SPECIALIZATIONS I N THE SKZIN O F THE SEAL (PHOCA VITULINA)

TWENTY-ONE V[GITRES

I N TR() 1)UCTION

Seals exhibit many anatomical and functional adaptations in their organ systems, and particularly the circulatory systelm, that arc undoubtedly associated with submarine life (Harrison and Tomlinson, '56). Their skin, like that of other aquatic manimals, has adaptations which protect it from the injurious action of water (Howell, '30 ; lCellogg, '28 ; Kiikcnthal, 1891). The histological features of the skin have bccn described by others (Bergersen, '31; Mohr, '5O), but its finer microscopic anatomy and the histochemical details have not been investi- gated. This paper deals with such details ; these may enlighten the biology of the skin of the sen1 and aid in understanding the significance of these particular adaptations. Many of the functions and rriodifications of nianimalian skin in general are obscure, and comparative anatomical studies of this type may shed light on soine of tlicnl.

Mamnials which spend much of their time in water have either largely lost their hair or, as in thp seal, their pclagc has become waterproofed. Several striking adaptations of the skin to a water environnicnt are found in the seal: (a) tlic

*Special Fellow of the United States Public Health Service at the London

* This work was supported in part by n grant from tllc United States Publir Hospital Medical College.

Health Service, RG2125 C6. 81

82 WILLIAM MONTAGNA AND RICHARD J. HARRISON

epidermis is thicker and more compact than thai of land mammals ; (b) hairs are flattened and grow in clusters ; and (c) the sebaceous glands are large and numerous ; their sccre- tion keeps the skin and hairs from becoming water-logged o r damaged. Like that of $many other aquatic mammals, the skin is heavily pigmented. The skin is rich in dilated venules arid capillaries. Seals have a characteristic odor (IIarnilton, ’56) ; after an animal has been handled the odor persists on the hands for a considerable time. These are the main modificn- tions of the skin of the seal ; the significance of some of these seems obvious, but that of others is not.

MATERIALS AND METIIODS

Skin for this study was collected from a number of newborn, adolescent, and adult Comrrion Seals (Phoca vitulina) of both sexes obtained from the Wash, Eng1anci3 All skin specimens were fixed imamediately after the animals were killed. The tissues of animals, kept alive for a period of time in the laboratory, were in general comparable to those of aniinals killed in the field. Since plucking of club hairs in othm mam- mals (Montagna, ’56) initiates tlie growth of their follicles, patches of club hairs from two captured half-grown seals were plucked at weekly intervals for 8 weeks. One week after the plucking of the last patch the skin was excised from each plucked area. Thus, this series of specimens contained hair follicles which had bccn growing from one to 8 weeks.

The following enzymes were studied : (1) succinic dehydro- genase activity was demonstrated in fresh frozen sections using the method of Seligman and Rutenburg ( ’51) as modified by Padykula ( ’52) ; (2) alkaline phosphatases and tween esterases were demonstrated in tissues fixed in chilled acetone, using sodhm glyeerophosphate and Tween 60 re- spectively as substrates ; (3) alpha esterases were studied in frozen sections of tissues fixed for 8 hours in chilled, un-

8 We are grateful t o the Eastern Sea Fisheries Committee in Kings T,gnn, Eng- land for assistance in obtaining specimens, and to the crew of M. V. “Seafish” for their hclpfulness in rwovcririg live and dead animals nndcr difficult conditions.

SKIN O F T H E SEAL 83

buff ereci 10 % formalin, neutralized with marble chips, using alpha naphtliyl acetate as a substrate. Lipids were studied in frozen sections of tissues fixed in 10% formalin, by observ- ing unstained sections under polarized light, by coloring them with Sudan T,lack, lop applying the Schaftz test, and by treating with digitonin and subsequently extracting with chloroform. Paraffin seetioils of Helly-fixed tissues were (I ) stained with toluidin blue buffered to pW 4.5; control sections were inca- hated in a solution of 0.1% ribonuclease at pH 6.0, before staining in tolnidin blue; (2) treated with thc Prnssian blue reaction for ionic iron; (3) treated with the periodic acid- Scliiff reaction for glycogen and other carbohydrates ; (4) treated with ainmoniacal silver nitrate to sliow reticular fibers ; (5) stained with resorcin fuchsin to demonstrate elastic fibers ; and (6) stained with van Geison's mixture to show elastic fibers and collagenous fibers. All of the methods mentioned above are described and critically apparised by Lillie ('34) and Gornori ( '52)' and no further reference 01- coninicnt about tlieni need be riiacle here. Sulfhydryl and disulfide groups were demonstrated ill skin fixed in a solution of 1% tri- chloracetic acid in 80% alcoliol (Rarrnett and Seligman, '52 and '54).

OBSERVATIONS

The epitlernzis. The epiderniis is very thick, measuring one-half to one millimeter over the entire body; it is thicker in adult animals than in young ones. The cells are usually small and low columnar in the basal laychr. The spinous layer is about 10 cells in tliiclrness : its cells become progressively larger as they rise toward the surface; in the upper three or four layers they are greatly flattened horizontally, and their volmne is apparently reduced. The spirious layer passcs imperceptibly into the corneal layer (fig. I), though rarely a discontinuous one-cell-thick granular layer may be fouiid. The stratum corneum is thick and compact; its 10 or more layers of cells arc not fully keratinized, and even those at the suri'acc show a stainable nucleus. Tn anirnals kept alive and

84 WILLIAM MONTABNA AND RICHARD J. HARRISON

out of water in the laboratory for a week or more, the epi- dermis is less compact and somewhat flaky.

The nuclei in the basal cells and the lower two or three layers of the stratum spinosum stain very strongly with tho Feulgen reaction, but they become progressively less reactive in the uppcr layers. All of the cells of the stratum corneum have a faintly Feulgen reactive nucleus ; even the flattened, desiccated cells in the surface layer have weakly reactive nuclei. Whethcr in the cpidermis of the young or adult, mitotic activity has been encountered rarely. Mitosis is found more often in the epidermis of bruised skin. Mitotic figures are normally found in the sebaceous glands and in the bulb of active hair follicles; hencc, if they had been present in the epidermis, it would have been possible to demonstrate them.

The cytoplasm of the cells in the lower layers of the epi- dermis contains matcrial which stains intensely with basic dyes. This material is removed by incubation in ribonuclease and probably consists of ribonncleic acid. The staining of the cytoplasm fades as the cells ascend to more superficial levels arid is absent in the straturn corneum. The line of divisioii between the spinous and the corneal layers is not distinct since a stratum granulosum is only rarely present (lig. 1). A granular layer I w o or three cells thick is always present in the wall of the pilosebaceous canal. These cells contain vcry srnall lieratohyalin granules. When a granular layer is present, the corneal layer above it is flaky and its cells show completc cornification and disappearance of nuclei. When the epi- dermis is irritated, the corneal layer becomes vcry thick, and either a granular layer appears o r a wide band of basophil cells becomes visible between the stratum spinosum aiid thc stratnm corneum.

The upper tmo-thirds of the corneal layer is intensely sudanophil in frozen sections colored with Sudan black (fig. S), but the lower third is only weakly colored. The cells in tlze spinous layer all take on a light grey color. Since the epidermal cells arc all pigmented (fig. l), it is not possible to

SKIN 03' THE SEAL 85

distinguish between rriclaiiiri granules and discrete lipid granules.

Non-specific esterase activity is demonstrable in the epi- dermis (fig. 14). The enzyme is concentmted in a band of cclls between tlie spinous and corneal layers. There is no phospliatase activity in the epidermis. Strong succiriic de- hydrogenasc activity is seen in the basal layer and thc deeper half of the spinous layer. The reaction fades quickly in the cells of tlie upper half of the spinous layer and disappears in its superficial cells (fig. 20). A moderate reaction for sulf- liydryl and disulfide groups can be demonstrated in the basal and spinous layers. Largp amounts of sulfhydryl groups are conccntratcd in ibe cells of the corneal layer (fig. 2).

Every cell in the epidermis contains melanin. Dendritic melanocytes, larger than the basal epidermal cells, are spaced at a ratio of roughly onc to 5 basal cells (fig. 1). The large, often branching, cytoplasmic processes are insinuated be- tween the cells of tlie spinous layer. Pigmentation seems to decrease gradually in the upper part of the spinous layer, but it must be relmernbered that the pigment becomes more thinly distributed or diluted as the epidermal cells increase in volume during the ascent to the corneal layer. The pigment is always localized in a shield o r cap over each nucleus in the super- ficial part of' the cell. The cells in the stratum corneum become gradually flattened, lose volume, and the melanin in them is more concentrated.

The hair folticles. Hairs grow in groups composed of one large, bristle-like overhair and 4 or 5 fine and short nnder- hairs (fig. 21), all of which emcrgc from a single canal. The overhairs as well as their folliclcs are flattened. The under- hairs grow on the ventral side of the overhair, they are very slightly flattened or riot at all, and they fill up the space beneath the overhairs.

The follicles of each hair group are all conjoined and their hairs share one pilary canal. The bulbs of the underhair fol- licles are nearer to tlie surface than those of the overhairs arid grow on the posterior side of the follicle of the overhair. The

S6 TVILLId1\1 NONTAGNA A N D RICHARD J. IIALXLISON

size of tliese adnesal follicles varies, those closer to the ovcr- hair are larger and longer than those farthest away, so that they are arranged in step-like fashion (fig. 2 l ) , a condition also found in other seals (Bergersen, '31).

Visible melaiiocytes and melanin granules are found throughout the hair folliclc, except in the lower part of the bulb. Melanocytes are iiurnerous in the wall of the pilary canal, aiid varying numbers of them can be found in the outer sheath all the %my to the base of the follicle, whether the follicle is active o r quiescent. The distribution of melanin in the bulb of active follicles will be described later.

The cells of the outer slieatli in growing Pollicles, but not those of the pilary canal, abound in glycogen. I n resting folliclcs neither thc cells of the outer sheath nor those of any other part of the follicle contain glycogen. When, a t the completion of the hair growth cycle, the follicles begin to form the club hair and become transformed from the active to thc quiescent state, the bulb begins to degenerate and all of its cells become laden with glycogen. Thesc cells degeneratc very rapidly, and even the debris they form in the lower part of the degenerating bulb coiitains largc globules of glrcogen (figs. 6, 7). Even the ground suhstance and thc cells of the dermal papilla contain glycogen at this time. This condition is similar to that found during the degeneration of human scalp hair follicles after they have been irritated (Montagna and Chase, '56). The ground substanc of tlie dermal papilla in active hair folliclcs is intensely periodic acid-Schiff re- active but saliva resistant, whereas that of quiescent folliclcs is not at all reactive (Montagna, '56).

Only the cells of the bulb of active hair follicles contain large amounts of substances stainable with toluidin blue buffered to pH 4.5. The lower part, of the bulb is intensely stained (fig. 4) ' but the staining of the upper part becomes gradually less intense, and a t the level of the liair root, in the keratogenous zone, it is no longcr evident. The cells of the outer root sheath in the upper third of the follicle slain lightly NTith basic dyes; those in tlie middle third stain a pale color,

S K I K O F THE SEAL 87

and those in the lower third stain inoclcratcly well. This basopliil substance is abolished by ribonuclease and is prob- ably ribonucleic acid, a substance which is particularly abun- dant in the bulb, or growing portion of hair follicles. There is an incipient breakdown of the cells into sinall globules which stain with basic dyes in the bulb of all growing follicles along the uppcr part of the dernial papilla. This is pronounced in follicles in which the tips of the papillae cxtcnd far into the upper part of thc bulb. The bulb is pigmented in the upper half but not in tlie lower. h line drawn across tlie bulk, to separate the pigmented from tlie non-pigmented por- tions would effectively separate the mitotically active, noii- pigmented lower part from the mitotically inert, pigmented upper part (fig. 4). TVhcn stained with the Fculgen technique for the demonstration of desoxyriboniicleio acid, the nuclei of the cells in the lo.l\-er. half of the bulb stain much more intenscly than those in tlie upper part (fig. 4). In the upper half only tlie nuclei of the melanocytes are intensely colored.

During catagen, the hair root forms a club hair and thc bulb degeiierat ~ s , leaving intenscly basophil cell fragments below the capsule of cells around the club hairs. In quiescent hair follicles, the cells of the outer sheath, or the epithelial sac and the epithelial strand, all stain moderately well with basic dyes .

The ground substance in the dermal papilla of active hair fo1liclc.s stains metachromatically with toluidin bluc. The long and acuminate dermal papillae havc the most intense metachromasia. The less wcll developed and dome-shaped dermal papillae stain more weakly. Xetacliromasia dis- appears from the dermal papilla during catagen. In quiescent follicles the dermal papilla is small, its cells are crowded together, and the scant amount of ground suhstance bctmeen them remains unstained.

The distribution of alkaline phosphatase in the hair follicles i s similar to that of other animals (Montagna, '56). Enzyme activity is evident in the dermal papilla of active follicles but not in that of quiescent, ones.

88 WILLIAM MONTAGNA AND RICHARD J. HARRISON

The cells in the outer root sheath of active follicles have strong succiiiic dehydrogenase activity. The richest enzyme reaction is concentrated in the cells of the lower half of the bulb, as in the follicles of other mammals (Montagna, '56). Moderately strong enzyme activity is also found in all the cclls of the epithelial elements of the quiescent hair follicles. The cells of the dermal papilla of active follicles always show strong enzyme activity. Those of quiescent follicles show a less intense reaction.

The dermal papilla of the seal is similar to that of the follicles of other mammals. It is very long in the active follicle (figs. 4, 5 ) and contains substances which stain metachroma- tically and which are strongly periodic acid-Schiff reactive ; it contains alkaline phosphatase, and its cells are large and rich in succinic dehydrogenase. The papilla of quiescent fol- licles is small, and it shows only traces of these substances o r none. The papilla of active follicles is much larger than that of quiescent ones, containing more ground substance, larger cells, and numerous capillaries. I n active follicles a tuft of capillaries runs through the center of the papilla and in the upper bulb it makes contact with the cells that face the papilla cavity. Capillaries are difficult to visualize in quiescent follicles .

This species of seal is born with an adult type of hair, since its wooly juvenile fur is shed in utero. The skin of new- born seals and that of animals a few weeks old contains only club hairs. The skin of all animals collected in the winter and spring months also had only club hairs. That of animals collected in late August contained hair follicles either in full growth or in the catagcn stage. Thus, moulting in these particular animals takes place during the summer months. The hairs from csch hair group are in the salnie phase of the growth cycle, and this sychrony is observed even in catagen. From observations of whole pelts it appears that hair growth occurs in waves o r patches, not unlike the pattcrns which occur in rodents (Bergersen, '31).

S K l N O F THE SEAL 89

When club hairs n-ere plucked from the two captured ani- ~iials, tlie skin responded in one of two ways. When the pelage was wet, the hair \\-as difficult to pluck and the skin became heniorrhagic and etlematous, a condition which persisted for several weeks. Two weeks later these plucked patches were covered with hyperkaratotic flakes. Biopsy specimens re- moved a t weekly intervals from such plucked areas showed that the hair follicles w x e still quiescent after 8 weeks. The surface epidermis was very thin but tlie stratum corneiim was thick and flaky, having none of the compactness of that found in the normal skin adjoining these plucked areas. When the pelage was dry, club hairs could he plucl<cd easily and cleanly, and none of the traumatic conditions described above de- veloped in the denuded areas. Plucking initiated growth of the quicscent follicles, and 6 weeks later the hairs had regrown enough to have just reached the surface of the skin. All of the follicles in each hair group were active and in phase. The establishment of a bulb is very slow after plucking of the club hairs, and it requires about three weeks. It is not known if this represents the amount of time required normally by follicles to establish a bulb.

The sebaceous .gla?zds. Several clusters of sebaceous glands surround the pilary canal (fig. 3). The larger clusters of glands are found in the obtuse angle of the follicle, and the smaller in the acute anglc. Each cluster is composed of a number of closely packed selmceons glands, each opening separately into the pilary canal by a long narrow duct (fig. 8). The glands in each group have a number of cornplcx acinar systems which converge toward the duct.

Variable numbers of dendritic melanocytes are found throughout the cells of the ducts and at the periphery of tlie acini, and most sebaceous cells contain some pigment (fig. 9). The undifferentiated cells in the duct and a t the periphery of the acini may be darkly pigmented like the surface epidermal cells. The pigment is considerably diluted by the sebum as the cells undergo sebaceous differenti a t’ ion

90 WILLlAM A10KTAGKA A K D E1ICFlTARi) J. HARRISON

arid their volume increases inany fold. In mature sebaceons cells nlelanin graiiules may be seen between the lipid droplets.

The cells of the ducts and the undifferentiated cells a t the periphery of each acinus stain intensely with toluidin blue buffered to pfI 4.5. When the cells increase in size during sebaceous diff erentiation, their cytoplasm acquires distinct basophil granules between the lipids. These seem to dis- appear, but actually they become diluted by the accurnulatioii of lipid; in the mature sebaceous cells only a few basopliil granules occur scattered between lipid globules.

The nuclei of the undifferentiated peripheral cells and those of the cells of the ducts stain intensely with the Feulgcn tech- nique and resemhle those of the basal layer of the epidermis. Mitotic figures are found often in both of these places. Thc nuclei become larger with sebaceous differentiation and ap- pear like delicate skeins of weakly Feulgen reactive clironia- tin. Before the mature sebaceous cells break down, the iiucleus collapses and becomes more strongly Feulgcn reactive. Later, when the cells degenerate, the nucleus breaks up aud is no longer Feulgen reactive.

Sebaceous differentiation niizst progress very rapidly sincc the area of transformation from the peripheral cells to thc mature sebaceous cells is very narrow. Mature cells arc crammed with small lipid granules so intensely suilanophil that they mask all of the details of cellular architecture when treated with Sudan black (fig. 10). The sebum in the ducts and in the pilary canal is strongly and uniformly sudanophil. Viewed under polarized light, the sebum is not birefringent. Treating the sebum with digitonin does not form digitonide crystals. The sebum is ncgative to the Schultz test for un- saturated sterols.

The distribution of succinic dehydrogenase is similar to that found in the glands of other mammals (Alontagna, '56). The undifferentiated peripheral cells and the cells of the ducts have the strongest reaction (fig. 11). The mature sebaceous cells have a small amount of enzyme activity, but these cells contain lipid, and tlic reactive dif ormazan granules dissolve

91 SKIN O F THE SEAL

in the lipid forming it reddish rrionoformazan (Forniisaiio and Blontagna, '55).

Sebaceous glands have strong alkaline phospliatase activity and in this respect resemble those of the cat and the dog (hlontagna, '56). The strongest enzyme activity is found in the differentiating cells; the mature cells have no enzyme activity and the peripheral indifferent cells have a moclcratc reaction (fig. 12) . Tween esterase activity is localized in tho disintegrating sehaceous cells and in the sebum. The reaction is similar to that in the sebaceous glands of the hamster (Mon- t agna and Harnilton, '49). Abundant non-specific esterase activity can he seen in the cells at the periphery of the glands where the undi-Cferentiated and the differentiating sebaceous cells have the strongest reaction (fig. 11). The mature sebacct- ous cells and the sebum have a somewhat weaker reaction.

One simple tubular coiled apocrine sweat gland opens into each pilary canal just below the entrance of the ducts of sebaceous glands (fig. 21). The glands lie within the bed of loose coniiectivr tissue and fat that surrounds the follicles of each hair group. The lowest coil of the glands in skin wi-itli resting follicles extends some distance below the base of the overhair follicle, but in active folliclcs the bulbs are almost as deep in the dermis as the lowest coil of the glands. The degree of tortuosity and dilatation of the glands is variable. The secretory portion is dilated and extends to about midway up the level of hair follicles where the gland contracts into a narrow duct. The dnct is also dilated when the secretory portion is dilated, and spaced contractions along its path give it the appearance of sausage links.

The cells of the secretory segment are columnar or culnoiclal. The apical part of the crlls often protrudes into the lumen, as in typical apocrine cells (fig. 11). A compact layer of large myoepithrlial cells lies between the base of the apocrine cells and the basement membrane. The dnct is composed of two layers of cuboidal cells, the superficial ones being slightly flattened and hyalin.

Xweut glarzds.

92 W I L L I A M M O N T A G N A A N D BLCI-IdRD J. HARRLSON

The roughly spllerical nuclei of the secretory cells a1-e stained moderately well with the Feulgcn technique. We have never observed mitotic activity in the glands.

The cytoplasin of the secretory cells contains small granules of pigment above the nucleus, but the apical part which pro- trudes into the lumen is free of thorn and is often hyalin in appearance. Regardless of the apparent degree of activity and of the amount of pigment present, noric of the glands contains iron. The pigment is non-lipoidal when preparations are colored with Sudan black. Fine basopliil granules are particularly numerous in tlie basal cytoplasm. &my reactive granules of different sizes are found above and alongside the nucleus after the application of the periodic acid-Scliiff mcthod (fig. 15). These and the secretion in tlie lumen, which is also strongly reactive (fig. 16), are saliva resistant. The gland cont,ains no demonstrable glycogen.

Succinic dehydrogenase activity is very strong in the cells of the duct but only moderate in the secretory coil. The distribution of this enzyme is like that found in human apocrine sweat glands ( X o n t a p a and Formisano, '55). A strong alkaline phosphatase reaction is localized in the luminal part of the secretory cells (fig. 17) ; thc base of the cells and the myoepithelial cells remain unreactive. The secretion in the lumen of the glands often shows strong phosphatase ; when this is so the cells are also reactive; when the content is unreactive the cells of the duct are unreactive. Non-specific esterases are widely distribtued in the cells of the secretory coil as well as in those of the duct. The strongest reaction is found in the luminal cytoplasm of the secretory cells

Sweat is poured into the pilary canal together with the sebum, but it does not mix readily with it. The yellowish, compact, twisted casts of tlie scnii-solid sweat stand out clearly in unstained frozen sections from the surrounding colorless, homogeneous sebum. The sweat casts are non-sudanophilic in preparations colored with Sudan black and can be seen extending practically to the orifice of the pilary canal against

(fig. 18).

SKIN O F THE SEAL 93

the intensely sudanophilic sebum. The sweat casts are strongly Schiff-reactive (fig. 19) but not metachromatic when stained with toluidin blue.

The dermis. The dermis is composed of a thick reticular layer and a thin papillary layer. As seen in vertical sections oriented parallel to the hair follicles, it seems to be composed of altcrnating columns of dense connective tissue amd loose, fatty tissue. The pilary units, sweat glands, arid arteries and veiiis grow through the columns. The blood vessels run a straight course parallel to the hair follicles, to the level of the sebaceous glaiids. The heavy fibrous portion of the dermis i.esembles a sieve, through thc rhombic meshes of which rise columns of fatty tissue which are continuous with the sub- jacent blubber.

The papillary layer contains few, delicate collagenoua fibers arid reticular fibers but no visible elastic fibers. The upper part of the reticular layer is composed of very coarse colla- genous fibers and a few elastic fibers ; the deeper portion con- tains inany elastic fibers aiid a thick layer of smooth muscle fiber bundles admixed with collagenous fibers. Obliquely ar- ranged collagenous fibers are interlaced with transversely oriented ones in such a way that they outline the rhombic patterns mentioned above (Terao, '40).

The dermis has a very profuse vascular bed. The upper part of the reticular layer and the entire papillary layer are riddled with dilated venules and capillaries which often form sinusoidal enlargemcnts. Nany of these thin-walled veins are so dilated that only traces of connective tissue separate them from the bases of the epidermal cclls. The endothelial cells of these vessels are reactive to the techniquc for suc- cinic deliydrogenase ; tissues thus prepared arc useful for the visualization of blood vessels. The tlerrnal blood vessels branch extensively (fig. 20).

More cells are prcscnt in the papillary than in the reticular layer. All of the fibroblasts in the reticular layer contain Jernonstralsle lipid granules in the cytoplasm. Histiocytes are invariably prescnt, though more numerous in some speci-

94 WILLIAM MOn’TAGNA AND RICHARD J. IIARRISON

mens than in others, and can be recognized by their foamy cytoplasm and by their strong reaction to non-specific esterase. Histiocytes containing large amounts of periodic acid-Schiff reactive substances and lipids are often aggregated around sweat glands. Histiocytes are very numerous in irritated skin o r around encysted parasites.

Variable numbers of dendritic melanocytes are present in the papillary layer close to, but not quite touching, the epi- dermal structures. They are very numerous in some speci- mens. Nearly every cell in thc papillary layer contains some melanin granules. Most of these cells resemble fibroblasts ; some, however, are round and smaller than fibroblasts, and their cytoplasm, which is periodic acid-Schiff reactive, is replete with melanin granules. These cells have no visible dendritic processes and are probably not melanocytes.

There are no demonstrable mast cells in the dermis. The assumption that the mast cell granules are so labile that they are not preserved by the fzative can be dismissed since mast cells can be demonstrated easily in the other organs of the seal fixed in the same fixatives.

The dermis is very fat. Even skin separated carefully from the blubber is so fatty that it is not possible to prepare good frozen sections colored with Sudan black. The melting point of this fat is low and oily droplets always float over the prepared sections.

DISCUSSIOS

This is the first segment of a study undcrtaken to see in what ways, if any, the rriorpliological and histochemical char- acteristics of the skin of the seal differ from those of other furred animals. In a sense, this is a catalog of the details we have found. One might expect that the skin of the seal possesses adaptations to protect it against the action of water. Some of the details described here are self-explanatory, but others give as yet no clue to their specific role in the aclapta- tion of the seal to a water environment.

SIiIh' O F THE SEAL 95

The skin of the seal departs from that of land mammals in the morphology of its epidermis. The tliick epidermis has a deep arid compact stratum coimeiim composed of cells with visible nuclei practically up to the surface and the entire layer is rich in sulfhytlryl groups. The stratum corncum is similar to that found in the mucous regions or in patliological paralieratotic lesions and, as in these conditions, there is no stratum granulosuni. This would seem to be an excellent adaptation since a surface of scaly keratin might easily become water-logged and flake off. This adaptation makes the stratum corneuni a relatively permanent layer, and thc absence of mitotic figures in the epidermis testifies to this. The rate of mitosis in the normal epidermis of other mammals is in quilibrium with the rate of cell loss on the surface; this loss must be insignificant in the seal. The abundance of pig- ment in the epidermis and its distribution above the nucleus of each cell almost suggests that melanin is distributed in suc:h a may as to shield the cells from injurious factors such as sunlight. It is possible also that melanin might act as a heat absorber. However, no one knows the biological significance of melanin.

Tn spite of apparciitly adverse conditions and tlic fact that many aquatic niarrirrials have lost it, the seal has retained a luxuriant pelage. The hairs of seals grow in groups ; this may have special adaptative significance. Each hair group is com- posed of one superficial guard hair wliich is flattened and fits over the basc of other guard hairs like a scale. The small undcrhairs, variable in numbers, pack the space beneath the guard hairs. Xumeroiw tiny bubbles of air adhcrc to tlie hairs and particularly to the unclcrhairs when the animal submerges. Such an insulating blanket of air could conserve the animal's heat, if only f o r a short timc, and could play a role in kceping the skin dry. Other mechanisms, such as the thick layer of surface lipids must also be effective in the conservation of heat and keeping tlie skin from becoming water-logged.

The scant information that we 'have does not permit us to interpret tlie special adaptation of the sebnrn. The sebum of

96 W I L L I A M MONTAGNA AND R I C H l R D J. HARRISON

most mammals is miscible with water (Rothman, '54), but such a property would scarcely serve a useful purpose in an animal which lives much of its life in water. It might be useful to know if cholesterol, a hydrophilic substance found in the skin surface fats of all mammals examined (Rothman, '54)' is present in the surface fat of the seal. The histochem- ical tests applied thus far show no cholesterol there, but this is not proof of its absence. If cholesterol and other hydrophilie substances are really absent from the surface lipids, the sebum of the seal would show particularly significant adaptative specializations.

An interesting morpliological adaptation to protect the skin against the erosion of water is found in the resting hair follicles. A thickened, partially keratinized collar grips tightly around the hair above the club and blocks the sepage of either the sebum o r the water into the lower part of the follicle.

The details of the histochemical findings in the sebaceous glands have little meaning in themselves. This body of in- formation may some day acquire some significance. For ex- ample, the presence of different enzymes in sebaccous glands exhibits striking species differences (Montagna, '56). We may understand the significance of these diffcrcnces when more is known about the chemistry of the sebum.

The presence of melanocytes in the sebaceous glands is curious. Nearly every sebaceous cell contains melanin gran- ules; these are more readily discernible in the indifferent peripheral cells than in the differentiated sebaceous cells. Cells which at the beginning of differentiation havc an ap- preciable amount of pigment, contain a rclatively small amount of it a t the end of sebaceous transformation. Sebaceous cells, then, acquire pigment when they are still uiidiff erentiated. This detail throws some light on a phenomenon observed by all who have studied the skin of the mouse, which nornially contains no visible mclanocytes other than those in the hair follicles. d f t e r it is painted with carcinogenic agents, melanin becomes visible in the epidermis and in thr sebaccous glands.

SHIN OF THE SEAL 97

Amelanotic melanocytes must be present in these sites at all times, and melanogenesis must be initiated by irritants.

Although sweat glands are numerous, they are small and their function seems to be somewhat paradoxical. The amount of periodic acid-Schiff reactive secretion stored in the lumen of the glands is out of proportion to the number of intra- cellular granules which have a similar property. The viscous and condensed secretion is probably manufactured slowly. The histochemical properties of the secretion give no clue to its composition other than that it probably contains mucoicl, non-fatty substances. The “sweat” casts are not readily miscible with sebuni in the pilary canal. Perhaps the secre- tion is a waste product, responsible for the niusky odor char- acteristic of the seal. The seals, and particularly the Otariicls, have a strong odor (Hamilton, ’56), but it is not known if the sweat glands are responsible f o r it. In some mammals the characteristic odor is traceable to the skin glands (Montagna, ’50).

The hair follicles have only a few peculiarities. The general morphology of the hair groups has been described by Berger- sen ( ’31), and the histochemical findings are concordant with those in the follicles of other mammals (Xontagna, ’56). The dermal papilla in active follicles is long and narrow and re- sembles that found in the follicles of rodents. Unlike that of rodents, however, it is richly vascularized. A long narrow papilla is always associated with rapidly growing hairs. In human follicles, for example, which grow more slowly, the dermal papilla is dome-shaped or onion-shaped. Like the other cutaneous structures (except sweat glands), the hair follicIe is rich in pigment. Nelanocytes are particularly numerous in. the wall of the pilary canal from which they extend into the sebaceous glands. Pigment extends along the outer root sheath all the way t o the bulb instead of stopping at this level, as in the follicles of other mammals. Melanocytes are not numerous in the outer sheath, and they are small, but some are always found there. The presence of melanocytes in the outer sheath

98 WILLIAM MONTAGNA A N D RICHARD J. HARRISON

gives support to the belief that these cells are normally pres- ent in the outer sheath of the follicles of other mammals as well, but that they remain pigmentless unless they are trig- gered by such agents as X-rays (Montagna and Chase, '56) or carcinogens.

One of the distinctive features of trhe dermis is its vascu- larity. In the papillary layer, and particularly in the skin of the hind flipper, enlarged venules forni spaces which re- semble cavernous sinuses. When one holds a struggling seal by the flipper, the skin of the flipper is often bruised and hemorrhagenous. The vessels of the skin may serve as a blood reservoir. The general enlargement of the abdominal veins, the development of venous networks and plexuses, and the presence of numerous anastomotic channels in the whole venous system (Harrison and Tonilinson, '66) are reflected in the vascularity of the skin. It is known that there is brady- cardia in the seal on diving (Scholander, '40) and that there is probably occlusion of the venous return to the right heart due to closure of a thick striated muscle sphincter that girdles the posterior vena cava immediately above the dia- phragm. The skin could play an important part in accommo- dating the venous blood, in which event the heat-retaining property of the pelage would become essential. Perhaps the persistence of the pelage in Pinnipedia has allowed the diving bradycardia to evolve without the seal suffering excessive heat loss under water. A similar bradycardia is not definitely known to occur in all Cetacea, and some Cetacea at least appear to combat heat loss by countercurrent vascular heat exchange in the fins (Scholander and Schevill, '55).

Mast cells have been found in the skin of other mammals, but the seal is an exception. If mast cells are present in the skin of seals they must be different from the easily demon- strable and relatively typical mast cells in the other organs of this animal. The fibroblasts and the histiocytes both show some peculiarities. All of the fibroblasts in the papillary layer contain some melanin granules. If it is admitted that these are fibroblasts, each of them demonstrates phagocytic

SKIN OF T H E SE.4L 99

properties. Zf it is denied that they are fibroblasts, then the dermis is left practically without cells except for the relatively numerous recognizable histiocytes particularly in certain places which may represent sites of endoparasites.

Most of tlir prololrms of adaptation in the skin of the seal remain unsolved, but this study at least iiidicatcs the impor- tance of comparative anatomy in attaining an understanding of the skin.

STTMM ART;

I. Tlir cpidcrmis of thc seal is very thick, it is heavily pignientecl, and i t has no straturn grariulosum ; the stratum cornemn rcsembles that of micoiis membranes, such as the esophagus and it contains large amounts of sulfhydryl groups.

Hair follicles grow in groups coinposed of one large overhair arid 4 01- more underhairs, all of which share oiic pilary canal. Thc histochemical features of the hair follicles a re similar to those of tcrrestial mamrrials. Wlien club hairs arr plucked, their follicles liecomc active ancl give rise to neu- hairs. Tlic overhairs are flattened so that they fit over each otlirr like imhricatcd scales.

3. Ni i r r i~ ro~s , largc scl)wceons glands encirclr each pilary caiial arid open into it by long, iiarrow ducts. T h e glands al,ounti in alkaline phosphatase, iion-specific csterases, tween estrrases and succinic dehyclrogenase. They are vcry active and their sccrction covers thc skin anti hair with a watei*proof, oily film.

Oiic rnoderately coilcd, apocrinc gland grows parallel to each follicle of overhairs a id o p t m into the pilary caiial. These gluntls contain succinic rlchytlrogt~nase, non-specific estcrasw arid alkaline phosphatasc. T h e secretory cells con- tain granules, which like the sccrction in tlic luriwn of the gluntls am pcJrioclic acid-Rchiff reactive. The secrrtioii is not miscible with tlie sehum in the pilary canal.

5. The architcciure of the dermis is sirriilitr to that of other P7tocitlirr. Practically all of tlir filiroblasts contain pig-

2.

4.

100 WILLIAM MONTAGNA AND EICHSRD J. HARXISON

ment granules, there are numerous histiocytes arid ihcw are no mast cells. Tho dermis is extremely vascular.

I JTERAT~RE c m m R ZRRNETT, R. J., kNn Ak. bf. SELIQUAN 1953 T h r histoclrcmical (listrihntioii of

protriii hound su l fh~-dry l groups. J. Nat. (':inter Inst., 13: 905-926. 1934 ITistochemical tlenionstiatioii of sulfhydrgl and ilisulfirltl

groups of protein. J . Nat . (‘anrer Inst., 14: 7(%-S03. l i b KGEESEPT, B. 1931 Reitrage zur Kciintiiis dei. Hnnt einigcr Pinnipetlicn

iuitrr IJcsoiiclerrr Herucksichtigung der Hnut tler P1ioc.a groiiltmtlica. Skriftei Utgit t av (let Noiske Videnslrali~ Akaderiii i 0510. I. M a t Saturv. Iilassc.

FORRIISANO, v. R., AND w. M O N T k G N S 1954 Surcinic debydrogennsP activity 111

the skin of the guinea pig. Annt. Rec., 3511: 893-906. (IonfORI, G. 1952 Micro\ropie Histocliemistr>. Priilciples ant1 Piactice. TTiii

vcr,sitj of Chicago Press, Chicago. T r u m m o ~ , J . E. 1956 Scent of Otariids. Nature, Lonrlon, 7.77: 900. HIRRISOX, R. J., AND J. D. W. TOMLIXHON 1956 Observatio~is on thc venons

system in certain Pinnil~edian and Cvtaccn. t‘roc. Zool. Sor. Tmntl., 196: 205-233.

IIOWELL, A . 13. 1930 Aquatic &IIamnials. Thrir Atlaptationx to L i f e j i i thr Water. C‘linilea C . Thomas, Baltiinore.

KhThOGG, R. The histor) of whalrs - tlirii adaptation to life in the !latcsi. Quart. Rev. Biol., 3: 29-76 and 174-208.

Ki liENTII.ir>, W. 1891 0 1 1 tlie adaptation of mmmials to aquatic lifc. Ann. Mag. Nat. Hist., scr., 6, 7 : 173-179.

LILLIE, R. D. 1964 IIistopatliologie Tcchnic nntl P!ncticsl Hisfoclinmstr~ . l3lIakiston Coinl~aiiy, h e , , New York.

MOHR, 8. 1950 Beliaarung uiid Ilaamverhsel der Rok11)eii. Neue Ergd) . €’rob leme Zool. (Kln t t Festschrift) I m p ~ i g , 605-614.

M o v ~ ~ c v a , W. 1950 Wie I1icr\211 iiiguinal g l i ~ n d ~ of the rnb1)lt. A m . J. Annt., 87: 215-235.

19% Tlic Rt i ucturr r i i id F u n c t i o n uE Skin. Ac :irlriiiiv Press, N r n York.

Histology wild c$tochrirli~tly of ~l~n1:tIl

1938

MONT \GNk, W., kN1) If. B. ( ’ H A W I%% skin. X. X irrsdiatioii of tlic scalp. bin. J. Anat., i n press”.

huriian skin. V11. The tlistributioii of succiiiic deli? ilrogenasc. xet ivi t~. . Anat. Ref., 123: 65-78.

The d i n c e o u s gl:rilds of the h;1InStPI’

i lm. .T. h a t . . 84: 365-396. The locahzntion of vlccinic dehydrogenasc in tissue we

Physiology ant1 Riochemi\trj of Skin. TJiii, el sity of C‘bicnpo

&rOlUTARUA, w., AND v. w. FORMISAY0 1955 kIi9tology aild ~ ~ t ~ l ~ h ~ l l l l 5 t l ’ ~ Of

S~ONTUIN~, W., AND d. B. H4MILTON

PADYRUL I, 14. A.

EOWIAI,\N, S.

1949

1953 tions of tlic ra t . Am. J . Anat., 0 1 : 107-146.

Prcss, Chicago. 1964

S K I N OP T H E SEAL 101

SCHOLANULIL, P. F. 1940 E q ~ r i m m t n l investigation on the icspiratory func- tion in tiiviiiy itiairiiii;d~ am1 hinls. HvwlrBd. Skr., Srientifir results Nnriiic Hiol. Res., Norske Vid~nskaps Akadeinl, Oslo, 82: 1-131.

SCHOT~AXDER, P. F., NU W. R. S:CHF\ILL 1955 C’ounter current vascular liest ex cliaiigr in tlie fins of w h d r s . J. Appl. Phgsiol., S: 279-282.

SELIGMAN, A . M., AND &I. RUTENBI KO The Iiistocheinieal demonstration of succinic doli~drogenase. Science, 115: 317-320.

SIVEKTRBN, E. (IT. Oslo) 1 9 i i ON tlic hiology of the harp w a l . Pliora gronlaiitlic*a Erxl. Norskr Virlriisk. 4 k a d . 040, Hvalrlcl. Skr. Sci. Re- sults Marine Hiol. Res., 26: 166.

~Jlicroxcopica1 cvnniiiiatioii of tlit, leather of aquatic: xnirnx’ls. 1. Btill. .Tapii. Soc. S c i . Fidieiivs, X : 343-346.

1951

TERAO, A. 1940

PLATE 1

EXPLANA'J'ION Oh' F I G U R E S

1 l'fre epideriniq of an adult femalc. It i8 \Pry thick, the spirious layer blends into tlie thick corneal layer (inclicnted by a bracket), and there is no granular layer. Each re11 contains inelanin above the iluclcus even in the eorrical lajer. Thr arrows indicate tlie dendritic iiiclaiioc~ t c s in the ba,xal lwycr. T h e section i i stained lightly with toluidiii blue. 500 x.

2 Tlic epidcrrriis of an adult fernale, showing the rlistrihrition of sulfhytlryl ginups, stroiiglg c.onrrntiated in the rntire corneal layer (inclicatccl b y a hraclret). U(4aniri is seen pnrtlcularly well in the cells of tbc basal layer. 450 x.

102

5 K I N OF SEAL V ILLIAAI MONTAONA A N D R I C H A R D J. H A R R I S O N

103

PLATE 2

EXPLANATION OF FIGURES

3 Skin from an adult female showiiig the general topography of tlie active hair follicles. The other follicles of each hair group are not completely included in this plane. The arro\ts point to follicles of uderha i i s . Sweat glands can he seen on the right of each follicle, particularly 011 tlw right cnriier of thr, figure. Heniatoxylin and eosin. 50 X.

4 The bulb of an active hair follicle stained \-,it11 toluitliri blue. Tlic cells of the lower half of the bulb :ire intensely hsoplii l , wliereas those farther up are riot. Tlic dermal papilla is typically liarrow aiitl v c ~ y loiig. A sweat gla~lcl is we11 to tlic left of tlic bulb. 125 x.

6 The bulb of an active hair follicle from an ailolerceiit male, stained mjtli t l x Fculgen method. The line separates tlie bulh into upper a ~ ~ d l o v w parts. The rells in the lower part below the line have clarklp stained nuclei; those farther up are staiuecl vcry pale. Since only the nurlei below thc: pigmented area are in mitosis, the non-pigrnentcd lower half is actually the matrix. The long arid riarrow dermal papilln is diown aell. 100 X.

104

105

PLATE 3

6 The lower par t of a hair follicle during catagen, from the skln of a n adult female, terxted with the periodic acid-Schiff terhriique. Most of the cells in this region of the follicle are dead or dgiiig slid are repletr \\itti glycogen, which is shown here. Even the dermal papilla contains glycogen. 100 x. Hair follicle in late extagen, showing the extablislirriciit of n club and the almost conipIete disappearance of the bulb. The outvr sheath still contxiiir abundant gljcogeri, which disappears later in telogen. From the skin of an adult female, treated with tlic periodic wiil-Seliiff riiethod. 100 X.

Skin from 5 juveiiilc male, colored with Sudan black. The straturn eoriieiiin is deep17 sudanophilir. individual glancls which open separately into tllc pilary canal by long, narrow ducts. 50 X .

7

8 Tlic sebaceous glaxds are conipo

9 Dendritic mclanocytes in a scbaeeous gland. F r o m the skin of a yoiing female, lightly stainpi1 with toluidin bli~u. 500 X.

106

1 0 7

( \'oiih gl:rnds, colored with Sudan black, showing tho extciit of the ncini aiicl the ducts. 100 x . The dis t r ihi i tm~i of s i i r~ i i i i~ l dc~hytlroge activity iir t l i r x k i t i froiii :in nrlnlt

i c activity is copious in the epiclornris, the outer qhcatlr of hair follielcs, iiiiij tlia suhnreous gldiid~. A xweat gla~ld ( anow) sliov~~s only modem tc activity. 75 X .

11

12 A1k:iliuc phohl~hAtasc a e t i v l t j 111 the Se~)aW(l lL~ gl:lntis of a youllg fenlalc. 500 x . Tween csterase activity ill the sebim ant? in tlic tlisintcgratlug belrrarcous cells. 150 X.

13

108

PLATE 4

109

1-1 Nowspecific esterase activity 111 the sebaceous glands of :I young female. The h a i r follivles have hern ru t p i : ~ t i ~ a l I y transversely iiiid tlir distribution of the sehceou.: glanils :u~oi incl it (>:IN Ijc we11 clcnriy in the gi oup jiic1ir:ited by the arrow. 35 X.

Intracellnlar d i s t r i h ~ i t i ~ n o f ppriotlic %liiff reactibe (tiot glycogen) graiiules in thr sweat glands of :t juvenile female. 500 X.

1.5

1 6 Deeply periodic acid f3sclliff 1 eactivt. (not glyoogc~i) retion pioduct a(>- cumulated in the luuieii of :L smeat ylirl~d of a n adult fenlxlc.

The distribution of allrdine phobphstase in the skin from R juvenile fcuinlc. The arrows indicate t h r paths of two strongly reactive slveat glandr. 35 X.

300 X.

1 7

11 0

PTAATE G

EXPLANATION O F I I G I J K X S

18 The distribution of non-sperific c s t c i i i ~ e s iri n sweat gland of an aclult feintdc. The surface cells arc rich i n en~yiiie act ivi ty hnt th r mywpitbrlial cells :ire niireactive. 500 x. The intact sn ra t c:ist within the pilary cn~iul, eolorc,cl dreply with thc, peiioilic ncid-Sclnif nrrthotl (not glycogeii). 125 x. ‘l’hc distribution of surviiiic dc~hydrogcwise activity in the skin of an :tdnlt fcmalc. Thc reaction is strong in tlic cpithrlial striwturc~s a r n l in tlir endo- thelial crlls of t l rc drrnial capillaiies and venules (a rmw). 200 X.

Schematic representatimi of a hair gronp shoming all of the elemcuts prcscnt. 1\11 of tlic hairs emerge 111 oiie pilary canal, into which a130 opcri tlir hingle, coiled sweat gland aid the scbaccouli glands.

19

20

2 1

PLATE 6