THE LOCALIZATION O F S35 I N THE SKIN O F THE RAT

WILLIAM MONTAGNA' AND C. RUTH HILL Department of Biology, Brown University, Providence, Rhode Island

and Department of Histology, London Hospital Medical College, London, England

FOUR FIGURES

INTRODUCTION

The fate of sulfur in the skin has been studied by several authors in animals injected with S3j either in the form of inorganic sulfur or as S35 labelled cystine or methionine (Bostrom, '52 ; Bostrom and Gardell, '53 ; Bostrom et al., '53 ; BBlanger, '54 and '56, and others). The radioautographs prepared by these and other authors, however, have not been sufficiently clear to permit a detailed study of the distribution of S35 in the skin appendages. In this paper the localization of S35 in the skin is more clear than in previous papers. We have studied the concentration of S3j from two to 48 hours after injection, paying particular attention to its distribution in skin with active and quiescent hair follicles.

MATERIALS AND METHODS

Each of 4 young adult female albino rats was treated in the identical way before it was injected with the radioactive material. An area from the skin of the back was plucked of its club hairs 8 days before, another area immediately anterior to it was plucked 4 days before and a third area was plucked 24 hours before. A fourth area contained quiescent hair

Special Fellow of the United States Public Health Service at the London Hospital Medical College.

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164 WILLIAM MONTAGNA AND C. RUTH HILL

follicles and was left unplucked. Since plucking of club hairs initiates the growth of their follicles, a t the time they were injected, all animals possessed 4 fields of skin in which the hair follicles were in different states of activity. Each animal was injected intraperitoneally with a dose of 15pc/gm of body weight of S35 labelled Na,SO, dissolved in 0.9% saline containing 0.02% Na,S04. One animal was killed after two hours, one after 4 hours, one after 24 and one after 48 hours. Pieces of skin from each of the 4 fields were fixed in a mixture containing one part formaldehyde and three parts absolute alcohol. Ten micron paraffin sections were mounted on gela- tinized slides, deparaffinized, hydrated and covered with Kodak autoradiographic stripping film (Pelc, '47). After an exposure of about 4 weeks the slides were developed in Kodak D-19b developer. Some of the sections were stained with celestin blue, others were left unstained. After drying, the sections were mounted in Canada Balsam.

OBSERVATIONS

The dermis amd epidermis . Two hours after its injection a great deal of S35 is localized in the interstices of the dermis, where the strongest concentration is found in the papillary layer. Numerous coarse granules indicate the presence of S35 in granules of mast cells. The fatty layer of the skin contains a very small amount of radioactive material. I n the epidermis, a scattering of sparse radioactive particles is found only in the lower two layers, the upper layers being free of Ibem.

After 4 hours S35 is more heavily concentrated throughout the dermis; the papillary layer contains the strongest con- centration (fig. 1). The granules of radioactive particles in the mast cells are more numerous than after two hours. I n the epidermis small radioactive particles are found through- out the Malpighian layer. I n specimens of skin removed 24 hours after the injection of S35 the concentration of radio- active particles in the dermis is sharply reduced and is fur-

S35 IN THE SKIN O F THE RAT 165

ther reduced after 48 hours. I n spite of its reduction, however, the papillary layer still shows a greater amount of S35 than the reticular layer. The mast cells show no reduction of S35. Only a few radioactive particles are still demonstrable in the epidermis after 48 hours.

The layers of the skin are thicker when the hair follicles are growing than when they are quiescent (Chase et al., '53). Concurrently, a much larger amount of SS5 is seen in the dermis of skin which contains hair follicles which have been growing for 6 to 10 days than in the dermis of skin containing younger follicles or skin with quiescent follicles.

T h e pilosebaceous system. In the skin of animals killed two hours after injection, the strongest amount of S35 in the skin is found in the dermal papilla and in the outer root sheath of hair follicles which have been growing 4 days or longer (figs. 2, 3, 4 ) . Just above the bulb of growing follicles, radioactive particles are particularly numerous in the periph- eral cells of the very thick outer sheath and in the connective tissue sheath. The lower half of the bulb, or matrix, contains diffuse, but distinct radioactive granules (fig. 4). In the upper part of the bulb, where the cells are differentiating into the layers of the inner sheath and the layers of the hair, radioactive particles are relatively numerous. The keratinized portion of the hair above the keratogenous zone and the kera- tinized part of the inner root sheath above the level of the bulb, however, show no S36 particles.

After 4 hours the distribution of SS5 is similar to that described above but it is more intense. After 24 and 48 hours the dermal papilla and the outer sheath contain as many radioactive particles as after 4 hours, but the bulb contains fewer and smaller particles.

The sebaceous glands accumulate very few radioactive particles. After 2 and 4 hours only the peripheral, undiffer- entiated cells contain some S35. After 24 and 48 hours a few particles may be found in the more central parts of the glands and in the sebum.

166 WILLIAM MONTAGNA AND C. R U T H HILL

During the early stages of hair follicle activity, radioactive particles are distributed evenly through all the parts of the follicles. In follicles which have been growing for 4 days, and which have already formed a bulb, the dermal papilla and the outer root sheath concentrate more radioactive particles than the rest of the follicles. This is coincident with the appearance of mucopolysaccharides in the dermal papilla and glycogen in the outer sheath (Montagna et al., '52; Ship- man et al., '55). When follicles are quiescent and these sub- stances are not present, S35 is no longer concentrated in the dermal papilla and in the outer root sheath.

The concentration of S35 is scanty in quiescent hair follicles and is no greater than that found in the reticular layer of the dermis. Even in the dermal papilla the accumulation is the same as that in the surrounding connective tissue.

DISCUSSION

Skin contains abundant mucopolysaccharides (Meyer and Rapport, '51). Since chondroitin sulfate is the fraction of mucopolysaccharides which most readily takes up S35 (Bos- trom, '52; Bostrom and Gardell, ' 5 3 ) , the radioactive particles visualized may represent the site of ester sulfate groups in chondroitin sulfates. Concordant with this, the concentration of S35 in the skin appendages is strongest in the dermal papilla and in the outer root sheath of growing hair follicles both of which contain metachromatically stainable substances (BBlanger, '54; Montagna, '56).

The radioactive particles in the dermis are located mostly in the ground substance. The papillary layer being richer in ground substance than the reticular layer, shows a greater concentration of radioactive material. Both layers of the dermis are thicker, and perhaps richer in ground substance when hairs are growing than when they are resting (Chase et al., ' 5 3 ) , and they concentrate more S35.

Although the hair bulb and the epidermis show particles of S35 two and 4 hours after its administration, none are found

S35 I N THE SKIN O F THE RAT 167

in the keratinized structures even after 48 hours. In contrast, S35 is concentrated in the spinous layer of the epidermis and in the keratogenous zone of the hair follicle a few hours after the injection of S35 labelled cystine or methionine. Two and 4 days after the injection of these substances the maximum concentration is found in the stratum corneum and in the free part of the hair (BBlanger, '56). Thus, the distribution of inorganic S35 in the skin is not associated with keratiniza- tion, but indicates the regions of great turnover and exchange. S35 in the dermal papilla is probably associated with the mucopolysaccharide present in its ground substance ; that in the outer sheath is similarly tied up with the mucopoly- saccharides stored together with glycogen in its cells (Mon- tagna, '56). Quiescent hair follicles which contain no demon- strable mucopolysaccharides do not concentrate S35 in such amounts.

SUMMARY

1. The concentration of S35 in the dermis is maximal at 4 hours and diminishes appreciably 24 and 48 hours after it is injected. S35 is localized in the ground substance, showing greater concentrations in the papillary than in the reticular layer. The dermis of skin with growing hair follicles is thicker than that with quiescent follicles and it contains greater amounts of S35.

2. In mast cells S35 is accumulated in coarse granules. The concentration does not seem to diminish after 24 and 48 hours.

3. Active hair follicles, coincident with the appearance of metachromatic substances, accumulate large amounts of S35 in the dermal papilla and in the periphery of the outer sheath. Small amounts of radioactive granules may be found in the bulb of hair follicles especially 4 hours after the injection of S35, but none are seen in the keratinized parts. During the early stages of the hair growth cycle o r in quiescent follicles the dermal papilla does not accumulate S35.

168 WILLIAM MONTAGNA AND C. RUTH HILL

LITERATURE CITED

B~LANGER, L. F. 1954 Autoradiographic visualization of S" incorporation and turnover by the mucous glands of the gastro-intestinal tract and other soft tissues of ra t and hamster. Anat. Rec., 118: 755-772.

1956 Autoradiographic visualization of the entry and transit of S" methionine and cystine in the soft and hard tissues of the growing rat. Anat. Rec., 114: 555-579.

On the metabolism of the sulfate group chondroitinsulfuric acid. J. Biol. Chem., 196: 477-481.

Uptake of sulphates in mucopolysaccharides esterified with sulphuric acid in the skin of adult rats after intra- peritoneal injection of SE-labelled sodium sulphate. Acta Chem. Scand.,

1953 A qualitative and quantitative autoradiographic study on the uptake of SE-labelled sodium sulphate in the skin of the adult rat. Acta Path. Microbiol. Scand., 3.2: 516-521.

CHASE, H. B., W. MONTAGNA AND J. D. MALONE Changes in the skin in relation to the hair growth cycle. Anat. Rec., 116: 75-81.

DURAN-REYNALS, F., H. BUNTING AND G. VAN WAGENEN 1950 Studies on the sex skin of Macaca mulatta. Ann. N. Y. Acad. Sci., 5.2: 1006-1014.

MEYER, K., AND M. M. RAPPORT 1951 The mucopolysaccharides of the ground substance of connective tissue. Science, 113 : 596-600.

MONTAGNA, W. 1956 The Structure and Function of Skin. Academic Press, New York.

MONTAGNA, W., H. B. CHASE, J. D. MALONE AND H. P. MELARAGNO 1952 Cyclic changes in polysaccharides of the papilla of the hair follicle. Quart. .J. Microseop. Sci., 93: 241-245.

PELC, S. R. 1947 Autoradiographic technique. Nature, London. 160 : 749-750. SHIPNAN, M., H. B. CHASE AND W. MONTAGNA Glycogen in skin of the

mouse during cycles of hair growth. Proc. SOC. Exptl. Biol. Med., 88:

BOSTRON, II.

BOSTROX, IT., AND S. GARDELL

1952

1953

7: 216-222. BOSTRON, II., E. ODEBLAD AND U. FRIBERG

1953

1955

449-451.

PLATE

PLATE 1

EXPLANATION OF FIGURES

1 Skin with growing hair follicles showing the concentration of S s in the dermis 4 hours after its injection. The radioactive particles are concentrated in the papillary layer in the upper part of the figure. I n the reticular layer, only a part of which is shown, the particles are more widely separated.

2 Longitudinal section through the bulb of a hair follicle which had been growing 8 days, 4 hours after the injection of S35. Radioactive particles are concentrated in the dermal papilla and in the outer root sheath. The arrows indicate hair keratin, which although black in this figure, contains no 5%.

Transverse section through the lower part of the bulb. Particles of Sa are accumulated in the dermal papilla and in the narrow outer root sheath at the periphery of the bulb. A few radioactive particles are visible in the matrix of the bulb two hours after the injection of S3’.

Longitudinal section through the bulb of an active follicle, two hours after the injection of 5%. The strongest concentration of Ss is in the dermal papilla and the connective tissue sheath to which the papilla is attached. Farther up in the bulb the outer rooth sheath, which a t this level is not very thick, also shows a heavy concentration of F5. Compare with figure 2.

3

4

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S& I N THE: SKIN OF THE R.1’1’ I\ ILIJA&I MONTAUNA AND C. RUT11 H I L L

PLA4TE 1

171