73

The demonstration of lysosomes in mouse skin

By J. V. DIENGDOH(From the Department of Pathology, Royal College of Surgeons of

England, Lincoln's Inn Fields, London, W.C. 2)

With 2 plates (figs. 1 and 2)

SummaryLysosomes, as demonstrated biochemically in the liver, are subcellular particles con-taining a group of hydrolytic enzymes enclosed by a membrane-like barrier. Theyare apparently inactive in the normal state, but when subjected to various formsof injurious treatments the enzymes associated with them are released. The existenceof lysosomes in skin is demonstrated in the present communication. The resistance ofthis tissue to homogenization makes the biochemical study of lysosomes very difficult.Yet, by the application of histochemical methods to sections of skin, it has beenpossible to use the same criteria as would be employed in the biochemical character-ization of these organelles. By using the controlled-temperature freezing-sectioningmethod it has been possible to obtain frozen sections in which cytoplasmic particlescould be demonstrated which were enzymically inactive for acid phosphatase untilthe sections were subjected to such injurious treatments as heat, repeated freezingand thawing, hypotonic solutions, distilled water, and 'triton-X-100'. Since the sub-cellular particles demonstrated behaved in the same manner as lysosomes preparedbiochemically from liver, it is concluded that the cytoplasmic organelles staining foracid phosphatase in mouse skin are lysosomes as biochemically defined.

IntroductionI T has been demonstrated by de Duve and his associates (Appelmans andde Duve, 1955; Gianetto and de Duve, 1955) that acid phosphatase in ratliver is associated with a special type of subcellular particle with sedimentationproperties intermediate between those of mitochondria and of microsomes.By differential fractionation procedures they were able to separate thesegranules from the true mitochondria, which were defined by their cytochromeoxidase activity, and from the microsomes, which possessed all the glucose-6-phosphatase of the cells. In contrast, these organelles could be characterizedby their content of hydrolytic enzymes and hence were named lysosomes(de Duve, 1959).

Fresh preparations of these particles suspended in 0-25 M sucrose showedlittle enzymic activity when tested with /J-glycerophosphate as substrate.However, osmolysis, heat, repeated freezing and thawing, or treatment withthe surface-active agent, 'triton-X-100', all released acid phosphatase fromthese organelles. De Duve, therefore, postulated that these particles werecytoplasmic sacs or capsules in which the enzyme was contained by a mem-brane which was impermeable to /?-glycerophosphate. The granules appearedto behave as osmotic systems.

[Quart. J. micr. Sci., Vol. 105, pt. 1, pp. 73-78, 1964.]

74 Diengdoh—Lysosomes in mouse skin

Cytoplasmic particles which resemble lysosomes in size and shape andin that they alone stain with the reaction for acid phosphatase, have beendemonstrated in rat liver and kidney (Holt, 1959). The method used, however,had so affected their membranes that they were immediately permeable to/?-glycerophosphate. The use of the controlled-temperature freezing-section-ing technique avoids this damage (Cunningham and others, 1962). ThusBitensky (1962) was able to show in such sections of rat liver that acid phos-phatase activity could be demonstrated only after prior alteration of themembranes of such particles. In consequence she was able to use criteriasimilar to those used by biochemists to define as lysosomes the cytoplasmicorganelles made visible in her sections.

During the course of a study of the histochemistry of mouse skin theGomori method for acid phosphatase revealed the enzyme as occurring onparticles in the cytoplasm of the cells of the epithelium and of the hair follicles.It was therefore considered advisable to test whether this localization indicatedthe presence of lysosomes in skin. Since it is notoriously difficult to homo-genize this tissue, the identification of these organelles would have to dependon the application of histochemical procedures alone, based on the biochemicalcriteria used on the liver (Appelmans and de Duve, 1955; Appelmans andothers, 1955; Gianetto and de Duve, 1955).

Materials and methodsMice of the A2G strain were used. They were allowed unlimited quantities

of MRC diet 41 and water. Frozen sections of mouse skin were cut at 8 fx,on a freezing cryostat microtome by the controlled-temperature freezing-sectioning method of Cunningham and others (1962). Acid phosphatase wasdemonstrated by Holt's modification of the Gomori method (Holt, 1959;Bitensky, 1963). The incubation medium consisted of 0-05 M acetate bufferat pH 5, to which o-i M sodium-/3-glycerophosphate (BDH) and 0-003 Mlead nitrate were added. The mixture was kept in the incubator at 370 C for18 h, after which the precipitate was filtered. Sections were incubated in themedium at 370 C for the required time, washed in water, and transferred to asolution of hydrogen sulphide. The sections were then rinsed in water andmounted in Farrants's medium (Gurr). A parallel control was also run, butwith the addition of o-oi M sodium fluoride as an inhibitor for the enzyme.

Fresh frozen sections were incubated for various times in the Gomorimedium to determine the minimum time required for a perceptible reactionto be obtained. It was found that at 10 min a reaction just began to appear inthe form of a few separate granules in the cytoplasm of the cells of the hairfollicles. Frozen sections were then subjected to various forms of pretreat-ments as given in table 1, and the sections so treated were then incubated for10 min, the minimum time for a visible reaction in untreated sections.

The reagents for pretreatments were prepared in Coplin jars and the sec-tions were kept in the solutions for the given time; excess of solution wasblotted off from each slide and the section transferred directly to the incubating

Diengdoh—Lysosomes in mouse skin 75

medium. Freezing and thawing were effected by placing the section, whichwas not allowed to dry, on a block of solid carbon dioxide, and packing piecesof this ice on the glass slide; the section was left for about 5 min beforebeing withdrawn and left to warm to room temperature. This procedure hadto be repeated two or three times to produce any appreciable change instaining response.

TABLE I

Effects of various treatments on acid phosphatase staining and localization inhair follicles*

Agents employed forpretreatments

none (i.e. untreated) .

acetate buffer pH 50-25 M sucrose0-125 M sucrosedistilled water .0-25% triton-X-100 .

heat (580 C)freezing and thawing

Time ofpretreatment

(min)0

1 0

1 0

1 0

1 0

52 0

2 0

I O

repeated(3 or 4 times)

Time ofincubation

(min)35

1 0

2 0

3°60

50

0

0

0

0

>o0

0

Particulatestaining

0

0

++ ++ +

?++

+ ++ ++ +

0

0

+ ++ +

Cyto-plasmicstaining

0

0

0

++ +

+ + ++0

+++0

0

+ ++

Nuclearstaining

0

0

0

0

++ + +

0

0

+++0

0

+ ++

* Similar results were obtained in the epithelium, except that the minimal incubation timewas 20 min.

Key. + + + = very strong reaction; + + = strong reaction; + = moderate reaction;? = indeterminate; o = no reaction.

ResultsThe results are summarized in table 1.No reaction was obtained when untreated sections of skin were incubated

for less than 10 min in the Gomori substrate for acid phosphatase. Incubationfor 10 min produced the first signs of a positive reaction, in the form of a fewseparate granules in the cytoplasm of the cells of the hair follicles (fig. 1, A) ;but cells adjacent to the root of the hair showed diffuse staining even with thisincubation. After 20 minutes' incubation there was an increase in the numberof stained particles with some diffuse staining of the cytoplasm (fig. 1, B).Extending the incubation time still further produced progressively increasedcytoplasmic coloration and marked staining of the nuclei. Indeed, afterincubating for 60 min, the intensity of cytoplasmic staining was too great toallow any cellular detail to be discerned and the particles themselves were nolonger distinguishable (fig. 1, c, D).

76 Diengdoh—Lysosomes in mouse skin

A pretreatment of the section in acetate buffer at pH 5 gave a reaction inthe form of numerous stained particles even after incubating for only 5 minin the substrate. This incubation time produced no reaction in untreatedsections (fig. 1, E).

Treatment with 0-25 M sucrose had no effect on the response of the sectionsto the Gomori procedure. This was in contrast to the marked change inducedby treatment with 0-125 M sucrose, after which the otherwise minimal 10 minof incubation produced many stained particles, and also diffuse cytoplasmicand appreciable nuclear staining (fig. 2, A). A similar enhancement of thereaction was observed after treatment with distilled water (fig. 2, B).

The effect of treatment with 0-25% triton-X-100 depended on the lengthof exposure. Thus 5 min in this solution induced a marked increase in stain-ing, even after incubation for only 10 min (fig. 2, c), whereas treatment for20 min apparently removed the enzyme completely from the sections, asevinced by a negative reaction even after incubation for 60 min.

Sections kept in the oven at 5 8° C for 10 min gave a very marked increase inthe reaction for the enzyme (fig. 2, D). Sections cut in the cryostat at about—25° C and kept at room temperature up to 1 h before staining showed noactivation of the enzyme. When the sections were repeatedly frozen andthawed 3 or 4 times, however, a marked increase in staining for acid phos-phatase was obtained (fig. 2, E).

DiscussionThe problem posed is whether lysosomes, as demonstrated in liver and

kidney, exist in skin. It is not feasible to demonstrate these organelles in skinby biochemical procedures, owing to the difficulty in homogenizing thistissue satisfactorily. An attempt has therefore been made to apply biochemicalcriteria to a histochemical demonstration of lysosomes in frozen sectionsof skin. If in such sections of skin it is possible, by histochemical proce-dures, to demonstrate cytoplasmic particles which, in their response tocertain treatments, behave in an identical manner to isolated lysosomes, thenit can be deduced that such cytoplasmic structures are lysosomes as definedbiochemically.

Fresh frozen sections of skin cut in the cryostat showed no activity for acidphosphatase when incubated in the Gomori substrate for short periods. It

FIG. 1 (plate). A, untreated section, incubated 10 min; shows separate black granules inouter sheath of hair follicles.

B, untreated section, incubated 20 min; numerous stained granules can be seen in the hairfollicle and a few in the epithelium.

c, untreated section, incubated 30 min; diffuse cytoplasmic staining and marked nuclearstaining, but the stained granules are still distinguishable.

D, untreated section, incubated 60 min; intense black staining of epithelium and hair folliclemasking all cellular detail.

E, section treated for 10 min with acetate buffer, pH 5, and then incubated for 5 min;numerous black particles with some diffuse cytoplasmic staining can be seen.

The scale on A applies to all the micrographs.

p

M l

50//

FIG. I

J. V. DIENGDOH

SOp

A B

FIG. 2

J. V. DIENGDOH

Diengdoh—Lysosomes in mouse skin 77

was only after incubation for 10 min in the medium that a reaction was obtainedin the form of a few separate granules in the cytoplasm. As the incubationtime was increased the number of stained granules became greater and this at20 min was accompanied by a diffuse staining of the cytoplasm, the granulesthemselves becoming less separate, until after about 30 min there was intensestaining of the cytoplasm and nucleus. It may be argued that the delay of10 min for the first reaction to appear was the time taken for the formationof sufficient reaction-product to permit its detection. Hence the increase inintensity of stain with the increase of incubation time would be due to theprogressive accumulation of reaction-product purely as a result of increasedtime of incubation. For such an explanation there would be no need to im-plicate any membrane effect. In order to test this suggestion the sections werepretreated with acetate buffer at pH 5, from which the substrate had beenomitted. It was found that sections so treated gave a reaction after incubationfor only 5 min, which normally would not give a reaction. This experimentindicated that the delay in reaction appears to be due to a barrier whichseparated the substrate from the enzyme in the particles and that the passageof the substrate through the barrier had been facilitated by the pretreatmentwith the buffer.

Biochemically, it had been demonstrated by de Duve and his co-workersthat fresh preparations of lysosomal granules show no enzymic activity, butthat the enzymes associated with them can be released by treatment withhypotonic solutions. Applying the same principle to frozen sections of skin,analogous results were obtained. Pretreatment of the sections with 0-25 Msucrose gave a reaction which was identical with that obtained in untreatedsections. This is in accordance with the biochemical finding that 0-25 Msucrose, which is used as a suspension medium for the isolated particles,affords them osmotic protection. Pretreatment of the sections with 0-125 Msucrose and with distilled water, however, resulted in a marked increase in thenumber of stained granules with diffuse staining of the cytoplasm and nucleus.This suggests that hypotonic solutions have an osmolytic effect on the granulesand may act by increasing the permeability of their membranes, thereby allow-ing quicker access of the substrate to the enzyme as well as escape of theenzyme or reaction-product from the particles into the cytoplasm, whencethey could be adsorbed on to the nucleus.

FIG. 2 (plate). A, section treated with 0125 M sucrose and then incubated for 10 min;many black particles occur in the hair follicle, with diffuse cytoplasmic staining.

B, section treated for 10 min and incubated for 10 min; note the strong nuclear and diffusecytoplasmic staining.

C, section treated with 0-25% triton-X-100 for 5 min and incubated for 10 min. Blackenedgranules with diffuse cytoplasmic and some nuclear staining are visible in the hair follicle;numerous granules are also seen in the epithelium.

D, section heated in oven at 58° C; incubated for 10 min. Note the intense diffuse stainingof the hair follicle.

E, section repeatedly frozen and thawed; incubated for 10 min. There is considerablecytoplasmic and nuclear staining.

The scale on A applies to all the micrographs.

78 Diengdoh—Lysosomes in mouse skin

The lysosomal membrane-barrier is presumably a lipoprotein complex, andthe action of triton-X-100 is to render the lipid fraction unstable, looseningthe bond and causing a release of the enzymes (Holt, 1959). A short treat-ment with the detergent gave an increased reaction for the enzyme acidphosphatase, whereas longer treatment resulted in a completely negativereaction. This might be due to the fact that a short treatment caused justsufficient damage to allow increased availability of the enzyme, whereas aprolonged treatment had so damaged the membrane that the enzyme hadescaped into the incubation medium and was no longer available for staining.The increased staining obtained in the sections after repeated freezing andthawing, and also the effect of heat, could be due to damage caused to thelysosomal membrane by these means.

Evidence presented here would indicate that the cytoplasmic particlesstaining for acid phosphatase in mouse skin are specialized organelles contain-ing the enzyme enclosed by a membrane-like barrier. Normally this membraneis impermeable to the substrate used in this study, and the particles appear tobe enzymically inert. However, after various forms of injurious treatments,which apparently alter this membrane, the substrate can penetrate into theorganelles, so permitting the detection within them of acid phosphataseactivity. Thus histochemical methods have permitted the identification ofthese particles with lysosomes as defined biochemically.

I wish to express my gratitude to Professor G. J. Cunningham for hisencouragement and to Dr. J. Chayen for his guidance in this work. I alsowish to thank Mr. E. K. Aves and Mr. A. Maple for skilful technical assistance,and Mr. A. L. E. Barron for the photomicrographs.

ReferencesAppelmans, F., and de Duve, C, 1955. Biochem. J., 59, 426.

Wattiaux, R., and de Duve, C, 1955. Ibid., 59, 438.Berthet, J., and de Duve, C, 1951. Ibid., 50, 174.

Berthet, F., Appelmans, F., and de Duve, C, 1951. Ibid., 50, 182.Bitensky, L., 1962. Quart. J. micr. Sci., 103, 205.

1963- Ibid., 104, 193.Cunningham, G. J., Bitensky, L., Chayen, J., and Silcox, A. A., 1962. Ann. Histochim., 7,

433.De Duve, C, 1959. In Subcellular particles, edited by T. Hayashi, p. 128. New York

(Ronald Press).Gianetto, R., and de Duve, C, 1955. Biochem. J., 59, 533.Gomori, G., 1941. Arch. Path., 82, 189.Holt, S. J., 1959. Exp. Cell Res., Suppl. 7, 1.