acid phosphatase distribution in the wool follicle. i. cortex and fiber cuticle

JOURNAL OF ULTRASTRUCTURE RESEARCH 55, 312-324 (1976)

Acid Phosphatase Distribution in the Wool Follicle

I. Cor tex and Fiber Cut ic le

D. F. G. ORWIN

Wool Research Organisation of New Zealand (Inc.), Christchurch, New Zealand

Received April 25, 1975, and in revised form, November 7, 1975

Acid phosphatase activity is demonstrated in some single membrane-bounded organelles of the cortex and fiber cuticle of the Romney wool follicle. These organelles include Golgi complexes, single membrane-bounded bodies with homogeneous contents, smooth vesicles, endoplasmic reticulum, and autophagic vacuoles and it is concluded that they form part of a lysosomal system. The types and numbers of organelles with acid phosphatase activity vary during differentiation. Lysosomal activity appears least in the bulb of the follicle (zone A) and greatest in the more differentiated regions (zones C and D). The relationship between lysosomal activity and other features of the differentiating fiber is discussed.

The p r e s e n c e of ac id p h o s p h a t a s e (Ac-

Pase ) h a s b e e n s h o w n in wool a n d h a i r

fo l l ic les by b o t h b i o c h e m i c a l a n d h i s to -

c h e m i c a l m e t h o d s (7, 18, 36, 37). D e s p i t e

t h e c o r r e l a t i o n of A c P a s e a n d l y s o s o m a l

s y s t e m s in m a n y m a m m a l i a n t i s s u e s (11, 12), t h e ro le a n d l oca l i z a t i on of A c P a s e in

fol l ic le ce l l s h a v e n o t b e e n de f i ned . T h e r e

is l i t t l e i n d i c a t i o n f r o m p r e v i o u s u l t r a -

s t r u c t u r a l s t u d i e s t h a t o r g a n e l l e s t yp i ca l

of a l y s o s o m a l s y s t e m a r e p r e s e n t in follicle cel ls (5, 6, 8, 15, 30, 35).

As p r e v i o u s u n p u b l i s h e d o b s e r v a t i o n s i n

t h i s l a b o r a t o r y h a v e i n d i c a t e d t h e p res -

ence of l y s o s o m e - l i k e o r g a n e l l e s in foll icle

cel ls , A c P a s e w a s u s e d as a m a r k e r en-

z y m e to d e t e c t w h e t h e r l y s o s o m a l s y s t e m s

w e r e p r e s e n t . T h i s p a p e r d e s c r i b e s r e s u l t s

o b t a i n e d for t h e co r t ex a n d f i b e r cu t ic le

cell l ines . P r e l i m i n a r y f i n d i n g s of p a r t o f

t h i s s t u d y h a v e b e e n p r e s e n t e d e l s e w h e r e

(26).

MATERIALS AND METHODS

Electron microscopy. Follicles in Anagen VI (9) producing nonmedullated fibers were obtained from midside biopsies of Romney sheep. They were fixed in Karnovsky's cacodylate-buffered (pH 7.4) glutar- aldehyde-formaldehyde fixative for 3-4 hr at 4°C. After fixation the follicles were washed with 0.1 M cacodylate buffer (pH 7.4) containing 7.5% sucrose, and postfixed in ice-cold 1% OsO~ for 2 hr in the

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

same buffer. The follicles were then stained with 1% uranyl acetate in 0.1 M acetate buffer (pH 5.0) for 30-45 min at 20 or 50°C, depending on the degree of staining required (20). Dehydration of follicles in a graded series of hexylene glycols (40) was followed by embedding in epoxy resin (TAAB Laboratories, Reading, England). Gray to gold sections were cut on a LKB ultramicrotome, stained with lead salts, and examined in a Philips EM300 electron micro- scope operating at 60 kV.

Cytochemistry. Follicles or strips of skin were fixed as above in Karnovsky's fixative. After the initial wash, the tissue was placed in 1.2 M sucrose buffered with 0.1 M cacodylate (pH 7.4) in order to reduce freezing damage (41). Fifty-micrometre sections were cut from pieces of skin frozen by means of a thermoelectrically-cooled microtome stage (De la Rue Frigistor Ltd., Langley, England). Alterna- tively, follicles were frozen in Freon 12 at -150°C and sectioned with a LKB cryokit until the cortex was exposed. This was found necessary as the incubation media would not penetrate through the hardened Henle's layer in whole follicles. After incubation in the appropriate medium, the sections and follicles were rinsed and processed for electron microscopy as above. Substrates for the media were obtained from the Sigma Chemical Co., St. Louis, Mo.

Acid phosphatase. Sections or follicles were incu- bated in the medium of Barka and Anderson (4) using sodium/3-glycerophosphate as substrate with the addition of 5% sucrose. The tissues were incu- bated for 10-20 min at 37°C. Controls consisted of the medium without substrate or the complete medium with the addition of 0.01 M sodium fluoride.

Thiamine pyrophosphatase. The incubation medium (Novikoff and Goldfisher, 22) was filtered be-

312

ACID PHOSPHATASE IN FORMING WOOL FIBER

fore use and replaced after 30 min. The incubation time was 2 hr at 37°C. Controls consisted of medium without substrate or lead nitrate.

Phosphotungstic acid (PTA) at low pH. Glutaral- dehyde-formaldehyde-fixed follicles were fixed and washed as above and embedded in glycol methacry- late according to the method of Leduc and Bernhard (19). Thin sections were floated on water, trans- ferred to 2% PTA-HC1 (pH < 0.5) prepared as described by Babai and Bernhard (3) for 15 min, rinsed in distilled water, and picked up on carbon-stabi- lized, collodion-coated grids.

Distribution oforganeUes. Data were obtained for eight follicles from three sheep. Counting of AcPase- labeled organelles in cell cross sections was carried out on the screen. All AcPase-containing organelles were identified in the cell section, although in some cases accurate identification proved difficult, e.g., elongated vesicles may be confused with short profiles of endoplasmic reticulum (ER). The results are therefore regarded as approximations.

Sections of the same cell line in the immediate vicinity of labeled cell sections were also examined. The ratio of cell sections without labeled organelles to sections with labeled organelles was taken as an indication of the overall degree of AcPase activity.

A minimum of five cell sections showing AcPase activity was counted for each cell line in each zone. The follicles were divided into zones (27) in order to detect changes occurring during differentiation (Fig. 1).

Zone A is the basal region of the follicle to the top of the dermal papilla and includes the mitotic zone.

Zone B covers from zone A to the level where Henle's layer hardens.

Zone C is the region from zone B to the level where a continuous layer of fiber cuticle protein has formed against the plasma membrane apposed to inner root sheath (IRS) cuticle cells.

Zone D is the region from the top of zone C to where the remaining IRS cells harden.

Organelles showing AcPase activity were counted for zones A, B, C, and D for the cortex and zones B, C, and D for the fiber cuticle.

L

RESULTS

General

Examination of standard preparations of wool follicle tissue revealed the presence of a number of previously unreported organelles in the cortex. In the relatively undifferentiated cells of zone A, multivesicular bodies (MVB), vacuoles, well-developed Golgi complexes, and rough endoplasmic reticulum (RER) were found (Fig. 2). In addition, single membrane-bounded

313

e

HEN. S ~YER ~DENED

e c ~ x H ~ E M I ~ KE~INIZED

APP~x. ~ , M ~ s l ~ s

Fro. 1. Diagrammatic representation of a longitudinal section through a Romney wool follicle. The zones used in this study are depicted in relationship to those commonly used in describing regions of the follicle. Zones E, F, and G are referred to in later papers in this series.

bodies with homogeneous contents and small vesicles were present. However, organelles were scarce in this zone except fo r Golgi complexes and RER, which were at their greatest development.

In the lower regions of zone B there was a marked increase in the number of vacu~ oles (Fig. 3). These varied considerably i n size; some were partially filled with mod- erately electron-dense material and: were often located near the plasma membran e (Fig. 3). Vesicles with smooth membranes (smooth vesicles) ranging in diameter from about 60-100 nm were more common and were sometimes closely associated with the larger vacuolar-like organelles

314 D. F. G. ORWIN

ACID PHOSPHATASE IN FORMING WOOL FIBER 315

(Fig. 3). Golgi complexes were less well developed bu t more numerous , while the RER was not as preva len t as in zone A.

During the fu r the r different ia t ion of cortex cells in zones upper B to D, the organelles present included single membrane- bounded bodies with homogeneous contents (d iameter ca. 100-250 nm); smooth vesicles, often in clusters; small Golgi complexes, commonly more t han one per cell; profiles of endoplasmic re t i cu lum (ER); and vacuoles (Fig. 4). The ER was not present to any major extent and was often closely associated with mi tochondr ia and vacuoles. Coated vesicles and autophagic vacuoles (cf. 24) were also observed.

In the fiber cuticle cell layer , at the level where it could be identified with cer ta in ty (zone B), s imilar organelles to those of the cortex were seen (Fig. 5). The ER was not present to the same ex ten t as in the cortex. Li t t le change in type and number of organelle was seen dur ing different ia t ion (Fig. 6).

AcPase Activity

Occasionally whole cells or major par ts of cells, both wi th a condensed appearance, were found in large vacuoles of cells in zone A. In these vacuoles AcPase act ivi ty was restr ic ted to membrane-bounded organelles wi th in the engulfed cells and it was not found in the vacuolar lumen (Fig. 7).

Golgi complexes showing AcPase activ- i ty were commonly labeled along the inner aspect of the stack (Fig. 8). In face view, this region consisted of connect ing tubules (Fig. 9). In contrast , t h i amine pyrophosphatase (TPPase) act ivi ty was found toward the outer aspect of the Golgi complex (Fig. 10).

Mult ivesicular bodies appeared to be of two types. In Type I the AcPase activity was associated with the mater ia l be tween the vesicles (Fig. 11). In Type II, i t seemed to be associated with the in te rna l vesicles (Figs. 12 and 13).

In zone B par t icular ly , AcPase activity was found in some vacuoles par t ia l ly filled with electron-dense mater ia l . In a few cases it appeared as if vesicles showing AcPase act ivi ty were a t tached to such vacuoles (Fig. 13). Larger unident i f ied Ac- Pase-posit ive organelles were also observed in close juxtaposi t ion to vacuoles (Fig. 14).

Autophagic vacuoles showing AcPase activity were present in var ious zones of the cortex (Figs. 14 and 15). Thei r contents were usual ly difficult to identify; for exam- ple, mi tochondr ia were never positively identified in such vacuoles.

Single membrane-bounded organelles with homogeneous contents often showed AcPase activity, and were identified as lysosomes (Fig. 16). They var ied considerably in size (d iameter ca. 110-230 nm) but seemed to form a distinct populat ion from the smaller vesicles (d iameter ca. 60-100 nm) containing AcPase act ivi ty (Fig. 16). These smaller, smooth vesicles often formed clusters in which only some of the vesicles showed AcPase act ivi ty (Figs. 16 and 17). Both lysosomes and vesicles were found th roughout di f ferent ia t ing regions of the cortex.

Profiles of ER showing AcPase act ivi ty were found main ly in more different ia ted regions (Fig. 18).

In the fiber cuticle, AcPase act ivi ty was found in the same organelles as in the cortex (Fig. 19).

The numbers of labeled organelles at

FIG. 2. Zone A. The cytoplasm contains relatively few organelles. The most prominent features are the Golgi complex (GC) and endoplasmic reticulum (ER). A multivesicular body (MVB) and a vacuole (Va) are present, x 24 000.

FIG. 3. Lower zone B. The cortex cells contain many single membrane-bounded vacuoles (Va) and vesicles (Ve). Some of these contain electron-dense material. Others are associated with the plasma membrane (arrowheads). Many extensive intercellular spaces (IS) are present. Macrofibrils (M) are in an early stage of development, x 24 000.

316 D. F. G. ORWIN

di f ferent s t ages of d i f f e r e n t i a t i o n of the cortex a n d cut ic le a re p r e s e n t e d in Tab le I. Severa l ma jo r t r e n d s a re appa ren t .

F i r s t ly , t he n u m b e r of cell sec t ions wi th AcPase -ac t ive o rgane l l e s i nc r ea sed f rom zone A to a po in t where in zones C a n d D

v i r t u a l l y al l cell sec t ions c o n t a i n e d l abe led

organe l les . Secondly, t he r e were possible inc reases i n Golgi complexes w i th AcPase in zones B a n d C a n d also i nc r ea se s in lead

phospha t e - l abe l ed vacuoles i n zone B in c o m p a r i s o n wi th zones A, C, a n d D. Auto- phag ic vacuo les w i th AcPase ac t i v i t y were not c o m m o n in the cortex. Th i rd ly , t he mos t de f in i t e t r e n d was the inc rease i n

n u m b e r s of lysosomes, smoo th vesicles, a n d ER profi les s h o w i n g AcPase ac t iv i ty

d u r i n g d i f f e ren t i a t ion .

In the f iber cut icle , t he ma jo r difference from the cortex was t h a t the l abe led

smooth vesicles a n d ER did no t i nc rease to

the s a m e e x t e n t i n zones C a n d D. Two o the r f ea tu re s i n the cortex m a y

have r e l e v a n c e to lysosomal sys tems , even

t h o u g h t h e y did no t show AcPase ac t iv i ty in th i s s tudy . S m a l l vesicles a p p e a r e d to be assoc ia ted w i t h r eg ions of ER (Fig. 20) a n d ER or vacuo les a p p e a r e d to p a r t i a l l y sur-

r o u n d reg ions of cy top l a sm c o n t a i n i n g ri-

bosomes (Fig. 21). The l a t t e r was more c o m m o n i n zones C a n d D.

A l t h o u g h AcPase ac t i v i t y was no t found in the i n t e r c e l l u l a r space of apposed corti-

cal cells i n zone A (Fig. 22), i t a p p e a r e d in u p p e r zone B (Fig. 16) a n d was a cons i s t en t f ea tu re of zones C a n d D (Fig. 23). In addi- t ion, th i s a c t i v i t y was p r e s e n t i n the i n t r a - ce l lu l a r spaces of apposed cortex a n d f iber cut ic le p l a s m a m e m b r a n e s in u p p e r zone B a n d zones C a n d D. I t was no t found be-

t w e e n apposed f iber cut ic le cells (Fig. 19) nor b e t w e e n f iber cu t ic le / IRS cut ic le cells.

P T A - s t a i n i n g showed the r eve r se t rend; i t was the mos t i n t e n s e in the i n t r a c e l l u l a r

space of apposed cort ical cell m e m b r a n e s in zone A (Fig. 24), b u t i t b e c a m e m u c h less i n t e n s e a n d more sporadic in u p p e r zone B a n d in zones C a n d D (Fig. 25).

Flo. 4. Upper zone C. This longitudinal section of cortex cells shows three small Golgi complexes (GC), endoplasmic reticulum (ER), a vacuole (Va), and single membrane-bounded vesicles (Ve) and bodies (Ly). No intercellular spaces are found between the apposed plasma membranes (PM) and the macrofibrils (M) are in a more advanced state of differentiation, x 21 000.

FIG. 5. Lower zone B. The fiber cuticle cell is flanked by cortex (Co) and inner root sheath cuticle (IRC) cells. Within its cytoplasm, a Golgi complex (GC), a vacuole (Va), a single membrane-bounded body (Ly), and a multivesicular body (MVB) are present, x 17 000.

FIG. 6. Zone C. The fiber cuticle cell contains a Golgi complex (GC) and several vacuoles (arrowheads) while other organelles such as single membrane-bounded bodies and vesicles and endoplasmic reticulum are absent. Parts of a cortex (Co) and an inner root sheath cuticle (IRC) cell are shown, x 24 000.

Fia. 7. Zone A. The lumen of this vacuole contains a degrading portion of another cell. Acid phosphatase activity is restricted to two organelles within the degrading cell (arrowhead). Acid phosphatase, x 18 000.

FIG. 8. Zone A. Golgi complex. Acid phosphatase activity is restricted to the inner aspect of the Golgi complex. Acid phosphatase, x 34 000.

FIo. 9. Zone A Golgi complex. This oblique section through the Golgi complex shows that acid phosphatase activity is present in interconnecting tubules. Acid phosphatase, x 34 000.

Fro. 10. Zone A Golgi complexes. Thiamine pyrophosphatase activity is present in elements located toward the middle of the Golgi stack. Thiamine pyrophosphatase, x 28 000.

FIG. 11. Zone A. A single membrane-bounded organelle containing vesicles shows acid phosphatase activity in its matrix. Acid phosphatase, x 57 000.

Fro. 12. Lower zone B cortex. Acid phosphatase activity appears to be located in one vesicle of this multivesicular body. Acid phosphatase, x 56 000.

FIG. 13. Zone A. Acid phosphatase activity is located in one vesicle present in a vacuole and also the electron-dense material present in another single membrane-bounded body (arrowhead). Acid phosphatase. Inset: A single membrane-bounded organelle which appears to have two acid phosphatase-containing vesicles attached to its membrane (arrowhead). Acid phosphatase, x 40 000; inset, × 57 000.

Fro. 14. Lower zone C cortex, An acid phosphatase-containing organelle is in close apposition to a vacuole. Two autophagic vacuoles showing acid phosphatase activity are present (arrowheads). Acid phosphatase, x 44 000.

320 D. F. G. ORWIN

Fm 15. Lower zone B Cortex. The two single membrane-bounded organelles showing acid phosphatase activity in their lumens are probably autophagic vacuoles (V). Two other bodies with homogeneous contents also contain acid phosphatase activity (Ly). One of these is closely associated with a vacuole. A profile of endoplasmic reticulum also shows acid phosphatase activity (arrowhead). Acid phosphatase, x 29 000.

FIG. 16. Upper zone B cortex. Two single membrane-bounded bodies with homogeneous contents show acid phosphatase activity. A cluster of smaller vesicles with similar contents is indicated (arrowhead). Acid phosphatase activity is present in some of these. The intercellular space shows acid phosphatase activity as well. Acid phosphatase. Inset: A single membrane-bounded body containing acid phosphatase. Acid phosphatase, x 29 000; inset, x 54 000.

FIG. 17. Zone D cortex. This transverse section shows acid phosphatase activity in a single membrane- bounded body (Ly) and several vesicles, including one in a cluster of vesicles (arrowheads). A profile of endoplasmic reticulum containing acid phosphatase activity appears to be continuous with a mitochondrial membrane (ER). M, macrofibril. Acid phosphatase, x 35 000.

Fro. 18. High zone C cortex. Profiles of endoplasmic reticulum contain acid phosphatase. One profile is closely juxtaposed to a vacuole (Va). M, macrofibrils. Acid phosphatase, x 54 000.

DISCUSSION

Prev ious s tud ies h a v e no ted the presence of Golgi complexes, t h e i r assoc ia ted vesicles, ER, a n d a s ing le m e m b r a n e - b o u n d e d o rgane l l e ( t h o u g h t no t to be lysosomal i n n a t u r e ) i n cortex a n d f iber cut icle cells (5, 6, 8, 15, 28, 30, 34, 35). This s tudy has iden t i f i ed the p re sence in the

follicle of MVBs, lysosomes, a n d smoo th vesicles. W h e r e a s p r e v i o u s l y o rgane l l e s have b e e n repor ted in the less differen- t i a t ed r eg ions of the follicle, ev idence is

p r e s e n t e d he re t h a t these o rgane l l e s occur t h r o u g h o u t d i f f e r e n t i a t i o n of the cortex a nd cuticle.

As m a n y o r g a n e l l e s were s h o w n to con- t a i n the ly sosomal e n z y m e acid phospha-

ACID P H O S P H A T A S E IN F O R M I N G WOOL FIBER

T A B L E I

MEAN NUMBER OF ORGANELLES SHOWING ACID PHOSPHATASE ACTIVITY PER 10 CELL SEcTIoNS a

321

Cortex , zone Fiber cuticle, zone

A B C D B C D

Labeled cell sec t ions ~ 33 47 41 35 45 40 35

Un labe led / l abe led c 29 4 1 1 20 1 2

Golgi complexes 5 10 10 5 10 5 3 Vacuoles 2 7 3 3 4 2 0 Au tophag i c vacuoles 2 3 2 1 1 2 1 Lysosomes 8 15 10 16 17 15 16 E n d o p l a s m i c r e t i c u l u m 1 4 16 22 1 8 7 Smal l s m o o t h ves ic les 2 11 25 28 8 12 9

In t e rce l lu l a r l abe l ing - + + + +~ +'~ +'~ (upper) (upper)

Coated ves ic les a n d m u l t i v e s i c u l a r bodies occas ional ly showed acid p h o s p h a t a s e ac t iv i ty . b Ac tua l n u m b e r of labe led cell sec t ions assessed . c N u m b e r of un l a be l ed cell sect ions to 10 labeled sect ions.

Apposed cut ic le /cor tex cells, b u t no t cut ic le /cut ic le cells.

tase (11, 12), the cortex and fiber cuticle may be considered to have a lysosomal system. The presence of AcPase activity in Golgi complexes, MVBs, smooth vesicles, single membrane,bounded bodies, and autophagic vacuoles in these cell lines is par- alleled by AcPase activity in similar organelles in the lysosomal systems of many other mammalian cells and tissues (see 11, 12 for review). AcPase activity in ER has been recorded less frequently in other tissues (31, 42). However, ER is regarded as an integral part of lysosomal systems (11, 23, 24).

The Golgi complex has characteristics similar to the Golgi of the neuron. AcPase activity is located in the inner aspect of the Golgi complex which has the form of interconnecting tubules in this region. It seems likely, therefore, that the lysosomal system of the follicle may be similar to the Golgi-ER-lysosomal system described for the neuron (10, 23, 24). The different loca- tion of TPPase within the Golgi complex supports the idea of a specialized region of the complex being involved in the lysosomal system. The changes in number and complexity of the Golgi complexes (28)

during cortex differentiation may reflect changes in lysosomal activity.

This study confirms and extends the general pattern of AcPase distribution described in histochemical studies of both hair and wool follicles (7, 3 7). The results of a biochemical study (18) of a-naphthyl acid phosphatase in the hair follicle also support the present findings.

The changes in numbers and types of organelles showing AcPase activity during differentiation can be considered against other developments in the follicle. While such a comparison must be speculative as it is based on morphological and cytochem- ical evidence alone (1, 43), it helps to pro- vide a more general perspective of the possible roles of the lysosomal system in the differentiating cortex and fiber cuticle.

In zone A, where cell division is the main activity, the lysosomal system does not appear highly developed, probably be- cause of the high turnover of cells (14, 16, 39).

Zone B, characterized by the initiation of differentiation and cell growth, shows an increase in numbers of vacuoles, often containing electron-dense material. These

ACID PHOSPHATASE IN FORMING WOOL FIBER 323

vacuoles may result from endocytosis. The close association of AcPase-containing vesicles with such vacuoles suggests that this may be the means by which lysosomal en- zymes are transported to them. Such a pathway is believed to be common to endo- cytic activity in many mammalian tissues (11, 17).

Zones C and D are characterized by the synthesis of keratin proteins. Coupled with this is a decrease in cytoplasm and, in the Romney wool follicle, a 25% decrease in fiber diameter (2). AcPase activity is also at a maximum in these zones. This suggests that the lysosomal system may be primarily concerned with autophagy. However, since lysosomes and vesicles showing AcPase activity in these regions are numerous relative to both vacuoles and autophagic vacuoles, they cannot be regarded solely as primary lysosomes. Some of them may represent residual bodies.

During differentiation of the cuticle, the lysosomal system develops in a manner similar to that of the cortex. However, ER showing AcPase activity does not appear to develop to the same extent. This may indicate a lower degree of autophagic activity.

The presence of AcPase activity in the intercellular space of cortex cells is paral- leled by similar findings in the intercellular space of mouse kidney tubules (21,38), though different substrates were used. In

the follicle, the enzyme is restricted to the intercellular spaces of apposed cortex cells and cortex-fiber cuticle cells. Its appearance in upper zone B is correlated with changes (25) such as decreased PTA staining probably of glycoproteins (3, 13, 32, 33) and others as part of the formation of the cell membrane complex (29). The bio- logical significance of this AcPase activity is not clear at the present time.

The excellent assistance of Mr. R. W. Thomson is gratefully acknowledged. Appreciation is expressed to Mrs. Joanna Orwin and Dr. L. F. Story for criti- cally reading the manuscript, and to Mr. M. Hedwig for the drawing of a wool follicle.

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Fie. 19. Zone D fiber cuticle. Acid phosphatase activity is present in both single-membraned bodies (Ly) and vesicles (arrows). The cortex (Co)-fiber cuticle (Cu) intercellular space contains acid phosphatase while the apposed fiber cuticle intercellular space does not, Acid phosphatase. × 40 000.

FIG. 20. Zone A. Vesicles appear to be associated with the endoplasmic reticulum (arrowheads). × 26 000.

FIG. 21. Zone D cortex. Endoplasmic reticulum appears to be sequestering a region of cytoplasm containing ribosomes. × 72 000.

FIG. 22. Zone A. No acid phosphatase is present in the intercellular space between the apposed plasma membranes. A vesicle does show the presence of acid phosphatase (arrow). Acid phosphatase. × 34 000.

Fro. 23. Zone D cortex. Acid phosphatase activity is clearly present in the intercellular space between apposed plasma membranes. Acid phosphatase. × 72 000.

FIG. 24. Lower zone B cortex. Staining with PTA has resulted in the intercellular material showing marked electron density. Phosphotungstic acid. × 57 000.

FIG. 25. Upper zone B cortex. Similar staining to Fig. 24 has resulted in only moderate electron density appearing in short regions of the intercellular material. Phosphotungstic acid. z 72 000.

324 D. F. G. ORWIN

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