JOURNAL OF ULTRASTRUCTURE RESEARCH 55, 325-334 (1976)

Acid Phosphatase Distribution in the Wool Follicle

II. Henle's Layer and Outer Root Sheath

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

A cytochemical and morphological study of the outer root sheath and Henle's layer of the Romney wool follicle reveals the presence of a lysosomal system. Using acid phesphatase as a lysosomal marker enzyme, reaction product is found in Golgi complexes, single membrane- bounded bodies, small smooth vesicles, and some types of vacuoles. The lysosomal system of these cell lines does not develop to the same extent as in the cortex. There is little evidence that the lysosomal system of outer root sheath cells is involved in degradation of the inner root sheath.

The wool follicle produces two other ma- jor cell types apart from those forming the fiber. These are the cell lines making up the inner root sheath (IRS) and the outer root sheath (ORS).

The IRS, of which Henle's layer is a part, synthesizes trichohyalin, whose transfor- mation results in the hardening of the cells (1, 4, 14, 22, 23). The hardened cells are later partially degraded during their passage towards the skin surface and are sloughed off into the pilary canal (1, 14).

The ORS undergoes neither keratiniza- tion nor hardening (1, 13, 14, 22). ORS cells move toward the skin surface and, except for those which are continuous with the follicle neck, undergo a form of cornifi- cation (14) just prior to their sloughing off into the pilary canal in the same region as the IRS cells (1, 6, 14, 22, 26).

Both IRS and ORS cells are known from histochemical and biochemical studies to show acid phosphatase activity (5, 12, 16, 24, 25). Although this enzyme is usually found in lysosomes and other organelles in most mammalian tissues (9, 10), it is not known which organelles are involved in these cell types.

This study was undertaken to determine the types and distribution of organelles with acid phosphatase (AcPase) activity in Henle's and ORS cells, following the simi-

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

lar study of cortex and fiber cuticle cells described in Part I (21). Evidence was sought for the role that these organelles might have in the differentiation and deg- radation of these cell types. In particular, an attempt was made to determine whether companion cells (19) of the ORS are involved in the degradation of the IRS.

Preliminary findings of part of this study have been presented elsewhere (20).

MATERIALS AND METHODS

Electron microscopy and cytochemistry. Romney wool follicles in anagen VI (7) were used. Their preparation is described in Part I of this series. As before, the method of Barka and Anderson (3) was used to show sites of AcPase activity. Similarly, sections of glycol methacrylate-embedded follicles (16) were stained with phosphotungstic acid at low pH (11).

Distribution of organelles showing AcPase activ- ity. The method of counting the different types of organelles showing AcPase activity is described in Part I. However, for Henle's layer, which hardens at the top of zone B, the zones were divided into A, lower B, and upper B. For the ORS (excluding com- panion cells) organelles with AcPase activity were counted in zones A, B, C, and D.

Three other zones higher in the follicle (21) were included in this study:

Zone E extends from zone D to the level where the cortex is no longer osmiophilic.

Zone F ends at the level where the IRS and ORS cells are sloughed into the pilary canal.

Zone G consists of the follicle neck. Both companion cells and IRS cells were studied

325

326 D .F .G .

in zones E and F to determine which organelles were present and the zones of AcPase activity. ORS cells in zones E, F and G were also examined.

RESULTS

General

In zone A of convent ional ly prepared fol- licles, Henle 's cells were re la t ively poor in cytomembranes and single membrane- bounded organelles. Of these, Golgi com- plexes and rough endoplasmic re t icu lum (rough ER) were the most f requent ly found (Fig. 1). Occasionally, mult ivesicu- lar bodies (MVBs), vacuoles, (Fig. 1), sin- gle membrane-bounded bodies, and small smooth vesicles were also present . In lower zone B (Fig. 2), the most notable fea ture was the development o f t r i chohya l in in the cytoplasm. In upper zone B, organelle type showed lit t le change, with the exception of the appearance of variably-sized, but usu- ally large, dilated s t ruc tures cont inuous with and derived from rough ER (Figs. 3 and 7). These dilations were often seen close to Golgi complexes. Thei r lumens usual ly contained membranous and/or fi- bri l lar mater ia l . In cells about to harden, the dilations had usual ly disappeared or become reduced in size.

Of all the cell lines in the follicle, the ORS had the most extensively developed cytomembrane and single membrane- bounded organelle system. In upper zone A (Fig. 4) ORS cells contained large Golgi complexes with m a n y associated vesicles. Both smooth and rough ER were common and vacuoles, MVBs, lysosome-like bodies, and vesicles were re la t ively numerous . Lit t le change was detected in the types and numbers of organelles present at sub- sequent stages of different iat ion as high as

ORWIN

zone D, by which stage these cells had accumulated large amounts of glycogen.

Acid Phosphatase Activity

In general , the types of organelles show- ing AcPase act ivi ty in Henle 's layer and the ORS were the same as those found in the cortex and fiber cuticle. AcPase activ- i ty in Golgi complexes was found in the inner aspect of Golgi stacks. Single mem- brane-bounded bodies with homogeneous contents, small smooth-membraned vesi- cles, vacuoles par t ia l ly filled wi th mate- rial (Fig. 5), and, rarely, MVBs and coated vesicles showed AcPase activity.

In Henle 's cells, jus t prior to hardening, larger residual-body-like organelles were usual ly the only ones to show AcPase ac- t ivi ty (Fig. 6). It is no tewor thy tha t ribo- somes (Fig. 6) and mitochondria were usu- ally present a t this stage.

The dilated regions of rough ER (Fig. 7) did not show AcPase activity, despite the i r association with Golgi complexes.

Another type of single membrane- bounded body, not previously reported, was found in Henle 's cells in upper zone B. These organelles, which were about 100- 180 nm in d iamete r and usual ly contained electron-dense mater ia l , were typical ly found near the plasma membranes (Fig. 8). They did not show AcPase act ivi ty al- though thei r contents were s ta ined with PTA (Fig. 9), indicat ing tha t they may contain glycoprotein (2, 11).

Changes in the numbers of organelles with AcPase act ivi ty dur ing differentia- t ion of Henle 's cells are shown in Table I. The numbers of cell sections showing Ac- Pase act ivi ty increased dur ing differentia- tion but even at the most advanced stages

FIG. 1. Zone A Henle's cell (He). Common features found in the cytoplasm of these cells are Golgi complexes (GC) and relatively rare profiles ofendoplasmic reticulum (ER). A few vacuoles (arrowheads) and a multivesicular body (arrow) are also present. The Henle's cell is flanked by companion (Comp) and Huxley's (Hu) cells, x 12 000.

FIa. 2. Lower zone B Hente's cell (He). In comparison with Fig. 1 there is little development of the cytomembranes. A Golgi complex (GC) and profiles of endoplasmic reticulum (arrowheads) are indicated. Much trichohyalin (T) is present. A single membrane-bounded body (Ly) is indicated in the neighboring Huxley's cell. Comp, companion cell; OR, outer root sheath cell. x 12 000.

327

FIG. 3. Upper zone B Henle's cell. At this stage of differentiation the endoplasmic reticulum has become dilated (DER). One dilation contains membranous material (arrowhead). A Golgi complex (GC) is closely associated with two regions of dilated endoplasmic reticulum. Trichohyalin (T) is abundant. A single membrane-bounded body (Ly) is present in a neighboring Huxley's cell. × 23 000.

FIG. 4. Upper zone A outer root sheath cell. This section shows the relatively extensive cytomembranes of this cell type. A Golgi complex (GC) and profiles of endoplasmic reticulum (arrows) are present. Vesicles (Ve) and a multivesicular body (arrowhead) are indicated. Comp, companion cell. × 21 000.

328

ACID PHOSPHATASE IN FORMING HENLE'S CELLS 329

FIG. 5. Lower zone B Henle's cell. Acid phosphatase activity is present in one aspect of a Golgi complex (GC). A single membrane-bounded body (Ly) and endoplasmic ret iculum (arrow) associated with another Golgi complex also show acid phosphatase activity. A small amount of reaction product is present in the mater ia l enclosed in a vacuole (arrow head). T, trichohyalin. Acid phosphatase. Inset: Acid phosphatase activity in a single membrane-bounded body with homogeneous contents and a smaller vesicle (arrow). Acid phosphatase. × 31 000; inset, × 46 000.

of development only about one out of two sections contained active organelles. Most of the AcPase activity was found in Golgi complexes ~nd single membrane-bounded organelles with homogeneous contents (ly- sosomes). Neither showed any major changes in numbers during differentia- tion. The numbers of other organelles with AcPase activity remained at low levels throughout differentiation. AcPase activ- ity was not detected in the intercellular spaces of plasma membranes of apposed Henle's cells.

Similar results were obtained for the ORS (Table I). Even in zone D only about one section in two contained AcPase-la-

beled organelles. As in Henle's layer, Ac- Pase activity was found mainly in Golgi complexes, lysosomes, and vesicles. The numbers of AcPase containing vacuoles, autophagic vacuoles, and profiles of ER were low, and AcPase activity was absent from the intercellular spaces of apposed ORS cells.

In higher regions of the follicle (zones E and F), Henle's cells in an advanced state of degradation (Fig. 10) or disintegrating and sloughing (Fig. 11) showed no evi- dence of AcPase activity.

Although AcPase activity was present in organelles of companion cells in zones E and F, it was rare in,the regions of convo-

330 D . F . G . ORWIN

TABLE I MEAN NUMBER OF ORGANELLES SHOWING ACID PHOSPHATASE ACTIVITY PER 10 CELL SECTIONS a

Zone Henle's layer Outer root sheath

A B low B high A B C D

Labeled cell sections ~ 33 35 4 31 45 39 34

Unlabeled/labeled c 19 10 7 17 17 13 8

Golgi complexes 7 9 5 8 8 6 4 Vacuoles 1 3 - - 1 1 1 Autophagic vacuoles - 1 1 1 1 1 2 Lysosomes 10 11 10 6 7 9 17 Endoplasmic reticulum - 1 1 - 1 1 3

Small smooth vesicles 2 4 4 3 5 7 6

Intercellular labeling . . . . . . .

a Coated vesicles and multivesicular bodies occasionally showed acid phosphatase activity. Actual number of labeled cell sections assessed.

c Number of unlabeled cell sections to 10 labeled sections.

l u t ed m e m b r a n e s where these cells ap- posed Hen l e ' s cells (Fig. 12). I f p r e sen t , the

r eac t ion p roduc t was found in the in te rce l - l u l a r space of the apposed p l a s m a m e m -

b r a n e s (Fig. 13). The i n t e r c e l l u l a r ma t e - r ia l was also s t a i n e d by PTA. I n add i t ion ,

bo th lysosome-s ized bodies w i t h homoge- neous c o n t e n t s a n d some w i t h v e s i c u l a r con t en t s were s t a i n e d by P T A i n compan- ion cells (Fig. 14).

I n zone F, some u n c o r n i f i e d ORS cells showed d e g r a d a t i v e c h a n g e s i n t h e i r ul- t r a s t r u c t u r e . T h e i r cy top l a sm c o n t a i n e d

m a n y vacuoles , a u t o p h a g i c vacuoles , re- g ions of swol l en ER, a n d la rge ( d i a m e t e r

ca. 300-500 nm) r e s idua l -body- type o rgan- el les c o n t a i n i n g AcPase ac t iv i ty (Fig. 15).

DISCUSSION

Both the morpho log ica l a n d the cyto-

chemica l ev idence p r e s e n t e d he re h a v e in- d ica ted t h a t the cell l ines of the IRS (as r e p r e s e n t e d by H e n l e ' s layer) a n d t he ORS c o n t a i n lysosomal sys tems . The p re sence of AcPase ac t i v i t y in s i m i l a r o r g a n e l l e s also has b e e n r epor t ed in the lysosomal sy s t e ms of m a n y o the r t i s sues (see 9 a n d 10 for reviews) . As in the cortex, the loca-

t i on of AcPase ac t i v i t y in the Golgi com- plexes sugges t s t h a t the lysosomal s y s t e m

FIG. 6. Upper zone B Henle's cell just prior to hardening. At this late stage of differentiation the cell contents consist largely of trichohyalin (T). Despite this, ribosomes (r), small areas of dilated endoplasmic reticulum (arrows), and a single membrane-bounded body with acid phosphatase activity (Ly) are still present in the cytoplasm. Acid phosphatase, x 27 000.

FIG. 7. Upper zone B Henle's cell. Two regions of dilated rough endoplasmic reticulum (DER) are shown. Their derivation from rough endoplasmic reticulum (ER) and association with a Golgi complex (GC) are apparent. Both membranous (arrowheads) and fibrillar material (arrows) are present in their lumens. The fibrillar material is often associated with the membrane of the dilated endoplasmic reticulum. Acid phosphatase. × 23 000.

FIG. 8. Upper zone B Henle's cell. Four single membrane-bounded organelles (arrows) are indicated. They contain varying amounts of an electron-dense material. × 26 000.

FIG. 9. Upper zone B Henle's cell. The electron-dense material found in the single membrane-bounded organelles shown in Fig. 8 shows enhanced electron density following phosphotungstic acid staining, x 31 000.

FIG. 10. Low zone F. Both Henle's (He) and Huxley's (Hu) cells show signs of degradation and extraction. There is no acid phosphatase activity. Acid phosphatase. × 24 000.

331

332 D. F. G. ORWIN

!e

FIG. 11. Zone F. There is no acid phosphatase activity in the sloughing inner root sheath cells IR) or in the cell debris (CD) in the pilary canal (PC). Acid phosphatase, x 24 000.

Fro. 12. Zone E. The intercellular material of both the infoldings of companion celt (Comp) plasma membranes (arrows) and apposed Henle's (He)-companion cell plasma membranes typically lack acid phosphatase activity. Acid phosphatase, x 85 000.

ACID PHOSPHATASE IN FORMING HENLE'S CELLS 333

of these cell lines may conform to the Golgi-ER-lysosomal (GERL) type of neu- rons (8, 17, 18).

The results indicated little development of the lysosomal system during differentia- tion of both Henle's and ORS cells (up to zone D). In contrast to the cortex (21), these cell lines showed fewer small smooth-membraned vesicles and profiles of ER with AcPase activity. Together with the general low level of AcPase activity this suggests that the lysosomal systems of these cell lines may not play as extensive a role in differentiation as in the cortex.

For both cell lines, the lower level of AcPase activity compared with the cortex confirms the findings of histochemical studies of both wool (25) and hair (5) folli- cles; a biochemical study of a-naphthyl acid phosphatase has also demonstrated a lower level of enzyme activity in these cell lines (15).

Although the morphological events leading to the hardening of Henle's cells are generally agreed upon (4, 13, 14, 22, 23) the fate of the cytoplasm and its organ- elles remains controversial. Cytoplasmic components have been observed just prior to hardening and during hardening (23). However, Parakkal (22) has stated that cytoplasmic components were degraded and partially eliminated prior to harden- ing. While the presence of AcPase in Henle's cells indicates that some degrada- tion probably occurs during differentia- tion, this study confirms that cytoplasmic components are present just prior to hardening. If these components are to be degraded, the means are not clear.

The lack of AcPase activity in the lumen of dilated rough ER in Henle's cells sug- gests that this form of ER is probably not part of the lysosomal system. The role that these regions of ER play in the differentia- tion of Henle's cells is also not clear.

Little evidence was found in this study to support lysosomal degradation of the IRS in the upper regions of the follicle (zones E and F). AcPase activity was not observed in degrading cells of the IRS. There was no evidence that the lysosomal system of companion cells (19) transported AcPase to Henle's cells. The infoldings of companion cell plasma membranes, which may be involved in the transport of ex- tracted material from the IRS to the ORS (14), very rarely showed AcPase activity. However, since the absence of AcPase ac- tivity cannot be taken as proof of the ab- sence of lysosomal enzymes (2 7), the possi- bility of lysosomal involvement in IRS breakdown cannot be entirely excluded.

The presence of large organelles con- taining AcPase activity in ORS cells in the region where the ORS sloughs (zone G) has been demonstrated in this study. They probably correspond to the granules show- ing AcPase activity found in histochemica! studies of similar regions of hair follicles (12, 24). These organelles may represent residual bodies resulting from previous au- tophagic processes of cornifying and sloughing ORS cells (1, 14, 26).

The skilled assistance of Mr. R. W. Thomson is gratefully acknowledged. Many thanks are due to Mrs. Joanna Orwin and to Dr. L. F. Story for criti- cally reading the manuscript .

Fro. 13. Zone E. On the rare occasion tha t acid phosphatase activity is present in the region shown in Fig. 12, i t is found in the intercel lular mater ia l of apposed Henle's (He)-companion cell (Comp) plasma membranes and the lower par t of companion cell p lasma membrane infoldings (arrows). Acid phosphatase. × 34 000.

FIG. 14. Phosphotungst ic acid s ta in ing has increased the electron density of the intercel lular mater ia l (arrowheads) of both companion (Comp) and Henle's (He) celL,~. Two other organelles which show phospho- tungstic acid uptake are indicated (arrows). Phosphotungstic acid. × 31 000.

FIG. 15. Zone F outer root sheath cell. Acid phosphatase activity is present in several large organelles (Ly). The cytoplasm shows signs of vacuolation and disorganization. Acid phosphatase. × 20 000.

334 D. F. G. ORWIN

REFERENCES

1. AUBER, L., Trans. Roy. Soc. Edinburgh 62, 191 (1950).

2. BABAI, F., AND BERNHARD, W., J. Ultrastruct. Res. 37, 601 (1971).

3. BARKA, T., AND ANDERSON, P. J. , Histochemis- try. Theory, Practice, Bibliography, p. 240. Harper (Hoeber), New York, 1963.

4. BIREECK, M. S. C., AND MERCER, E. H., J. BiG- phys. Biochem. Cytol. 3, 223 (1957).

5. BRAUN-FALcO, O., in MONTAGNA, W., AND EL- LIS, R. A. (Eds.), The Biology of Hair Growth, p. 65. Academic Press, New York, 1958.

6. CHAPMAN, R. E., J. Cell Sci. 9, 791 (1971). 7. CHASE, H. B., RAUCH, H., AND SMITH, V. W.,

Physiol. Zool. 24, 1 (1951). 8. DECKER, R. S., J. Cell Biol. 61, 599 (1974). 9. DE DUVE, C., AND WATTIAUX, R., Ann. Rev.

Physiol. 28, 435 (1966). 10. DE DUVE, C., in DINGLE, J. T., AND FELL, H. B.

(Eds.), Lysosomes in Biology and Pathology, Vol. I, p. 3. North-Holland, Amsterdam, 1969.

11. DERMER, G. B., J. Ultrastruct. Res. 45, 183 (1973).

12. DIENGDOH, J. V., Quart. J. Microseop. Sci. 105, 73 (1964).

13. FRASER, R. D. B., MACRAE, T. P., AND ROGERS, G. E., Keratins, Their Composition, Struc- ture and Biosynthesis, p. 165. Thomas, Illi- nois, 1972.

14. GEMMELL, R. T., AND CHAPMAN, R. E., J. Ultra- struct. Res. 36, 355 (1971).

15. IM, M. J. C., AND HOOPES, J. E., J. Invest. Der- matol. 57, 184 (1971).

16. LEDUC, E. H., AND BERNHARD, W., J. Ultra- struct. Res. 19, 196 (1967).

17. NOVIKOFF, A. B., ESSNER, E., AND QUINTANA, N., Fed. Proc. 23, 1010 (1964).

18. NOVIKOFF, P. M., NOVIKOFF, A. B., QUINTANA, N., AND HAUW, J-J. , J. Cell Biol. 50, 859 (1971).

19. ORWIN, D. F. G., AuNt. J. Biol. S c i . 24, 989 (1971).

20. ORWIN, D. F. G., in SANDERS, J. V., AND GOOD- CHILD, O. J. (Eds.), Proe. 8th Internat. Con- gress on Electron Microscopy, Vol. If, p. 380. Australian Academy of Science, Canberra, 1974.

21. ORWIN, D. F. G., J. Ultrastruct. Res. 55, 312 (1976).

22. PARAKKAL, P. F., Adv. Biol. Sk in 9, 441 (1969). 23. ROTH, S. I., AND HELWIG, E. B., J. Ultrastruct.

Res. 11, 33 (1964). 24. ROWDEN, G., J. Invest. Dermatol. 49, 181 (1967). 25. RYDER, M. L., Quart. J. Microscop. Sci. 99, 221

(1958). 26. STRAILE, W. E., J. Exp. Zool. 150, 207 (1962). 27. VAN DUIJN, P., in WISSE, E., DAEMS, W. TH.,

MOLENAAR, I., AND VAN DUIJN, P. (Eds.), Electron Microscopy and Cytochemistry, p. 3. North-Holland, Amsterdam, 1974.