O o d h e a d p u b L i s h I n g L i m I t e d

1.4
Increasing the yield of wool per animal
The quantity of wool fibre that grows on a sheep is determined by a complex
of cellular events but primarily depends on the initiation of primary (P)
follicles and secondary (S) follicles and most importantly on the additional
population of follicles that derives from the secondaries, so-called secondary-
derived (S
D
) follicles. Moore et al. (1998) proposed that embryonic skin
Ectal side
Ental side
Fibre
Stratum corneum
Epidermis
Duct of
sweat gland
Arrector
muscle
Sebaceous
gland
‘Swelling’
Sweat gland
Papilla
Undifferentiated
region of bulb
Outer
root-sheath
Inner
root-sheath
Proximal
(lower)
regions
Distal
(upper)
regions
Upper limit of
inner root-sheath
Transverse folds
1.1
 The wool follicle showing the main layers and associated
sebaceous and sweat glands (Auber, 1951). Reproduced with
permission.
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Improvement of wool production through genetic manipulation
7
generates initiation sites in skin with a defined number of specialised dermal
(pre-papilla) cells. These cells induce follicle formation and finally form the
dermal papilla in each follicle. According to that proposal and supported by
sheep selection data, if a fixed population of the pre-papilla cells induces
more primary follicles, the total number of secondary follicles induced would
be less and then the secondary/primary (S/P) ratio would be lower. Conversely,
if a lower number of primaries are induced, more secondaries would form
and the S/P ratio would be higher. Such a process could account for the
apparent relationship between follicle population, wool yield and fibre diameter
that is the basis of a selection methodology (‘soft-rolling skin’, SRS™) that
is used by some woolgrowers.
It is broadly accepted that reaction–diffusion mechanisms in which diffusing
morphogens (Nagorcka and Mooney, 1989) act across the epidermal/dermal
basement membrane in the follicle bulb are responsible for the establishment
1.2
 The human hair follicle showing the main layers with cellular
detail of the seven cell lineages that differentiate from the bulb
(Rogers 
et al
., 2006). Reproduced with permission.
Connective tissue sheath
Outer root
sheath (ORS)
Companion layer
IRS henle
IRS huxley
IRS cuticle
Inner root
sheath (IRS)
Hair fibre
Hair cuticle
Hair cortex
Medulla
Dermal papilla
Keratinising
zone
Hair cell matrix region
Pre-cortex
Cortex
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Advances in wool technology
8
of the pattern of follicle distribution. Studies mainly in mice have identified
many signalling factors that can influence the initiation and pattern follicles
in the skin, and undoubtedly such factors operate similarly for wool follicles.
The Wnt signalling pathway for example is essential to the dermal–epithelial
initiation of hair follicles (Andl et al., 2002; Reddy et al., 2004). Further
experimental evidence for the importance of WnT and its inhibitor for the
establishment of follicle spacing in the skin has recently been provided (Sick
et al., 2006). Over-expression in transgenic mice of 
β-catenin, a component
in the Wnt pathway, and of another factor, Sonic Hedgehog, can initiate
formation of follicles and also induce fusion of follicles (Lo Celso et al., 2004).
Initiation of the follicle pattern also depends on the localisation of lymphoid
enhancer factor (Lef-1) at focal points in the epidermis (Zhou et al., 1995).
A reaction–diffusion mechanism involving the factor ectodysplasin-A1 (Eda)
and its receptor Edr (Barsh, 1999) with another factor BMP for establishing
patterning of hair follicles has been discussed by Barsh (1999). Furthermore,
when the factor Eda was over-expressed in transgenic mice many hair follicles
became fused at the level of the infundibulum (the neck of the follicle)
instead of being separated by interfollicular epidermis (Zhang et al., 2003),
a feature resembling the formation of S
D
 follicles in sheep. It can be concluded
from these examples that if the interplay of such signalling molecules in
developmental pathways in the growth of wool were better understood, it is
likely that their function could be manipulated by transgenesis to change
wool follicle population and wool production.
Another feature of wool growth is that measurements of cells differentiating
from the follicle bulb have indicated that up to 80% of cells that leave the
bulb differentiate into the inner root sheath layers and not wool fibre (Hynd
et al., 1986). Theoretically, more fibre could be produced if that differentiation
pathway could be biased to allow differentiation into cortical and cuticle
cells. Considering what is known of the involvement of specific regulatory
genes in establishment of inner root sheath cell fate in mouse follicles,
notably the transcription factors GATA3 (Kaufman et al., 2003) and TCF3
(Merrill et al., 2001), it might be possible to divert the commitment of wool
follicle bulb cells from an inner root sheath cell fate towards a fibre fate by
transgenesis. This would improve the so-called production ratio, diverting
protein synthesis to the fibre-producing cells and would lead to increased
yields of wool per follicle. The desirability of such a manipulation would
depend on how much it would influence the IRS structure that is part of the
fibre growth process and whether the effect might result in an increase in
fibre diameter.
The possibility of increasing the rate of wool growth by over-expressing
growth factors such as growth hormone (GH) or insulin-like growth factor
(IGF) has been investigated. Transgenic sheep have been produced expressing
a transgene for ovine GH by microinjection of a gene construct possessing
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Improvement of wool production through genetic manipulation
9
the coding region linked to a metal-activated (metallothionein) promoter for
controlling expression. The promoter was activated by providing zinc to the
sheep via drinking water. Some of the transgenic sheep grew faster and
produced more wool than control sheep but the growth effects varied and it
was clear that the expression of the gene depended on its location in the
genome (Adams and Briegel, 2005; Adams et al., 2002). A serious disadvantage
of over-expression of GH is its potential to cause health problems in the
animal later in life. In other experiments transgenic sheep have been produced
by pronuclear microinjection with a mouse keratin gene promoter linked to
an ovine insulin-like growth factor 1 (IGF1) cDNA. Although it was reported
that fleece weight increased in transgenic animals compared with non-
transgenic, the results were not reproducible (Damak et al., 1996; Su et al.,
1998).
Certain biochemical pathways that do not normally function in sheep
have been suggested as a means to increasing wool production and have
been subjected to initial investigation. If these pathways could be introduced
by transgenesis using appropriate genes they could provide metabolites that
are essential for wool growth but can be in short supply. For example, adequate
supplies of cysteine to the follicles are essential for wool growth. Inadequate
supply results in depressed fibre growth and ‘breaks’ in the fibres appear
with consequent fibre weakness. That supplying cysteine, or its precursor
amino acid methionine, can increase fleece weight has been demonstrated
empirically (Reis, 1979).
The proteins of plant material ingested by sheep are degraded to amino
acids by the microflora of the rumen and a significant amount of the cysteine
is further degraded with the release of sulphide into the ruminal fluid and
excreted after conversion to sulphate. It was postulated that if the genes for
two enzymes that utilise sulphide for cysteine synthesis, serine transacetylase
and  O-acetyl serine sulphydrylase, were targeted to, and expressed in, the
rumen wall then it might be possible to ‘capture’ the sulphide as cysteine that
would then be absorbed into the blood and increase the amount of available
cysteine. The necessary enzymes constitute the cysteine synthesis pathway
in bacteria and the two genes have been characterised. These genes were
linked with promoters and their expression successfully tested in cultured
cells and mice (Ward, 1994). Microinjection of similar genes into sheep
under the control of a viral promoter (Rous sarcoma virus long terminal
repeat) had limited success in producing transgenic sheep with the two enzymes
expressed in the tissues (Bawden et al., 1995). Experiments have not
been conducted in targeting to the rumen although candidate promoters are
known. It should be noted that if general or systemic expression of the
genes were to occur in the transgenic sheep it would not be a problem since
cysteine synthesis is dependent on sulphide, which is present only in the
rumen.
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Advances in wool technology
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Another novel pathway suggestion that has been investigated is based on
the knowledge that cellulose digestion in the sheep’s rumen produces high
levels of acetate. If acetate could be converted to glucose by a metabolic
process not normally present, it could provide a useful metabolic supplement
for the energy requirements of wool growth. Preliminary investigations have
demonstrated that gene constructs possessing the bacterial genes encoding
the two enzymes needed for the pathway can be expressed in mammalian
cells and also in transgenic mice, but their functioning in sheep has not been
pursued (Ward, 2000).