"• 2.7

\E6

2 5 *<*•».„„„. .^•'••••^J

TEXT-MGS. 23-27.

Fig. 23. Cell of 17-day cerebellum; ascorbic acid apparently local-ized in Golgi substance.

Fig. 24. Cell of 17-day cerebral hemisphere.Fig. 25. Cell of 23-day cerebral hemisphere.Fig. 26. Cell of 17-day cerebrum, showing ascorbic acid deposited

on an open network.Fig. 27. Cell of 16-day cerebrum, with ascorbic acid associated with

scattered material resembling Golgi substance.

common until the tenth day, but after this age the number ofcells showing it becomes progressively greater; it occurs in allparts of the brain and spinal cord, but not in the cells ofperipheral ganglia. In detail it takes various forms: sometimesit is reticular (Text-figs. 23, 24), quite often it takes the form ofclosely packed granules (Text-figs. 25, 28); in some cells thenetwork is open (Text-figs. 26, 28 c); in such cases the picture


is closely similar to those given by Alexenko (1930) of Golgisubstance in the spinal ganglion cells of the fowl embryo; ina few cells the reticulum appears to be partly broken up andscattered over the surface of the nucleus (Text-fig. 27).

The second type of localized distribution is shown by cells ofwhich the axon and its cone of origin are impregnated (Text-figs. 29-31, and figs. 6, 8, PI. 15). In this case the deposit isalways granular, and may be wholly confined to these sites; onthe other hand, it may be combined with any of the other types

JT

/ -. >K C

/ ^ )

TEXT-EIG. 28.

Cells from the 10-day spinal cord, showing localized deposits ofascorbic acid.

of distribution. Ascorbic acid appears in some axons at thefourth day, at which age little is to be seen in other parts of thenerve-cells (Barnett and Bourne, 1941). It is present in axonsat all later ages and in all parts of the brain and spinal cord,but not in all axons and to varying extents in different regions(see below). The third type of localization is less well definedthan the other two: the reaction is perinuclear, and so closelyconfined to the cytoplasm immediately surrounding the nucleusthat there is a definite area of cytoplasm which is clear. Thisarrangement has only been observed in cells which have im-pregnated axons. It is not sharply marked off from the diffusetype of arrangement, in which the whole of the cytoplasm of thecell-body contains granules. This last type is comparativelyrare (Text-figs. 30, 32).

Telencephalon.—Well marked deposits appear com-paratively late. At the tenth day local impregnations occur in

ASCORBIC ACIB IN CHICK 287

a few cells, and at later ages the number of such deposits in-creases steadily; at 16 days very many cells in all layers havelocal impregnations (Text-figs. 24, 27). Axon impregnationoccurs in the telencephalon, but it has not been observed earlierthan the seventeenth day (Text-fig. 30). By the second dayafter hatching local impregnations are exceedingly numerousin all regions (Text-fig. 25).

I J

j j

'^'-••^.^i^x'^'^* '---•f:"

SOTEXT-JIGS. 29-31.

Fig. 29. Neuron from 12-day optic tectum, with axon containingascorbic acid.

Fig. 30. Neuron from 17-day cerebrum, with ascorbic acid distributeddiffusely in cell-body, as well as in axon.

Kg. 31. Neuron from 12-day optic tectum, with axon containingascorbic acid.

Diencephalon.—The diencephalon is of interest for thereaction shown at the fourth and fifth days. A marked reactionoccurs at the fourth day: the lateral walls show a light, diffuseimpregnation, which becomes more marked ventro-laterally atthe level of the optic chiasma; at other levels, also ventro-laterally, the axons of some of the cells are seen to containascorbic acid. At 5 days this phenomenon is more widespread;

288 ' S. A. BAENETT AND G. BOURNE

ventro-laterally there is a layer of cells in the inner part of themantle layer, i.e. next to the ependymal layer, with the axonsimpregnated; farther up, in the greater part of the Hteral wall,there is a similar zone of nerve-cells in the middle of the mantlelayer. After 5 days the amount of ascorbic acid present de-creases, and no distinctive reaction is shown at later stages.

Mesencephalon.—As in the diencephalon the cells of themid-brain contain ascorbic acid from the fourth day: at thisage most of the cells show some degree of diffuse impregnation,and a few axons contain granules. After this there is anincrease in the amount present, and at the twelfth day the mid-brain contains more ascorbic acid than any other part of thenervous system. The greater part of the mid-brain is made upby the optic tectum, in which the cortical layers are at this agealready developing. Corresponding with them the distributionof ascorbic acid is also zoned. This distribution is of someinterest, and will be described in detail. In the superficial layersthere is a heavy deposit of a diffuse character in the greatmajority of cells; a fairly small number have their axonsheavily impregnated. Within these layers is a wider zone, alsomainly cellular, in which there are no impregnated axons andmuch less ascorbic acid. The next layer, also wide, is one,containing very few cell-bodies, across which migration istaking place; here the impregnation of axons is general andvery conspicuous, while the cell-bodies contain little or noascorbic acid (Text-figs. 29, 31). Between the migratory andependymal layers there is a narrow zone in which a few axonscontain ascorbic acid and many cells show a diffuse deposit.

In older embryos the numbers of impregnated axons showa decrease, but local deposits appear in many cells; these arecomparatively few at 16 days, but are numerous by thetwenty-third day; at the latter age numerous cells continue toshow a strong reaction in their axons, and these sometimesaccompany the local deposits.

Cerebellum.—At the seventeenth day local impregnationsare very numerous and striking in the cells of the nuclear layer,and as far as can be seen few of these cells do not contain them.In the molecular layers many of the axons contain ascorbic


acid; in addition there are cells, which may be nenroglial, con-taining a diffuse deposit around the nucleus; the latter areespecially numerous in a zone at the extreme periphery of themolecular layer (Text-fig. 32). Two days after hatching thedistribution of ascorbic acid is similar, but the amount isgreater; axon impregnations are more conspicuous and morenumerous, and more cells with well marked diffuse impregna-

//""Y\**"v-v-......

1

10 p-

'' *4-—' 34

.,.53

35

4JL

^..^—....

V . - ; •:-: :

TEXT-ITGS. 32-35.

Fig. 32. CeE from 17-day cerebellum, with diffuse distribution ofascorbic acid.

Fig. 33. Purkinje cell at 23 days; very slight reaction.Fig. 34. Ependymal cell from the 16-day nerve cord, with ascorbic

acid on lumen side of nucleus.Fig. 35. Cell of 16-day dorsal root ganglion, in lumbar region.

tions can be seen. In addition, Purkinje cells are now identifi-able, but they contain only a few scattered granules (Text-fig. 33).

Sp ina l Cord .—At 4 days many cells of the mantle layercontain a few granules, and some of their axons have begunto show a reaction (see Barnett and Bourne, 1941). At 6 daysgranules also appear in the ependymal cells. After this age theependymal layer always contains some ascorbic acid, generallyorientated at one end of the cell (Text-fig. 34). The mainchange which takes place after the sixth day is the disappear-


ance of the granular type of deposit, except in some axons, andthe appearance of local impregnations in increasing numbers ofcells (Text-fig. 28). This process is complete by the tQnth day,after which, apart from the ependymal cells of the canal, onlyaxons and small areas close to the nucleus show well markeddeposits. Axons in both the dorsal and ventral horns containascorbic acid.

Ganglia.—A little ascorbic acid is detectable in the cellsof the dorsal root ganglia at the sixth day; the deposit is diffuse.The type of distribution observed in ganglion cells is inter-mediate between the diffuse arrangement and strict localization(Text-fig. 35). There is much variation, even amongst theganglia of a single embryo, in the reaction shown. The gangliaof the cranial nerves have, on the whole, very little; theanterior sympathetic ganglia have fairly light deposits similarto those of the dorsal root ganglia, but those in the abdominalregion contain more than any of the others. This observationis in concordance with the titrimetric studies of Giroud (1938 a,p. 65). The variation seems to be due primarily to the presenceof different amounts in the nerve-fibres passing through theganglia, and not to great differences between the ascorbic acidcontents of the cell-bodies themselves.

Membranes.—Finally, it may. be mentioned that themesenchyme which gives rise to the membranes of the centralnervous system contains ascorbic acid from the sixth day. Theamount present is considerable even at this age, and it increases.The concentration is particularly high in the embryonic arachnoidand pia mater; this can be seen very well, for example,in the fissures of the cerebellum from 16 days onwards.At the same time, it is a general rule that the surface layers ofthe parts of the central nervous system adjacent to the pia-arachnoid show heavier deposits than the rest; and these heavierdeposits are not within the cells, for the most part, but seem torepresent the presence of ascorbic acid in the tissue fluid. Theappearance is as if ascorbic acid were being transported fromthe meninges into the brain tissue. Corresponding phenomenaare observed in the choroid plexuses. Their epithelial cells, atall ages examined from 10 to 23 days, show deposits which

ASCOBBIC ACID IN CHICK 291

appear to indicate a passage of ascorbic acid into, or from, thecerebro-spinal fluid.

Di scuss ion .

Although many estimates have been made of the quantitiesof ascorbic acid in the parts of the nervous system, there seemto be no data regarding its cellular distribution. It is conse-quently not possible to make a comparison between the em-bryonic and adult conditions. It can only be said that theincreasing amounts of ascorbic acid observed histologically arewhat would be expected from the quantitative studies of Eay(1934) on embryo chicks.

It seems likely, from the results reported above, that mostadult nerve-cells, and perhaps neuroglia as well, contain ascorbicacid localized in a specialized region of the cytoplasm; thisregion is probably also the site of the Golgi substance (seeHirsch, 1939, pp. 233 et seq.). The diffuse distributions ob-served seem for the most part to be transitory, and in a majorityof cases to be associated with impregnation of the axon. Whetherthe latter phenomenon occurs in axons which are not growingcannot be decided without further investigation. Their con-spicuous impregnation in the zone of migration in the optictectum, and the later reduction in the number of axons inwhich the phenomenon is seen, seem to indicate that thepresence of ascorbic acid is connected with their growth; thisis also in accord with the appearance of the vitamin in axonswhen they first develop during the fourth day.

IV. GENERAL DISCUSSION.

Although the results obtained for each organ have been dis-cussed in the sections devoted to them, there are two generalquestions to be considered here. One concerns the cellulardistribution of ascorbic acid, the other its role in the develop-ment of the various cell-types. A difficulty which attends boththe special and the general discussions arises from the fact thatnearly all the work previously done has been on mammals;in all comparisons based on work done on different species only

NO. 331 X


provisional conclusions can be drawn. Nevertheless, thereare some resemblances.

Cytology of Ascorbic Acid.There can be little doubt that the localized deposits of ascorbic

acid, observed in the cells of the liver, ovaries, nephroi, andnervous system, are the result of its presence in the Golgi sub-stance. The association of vitamin C with the Golgi materialhas been recognized for some time (Bourne, 1935 c), and hasrecently been considered in detail by Hirsch (1939, pp. 233et seq.). Owing to the absence of comparative preparations thisassociation has not been directly demonstrated in the presentwork, although Dalton's figures for liver make it sufficientlyclear in that tissue. Nevertheless, it must be admitted thatmany of the acid silver nitrate preparations could easily bemistaken for the results of the application of one of the elaboratetechniques for the demonstration of Golgi substance.

On the subject of the supposed association with mitochon-dria (see Bourne, 1935 c; Giroud, 1938 a, pp. 32 et seq.) quitedifferent conclusions can be drawn. The results reported heredo not support the view that large intracellular quantities ofascorbic acid are commonly associated with the ' chondriome'in the fowl embryo. Mitochondria are not, in most instances,spherical; for example, in the mesenchyme, chondroblasts, andosteoblasts of chick embryos they take the form of long threads(Fell, 1925), yet the silver deposits observed in these cells aregranular. What is more, mitochondria, according to Kappers(1936, p. 17), do not occur either in the axon or axon hillock ofnerve-cells; if this is true, the reaction observed in them con-stitutes a case of the presence of intracellular ascorbic acid notassociated either with Golgi substance or with mitochondria.It seems reasonable to suppose that the granular form of thesilver precipitates is a product of mechanical conditions only,and need not be taken to represent any structure existing invivo (see Bourne, 1933 c).

F u n c t i o n of Ascorbic Acid.The general result of this survey is clearly to lay emphasis,

in the first place, on the importance of ascorbic acid in the

ASCOEBIC ACID IN CHICK- 293

histogenesis of mesenchyme derivatives. In connexion withbone and cartilage this point has already been fully discussed.It is worth noting that, in addition to skeletal tissues, thefollowing mesenchymal derivatives show the presence of markedquantities of ascorbic acid in the early stages of their differentia-tion: muscle, dermis, vitreous humour, adrenal cortex, kidney,meninges. Nevertheless, even in these tissues, we can say nothingof the chemical mechanisms in which ascorbic acid plays apart, while in the case of other types of cell there is hardly anysuggestion even regarding the end-products of its activity; yetits widespread, though highly specific, distribution cannot befortuitous. In plants ascorbic acid is said to take part in cellularrespiration (Szent-Gyorgyi, 1931); in animals this possibilityseems to be precluded by the fact that it is present almost solelyin the reduced form (Borsook, et al., 1937; Green, 1940, p. 158).

Hirsch (1939) lays emphasis on the evidence, provided bycytological studies, for the view that ascorbic acid plays a partin the synthesis of many glandular products. There is probablyno justification for arguing from one synthesis to another, butit is worth noting that where there is evidence for the partplayed by ascorbic acid in animal metabolism, that evidencetends to indicate involvement in synthetic processes. Thisapplies to the deposition of intercellular matrices as well as toglandular secretion. There is some evidence for an associationbetween the presence of ascorbic acid and of a high concentra-tion of sterols; three endocrine organs whose hormones aresterols, the adrenal cortex, the interstitial cells of the testis, andthe corpus luteum are among the organs which contain thehighest concentrations of the vitamin. It is, therefore, interest-ing that, according to Page (1937, p. 34) the brain contains morecholesterol than any other organ; the cholesterol is largelypresent in the axons, and it may be suggested that the presenceof ascorbic acid in growing axons is related to this fact. It wouldbe interesting, in this connexion, to study the effect of experi-mental scurvy on neurogenesis; there appear to be no data onthis subject; until there are, any interpretation of the distribu-tion of ascorbic acid in the embryonic nervous system is likelyto be mere guess-work.


Finally, it is necessary to emphasize that there is every reasonto suppose that ascorbic acid has a wide variety of differentfunctions, even within one tissue. The observation^ on cartilagemake this clear: apart from the association of ascorbic acid withprocesses of differentiation, the vitamin appears also to beinvolved, in this tissue only, in cellular proliferation. It wouldindeed be surprising if a very strong reducing agent were foundto have a highly specific action in the body. It is consequentlyunlikely that it will be possible to fit observations of its dis-tribution in diverse tissues into a single scheme.

V. SUMMARY.

1. The distribution of ascorbic acid (vitamin C) in the cellsand tissues of chick embryos, from the fourth day of incubationto 2 days after hatching, has been studied by the acid silvernitrate method.

2. Mesenchymatous cells show a reaction when undergoinghisto-differentiation into cartilage, bone, muscle, dermis,vitreous humour, adrenal cortex, meninges. Differentiatedcartilage cells do not contain ascorbic acid except in areas ofproliferation; the cartilaginous matrix may contain it where thecartilage is about to be replaced by bone. Osteoblasts show noreaction at first, a slight reaction when they become surroundedby calcified tissue, and in some cases a strong reaction in olderbone. Ascorbic acid is associated with the 'osteogenic' fibresimmediately before calcification, and is also present in newlycalcified trabeculae.

8. In blood ascorbic acid is present in erythrocytes andplasma; evidence is presented for the view that it accumulatesin the older erythrocytes. Cells resembling histiocytes havebeen observed in muscle, containing high concentrations; noother leucocytes have been observed to show a reaction.

4. Liver shows a strong diffuse reaction in early embryos,but none at all after the tenth day. At the latter age ascorbicacid has been detected in a localized area of cytoplasm whichappears to be the Golgi substance.

5. The thyroid shows no reaction at the tenth day, or afterhatching, but has been seen to give a reaction at the twelfth day.

ASCOBBIC ACID IN CHICK 295

6. Various epithelia of the organs of special sense, and of thealimentary canal, contain ascorbic acid.

7. The adrenal, both cortex and medulla, first shows areaction at the twelfth day; by the fourteenth day typicaldistributions appear, though the amount of ascorbic acid presentcontinues to increase after that age. The reaction shown bythe medullary cells is much stronger than that shown by thecortical elements, a reversal of the condition in the mammalianadrenal.

8. Ascorbic acid was not detected in the testis. In the ovaryit appears at the fourteenth day, in a very restricted area of thecytoplasm of certain large cells in the cortex; at later ages thedistribution is similar, but the impregnated area in each cellis larger. The cells concerned are believed to be the germ-cells.

9. The mesonephros contains ascorbic acid from the fifth tothe sixteenth days in the tubule cells, the glomeruli, and thelumina of the tubules. In the tubule epithelia the deposit maybe localized at one pole of the cell. The metanephros beginsto show a reaction at the twelfth day; here, too, the tubule cellsfrequently have a deposit at one pole.

10. In the cells of the central nervous system ascorbic acidmay be localized in a small area near the nucleus (probably theGolgi material), round the whole of the nuclear surface, or inthe axon and axon hillock; or it may be diffusely distributedthrough the cytoplasm. A reaction is shown at the fourth day,especially in the axons of certain cells of the brain and spinalcord; later, localization in the Golgi substance becomes verygeneral in most parts of the brain and cord. A reaction is alsoshown in ganglion cells, the meninges, and the choroid plexuses.The specific distributions in the principle parts of the centralnervous system are described.

11. Results are discussed in the sections on each organ ortissue. In the general discussion it is pointed out that theobservations described are concordant with the view thatascorbic acid is frequently localized in the Golgi material. Onthe other hand, the evidence is that there is no specific relationwith the ' chondriome' in the embryonic cells of the fowl. Therole of ascorbic acid in development is also briefly discussed.


The authors are indebted to Prof. W. B. Le Gros Clark forhis advice and assistance. They also wish to thank Mr. W.Chesterman for taking a great deal of trouble with thephotographs.

VI. BIBLIOGRAPHY.

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Butler, A. M., and Gushaman, M., 1940.—'Journ. Clin. Investig.', 19.Dalldorf, G., 1939.—In 'The vitamins', 'Am. Med. Assn.' New York.Dalton, A. J., 1934.—'Anat. Rec.', 58.Dantschakoff, W., 1908.—'Anat. Hefte', 37.Eddy, W. H., and Dalldorf, G., 1937.—'The Avitaminoses.' Baltimore.Euler, H., and Malmberg, M., 1938.—'Zeit. physiol. Chem.', 252, 256.Fell, H. B., 1925.—'Journ. Morph. and Physiol.', 40.Fish, E. W., and Harris, L. J., 1934.—'Phil. Trans. Roy. Soe. B ' , 223. 'Fleischhacker, H., and Schurer-Waldheim, F., 1938.—'Wien. Klin.

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Berlin.1938 b.—'Personal communication.'

Giroud, A., and Leblond, C. P., 1934.—'C.R. Soc. Biol.', 115.1936.—'Nature', 138.1937.—'Anat. Rec.', 68.

Giroud, A., Leblond, C. P., Ratsimamanga, R., and Rabinowicz, M., 1936.—'Protoplasma', 25.

Glick, D., and Biskind, G. R., 1935.—'Journ. Biol. Chem.', 110.1936.—'Arch, of Ophthahn.', 16.

Green, D. E., 1940.—'Mechanisms of Biological Oxidations.' Cambridge.Ham, A. W., 1932.—In Cowdry's 'Special Cytology'. New York.Ham, A. W., and Elliott, H. C , 1938— 'Am. Journ. Pathol.', 14.Hanke, H., 1935.—'D. Z. Chirurg.', 245.


Harris, L. J., and Ray, S. N., 1933.—'Bioch. Joum.', 27.Heinemann, M., 1936.—' Acta brevia Neerlandica', 6,

1938.—'Joum. din . Investig.', 17.Heinemann, M., and Hald, P . M., 1940.—Ibid., 19.Hirsch, 6 . C , 1939.—'Form and Stoffwechsel der Golgi-Korper.' Berlin.Holmes, A. D., Tripp, F., and Satterfield, G. H., 1938.—'Journ. Nutr.', 16.Jessey, A. v., and Toro, E., 1936.—' Virchows Arch.', 298.Kappers, C. U. A., Huber, G. C , and Crosby, B. C , 1936.—'The Compara-

tive Anatomy of the Nervous System of Vertebrates.' 5LY.Klein, L., 1938.—'Anat. Anz.% 87.Leblond, C. P., 1934.—'La Vitamine C dans l'Organisme.' Paris.Lillie, F. R., 1908.—' The Development of the Chick.' New York.MacClean, D. L., Sheppard, M., andMcHenry, E. W., 1939.—'Brit. Joum.

exp. Pathol.', 20.Mazoue, H., 1937.—' C.R. Soc. Biol.', 126.Menshikov, F . K., 1938.—'Trans. Novosibirsk Inst. Sci. jShitrit.', 2, 17;

Abstract in 'Nut, Abs. and Revs.', 1939, 9, 338.Muller, H. K., 1935.—'Arch. Augenk.', 109.Needham, J., 1934.—'Biol. Rev.', 9.Page, I . H., 1937.—'Chemistry of the Brain.' London.Querido, A., and Gaillard, P. J., 1939.—'Acta brevia Neerlandica', 9.Ray, S. N., 1934.—'Bioch. Joum.', 28.Rohmer, P., Bezzsonoff, N., et al., 1938.—'C.R. Soc. Biol.', 127.Svirbely, J . L., 1935.—'Bioch. Joum.', 7.Szent-Gyorgyi, A. v., 1928.—Ibid., 22.

1931.—'Journ. biol. Chem.', 90.Tonutti, E., 1939.—'Z. Vitaminforsch.', 9.Uotila, U. U., 1939.—'Anat. Rec.', 75.Waugh, W. A., and King, C. G., 1932.—'Joum. biol. Chem.', 97.Willier, B. H., 1939.—In Allen's 'Sex and Internal Secretion'. London.Wolbach, S. B., and Howe, P. R., 1926.—'Arch. Pathol.', 1.

EXPLANATION OF PLATES

PLATE 14.

Fig. 1.—Growing cartilage in cranium at 10 days, showing precartilageand some differentiated cartilage cells impregnated. The sub-epidermalmesenchyme also contains ascorbic acid.

Fig. 2.—Cartilage of centrum at 12 days; in matrix near notochord,among hypertrophied cells, there is a deposit of silver. Sectionstained with haematoxylin and eosin after the usual treatment.

Fig. 3.—Group of heavily impregnated cells hi cartilage of 12-dayembryo.

Fig. 4.—Cells, similar to those in fig. 3, at higher magnification. Noteposition at edge of cartilage.

2 9 8 S. A. BAENETT AND G. BOURNE

PLATE 15.

Fig. 5.—Thyroid at 12 days, with deposits in some of the vesicles andsome cells.

Fig. 6.—Neuron of optic tectum at 12 days, with deposit in axon anda localized impregnation near the nucleus.

Fig. 7.—Early bone and ossifying tissue; preparation stained withhaematoxylin and eosin. The cells appear black. 'Osteogenic' fibres arevisible in the unossified area, with black granules spread along them; thesegranules indicate the presence of ascorbic acid.

Fig. 8.^-Cell, similar to that in fig. 6, from dorsal horn of spinal cord at12 days.

LETTERING OF PLATES.

A A, impregnated cartilage cells; AX, axon; B, bone; CART, unim-pregnated cartilage cells; GB, cell-body; CV, colloid vesicles containingascorbic acid; CW, colloid vesicle containing no ascorbic acid; HC, hyper-trophied cartilage cells; LD, local deposit in cell-body; MAT, cartilaginousmatrix containing ascorbic acid; ME8, sub-epidermal mesenchyme;N, nucleus of neuron; NOT, notochord; OB, osteoblast; OF, 'osteogenic'fibres; PREC, precartilage.

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