HISTOLOGY AND CYTOCHE,I\llSTRY O F HUMAN SKIN

VII. THE DISTRIBUTION O F SUCCINIC DEHYDROGENASE

-4CTIVITY

WILLIAM MOKTAGNA AND VICTOR FORMISANO Arnold Biological Laboratory, Brown Universi ty ,

Providence, Blbode Island

ELEVEN FIGURES

INTRODUCTION

The epidermis and its appendages contain relatively large amounts of demonstrable succinic dehydrogenase activity, although not all of the appendages have equal amounts of it. The distribution of succinic dehydrogenase in skin sug- gests that the enzyme is involved in both metabolic and proliferative functions. In the matrix of growing hair follicles and in the basal layer of the epidermis, the enzyme must be concerned mainly with proliferative activity, whereas that in the outer sheath of hair follicles and in the eccrine sweat glands, both of which are niitotically inert, must be related to metabolic functions.

This paper reports the localization of succinic dehydro- geiiase activity in fresh pieces of normal human skin. Pre- viously, only Rogers ('53) made some observations of the distribution of the enzgmc in human skin. Ellis et al. ('51), and Padykula ( '52) refer briefly to succinic dehydrogenase in the skin of laboratory animals, and Rogers ('53) and For- rnisano and JIonttigna ('2) made thorough studies of the en- zyme in the skin of the shclep and the guinea pig, respectively.

'This work was nipportcd in par t by a grant f rom the Vnited States Public Health Service, RG 2125 C i .

65

66 WILLIAM MONTAGNA A N D VICTOR FORMISANO

MATERIALS ,4KD RlETIiODS

Sixteen pieces of skin were used in this study. The specimens were all removed without anesthesia with a motor- driven rotary biopsy punch 5 mm (Urbacli and Shelley, '51 ). Five pieces of axilla, three pieces of scalp, three pieces of chest, 4 pieces of palm, and one of back were rcniorc3d from normal male and female subjects ranging from 24 to 34 pears of age. Each piece of skin was immediately pinned, epidermis down, on a paraffin l)lock, and as much fa t as possible way trimmed away from the hypodermis. The operation, which was never proloiigcd beyond two minutes, was performed under a dissecting microscope, and the tissue was kept bathed with warni physiological saline. The bulbs of hair follicles and the coils of sweat glands were partially freed from the surrounding connective tissue and fat (Formisaiio and Mon- tagna, '54). Each piece w a s cut into two, and both fragments were immersed in the incuhatiiig mixture.

The incubating mixture was prepared according to Pad).- kula's ('5%) inodificntion of the method of Seligman and Kutenburg ( '51), using neotetrazolium (NT) as the hydrogen acceptor. CYalciuni chloride and aluminum chloride mere acldcd to the mixture since the ions of these salts are said to activatc the succinic ciehydrogenase system (Padykula, ' 32 ) . 13cforc. the solution was uscd it w a s boiled, the flask \vas st opperecl and allowed to cool to 37°C. When the pieces of tissue were placed in the solution the flask n7as closed tightly with a rubher stop- per fitted with two glass tubes, one of which was attached to a nitrogen tank. The tissues ivere incubated for two hours a t 37°C. with nitrogen gas lmbhling slowly through tlic mix- ture.

After incubation the tissues were placed in 20% formalin and stored in the refrigerator for 24 hoiirs. The tissues were then cut in sections 15 p thick with the freezinq microtome; the sections were hardened in 10% formalin and then mounted in glycerin.

SUCCINIC DEHYDROGENASE I N HUMAN S K I N 67

OBSERVATIONS

The epidermis . I n the epidermis of the scalp, the axilla, the back and the chest, the strongest enzyme reaction, indica- ted by the presence of intracellular, purple diformazan crys- tals, is found in the basal cells. The lower two or three layers of the stratum spinosum contain variable amounts of reactive crystals but the reaction fades gradually upwards and disappears completely in the cells just below the stratum granulosum (fig. 1). The rest of the epidermis is unreactive. The cytoplasmic radicles on the lower surface of the basal cells arc strongly reactive. In the thick epidermis of the palms, tlie reaction is soniewhat stronger and more ex- tensive but it also disappears below the stratum granulosum. 14s in the other epidermis, tlie reaction is strongest in the basal layer. Since the reaction in the epidermis is much stronger in some preparations than in others, the penetration of the sub- strate and dye may have been uneven. Penetration seems to take place through the dermis, the stratum corneum serving as a barrier.

In the intraepidermal portion of tlie ducts of the eccrine sweat glands, only the lower segment is reactive. The peri- ductual and the basal cells of the duct show formazan granules, but the luniinal cells arc’ unreactive. The weak reaction in the “eccrine sweat duct unit” of Lobitz et al. (54) may be due to a lack of proper penetration by the substrate and dye through the epidermis.

The distribution of the for- mazan granules in the wall of the pilosebaceous canal is similar to but stronger than that in the epidermis

In growing hair follicles, the entire outer sheath is rich in enzyme activity (fig. 2). The reaction is particularly brilliant around the lower third of the follicle, the cells of which are riddled with vacuoles which contain glycogen and mucopoly- saccharide. The cells in the periphery of the outer sheath are always more strongly reactive than the others (fig. 3). I n the upper part of the hair bulb, the non-keratinized portion of the inner sheath and of the cortex show moderate enzymatic

The pilosebnccous S ! ~ S ~ C I W .

GS IVILLIAM MON TAGKA A N D VICTOR FORMISANO

reaction. Thc nial~- is , or blie lowei- bulb, which is easily re- cognized from the upper bulb by the absence of pigment, is intensely reactive. This is not shown well by figure 2. The fibroblasts ;md cndotlielial cells in the dcriiial papilla, and the fihroblasts in thc coiinectivc tissue sheath contain rcactive granules.

Thc follicles of club hairs contain much less succinic de- hydrogenase activity. The entire outer sheath and the epi- dermal sac arc only nioderatcly rcactive. The cord of cells hetween tlic epidermal sac and the hall-like dermal papilla, often referred to as the “hair germ,” has only some enzyme activity. The deimiil papilla also sliows reduced enzyme action.

In the sebaceous glmids, the cells of the duct system, and the indiff’erent sebaceous cells usually found a t the periphery of the glandular acini show strong reaction (fig. 4). The f ormazan crysials gradually diminish as the cells accuniulate lipid globules, and they disappear in niatuwe sehaceous cells. The s.ebum is co lo rd pink but this may he due to a diffusion of tlic formazan crystals which are solulnle in fats.

Siucnt gltrntls. The eccrine sweat glands and their ducts a r e tlic most reactive eleineiits in huniari skin. In tissues which liave heen teased rat her. than sectioned, entire sweat glands can he seen against the unstained background of the dermis (fig. 3 ) . These ~~repai .a t ioi is are useful f o r the demonstration of thcl gross niorpholog:.y of the glands, and the glands can he traced from the epidermis d o ~ w to the secretory knot. These preparations show that the eccririe sweat glands are considerably longer than is usually aclinowledged.

I n the straight clscretory duct, which extends from the epidermis to the secretory coil, the cytoplasm of the basal cells is laden with foriiiazan crystals. I n the luntinal cells, rcactive graiiules are gatlimed around the nuclcus and a few may he fourid in the cuticle, midway hctwecn the surface of the cell and the lateral surface of the nucleus. The ducts with a well-dcvclopcd cuticle havc fewer reactive granules in the luniinal cells.

ST'CCIKIC DEHYDROGENASE I N HVlRIhN SKIN 69

The coiled par t of tlie gland is coinposed of equal parts of duct and secretory tuhule. The coiled duct usually, but not always, has a poorly developed cuticle, and a segment of the duct seems to be the ti.ansition zone (,1Tontagna et al., '53). Both the hasal illid luniinnl cells of tlie coiled and transition part of tlic duct show very strong exizyrne activity, although the surface of the luniiiial cells is less reactive (figs. 6, 7 ) .

The secretory coil of eccrine glands is the most intensely reactive tissue in human skin. The reaction is occasionally uneven, some cells showing greater. activity thaii others, but as a rule, each cell is virtually 1;idcn with very dark purple forinazari granules. The luniiiial part of the cells has fewer granules than the hasal portion. The niyoepithelid cells seem to be unreactivc. I n the axilla a i d in the palm, the cccrine glands are always iiiuch more iiiteiisely co lo rd than those in the other skin studied. This seems to be a distinctive feature of the glands in the pal111 aiid axilla.

I n contrast with the cccrine glands, tlie axillai-y apoc~ine glands contain only small amouiits of succinic dehydrogeiiasc (fig. 9) . The excretory ducts have a similar but weaker rc- action than the eccrine tlucts. 1x1 the secretoi-y cells, formaxan granules are inversely proportional to the amount of secrc- tioxi material within the cells, regard1 of the size of thc cells (figs. 10, 11). \Then the cells are full of secretion gran- ules, formazaxi crystals are the least abundant and they ai*e concentrated iii a region above the nucleus (fig. 11 ). Formazan granules are usually clustered around the nucleus or above it. The basal part of the cells and the terminal cytoplasmic border are usually free of i*eactive granules. The myoepi- thelial cells are consistently unveactive.

IlIisceZZcizzeous. The arrectorcs pilorum muscles contain iiumerous reactive granules alligned orderly on the long axis of the muscle fibers. The endothelid cells in the dermis are always strongly reactive. The cytoplasni of fibroblasts has a sparse granulation. In the mast cells, some forxiiazan gran- ules may be formed between the mast granules, above the nucleus.

70 WILLIAM MONTAGKA AND VICTOR FORMISANO

DISCUSSION

This test cannot be performed adequately in fresh, frozen sectioiis of skin. Enzyme activity in sections is minimal. When wholc pieces of the saiiie skin are used, enzyme activity is al)undant, provided that the f a t is removed from its under- iide (cf. Formisaiio and Nontagiia, '54). Since it is not pos- sible to remove all of the fat speedily enough so that the remaining tissue is alive anti uiidaniaged when jt is placccl in the incubating mixture, tlie penetration of the suhstr*ate aiid dye through tlie tissue is not always optimal. Itefine- nicrits are needed in this method hefoi*e perfect result.: are o1)tained.

The abundaiit succinic dehydrogenase activity in the eccrine + w a t glands is in accord with the fact that succinic dehy- di-ogenase forms a link in the respiratory processes concerned ivith the oxidation of lipids, carbohydrates, arid proteins (Summer and Soinei.~, '47). The iich glycogen storage iii the secretory tubule of sweat glands (Montagna et al., '52) can bc exhausted temporarily hy prolonged stimulation (Shelley arid AIesco~i, '52), and it should be interesting to know if the distributioii of succiiiic dehydrogeiiase undergoes shifts corn- paruhle t o those of the glycogc~ri. Finding succinic deliydro- genasc activity in the ducts of sweat glands adds soine support to the already existing presumptive evidence that the eccrine sivpat ducts have a physiologic function (Montagna ct al., '3).

The secretory activity of cccrine glands is practically in- cshaustible, but that of the apocrine glands is of very short duration (Shelley, '51). One of the bases for thew differences might be the great disparity in succinic dehydrogenase ac- tivity between these t\vo glands. Different segments of the balm apocrine tubule oftcii show differences in the content of the enzyme, depending upon thc ainourit of secretion gran- ules within the cells. A similar situatioii exists in the sebace- ous glands where the indiffcrent cells 211-e rich in enzyme activity lmt the differentiated cc.11~ are not.

SUCCIPI’IC IIEHYDROGENASE IN H I J M A N SKIN 71

The possihle significaiice of succiiiic dehydrogenase activity in tissues of high mitotic activity has been discussed by B’ormisaiio and Rlontagiia (’51) and will not be repeated here.

The distribution of the forniazari crystals in tissues is simi- lar to that of the mitochondria. I n tlie eccrine sweat glands, however, which :we particularly rich i n enzyme activity, the number of mitochondria denionstruble with standard methods is much smaller than that of the formazan crystals. I n apo- crine cells, the correspondence is much closer. I n spite of this discrepancy, it seems certain that the mitochondria arc’ the reactive foci of the enzyme as stated before by other authors (Padyliula, ’52 ; Shelton aiid Schneider, ’52 ; lllalaty arid Bourne, ’53 ; Forrriisaiio arid Xontagna, ’54).

S U h f M h R V

1. Succinic dehytlrogenase activity is demonstrated more successfully in small, x7hole pieces of sliiii than in fresh frozen sections. In order that the substrate and dye penc- tratc readily, it is necessary to remove as much fat from the tcla subcutanea as possible.

2. Succinic dehydrogenase activity is present in the basal layer of the epidermis, in the outer sheath and bulb of hair follicles and in m e a t glands. The reaction is most intense in the eccrine sweat glands aiid iii their ducts. The apocriiie glands show only moderate activity.

3. The distribution of the enzyme in skin strongly suggests that the cnzyine is concerned with both metabolic and pro- liferative functions.

LITERATlTRE C I T E D

ELLIS, W. J., J. M. GILLESPIE , m n H. T d m r . E Y 1948 Biocheniical studics of the wool root. Nature , 165: 545-548.

FORXIS~NO, V., BNn W. MONT I O X L Succinic drliydrogenaxr activity in the skin of tlie guinea pig. Anat. Rec., 1W: 893-906.

LOBITZ, W. C., JR., J. B. HOLYOKE 4 ~ n TqT. >fONTAGN4 1951- “ T h e epidermal eccrine sweat duct unit.’ ’ A morpliologic and biologic entity. J. Invest. Drrmatol., 52: 157-158.

19.53 Historlicniistrv of succinic dehydro- genase. Nature , 77 I : 295-297.

1951

M4LATY, 1%. A,, ANI) G. 11. B O U R S E

72 W I L L I A M M O N T A G N A AND VICTOR P O R M I S A N O

MONTAGNA, W., H. B. CHASE BND TIT. C. LOBITZ, JR. 19.72 Histology a d eyto- clieinistry of human skin. 11. The distribution of glycogcn in the epidermis, hair follicles, sebaceous glands aiid eceriiie sweat glauds. Anat. Rec., 114: 231-248.

PADYKULA, H. A. 1952 The localization of succinic dehydrogennse in tissne sections of the ra t . Am. J. Anat., 9 1 : 107-146.

ROGERS, G. E. 1953 The localization of deliyclrogenase activity xird sulfhytlryl groups in wool arid hair follicles by the use of tetrazoliuni salts. Quart. J. Micro. Sci., 94: 253-268.

The histolocliemical dcnioiist r:i- tion of succinic dehydrogenase. Science, 115 : 31 7-320.

SELIGMAN, A. M., AND A. M. RUTENBURG

SHELLEY, W. B. 1951 Apocrine sweat. J. Invest. Dermatol., 1 7 : 255. SHELLEY, W. B., AND If. MESCON

1951

1952 Histochemical demonstration of secretory activity in human eccriiie sweat glancls. J. Invest. nermatol., IX: 289-301.

RHELTON, E., AND W. C. SCHNEIDER 1952 On the usefulness of tctrazoliuni salts as histochemical indicators of dehydrogeriase activity. Anat. Rec., 112: 61-82.

1947 Chemistry aiid Mcthods of Enzymes. 2nd ed. Academic Press, New York.

A rapid and simple method for obtaining punch biopsies without anesthesia. J. Invest. Dermntol., 27 : 131-1 34.

SUMNER, J. B., AND G. F. SOMERS

URBACH, F., AND W. B. SHELLEY 1951

PLATE 1

EXPLANATION O F FIGURES

1 Suecinic dehydrogenase activity in the epidermis of the scalp. Formazaii crystals concentrated in the basal layers of the stratum Malpighii. The stratum granulosum (arrow) and the stratum corneuni are unreactive. X 550.

Enzyme reaction in a hair follicle from the scalp. With the exception of the dark cone above the dermal papilla (P) which is pigment, all otlicr dark material represents enzyme activity. Poorly oriented f olliclc not slio\viiig an optimum amount of activity. X 250.

Formazan crystals i n the outer sheath of active hair follicle. Heaviest con- centration in the peripheral cells next t o the glassy membrane (arrow). X 500.

Distribution of the enzyme in a sebaceous gland from the back. The enzyme is concentrated at the periphery of the aciiius and in the duct. X 500.

2

3

4

SCCClNlC DEIIYDROUENABE I N HUMAN SKIN WIIILUY MONTABXA AND VICTOB FOBYISANO

73

PLATE 2

EXPLANATION OF FIGURES

5 Whole eccrine sweat gland from the axilla. X 60.

6 Eccrine sweat ducts from the palm. The heaviest concentration of the enzyme is in the basal cells; the cuticle is nearly free of it. X 500.

Transition portion of the eccrine sweat duct. Reactive granules are abundant up to the luminal border. From the palm. X 750.

Eccrine sweat gland; secretory tubule from the axilla. Extremely high eon- centration of reactive granules. X 750.

7

8

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SUCC.rN10 DEHYDROBENASE IN HUXAN BKIN V I l J ~ I A M MONTAOIA AND VIDTOK XYIRMISANO

PLATE 2

75

PLATE 3

EXPLANATION O F FIGUltES

9 Wliolc tubule of axillary apouine yl:md. Contrast with figure 5 . X GO.

10 Ahiiiitlant rrizyiiie activity iii npocriiie cells which arc nearly free of secre- tion grmules. x 750.

11 Moilcrate iiiiniher of graiiulcs roiiceiitr atecl above the nucleus of apocriiic cellz modrratrly laden with seeretioii grmiules. X 750.

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SUCCINIC DEHYDROBENASE IN HUJdAN SKIN W I l r b I A I XONTAUBA AKD VICTOR. FORMIMNO

PLATE 8

77