JOURNAL OF ULTRASTRUCTURE RESEARCH 55, 335-342 (1976)

Acid Phosphatase Distribution in the Wool Fo!licle

III. Fate of Organel les in Keratinized Cells

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

Recognizable cell organelles are still present in both the keratinized fiber and the hardened inner root sheath of the Romney wool follicle. In the cortex, single membrane-bounded bodies and vesicles can be detected in regions of cytoplasmic remnants. The size of these organelles is similar to that of the lysosomes of the differentiating cortex. The fiber cuticle also contains single membrane-bounded organelles in the endocuticle. These are less numerous than in the cortex. Lysosomes, mitochondria, gap junction-bounded vesicles, and vacuolar-like organelles are identified in hardened inner root sheath cells.

E v e r s ince t h e e a r l y s t u d i e s of t h e u l t r a - s t r u c t u r e of k e r a t i n i z i n g a n d k e r a t i n i z e d wool a n d h a i r f ibe r s , t h e i n c o r p o r a t i o n of c y t o p l a s m i c a n d n u c l e a r d e b r i s i n to t h e f ibe r h a s b e e n r e c o g n i z e d (3, 4, 6, 7, 11, 2 1 - 2 3 ). I n o s m i u m - s t a i n e d f i be r s t h i s de- b r i s fo rms a n e l e c t r o n - d e n s e a m o r p h o u s m a t e r i a l l y i n g b e t w e e n m a c r o f i b r i l s . L i t - t l e a t t e n t i o n h a s b e e n p a i d to t h e c o n t e n t or o r i g i n of t h i s d e b r i s a s i t h a s b e c o m e accep t ed t h a t c y t o p l a s m i c c o m p o n e n t s a r e d e g r a d e d a n d m o s t l y r e m o v e d be fore k e r a - t i n i z a t i o n is c o m p l e t e (14).

T h e p r e c e d i n g p a p e r s in t h i s s e r i e s (1 7, 18) h a v e p r e s e n t e d e v i d e n c e for a lysoso- m a l s y s t e m in b o t h f i b e r - f o r m i n g a n d in- n e r roo t s h e a t h cel l l i ne s p r i o r to t h e i r

k e r a t i n i z a t i o n or h a r d e n i n g . A s i t b e c a m e a p p a r e n t d u r i n g t h e s e s t u d i e s t h a t t h e s t a i n i n g m e t h o d s e m p l o y e d d e l i n e a t e d ly- s o s o m e - l i k e a n d o t h e r s t r u c t u r e s in fu l ly k e r a t i n i z e d a n d h a r d e n e d cel ls , a m o r e de- t a i l e d s t u d y w a s u n d e r t a k e n of t h e f a t e of v a r i o u s o r g a n e l l e s d u r i n g a n d a f t e r k e r a - t i n i z a t i o n or h a r d e n i n g .

MATERIALS AND METHODS

Romney wool fibers were fixed in Karnovsky's and osmium fixatives, stained with uranyl acetate, and embedded in epoxy resin as described in Part I of this series (17). Some follicles were incubated to demonstrate acid phosphatase (AcPase) activity as before. This was done not to show sites of AcPase

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

activity in particular, but because the treatment resulted in marked nonspecific lead deposition in certain components of keratinized fibers.

Phosphotungstic acid (PTA) staining at low pH was carried out on glycol methacrylate-embedded follicles as described previously (17).

The zones used in this paper are the same as those described in Parts I and II (17, 18). For the cortex and fiber cuticle, zones E, F, and G were examined, while for Henle's layer of the inner root sheath (IRS) zones C, D, E, and F were studied.

RESULTS

Cortex a n d F iber Cut ic le

I n l o w e r r e g i o n s of zone E, c o r t e x cel ls s t i l l c o n t a i n e d i d e n t i f i a b l e c y t o p l a s m i c c o m p o n e n t s a m o n g s t t h e c o a l e s c i n g mac - ro f ib r i l s (Fig. 1). A l t h o u g h t h e r e w a s dif- f i cu l ty in i d e n t i f y i n g some c o m p o n e n t s , such s t r u c t u r e s a s r i b o s o m e s , s i n g l e m e m - b r a n e - b o u n d e d bod ies , a n d a u t o p h a g i c v a c u o l e s cou ld be d e t e c t e d (Fig . 1). A t a l a t e r s t a g e of m a c r o f i b r i l fusion.~ r ibo- somes , s i ng l e m e m b r a n e - b o u n d e d bod i e s ( d i a m e t e r s ca. 110-200 nm) , a n d ves i c l e s ( d i a m e t e r s ca. 60-90 nm) could s t i l l be i d e n t i f i e d b e t w e e n t h e m a c r o f i b r i l s (F ig . 2).

By t h e m i d d l e of zone E, t h e f i n a l d e g r e e of m a c r o f i b r i l l a r f u s ion s e e m e d to h a v e b e e n r e a c h e d . A t t h i s s t age , i d e n t i f i c a t i o n of c y t o p l a s m i c c o m p o n e n t s b e c a m e m o r e d i f f i cu l t due to c h a n g e s in t h e a p p e a r a n c e

335

336 D . F . G .

of m e m b r a n e s . These resul ted in only the nonosmiophil ic l amel lae of the un i t m e m - b rane being dis t inguishable . Despi te this, s ingle m e m b r a n e - b o u n d e d tubules , bodies, and vesicles of va ry ing size (d iameters 60- 1500 nm) could be seen embedded in an electron-dense a m o r p h o u s ma te r i a l , pre- sumab ly der ived f rom cytoplasmic compo- nents such as r ibosomes (Fig. 3). In gen- eral , these a reas out l ined the more elec- t ron- lucent macrof ibr i l s in both pa racor tex and or thocortex cells.

Single m e m b r a n e - b o u n d e d s t ruc tu res could be seen in the endocut icular regions of kera t in ized cuticle cells. They tended not to be as n u m e r o u s nor as well defined as in the cortex (Fig. 4).

The enhanced cont ras t resu l t ing f rom incuba t ing fibers for AcPase ac t iv i ty is shown in Fig. 5. Cytoplasmic r e m n a n t s were heav i ly s ta ined and single m e m - brane-bounded bodies and other organel les were del ineated. Some of these organel les could be t raced th rough three or more se- r ial sections. In the ful ly kera t in ized fiber (zone G), the e n h a n c e m e n t of the ma t r i x of macrofibr i ls was not so marked . Never the - less, the cytoplasmic r e m n a n t s showed va ry ing degrees of electron densi ty and m a n y single m e m b r a n e - b o u n d e d bodies were presen t (Fig. 6). Areas of low electron densi ty which m a y represen t fo rmer vacu- olar spaces were occasionally found.

Phosphotungs t ic acid s ta in ing demon- s t ra ted discrete bodies (d iameter 70-130 nm), often grouped toge the r in the cortex. Smal l regions of the in te rce l lu la r ma te r i a l

ORWIN

showed some e n h a n c e m e n t of electron densi ty (Fig. 7).

The in te rce l lu la r ma te r i a l be tween ap- posed cortex cells r e ta ined AcPase ac t iv i ty in zones E (Fig. 8), F, and G (Fig. 6). This act ivi ty was inhibi ted by the addi t ion of sodium fluoride to the incubat ion m e d i u m (Fig. 9).

Inner Root Sheath

In the ha rdened IRS, both the conven- t ional and the cytochemical p repa ra t ions revealed the presence of organel les in all cell layers . These var ied f rom single mem- brane-bounded bodies (d iamete r ca. 160 nm) still showing AcPase ac t iv i ty a f te r ha rden ing (Fig. 10) to those lacking such act ivi ty and found dur ing more advanced s tages of degrada t ion pr ior to sloughing. In this ca tegory were single m e m b r a n e - bounded bodies (Figs. 11 and 14), mito- chondria (Fig. 12), vacuolar - l ike bodies wi th f ibr i l lar contents (Fig. 13), and gap junct ion-bounded vesicles (Figs. 14 and 15), some wi th r ibosome-l ike contents (Fig. 15).

DISCUSSION

Dur ing the l a te r s tages of cortex differ- en t ia t ion (lower zone E), cytoplasmic com- ponents become increas ingly condensed and even tua l ly fill the varying-s ized spaces be tween the macrof ibr i ls (3, 12, 13, 23). In genera l , the presence of r ibosomes be tween the macrof ibr i ls has been noted dur ing this process. The p resen t s tudy can add lysosome-l ike bodies, vesicles, and au-

FIa. 1. Cortex cell in lower zone E. This cross section shows macrofibrils (M) in various degrees of fusion. Many ribosomes (R) appear to be condensed in the intermacrofibrillar spaces. An autophagic vacuole (AV) containing ribosomes and an unidentifiable organelle (arrow) are present. CM, cell membrane complex, x 57 000.

FIG. 2. Cortex cell in lower zone E. This cross section shows a later stage of cortex differentiation than Fig. 1. The intermacrofibrillar spaces contain condensed ribosomes (R) and single membrane-bounded organelles (arrows). Some ribosome-like components (arrowheads) appear trapped between the fusing macrofibrils (M). × 77 000.

Fia. 3. Cortex cell in mid zone E. Fusion of the macrofibrils (M) has been completed and the intermacro- fibrillar spaces contain an electron-dense material (arrowhead). Within this material, single membrane- bounded organelles of varying size are present (arrows). The appearance of the single membranes is similar to those of the cell membrane complex (CM). × 48 000.

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338

ORGANELLES IN KERATINIZED WOOL CELLS 339

tophagic vacuoles to the organelles pres: ent. Mitochondria and endoplasmic reticu- lum-like structures were present but were not positively identified.

The manner in which any degradation of the cytoplasm and its organel!es: takes place (14, 20) has not been resolved by this study. Evidence of significant lysosomat autophagic processes as found in the lower cortex (17) and other tissues (8, 9) was not observed in lower zone E. However, other degradative mechanisms may be operative (16).

The presence of lysosome-like organelles in the keratinized fiber suggests that some of these at least are probably residual bod- ies derived from lysosomal activities prior to zone E in the differentiating cortex. Their presence in the fiber represents a possible pathway by which substances could be eliminated from the body. Sup- port for the presence of nonkeratinous structures in the fiber is given by the up- take of PTA by discrete bodies in the kera- tinized cortex. As PTA uptake indicates glycoproteins in part (2, 10), these bodies may represent lysosomes since lysosomal enzymes may be glycoproteins (15).

The exact nature of the apparently mod- ified membranes of entrapped organelles in keratinized cells is unknown. How- ever, the presence of membranes other than the plasma membrane this high in the follicle suggests that assigning lipids extracted from wool fibers to cell mem- brane complexes alone may be misleading (5).

The evidence presented in this paper suggests that the whole of zone E should be regarded as the zone where consolida- tion of the fiber takes place. In the region where the IRS becomes completely har~ened, i.e., at the beginning of zone E, keratinization of the cortex occurs or is at least initiated (1, 7). This region can be termed the level of histochemical keratini- zation. It marks the beginning of the zone (lower E) where the remaining cytoplas- m i c organelles appear to become en- trapped by the ~fusion" of fully formed macrofibrils (3, 12, 13, 23). A further zone (upper E) is characterised by osmium up- take (consolidation zone of (7)), indicating that the macrofibrils are still undergoing chemical modification. The upper level of' this zone is considered the limit of ultra- structural keratinization as further differ- entiation of the fiber has not been detected by electron microscope techniques.

Although the differentiation of the cuti- cle is somewhat similar to that of the cor- tex, macrofibrillar fusion does not occur as the cuticle proteins are laid down in a different manner (4). The less extensive development of the lysosomal system and the lower degree of autophagy are re- flected in the smaller number of single membrane-bounded organelles found in the cytoplasmic remnants, i.e., the endo- cuticle (4, 22).

In the IRS, organelles present in the cells prior to hardening (18) are trapped in the transformed trichohyalin. In the ini- tial stages, the lysosomes can retain Ac-

FIG. 4. Fiber cuticle cell in mid zone E. Membrane-bounded organelles (arrows) are present in the endocuticle (En) close to the exocuticle (Ex). Co, cortex. × 57 000.

FIG. 5. Upper zone E cortex cell. Incubation for acid phosphatase activity has increased the electron density of the matrix of macrofibrils (M) and the cytoplasmic remnants (arrowheads). Single membrane- bounded organelles of varying size are present in the cytoplasmic remnants (arrows). CM, cell membrane complex. × 82 000.

Fro. 6. Keratinized cortex cell in zone G. Incubation for acid phosphatase does not enhance the electron density of the matrix of macrofibrils (M) to the extent that it does in zone E. The cytoplasmic remnants show a vacuolar-like region (Va) and regions of varying electron density. Single membrane-bounded bodies can be detected in some of these regions (arrows). The intercellular material (arrowhead) of apposed cortex cells shows acid phosphatase activity. Acid phosphatase. × 60 000.

Fro. 7. Cortex cell in upper zone E. Phosphotungstic acid has stained three bodies in the cell. There are also indications of uptake in the intercellular material of apposed cortex cells (arrowhead). Phosphotungstic acid. x 46 000.

340 D. F. G. ORWIN

ORGANELLES IN KERATINIZED WOOL CELLS 341

FIG. 12. Hardened Henle's cell in lower zone F. A mitochondria-like organelle is present (arrow). N, nucleus. Acid phosphatase, x 64 000.

FIo. 13. Hardened Henle's cell in zone E. This cell contains two vacuolar-like organelles (arrowheads), whose contents differ markedly from the transformed trichohyalin surrounding them. × 38 000.

FIo. 14. Hardened Henle's cell in lower zone E. A gap junction-bounded vesicle (arrowhead) and a single membrane-bounded body (arrow) are present. Comp, companion cell. x 48 000.

FIG. 15. Hardened Huxley's cell in lower zone E. This gap junction-bounded vesicle contains ribosome- like material, x 45 000.

FIG. 8. Cortex cell in lower zone E. Acid phosphatase activity is present in the intercellular material (arrowhead) of apposed cortex cells. The enhanced electron density of the matrix and cytoplasmic remnants as a result of this t rea tment is apparent. Acid phosphatase, x 57 000.

FIG. 9. Cortex cell in lower zone E. The effect of sodium fluoride on sites of acid phosphatase activity is demonstrated in these cells. While the intercellular material (arrowhead) shows inhibition of acid phospha- tase activity, the matrix of macrofibrils and the regions of cytoplasmic remnants (CR) still show nonspecific uptake of lead. Acid phosphatase, x 61 000.

FIG. 10. Hardened Henle's cell in zone C. Acid phosphatase activity is still present in the single membrane-bounded body (Ly). N, nucleus. Acid phosphatase, x 57 000.

FIG. 11. Hardened Huxley's cell in lower zone F. The degradation and extraction of the cell contents has revealed the presence of two organelles (arrows). Acid phosphatase, x 40 000.

342 D. F. G. ORWIN

Pase activity, but the enzymes must be modified during the passage of the organ- elleN towards the skin surface as such ac- tivity has not yet been detected in the upper regions of the IRS. In regions where degradation of hardened IRS cell contents is apparent, mitochondria-like, vacuolar- like, and gap junction-bounded vesicles can be detected as well. All these organ- elles are presumably eliminated into the pilary canal when the IRS cells slough. As in the cortex, they represent possible path- ways by which substances can be elimi- nated from the body.

The presence of gap junction-bounded vesicles, often with recognizable contents, is unusual. Their presence can be taken as evidence that they pinch off from gap junc- tions during differentiation of the IRS as has been suggested (19).

The author is pleased to acknowledge the skilled assistance of Mr. R. W. Thomson. The editorial ser- vices of Mrs. Joanna Orwin and Dr. L. F. Story are gratefully acknowledged.

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