Arch Dermatol Res (1996) 288: 373382

Springer-Verlag 1996

O R I G I N A L PA P E R

J. J. Bond P. C. Wynn G. P. M. Moore

Effects of epidermal growth factor

and transforming growth factor alpha on the function
of wool follicles in culture

Received: 19 June 1995

Abstract The development of a procedure to culture

wool follicles from Merino sheep in serum-free conditions has enabled us to investigate the actions of epidermal growth factor (EGF) and transforming growth
factor alpha (TGF) on follicle function, including fibre growth. Follicles grown in the absence of growth
factors maintained their anagen morphology for 6
days as determined by light microscopy. During this
time they incorporated [3H]thymidine into the DNA of
the bulb matrix and outer root sheath (ORS) cells and
produced fibre keratins as detected by immunohistochemistry. In the presence of EGF and TGF, fibre
production ceased after 4 days, as it does following the
administration of EGF in vivo. Cessation of fibre
growth was not accompanied by regression of the follicle bulb which occurs in vivo. Follicle length growth
did not differ significantly from controls and cells in
the bulb continued to proliferate. Usually, the structure of the dermal papillae resembled that in control
follicles, which was also in marked contrast to changes
reported in vivo. In EGF- and TGF-treated follicles,
[3H]thymidine continued to be incorporated into DNA
of the ORS and bulb after fibre growth ceased. Although wool keratin synthesis ceased, cytokeratins of
the epidermis and ORS continued to be produced in
the bulb as detected by immunochemistry. These bulb
cells were also positive for the periodic acid-Schiff
(PAS) reaction indicating the presence of glycogen, a
normal component of ORS cells. The observations that

J. J.Bond P. C. Wynn G. P. M. Moore

Department of Animal Science, University of Sydney,
Camden, NSW 2570, Australia
J. J. Bond (Y)
CSIRO, Division of Animal Production, Locked Bag 1,
Delivery Centre, Blacktown, NSW 2148, Australia
G. P. M. Moore
Department of Biological Sciences,
University of Western Sydney, Nepean, Kingswood,
NSW 2747, Australia

cell proliferation continued in the bulb, that glycogen

was present and that soft keratins were expressed in
these cells suggest that the bulb cell population was induced to differentiate into an ORS phenotype by EGF
and TGF.
Key words Wool follicle culture EGF TGF

Introduction
In the mature wool follicle, cells of the bulb matrix undergo active proliferation and then move distally, differentiating into the layers of the fibre and follicle sheaths.
These include the cortex and cuticle of the fibre, and the
inner (IRS) and outer root sheaths (ORS) of the follicle. It
is becoming increasingly clear that a number of growth
factors are involved, not only in the proliferation of the
bulb cells, but also in the complex pathways of differentiation that give rise to the fibre and the root sheaths.
Members of the epidermal growth factor (EGF) family
have been implicated in normal follicle function. Expression of transforming growth factor alpha (TGF) has been
identified in the IRS [24] and inactivation of the TGF
gene in the mouse, by homologous recombination, gives
rise to a waved hair phenotype [23, 25]. Furthermore, an
EGF-like molecule has been detected in the ORS of the
wool follicle [7] and TGF in the human hair follicle [10]
by immunochemistry. Large polypeptides with a number
of EGF-like repeats, such as Notch, are also expressed in
hair follicle bulb cells of the mouse [21]. The EGF receptor (EGF-R) which binds both EGF and TGF [26] has
been detected in the cells of the root sheaths and bulb of
the wool follicle [49]. More recently, expression of the
EGF-R has also been localised to the ORS and follicle
bulb [24].
In mice, which exhibit pronounced patterns of cyclical
hair growth, injections of EGF or TGF after birth inhibit
the rate of hair growth and reduce hair diameter [28, 30,
46]. Similarly, in transgenic mice which overexpress TGF,
the epidermis is thicker and hair growth inhibited [47]

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when compared to control mice. Administration of EGF

to sheep induces thickening of the epidermis [29, 31] and
inhibits fibre formation and growth in anagen follicles. A
transient regression of the wool follicle population is observed and the fleece is shed [28]. Morphologically, the
wool follicles appear to enter a phase of the growth cycle
similar to catagen [5], causing a weakness to develop in
the fibre [18]. EGF-induced depilation of the angora rabbit [32] probably occurs by a similar mechanism.
Previous studies have investigated the effects of EGF
on cells derived from the skin in tissue culture. These
have shown that EGF and TGF are potent mitogens for
epidermal keratinocytes [42]. The development of culture
procedures for human hair [41] and wool follicles [2]
which support fibre growth has facilitated studies of the direct influences of growth factors, known to occur in the
skin, on follicle function.
In the present study we examined the effects of EGF
and TGF on the proliferation and differentiation of bulb
cells into wool fibres in isolated Merino sheep follicles in
vitro. The results of this study are more directly comparable to those observed in vivo for the sheep, since it is in
this species that the role of EGF in the regulation of follicle function has been studied in greatest detail. Harmon
and Nevins [15] and Philpott + Kealey [41] have reported
responses to EGF of isolated human hair follicles.

Experimental design and analysis of data

Effects of different concentrations of EGF
The effects of two different concentrations of EGF on fibre growth
and follicle morphology were analysed over 6 days. Hollis and
Chapman [18] reported marked between-sheep variation in response to low doses of EGF administered in vivo. Therefore, follicles were harvested from six animals using a balanced randomized
block design for statistical analysis of the data [44]. Follicles (n =
24) dissected from skin samples were transferred to a 24-well culture plate divided into three treatment groups (0, 1 and 10 ng/ml
EGF). Four skin samples were collected from each sheep.
Comparison of effects of EGF and TGF
In a subsequent experiment to compare the effects of EGF and
TGF on fibre growth and follicle morphology, skin was harvested from four sheep. Follicles dissected from each skin piece
were divided into three treatments per culture plate (0, 10 ng/ml EGF
or 10 ng/ml TGF). The protocol was repeated three times for
each sheep, using the design described above.
Histology and immunohistochemistry
Fresh and cultured wool follicles were fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4, dehydrated in
ethanol, cleared in propylene oxide and embedded in Araldite. Sections were cut at a thickness of 1 m on an ultramicrotome (LKB)
and stained with a 1:1 mixture of 1% azure II and 1% methylene
blue.

Materials and methods

Detection of glycogen

Isolation, culture and assessment of wool follicles

Glycogen was detected in follicular sections by the periodic acidSchiff (PAS) reaction and azure II and methylene blue were used
as counterstains. For the determination of glycogen, control sections were digested with 1% malt diastase solution at 37 C prior to
the PAS reaction.

Follicles were microdissected from midside skin samples of medium wool Peppin Merino wethers (fibre diameter 2223 m)
aged between 3 and 4 years. The dissection method was similar to
that described previously [2], except that skin pieces were not initially immersed in 70% alcohol. Individual follicles were placed
into 24-well tissue culture plates (Flow laboratories, Costamesa,
CA), containing 1 ml serum-free Williams E medium supplemented
as described previously [2]. The cultures were maintained at 37 C
in an atmosphere of 5% CO2 and 95% air. Follicles were examined
daily using an inverted microscope (TMS, Nikon). Follicle viability over 6 days in culture was assessed on the basis of morphology
and length growth. Follicles that were obviously damaged during
dissection, were morphologically abnormal or did not grow more
than 200 m over 6 days in culture, were discarded.
Changes in follicle length were measured using a graduated
eyepiece at a magnification of 10 . Follicle length was defined as
the distance between the tip of the protruding fibre and the base of
the follicle. Fibre growth was assessed by the difference in follicle
length between the day of dissection (day 0) and the end of the culture period (day 6). The diameter of the follicle bulb was also measured at its widest point, at right angles to the axis of the dermal
papilla.
Growth factors
EGF was isolated from the submandibular glands of adult male
mice according to the method of Savage and Cohen [43]. Human
recombinant TGF was purchased from Collaborative Research
(Bedford, Mass., USA). EGF or TGF were added on day 1 of culture. The culture medium supplemented with growth factors was
renewed on day 4.

Immunohistochemistry of keratins
For immunohistochemical detection of keratins, Araldite sections
were etched with sodium ethoxide, rehydrated and placed in Trisbuffered saline and Tween-20 (TBST) solution. The tissue was
then treated with 3% bovine serum albumin for 1.5 h at room temperature to block nonspecific protein binding and incubated with
the low-sulphur keratin monoclonal antibody K6 (1:1) [12] or the
epithelial soft keratin antibody AE1/AE3 (1:10; Boehringer,
Mannheim, Germany) for 18 h at 4 C in a humidified chamber.
Primary antibody binding was detected following incubation with
a fluorescent antisheep IgG marker (1:30; Wellcome, UK).
Western blots of keratins
Follicular keratins were extracted according to the procedure of
Marshall [27]. They were S-carboxymethylated with 3 M iodoacetic acid, dialysed against 0.05 M NH4OH, freeze dried and resuspended in 5 M Tris-HCl buffer (pH 6.8) containing 4% SDS
and 5% 2-mercaptoethanol. The samples were heated in a boiling
water bath for 3 min, centrifuged and the supernatant analysed by
SDS PAGE [22] using the mini-protean II (BioRad, Hercules,
Calif., USA) system. The proteins were separated on a 12.5%
polyacrylamide gel with a 3% stacking gel and stained with
Coomassie Brilliant Blue or silver.
Immunoblot analysis of cytokeratins was carried out by electrophoretic transfer from the SDS gels to a PVDF membrane (BioRad) using an LKB Novablot transfer unit (Pharmacia, Uppsala,

375
Sweden). Nonspecific binding was blocked with 3% bovine serum
albumin (fraction V) in TBST. Sections were then incubated with
monoclonal antibodies to cytokeratin proteins, AE1/AE3 (Boehringer)
for 2 h. This antibody has been shown to be a marker for differentiating epidermal cells in the human epidermis [9, 48] and ORS
[17]. Antibody binding was visualized after incubation with alkaline phosphatase-conjugated antimouse IgG marker (Wellcome).
3H-Thymidine

labelling of follicles

The distribution of mitotically active follicle cells was examined

by incubating follicles for 8 h with 2 Ci/ml [methyl-3H]thymidine
(Amersham, Amersham, UK). Radioactive incorporation was assessed on days 2, 4 and 6 of culture. To trace the movements of
cells, follicles were pulse labelled for 1 h with 3H-thymidine (2 Ci/
ml) on day 2 of culture and, following removal of the label, were
harvested on day 6. Labelled follicles were processed for autoradiography following embedding in Araldite. Slides were coated with
Ilford K2 nuclear emulsion and stored in light-tight boxes. After
development and fixation of the emulsion, the sections were
stained with azure II and methylene blue.

Results
Growth of wool follicles in culture
Isolated wool follicles maintained their anagen morphology and grew a fibre in serum-free medium for 6 days in
culture (Fig. 1). The mean length of control follicles (Fig.
2) increased in culture, linearly during the first 4 days and
at a decreased rate over the remaining 2 days. Approximately 6070% of the follicles dissected and placed in
serum-free medium maintained their morphology, and increases in follicle length were associated with fibre
growth. The diameter of the follicle bulb increased during
day 1 and then remained relatively constant for the remainder of the culture period (Fig. 3).

Fig. 1 Wool follicle grown in serum-free supplemented Williams

E medium for 6 days. Araldite section (1 m) stained with azure II/
methylene blue (IRS inner root sheath, ORS outer root sheath, DP
dermal papilla, bar = 50 m)

Effects of EGF and TGF on cultured follicles

Morphological changes
In the presence of 1 or 10 ng/ml EGF, follicle length increased and was not significantly different from that of
controls during the culture period (Fig. 2). Fibre growth
from EGF-treated follicles ceased but this was not accompanied by regression of the follicle bulb. Bulb diameter
increased in controls and in EGF-treated groups during
culture, but not significantly (Fig. 3). The dermal papilla
did not usually change in appearance or location (Fig. 4)
and increases in follicle length in the presence of EGF
were due to continued cell division in the bulb and the
progressive movement of the fibre out of the follicle
sheath. Apoptotic bodies were not observed by light microscopy in the bulb of these follicles. The fibre developed a tapered end after 4 days (Fig. 5).
Although EGF did not greatly affect the follicle length
compared with controls, inhibition of fibre growth always
occurred with 10 ng/ml EGF. The results using 1 ng/ml
EGF showed greater between-sheep variation. Some folli-

Fig. 2 Changes in mean length (m SEM) of follicles grown in

the absence (squares) and presence of different concentrations of
EGF (triangles 1 ng/ml, diamonds 10 ng/ml) for 6 days

cles in this treatment group did not completely cease fibre

production.
Comparisons of the effects of 10 ng/ml EGF and
TGF on follicle length are shown in Fig. 6. The length of
TGF-treated follicles did not differ significantly from
those treated with EGF and increases in follicle length
were not associated with continued fibre growth. Bulb diameter tended to increase during the latter part of the culture period and a significant increase was observed in

376

Fig. 3 Effects of EGF concentration on bulb diameter (m SEM)

of follicles cultured for 6 days. The bulb did not regress although
fibre growth ceased control crosshatch, EGF 1 ng/ml stripe, EGF
10 ng/ml black

Fig. 5 Median longitudinal section of a follicle cultured with EGF

(10 ng/ml) stained with azure II/methylene blue displaying a fibre
with a tapered end (F) (bar = 70 m)

Fig. 4 A follicle treated with EGF (10 ng/ml) for 6 days of culture,
demonstrating formation of a tapered-end fibre (F) and retention of
the lenticular structure of the dermal papilla (DP) outlined at the
base of the follicle (bar = 50 m)

Fig. 6 Comparison of length (m SEM) of follicles cultured for

6 days in the presence of EGF (10 ng/ml, triangles), TGF (10
ng/ml, diamonds) and controls (squares). There were no significant differences between the treated follicle groups and controls

EGF-treated follicles on day 6 when compared with control and TGF treatment groups (Fig. 7). The changes in
morphology of TGF-treated follicles at the end of 6 days
of culture (Fig. 8) were similar to those observed in the
presence of EGF (Fig. 4).
Outgrowths of ORS cells on the surface of the culture
dish were usually evident by day 3 of culture in all treatment groups. The sizes of the outgrowths and the fre-

quency with which they occurred were greater in EGFand TGF-treated follicles than in controls.
Intermediate filament keratins
The intermediate filament keratin antibody K6, which
binds to low-sulphur wool keratins, was detected in

377

tured for 6 days, the PAS reaction was observed in the majority of the bulb cells (Fig. 16), in addition to the ORS
characteristic staining. The presence of PAS-positive bulb
cells suggested either an ORS phenotype differentiation
or a repopulation of the bulb.
3H-Thymidine

Fig. 7 Bulb diameter (m SEM) of follicles cultured in the presence of 10 ng/ml EGF or TGF in comparison with controls. (control stripe, EGF 10 ng/ml hatched, FGF 10 ng/ml black)

suprabulbar cortical cells by immunofluorescence in sections of freshly isolated follicles and control follicles cultured for 6 days (Fig. 9). However, K6 reactivity was not
found in the lower bulb region of EGF- and TGF-treated
follicles. In these follicles staining was confined to the fibre-ends in the keratogenous region on day 6 (Fig. 10), indicating that synthesis of intermediate filament keratins
had ceased.
Cytokeratins
Expression of specific cytokeratins recognized by antibody AE1/AE3 in the remaining bulb cells was also investigated. In the present study the antibody specifically
reacted with the ORS and epidermis in sections of control
sheep skin and ORS cells of control follicles cultured for
6 days (Fig. 11).
After 6 days of culture with EGF and TGF, however,
bulb cells were found to bind this antibody (Fig. 12). The
identity of cytokeratins detected by AE1/AE3 was confirmed by western blot analysis of extracted follicles cultured with and without EGF. The antibody identified
bands between 40 000 and 68 000 Da in each extract (Fig.
13) which are comparable with the characteristic bands of
cytokeratins observed in human skin extracts [9, 17, 48].
The intensity of the staining in the sheep skin extracts was
greater than that of follicle extracts but the number and
size of the bands was the same in each extract.
Glycogen synthesis
The cytokeratin immunohistochemistry suggested that
cells in the proximal part of the follicle expressed an ORS
phenotype in the presence of EGF and TGF but not
without. We investigated this further using the PAS reaction for the detection of glycogen, a component typically
expressed in the ORS [39]. In control follicles after 6 days
in culture, glycogen was localized only in the ORS (Fig.
14). Follicles pretreated with malt diastase showed almost
no PAS staining (Fig. 15). In EGF and TGF follicles cul-

incorporation

Autoradiography revealed that the cells of the ORS and

bulb matrix of cultured follicles incorporated 3H-thymidine on day 2 (Fig. 17). The distribution of 3H-thymidinelabelled cells in control follicles on day 6 of culture was
the same as that on day 2. Since the bulb cells appeared to
have an ORS phenotype in the presence of EGF, pulse-labelling studies were carried out to determine whether they
were derived from either the upper ORS by migration or
from existing cells in the lower portion of the follicle
which had proliferated and differentiated. Follicles were
labelled for 8 h on day 2 of culture and harvested on day
6 for autoradiography and examination. In control incubations, label was observed in the bulb matrix, fibre cortex
and ORS (Fig. 18). This confirms that some of the cells
from the proliferative zone had migrated distally into the
keratogenous zone. In EGF- and TGF-treated follicles,
labelled cells were localized in the bulbar and suprabulbar
cells, indicating continued proliferation of the bulb cell
population (Fig. 19). The fact that the majority of labelled
cells were found in the bulb on days 2 and 6 of culture and
in the bulb of pulse-labelled follicles suggests that at least
some of the ORS-like cells of the bulb did not originate
from the upper ORS but proliferated in the matrix. That is,
EGF and TGF induced the continued proliferation of
cells in the lower portion of the follicle which differentiated and assumed an ORS phenotype after fibre growth
ceased.

Discussion
We have previously reported a procedure for the isolation
and culture of active wool follicles from Merino sheep
skin [2]. The method has been further refined to permit
culture in serum-free medium. This procedure enabled us
to exert greater control of the examination of the direct effects of hormones, growth factors and nutrient substrates
on wool follicle function. The mean length growth (Fig. 2)
of control follicles cultured in serum-free medium was
similar to that of follicles maintained in the presence of
serum [2]. We have previously shown that increases in
length of cultured follicles are associated with fibre elongation [2]. In this study, we investigated the effects of two
growth factors, EGF and TGF, on wool follicle function
and fibre synthesis in vitro.
Previous in vivo studies [31] have shown that EGF
inhibits fibre production in sheep. In this study we confirmed that both EGF and TGF elicit similar responses
when administered exogenously. Cessation of fibre growth
was not, however, accompanied by a significant reduction
in follicle length, and the follicle bulb did not undergo the

378

FIBRE

10

11

379
F Fig. 8 Longitudinal 1-m section of a cultured follicle grown for
6 days in the presence of TGF (10 ng/ml) showing that the
changes in morphology induced by TGF were similar to those
seen in EGF-treated follicles (Fig. 4). Stained with azure II/methylene blue (DP dermal papilla; bar = 50 m)
Fig. 9 Immunohistochemical detection of intermediate filament
keratins using monoclonal antibody K6 in a control follicle on
day 6 of culture. Fluorescent labelling in the suprabulbar and cortical cells (arrowhead) indicates that keratin synthesis has occurred (bar = 50 m)
Fig. 10 Immunohistochemical demonstration of hard keratin distribution in a follicle cultured with TGF (10 ng/ml) for 6 days using monoclonal antibody k6. Fluorescent labelling is confined to
the fibre-ends in the keratogenous region (arrow) (bar = 50 m)
Fig. 11 The localization of cytokeratin specific antibody (AE1/
AE3) (arrowhead) in the ORS of a control follicle on day 6 (bar =
50 m)

morphological changes usually associated with regression

in vivo. Indeed the cells of the bulb continued to proliferate and the keratinized fibre moved progressively further
out of the root sheath. Similar morphological observations
were recorded by Harmon and Nevins [15] and Philpott
and Kealey [40] in cultured human hair follicles. However, in these studies a significant increase in follicle
length was observed in EGF-treated follicles compared to
controls, but again this was not a result of continued fibre
growth. In addition, we also observed that some follicles
in the 1 ng/ml EGF treatment group did not completely
cease fibre production, an effect we have previously
found to be comparable to the development of a partial
break in the fleece, in vivo [19].
Following administration of EGF in vivo, mitotic activity of the bulb ceases 34 days after treatment [31] and
the dermal papilla contracts distally forming a rounded
aggregate [18]. Furthermore, follicle cells are removed by
the process of apoptosis during follicle involution [18].
These reductions in follicle cell numbers, together with

Fig. 12 Distribution of epidermal cytokeratins using monoclonal antibody AE1/AE3 in

the bulbar (B) and suprabulbar
(arrowhead) cells of an EGFtreated follicle on day 6 of culture (bar = 50 m)
Fig. 13 Western immunoblot of
S-carboxymethylated keratins
from skin and cultured follicles
using monoclonal antibody AE1/
AE3 to detect cytokeratin proteins. Prestained molecular
weight markers: myosin 200 kDa,
-galactosidase 118 kDa, bovine
serum albumin 78 kDa, ovalbumin 47 kDa, carbonic anhydrase
31.4 kDa, soybean trypsin inhibitor 25.5 kDa, lysozyme 18.8
kDa. Lane 1 Sheep skin extract,
lane 2 follicles (n = 40) grown
in culture in the absence of
EGF and lane 3 in the presence
of EGF (10 ng/ml) for 6 days

the fact that the follicle is anchored in the epidermis, contribute to the contraction of the proximal part of the follicle structure towards the surface of the skin.
It seems clear that factors other than EGF and TGF
cause the bulb to regress and the follicle to shrink after fibre growth ceases. The absence of apoptotic bodies in
EGF- and TGF-treated follicles in culture indicates that
the process of apoptosis was not initiated. Perhaps surprisingly, the cessation of fibre growth in vitro was not accompanied by the withdrawal of the dermal papilla and
regression of the follicle bulb. Interactions between the
dermal papilla and matrix are essential for fibre growth to
occur [3638]. In many instances the dermal papilla retained its lenticular shape and its position at the base of
follicles cultured in the presence of EGF and TGF. Furthermore mitotic activity was observed in the bulbar cells
even after fibre growth had ceased. As observed previously [31], there was a decline in mitotic activity of the
bulb cells in response to EGF and this coincided with an
increase in basal epidermal cell proliferation.
Our in vitro labelling studies indicated that proliferation of cells in the EGF- and TGF-treated bulbs continued after fibre growth had ceased. Pulse chase studies revealed that the majority of labelled cells originated from
cell division at the base of the follicle and the lower portion of the ORS. Similarly, Jiang et al. [20] reported a
thickening of the ORS in human hair follicle cultures
maintained in the presence of similar concentrations of
EGF. Although fibre growth in EGF-treated follicles
ceased, it is likely that the continued movement of the fibre out of the root sheaths was due to the upward and coordinated movement of the IRS and ORS cells. Labelling
studies in vivo [4] have shown that normal fibre growth
appears to be assisted by this mechanism. In addition, the
fibre-end moves upwards with the root sheaths to form a
club end proximal to the sebaceous glands during a characteristic catagen phase.

kDa
200
118
5

78
47

31
B

25
18

12

13

14

15

16

17

18

19

DP
B

381
F Fig. 14 Longitudinal section of a control follicle cultured for 6
days showing the detection of glycogen by the periodic acid Schiff
(PAS) reaction in the ORS (arrow) and counterstained with azure
II/methylene blue to define the fibre and bulb cells of the follicle
(bar = 50 m)
Fig. 15 Longitudinal section of a follicle treated with malt diastase prior to oxidation of carbohydrates showed no significant PAS
reactivity (bar = 50 m)
Fig. 16 Distribution of PAS-positive material indicating glycogen
synthesis in the bulbar (B) and suprabulbar (arrow) cells of an
EGF-treated follicle cultured for 6 days (bar = 40 m)
Fig. 17 Autoradiograph of the lower portion of a control follicle
on day 2 of culture showing incorporation of [3H]thymidine in the
bulb cells (lower arrow) and ORS (upper arrow) (bar = 50 m)
Fig. 18 An autoradiograph of a control follicle labelled with
[3H]thymidine for 1 h on day 2 of culture and harvested on day 6.
Labelled cells (arrows) are located in the bulb matrix (lower arrow), fibre cortex (C) and ORS (upper arrow) (bar = 50 m)
Fig. 19 An autoradiograph of an EGF-treated follicle pulse labelled with [3H]thymidine for 1 h on day 2 of culture and the follicle harvested on day 6. Labelled cells are localized in the bulbar
(B) and suprabulbar (arrow) regions of the follicle. No label is observed in the dermal papilla (DP) (bar = 40 m)

Furthermore, glycogen and keratins typical of the ORS

were detected in the bulb cells of EGF- and TGF-treated
follicles after fibre growth ceased. EGF and TGF exert
their action through EGFR which are concentrated in the
epidermis, ORS and matrix cells surrounding the dermal
papilla [13, 14, 3335, 49]. Recently, Luetteke et al. [24]
reported transcripts of EGFR in the bulb matrix of anagen
follicles. Expression of EGF and TGF has been detected
in the IRS and ORS of the follicle [24], which colocalizes
with glycogen deposits [39]. The stimulation of glycogen
synthesis by EGF has been demonstrated in cultured skin
cells and hepatocytes [3, 11]. Therefore it is possible that
EGF was involved in glycogen synthesis in some of the
bulb cells of EGF- and TGF-treated follicles.
The expression of keratins 6 and 16, which are consistently found in ORS cells in vivo and in vitro [45], are
specifically induced by EGF and TGF [20]. The detection of soft epithelial cytokeratins in the bulb cells after
EGF and TGF treatment indicates that these growth factors specified an epidermal pathway of differentiation.
It has been suggested that the regeneration of the follicle bulb during the transition from telogen to anagen of
the hair cycle is initiated by the proliferation of stem cells
residing in the bulge area of the follicle adjacent to the upper ORS [6]. Similarly Philpott and Kealey [40] suggested that ORS cells in EGF-treated human hair follicles
in vitro undergo a downward migration to repopulate the
bulb. A distinct bulge region has not been identified in
wool follicles. Labelling studies in vivo [4] do not localize characteristic label-retaining cells in the ORS, and
other stem cell-specific markers are not available. Although cell division continued in the bulb, our results did
not suggest that this was a result of stem cell division. Our
labelling studies revealed that the majority of labelled
bulb cells in fact originated from cell division at the base

of the follicle and not the upper ORS surrounding the

original fibre.
The changes induced by EGF and TGF in vitro compared with those in vivo, are significant to our understanding of the role of these growth factors in regulating
follicle function. Our in vitro results indicate that there
may be interactions with other hormones or growth factors, causing the distinct catagenic changes associated with
cessation of fibre growth and regression of the follicle
structure seen in vivo. These may include members of the
FGF [8, 16] and TGF [1] families that are expressed in
the ORS and are believed to control proliferation in the
follicle bulb. We have shown that EGF and TGF cause
cessation of fibre growth, but proliferation of cells in the
bulb of wool follicles in culture continues. Their effects
on bulb cell phenotype demonstrated here together with
their localisation in the ORS in vivo suggests that the role
of EGF or a related molecule is to perpetuate and regulate
the ORS layer of the follicle, presumably through autocrine and paracrine mechanisms.
Acknowledgements The authors thank K. Isaacs for technical assistance with immunohistochemistry. This work was supported by
a postgraduate scholarship and a research grant from the International Wool Secretariat.

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