histochemical observations on the sebaceous glands of the rat

HISTOCHEMICAL OBSERVATIONS ON THE SEBACEOUS GLANDS O F THE RAT1

WILLIAM MONTAGNA AND CHARLES R. NOBACK Long Island College of Yedioine, Department of Anatomy, Brooklyn, iVew Pork

EIGHT FIGTJRES

Sebum is composed of lipid substances, cellular debris, and proteins. Hammersten (1889) believes that the lipids are composed essentially of cholesterol. Jausion, Calop, and Chalopin ('42) state that sebum, like sex hormones, is a sterol. Ameseder ('0'7) asserts that sebum contains only traces of cholesterol, but that it is composed largely of a saturated, monobasic, high-molecular alcohol, which he iden- tifies as eicosyl alcohol, bound in ester form to fatty acids of the secretion. Whatever the exact nature of the sebum may be, it contains several chemical substances. In the present investigation, we have studied the localization of lipids, enzymes, and proteins in the sebaceous glands of the rat. With the exception of the work of Melczer and Deme ('42) on human sebaceous glands, no systematic histochemical study of the sebaceous glands has been reported previously. There are species differences in the sebum of animals (Hammersten, 1889). The observations of this paper apply only to the sebaceous glands of the rat.

MATERIAL AND METHODS

This investigation is based principally on the sebaceous glands of the anus, these being chosen in preference to sebaceous glands of other regions because they are large and

'This work was partially supported by a grant from the Gans Fund, Bethany College, Bethany, West Virginia.

39

40 W. MONTAGNA A N D C. R. NOBACK

extremely active, However, they present no marked histochemical distinctions under comparison with sebaceous glands of the general skin. The rats used were normal, healthy adults of both sexes. There appear no significant differences between the glands of males and females.

For the detection of lipids, several techniques were em- ployed. F o r most of these methods, tissues were fixed in Raker’s ( ’44) f ormal-calcium-cadmium and embedded in gelatin. Other tissues were fixed in 10% neutral formalin. The following methods were applied to frozen sections 15 p in thickness: (1) for total fat content, Sudan IV and Sudan black B ; ( 2 ) for phospholipids, the Smith-Dietrich method (Baker, ’44) ; (3) for unsaturated glycerides, the Nile blue sulphate method of Lorrain Smith (Lison, ’36) ; (4) for birefringent and fluorescent lipids, examination of fresh and fixed sections under polarized and ultraviolet light; (5 ) for sharp differentiation of birefringent lipids, supravital staining with chrysoidin (Monn6, ’39) ; (6) for the 3-beta-hydroxy sterols, treatment of the tissues with digitonin (Bennett, ’40; Lison, ’36) ; (7) for unsaturated sterols, the Liebermann-Burchardt (Schultz) sulphuric acid test (Lison, ’36) ; (8) for the detection of the “plasmologens” of Feulgen and Voit ( ’24), the Schiff reagent and the 2,4-dinitrophenylhydrazine techinque (Albert and Leblond, ’46). Some tissues were fixed in Flem- ming’s fluid; frozen sections were further osniicated in 1% osmic acid in alcoholic solution, differentiated in 70% alcohol (Hoerr, ’36a), and stained with Sudan IV or with Nile blue sulphate.

Fo r the study of alkaline phosphatase, the technique of GOmori ( ’41a) was used; for acid phosphatase, Wolf, Kakat, and Newman’s ( ’43) modification of‘ Gomori’s method ( ’41b). Lipase was detected by Gomori’s technique ( ’45, ’46). For cytochrome oxidase, fresh and formalin-fixed tissues were treated with the M-nadi reagent (Lison, ’36).

The basophila was revealed in tissues fixed in Zenker-formol and stained with eosin-methylene blue. Sets of 2 consecutive sections were used for the determination of the effect of

HISTOCHEMISTRY OF SEBACEOUS GLANDS 41

ribonuclease upon the basophilia. The first of these sections was incubated for 3 hours at 60°C. in a solution of 0.1% ribonuclease buffered to pH 6.7. The second section was incubated for the same length of time in the buffered solution without the ribonuclease. The sections were stained and differentiated simultaneously.

Tlie authors wish to express to Miss June Galm their ap- preciation for her excellent technical help. They also wish to thank Miss Josephine Kristan for the preparation of the photomicrographs.

DESCRIPTION OF GLdNDS

Tlie sebaceous glands of the rat are small holocrine acinar glands of variable size which lie in the connective tissue of the dermis. The acini are composed of densely packed epithelial or epithelioid cells. Each acinus is surrounded by a complex network of arterial capillaries which adhere very closely to the basement membrane, but are never seen pene- trating the acinus. The vascular network was demonstrated by perfusing whole animals with a colored gelatin mixture. Surrounding the glands is a bed of areolar tissue rich in fibroblasts and mast cells.

Three gross morphological types of sebaceous glands are found in the rat. (1) The glands of the general body skin are small single acini which may either possess an attenuated duct or open directly into hair follicles. These glandular acini are composed of 20 or fewer cells as counted in serial sections. The center of the acinus usually contains a small amount of sebum. (2) The glands in the perineum are larger tha.n those of the general body skin. The acini are us’ually spherical, and may be simple or lobed. As a rule the acini contain appreciable amounts of sebum in the center. (3) Beneath the epithelium of the anal canal are many macro- scopic tubercles which, when pressed with the forceps, exude sebum. These tubercles represent aggregates of complex sebaceous glands, the largest except the preputial glands.

42 W. MOKTAGRA AND C. R. ROBACK

In spite of the general morphological differences of the glands in different parts of the body, they appear to be essentially similar histochemically, as will be discussed below. The observations in this paper apply to all 3 types of glands.

Each anal gland consists of 20 o r more elongated sebaceous acini which open into a central duct. The acini are embedded in the submucosa. I n each glandular cluster the acini vary in length and in activity. Some of them are elongated to such an extent that they penetrate and displace the sphincter ani muscle, while others are small and rounded. The long acini often contain sebum, but the small rounded ones do not. The glands near the orifice of the anus are associated with tufts of fine hairs; those in the inner part appear “free” of hairs. Vestigial hair follicles which do not penetrate the surface of the anal canal are usually found near the “free” sebaceous glands.

The small rounded acini represent the immature stage, and the large secular ones the definitive, actively secretory stage. In the immature acini, only a few central cells are enlarged and contain lipid droplets. I n the mature acini, practically all the cells are enlarged and contain many lipid droplets; the cytoplasm of these cells is reduced to delicate filaments between the lipid globules. .The central part usually contains sebum. In some acini tlie peripheral cells which rest upon the basement membrane are flattened and contain no demonstrable lipid droplets. In others even the peripheral cells are enlarged and engorged with lipids. These fully mature glandular acini contain much sebum. There are many intermediate types of acini in every gland (fig. 1).

Much controversy exists concerning the mode of replace- ment of sebaceous cells lost in acini in sebum formation. Most histolo,T textbooks present the view that new cells proliferate from the basal cells, and shift centrally to take the place of those lost in secretion. Mitotic figures in the basal acinar cells are reported (Bizzozzero and Vassale, 1887 ; Shaffer, ’26, and others) but are denied by some authors (Bab, ’04; Clara, ’29, and others). We have rarely found

HISTOCHEMISTRY O F SEBACEOUS GLANDS 43

mitotic figures in the basal cells, but they are more common in the deeper layers of the epithelium of the ducts where they a re joined by the acini. Clara ('29) also has made this observation, stating that new cells shift from the duct into the acini to replace cells degenerated in sebum formation.

D Fig. 1 Portion of anal sebaceous gland showing 4 acini in different states of

maturity. A. Young acinus developing from the epithelium of the central duct. The central cells only are enlarged. B. Young mature acinus. Sebaceous degeneration is occurring in the central and proximal portion of the acinus. The peripheral acinar cells are all small and do not contain discernible lipid droplets. C. Mature acinus. The cells in the proxinial half of the acinus are undergoing sebaceous degeneration. The peripheral cells are large and lipid-laden. D. Fully mature acinus a t peak of sebum formation. Only the distal par t of the acinus contains a f e v intact cells; these are about to break down.

44 W. MONTAGNA A N D C. R. NOBACK

Although this concept is reasonable, we find that exhausted acini in the rat degenerate. This is accompanied by a frag- mentation which is accomplished through invasion of the old acini by the surrounding connective tissue. The immature acini, which expand and replace the degenerating acini, are always found near the duct (fig. 1). Since mitotic figures are often found in this region, it appears that this map be the site of formation of new acini.

THE LIPIDS

We follow Lison's ('36 )nomenclature for the lipid substances.

Lipids (fatty substances)

I. Glycerides (neutral fats) : glycerol and fatty acids. 11. Cholesterides (esters of cholesterol) : cholesterol and

Lipines (lipoids) : phospholipins (phosphatides), galac- fatty acids.

tolipins (cerebrosides) . 111.

1. Sudnnophilic substances

Sebaceous glands, when stained with Sudan IV, show a rich content of red to orange lipid droplets (fig. 3 ) .

In the immature acini, sudanophilic droplets are abundant only in the enlarged central cells. They diminish peripherally and are absent in the small outer cells. In some of the large mature acini, all of the sebaceous cells are laden with sudanophilic droplets; in others, the peripheral cells are small and do not contain stainable lipids. The sebum in the center of the mature acini is highly sudanophilic. The distribution of the sudanophilic substances in the sebaceous glands suggests that the lipids are stored first in the central cells; the accumulation progresses toward the periphery until all of the cells are fully engorged.

Sudan black reveals essentially the same distribution of lipids as Sudan IV (fig. 4). However, the staining is more intense, and all of the cells, including the peripheral ones

HISTOCHEMISTFtY O F SEBACEOUS GLANDS 45

which do not contain droplets are stained with this dye, regardless of the maturity of the acini.

The lipid substance contained in the peripheral cells of the sebaceous acini is not dissolved by lipid solvents. When sections are immersed 48 hours or longer in acetone, alcohol, ether, chloroform, xylol, and other solvents at 60°C., and then stained with Sudan black, these cells stain blue-black with undiminshed intensity. The lipid droplets in the sebum and in the large sebaceous cells are dissolved, leaving a quan- tity of homogeneous sudanophilic substance less abundant than that in the peripheral cells.

Some stromal elements surrounding the sebaceous glands are sudanophilic. The basement membrane stains only with Sudan black, as do the granules of the mast cells. Lipid droplets in the interstices of the connective tissue stain with all Sudan dyes.

2. Phospholipids

The peripheral acinar cells contain a lipid substance which is stained with Sudan black but not with Sudan IV. This substance is not destroyed by immersion in lipid solvents. These facts suggest that this lipid may be a phospholipid. Accord- ing to Lison ( '36) and Baker ( '44), the Smith-Dietrich method when positive (black) appears to indicate the presence of phospholipids. The method consists of chromating frozen sections, staining them with Kultschitzky 's hematoxylin, and differentiating in Weigert 's borax potassium ferricyanide solution. Lipins resist differentiation in Weigert 's solution more than the other lipids do.

Sections stained by the Smith-Dietrich technique reveal a blackening of those peripheral acinar cells which contain the solvent-resistant lipids (stained previously only with Sudan black) (fig. 5). It has been mentioned above that not all of the acini are in the same state of maturation. The younger acini possess proportionately a greater number of flattened peripheral acinar cells whose cytoplasm is not loaded with lipid droplets. Concomitantly, in the immature acini a greater

46 W. MONTAGNA AND C. R. NOBACK

area of the periphery blackens. In those acini which are at the peak of sebum formation, and whose cells are all engorged with lipid droplets, there is a minimal blackening of the outer border of the peripheral cells. Thus, this reaction is obtained primarily in those acini which are storing lipid substances within their cells. The basement membranes of all the acini stain black. I n the smaller acini, some of the connective tissue adjacent to the basement membrane also stains black. I n the surrounding connective tissue, the capillary endothelium and red blood corpuscles, and the myelinated nerve fibers stain black to blue. In the hair follicles, the outer root- sheath blackens. I n the surface epithelium of the anal canal and skin, the stratum corneum is always black, and the basal cells bluish to black.

3. Unsaturnted glycerides When sections of sebaceous glands are stained with the

Lorrain Smith Nile blue sulphate technique, 2 sharply different color reactions are obtained: blue and pink. Lison (’36), Baker (’44), and others have pointed out that the “rose” color forms in sites of unsaturated glycerides, while the blue color has no histocl~emical significance.

I n the sebaceous acini, the sebum and those cells which contain visible lipid globules stain pink, while the flattened peripheral cells stain blue. On this basis we conclude that the lipid droplets in the sebaceods cells consist primarily of unsaturated glycerides. The progression of lipid accumulation in the acini in different states of maturation is thus easily traced. Small immature acini contain rose-colored droplets only in the central cells, and the rest of the cells are blue; all the cells of fully mature acini, even those along the periphery, contain rose droplets.

The basement membrane of the acini is blue. The surrounding connective tissue is usually colorless, but the interstices contain pink streaks and granules. The granules of the tissue mast cells stain brilliant blue. No other connective tissue element stains with such intensity.

HISTOCHEMISTRY OF SEBACEOUS QL-4XDS 47

The stratum corneum of the surface epithelium of the anal canal and skin stains pink, while the basal layers stain blue.

4 . Biref ringent lipids

Abundant anisotropic globules, showing the Maltese cross, are revealed principally in the sebum and the cells undergoing sebaceous degeneration (figs. 7 and 8). The larger mature acini contain more birefringent elements than the smaller, less mature ones d o ; some small acini contain none. The anisotropicity can be enhanced by staining the glands supravitally with chrysoidin, according to the method of Monn4 ( '39). This stain sharpens the resolution of the birefringent lipids, which appear as bright yellow spherules under crossed Nicols.

Acetone, chloroform, ether, alcohol, and other lipid solvents abolish the anisotropicity.

The anisotropic globules in the sebaceous glands appear to be true spherocrystals. This is demonstrated by the fact that they show the Maltese cross, and that they are not destroyed by alternate warming and cooling. When sections are warmed to 80"C., the spherocrystals are reduced to liquid droplets and are no longer birefringent (cf. Weltniann, '13, for the suprarenal gland). If the sections are then cooled quickly to approximately joC,, the anisotropicity reappears virtually unchanged. This process my be repeated many times without appreciable alteration. If, however, the sections are warmed and allowed to cool slowly at room temper- ature, the physical structure of some of the crystals seems to change. There are fewer sphcrocrystals, and in their place acicular crystals are formed. This physical behavior of the birefringent spherules, although strongly suggestive of cholesterides (Lison, '36), does not exclude entirely the presence of glycerides which may also show such polymor- phism (Ferguson and Lutton, '41).

Sections were treated with digitonin (the aqueous digitonin method of Bennett, '40, and alcoholic digitonin suggested by

48 W. MONTAGNA AND C. R. NOBACK

Lison, ’36, and Baker, ’44), immersed for a few minutes in acetone or chloroform, mounted in glycerine, and studied under polarized light. Digitonin precipitates with the 3-beta- hydroxy sterols to form anisotropic crystals which are insoluble in acetone and chloroform (Fieser, ’37 ; Sabotka, ’38). In the sebaceous glands no insoluble digitonides were formed.

5. Schultx reaction. The Schultz test for unsaturated sterols, when applied to

sebaceous glands, produces a positive but rather weak reaction. It is believed that the specific color reaction evoked by this test (red, or blue, or purple subsequently turning to green) indicates the presence of steroids which possess some degree of unsaturation. A positive reaction was observed primarily in the sebum and in the degenerating sebaceous cells. The rest of the cells were always negative, exhibiting a yellowish brown color. The small immature acini which contain little or no sebum were consistently negative. It appears, then, that the reaction of this test is positive only in those sites where the anisotropic spherules are most abundant.

6. “Plnsmaloge%s ” The recent work of Albert and Leblond (’46) indicates that

the “plasmalogens, demonstrated by Feulgen’s leucofuchsin reagent, may be “loosely bound aldehydes of fatty acids.” I n addition these authors correlate with “plasmalogens ” the color reaction obtained by the use of 2,4-dinitrophenylhydrazine (for technique see Albert and Leblond, ’46). Gomori (’42) and others are also of the opinion that both reactions indicate the same substances. For this reason we shall con- sider as one the results from these two reactions. When either Feulgen’s reagent o r 2,4-dinitropheriylyhydrazine is used, a weak reaction is obtained in the sebaceous glands. The sebum and the degenerating sebaceous cells show mild color reactions, but the rest of the glandular elements contain only traces of the “plasmalogens. ” This is in sharp coritrast


with the preputial glands of the rat in which the sebum has a very strong reaction (Montagna and Noback, '46b; Albert and Leblond, '46).

7. Osmiophilic lipids Although it is not advisable to use osmic acid as a histo-

chemical reagent because of its lack of specificity (Lison, '36 ; Hoerr, '36a, '36b; Bensley and Bensley, '38), some authors have attached considerable importance to this reagent (Ameseder, '07 ; Stern, '05 ; Melcser and Deme, '42, and others). When sebaceous glands are treated with osmic acid, the results are so erratic that it is impossible to evaluate their significance. The sebum and the lipid globules in the degenerating cells usually blacken, but the rest of the glandular cells, regardless of the amount of lipids they contain, may or may not show osmication in varying degrees. The reagent appears to be so capricious that the results do not warrant serious consideration.

8. Autofluotrescence The sebaceous glands, when studied under ultraviolet light,

display little autofluorescence. The sebum and those cells richest in birefringent lipids emit a greenish-yellow light of medium intensity, whereas the rest of the glandular sub- staiices give a pale grayish-yellow light of very low intensity. The stromal elements show a weak lead-gray color, and the stratum germinativum of the epithelium of the skin emits a dull red fluorescence (cf. lllontagna and Noback, '46, for the fluorescence of the preputial gland).

THE ENZYMES

1. Alkal ine pkosphatase Tlie sebaceous glands of the anal canal are foci of strong

phosphatase activity (fig. 6). The enzyme is not as abundant in the glands of the body skin. Alkaline phosphatase is par- ticularly abundant in the periphery of the acini, diminishing

50 W. MONTAaNA AND C . R. NOBACK

slightly toward the center, where the cells are large and lipid-laden. No glandular elements lack this enzyme ; even the sebum shows a considerable amount of the active phosphatase.

The epithelium of the sebaceous ducts lacks the enzyme, as does the epithelium of the surface of the anus and of the skin. The stratum corneam of the epithelium of the anal canal, however, usually reveals strong enzyme activity.

The connective tissue stroma usually lacks phosphatase activity. In the following structures, however, enzyme activity is noted. The basement membrane blackens readily. Tufts of connective tissue underneath the surface epithelium on occasion show intense staining. The endothelium of the capillaries blackens, as do the granules of the mast cells (cf. Koback and Riontagna, '46 ; Wislocki and Dempsey, '46).

For the study of the distribution of this enzyme we em- ployed principally Gomori's technique ( '41a). Following the suggestions of Dempsey and Deane ('46) and Deane and Dempsey ('45) we studied the effect of other substrates (only adenylic acid, nucleic acid, and lecithin were available in our laboratory), and the pH a t which optimal phosphatase activity is revealed. For the sebaceous gland the standard substrate, sodium glycerophosphate, buffered to pH 8.5 to 9.5, gave uniformly good results. Mediocre staining was obtained with adenylic acid and nucleic acid. Lecithin as a substrate was completely unsatisfactory.

8. Acid phosphatase nnd lipase Repeated tests for these 2 enzymes in the sebaceous gland

of the rat have failed to show their presence in appreciable amounts. Traces are occasionally observed in the sebum. The glandular substance is always completely negative.

The granules of stromal mast cells become brown or black,

3 . Tirdophe~aol ozidase This enzyme system is found diffusely in the sebaceous

glands. The reaction is usually so weak that it cannot be

HISTOCHEMISTRY OF SEBACEOUS GLAFDS 51

observed critically. This is in contrast with the preputial gland (Montagma and Noback, ’46b), where indophenol blue granules are found in large numbers.

The stromal elements, with the exception of the mast granules, lack the “stabile” cytochrome osidase.

THE BASOPHILIC ELEMENTS

It has been demonstrated recently that the presence of basophilia in the cells of some organs appears to be associated with the presence of ribonucleic acid (in the pancreas, Brachet, ’40; in motoneurons, Gersh and Bodian, ’43; in the placenta and pituitary, Dempsey and Wislocki, ’45 ; in the liver, Deane, ’46).

Sections of anal canal and skin fixed in Zenker-formol were incubated at 60°C. for 3 hours in a 0.1% solution of ribonuclease buffered (veronal acetate) to pH 6.i, then stained with eosin-methylene blue. Control sections were incubated in the buffer solution without the enzyme. The digested and control sections were stained and differentiated simultaneously in a solution of alcoholic colophons-.

In control sections the peripheral cells of immature glandular acini show a pronounced basopliilia in the form of blue granules. I n mature acini whose peripheral cells, like the central ones, are engorged with lipid globules, there is little basophilia. Those cells whose cytoplasm contains large numbers of lipid granules show only traces of basophilia. The sebum and the degenerating sebaceous cells hare none. I n sections incubated in the enzyme, these basophilic elements are completely lost. For this reason the basophilia is inter- preted as ribonucleic acid.

The outer root-sheaths of bair follicles, the epithelium of the sebaceous ducts, and the Malpighian layer of the surface epithelium of the anal canal and skin also display basophilia which is destroyed by ribonuclease. I n the connective tissue stroma, the many mast cells have conspicuous basophilic granules which are not digested by ribonuclease (cf. Dempsey, Wislocki and Singer, ’46 ; Wislocki and Dempsey, ’46).

52 W. MONTAQNA AND C. R. NOBACK

Xanthoproteic test

Frozen sections were mounted on slides, blotted dry, and covered with a few drops of concentrated nitric acid. After a few minutes the acid was rinsed off, and the tissue blotted and covered with concentrated ammonia. A bright yellow color developed along the periphery of the immature acini. The color reaction was present in inverse ratio to the number of lipid globules in the sebaceous cells. The lipid-laden cells showed very little of it, and the sebum remained colorless. The coloration evoked by this test is supposed to indicate the presence of tyrosine, tryptophane, and phenylalanine (Baker, '44 ; Lison, '36).

DISCUSSION

The l ipids . The available histochemical techniques for the lipids have been reviewed critically by Lison ( '36) and Baker ( '44). It is the general belief of. these authors and others that few of the existing tests for lipids are specific. However, when several tests of uncertain specificity are used in combination with one another, it is possible to obtain some degree of assurance if the tests used point toward the same general conclusion.

Osmic acid as a histochemical reagent has been discredited, because of its total lack of specificity, by Lison ('36), Baker ( '44) and others. We have used this reagent repeatedly with the sebaceous glands. The erratic results which we have obtained have made it impossible to interpret the significance of the osmiophilic substances. Melczer arid Deme ( '42) have studied human sebaceous glands by fixing tissues first in for- maline and then in Flemming's fluid. Frozen sections of these tissues were further osmicated, or stained with Nile blue sulphate, or stained with Stern's reagent ('05), which is a combination of osmic acid and scarlet red. These authors have obtained interesting results with these procedures, but their histochemical interpretations should be accepted with caution. If osmic acid must be used as a cytochemical reagent,


formaldehyde should be avoided because it forms additive compounds which also reduce osmic acid (Bensley and Ben- sley, ’38).

The following facts suggest that the peripheral acinar cells of the sebaceous glands of the rat contain phospholipids. Sudan IV does not stain these cells, whereas Sudan black does. The lipid substance in the cells is not dissolved by lipid solvents, and can be stained with Sudan black even after prolonged immersion in such solvents. Baker ( ’44) has demonstrated that, of the 2 dyes mentioned above, only Sudan black stains phospholipids clearly. He has further demonstrated that phospholipids in tissues fixed in formal-calcium- cadmium are not destroyed by lipid solvents. The application of the technique of Smith-Dietrich, demonstrated by Baker (’44) to be specific for sphingomyelins, cephalins, and leei- thins, stains the peripheral acinar cells black.

The peripheral phospholipids diminish in abundance with the maturation of the acini. Sebaceous glands, when stained with Sudan IV and Nile blue, show that lipid substances are deposited first in the central core of cells, and then progress toward the periphery. Concomitant with this accumulation of lipid substances toward the periphery of the glands, there is a diminution of phospholipids, until in fully mature acini practically none is seen. The phospholipids, located as they are along the periphery of the glands, and absent in the mature acini, appear to be lipids of passage in the sebaceous glands. They may be the medium of transport of fatty acids in the deposition of neutral fats (Sabotka, ’38) in the sebaceous cells.

Nile blue sulphate reveals that the mass of lipid substance in the sebaceous glands and sebum consists primarily of unsaturated glycerides. With this technique the unsaturated glycerides stain pink, whereas other Substances, not necessarily of a lipid nature, stain blue. Kaufmann and Lehmann ( ’26) question the specificity of this technique, but Lison ( ’36), after critical consideration of these authors’ protocols, concludes that “une coloration rose . . . signifie prksence d’un

54 W. MONTAGNA AND C. R. ROBACK

glyckride non satur6 (triolbin) ; une coloration bleue n’a aucun signification ; un r4sultat n4gatif n’a aucun signification.”

In addition to unsaturated glycerides, the sebum and the large sebaceous cells contain large quantities of antisotropic lipids. These lipids are more abundant in mature acini (es- pecially in the sebum).

Monn6 ( ’39) has demonstrated that chrysoidin applied supravitally to tissues intensifies the birefringence of the Golgi elements. When sebaceous glands are treated supravitally with chrysoidin there is a remarkable increase in amount and in resolution of the birefringent substances. The significance of this reaction is not understood.

When sections of sebaceous glands are heated to BO’C., the birefringent globules are no longer visible under polarized light. They reappear brilliantly when the sections are cooled quickly to approximately 5” C. This experiment gives strong indication that these are real spherocrystals, for they reform even when the experiment is repeated several times.

According to Lison (’36), lipids which exist in the form of spherocrystals (showing the Maltese cross) are either cholesterides or lipins. Glycerides and fatty acids are never found in this form. Ferguson and Luttori ( ’41), however, point out that glycerides may also be in the form of birefringent spherocrystals. This characterization, then, has little value in itself and may be indicative of any and all of the lipid substances.

The birefringent lipids of the sebaceous glands are not phopholipids, but appear to be cholesteryl esters or a mixture of cholesteryl esters and glycerides (previously demonstrated by Nile blue sulphate). When phospholipids are fixed in formaldehyde, they resist the ordinary fat solvents. Baker ( ’44) demonstrated that lipins in tissues fixed in his “formal-calcium-cadmium” are virtually insoluble. The spherocrystals of the sebaceous glands, in fresh tissues or tissues fixed in f ormalin or in f ormal-calcium-cadmium, are easily dissolved by acetone, alcohol, ether, and other solvents. When the Smith- Dietrich test fo r phospholipids is applied to these tissues,

HISTOCHEMISTRY OF SEBACEOUS GLANDS 55

there is no positive reaction with the anisotropic material. These facts seem to justify the consideration that the bire- fririgent lipids in the sebaceous glands are not lipins but per- haps a mixture of cholesterides arid glycerides.

Cholesterol occurs in tissues in free form o r in the form of cholesteryl esters of palmitic, stearic, and other higher fatty acids. These 2 groups can be separated chemically by the use of digitonin, which precipitates with the 3-beta- liydroxy sterols, as anisotropic crystals which are insoluble in chloroform and acetone (Fieser and Fieser, '44). Digitonin does not precipitate with cliolesteryl esters because the presence of the free hydroxyl group is necessary for the reaction (Fieser, '37, and Sabotka, '38). Since the birefringent lipids in the sebaceous glands do not yield insoluble digitonides, they probably represent cholesteryl esters (cf. Everett, '45, on the birefringent elements of the corpus luteum). This conclusion is in agreement with the fact that cholesterol, partic- ularly when combined with fatty acids, is nsually highly birefringent (Hoerr, '36a). It is an interesting fact (and this may give added weight to the supposition that the birefringent elements are cholesteryl esters) that when the Schulz re- agents are applied to tissues, a greenish positive reaction is evoked in the general region of the anisotropic lipids. It- must be pointed out, however, that Schultz-positive lipids are not necessarily birefringent (cf. Knouff, Brown, and Schnei- der, '41). The Schultz reaction is obtained not only with cholesterol, but also with cholesteryl esters. Although this test may be negative when these substances are present in small quantities, it is never positive when they are absent.

We have mentioned earlier that when the sebaceous glands are immersed for long periods of time in solvents and then stained with Sudan black, the whole gland, including the sebum, retains some detectable sudanophilia. We have, in addition, shown that only the peripheral cells of the glands appear to contain large amounts of phospholipids. The solvent-resistant sudanophilia in other parts of the glands, including the sebum, must, then, be some other substance.

56 W. MOSTAGNA AND C. I;. XOBACIE

?lTe liave not established the identity of this substance. Per- haps it represents eicosyl alcohol (reported in abundance in sebaceous glands by Ameseder, '07; Melczcr and Deme, '42) or some alllied substaiice.

Elzxynzes. There is abundant alkaline phospliatase activity in the sebaceous g1a:ids. ?Vliatever the specific role of this enzyme may be in tlie deposition of lipids in the sebaceous cells, its presence seems correlated with the activity of the glands. It has been stated above that the glands of the skin are less active than those of the anal region, judging by tlie amount of sebum within them and the nuniber of cells wliich are undergoing sebaceous degeneration. Comparably, the glands of the anal canal always contain more demonstrable phospliatase than the glands of the skin. The known functions of the phosphatases liave been discussed extensively by other workers, so they will not be repeated here.

The other enzynies studied, acid phosphatase, lipase, and cytochrome oxidase, were either absent or found in minute amounts.

SUMM,4RY

1.

2.

The pattern of distribution of the substances studied in the sebaceous gland lias been plotted (fig. 2) .

Sudan I V and Sudan black dernoiistrate abundant lipid substances in the sebum and aciiiar cells. The peripheral acinar cells stain only with Sudan black and, from other evi- dence, appear to contaiii phospholipids.

The large, more centrally located acinar cells, which possess abundant lipid droplets in their cytoplasm, contain large quantities of unsaturated glycerides.

The mature, clying acinar cells which are about to un- dergo, or are undergoing sebaceous degeneration, contain large quantities of unsaturated glycerides and cholesteryl esters.

5. The sebum is coniposed primarily of unsaturated glycerides and cholesteryl esters.

3.

4.


6.

7.

‘Plasmologeiis” were found in limited amounts, primarily in the sebum.

The sebum and the large acinar cells contain, in addition, traces of a lipid substance which resists lipid solvents. The identity of this substance has not been established.

holesf eryl es f er: hnkJ and unsof. g/yc erides

unsa f. g/yc erides

-

VQXC 1 lka /in e Ihosphafase

hosphollbids ana %on uc /ek acid

Fig. 2 Schematic representation of the distribution of chemical substancco in n sebaceous acinus.

8. Alkaline phosphatase activity is abundant in the sebaceous acini. The peripheral acinar cells contain a greater concentration of the enzyme than the central cells.

R,ibonucleic acid is present in those peripheral acinar cells which possess few or no lipid droplets. It is not revealed in the mature acinar cells and sebum.

9.

58 W. MONTAGNA AKD C. R. XOBACK

LITERATURE CITED

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1946

~VOLF, A., E. 8. KasaT AND W. NEWMAN

PLATE 1

EXPLANATION OF FIGURES

3 Nest of sebaceous aciiii of the anus, stained v i th Sudan IT. The peri-

4 Anal sebaceous glaiids stained with Sudan black. Note that the peripheries

5 Anal sebaceous glaiids stained with the Sniitli-Dietricli method for phos-

6 Anal sebaceous glaiids revealing intense alkaline phosphatase activity. 7 and 8

pheries of the aciiii do iiot stain.

of the aciiii stain iiiteiisely like the centers.

pholipids. Only tlic peripheral cells stain black.

Frozen section of aiial sebaceous glands seen under ordinary light (fig. 7) and under polarized light (fig. 8 ) . The anisotropieity is particularl? abundant in the centers of the elongated acini. Note that all of the birefringeiit particles show a Maltese cross.

IIISTOCHEMISTRY OF SEBICEOrS GLANDS \YII.LIA31 M O N T A G N A AND CHAIILES R. NOBAPK

PLATE 1

61

histochemical observations on the sebaceous glands of the rat

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