Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.

Many farmers in New Zealand have moved towards using composite-breed sheep flocks to increase the meat production and fecundity of their stock. We aimed to determine whether flocks with a wider genetic base had greater between and within-fleece variation than single-breed flocks. Wool from seventeen body sites of sheep from five flocks of diverse genetic background (Romney, Coopworth, and crosses with Texel, Finnish Landrace and East Friesian breed lines) were compared. Both mean fibre diameter and mean fibre curvature differed between flocks (P <0.001), and across the body (P <0.01). While fibre diameter was more consistent, fibre curvature was more variable across the finer fleeces of flocks with some Finnish Landrace influence. Wool was shorter in Texel crosses, resulting in more consistent length across body sites. Our data suggests that composite-breed sheep exhibit increased within-fleece variation of some wool traits and decreased variation of others. Of most significance was that the extent of wool variation across body sites varied considerably between sheep, regardless of their genotype. In general, there was little association between wool traits with respect to this across-fleece uniformity. Fleece uniformity can be assessed by sampling across the anteriorposterior axis. Selection of stock with more uniform fleeces for specific traits may aid the production of wool better specified to satisfy differentiated niche markets.

Wool fibres are formed from the germinative epithelium, a population of keratinocytes at the base of wool follicles. In order to test the function of candidate wool growth genes and pharmaceuticals, we have developed methods to isolate and maintain these cells in culture. Pure, clonal lines of ovine keratinocytes were isolated from both vibrissae and wool follicles. Vibrissa-derived cells proliferated more extensively before senescing, but cells from both follicle types possessed ample proliferative potential for further experimentation. Standard assays have been established to quantify keratinocyte proliferation and apoptosis in culture. Differentiation of keratinocytes could be induced by maintaining them at high density for a week. Keratinocytes expressed differentiation markers for epidermis and inner root sheath. Expression of a chosen gene could be up-regulated by transfection with a liposome-borne plasmid. A transfected plasmid encoding a fluorescent reporter protein was expressed in 68-100% of cells. Similarly, expression of a gene of interest could be suppressed by RNA interference. The experimental tractability of these keratinocytes suggests that they will be a valuable model for investigating the cell biology and molecular genetics of wool growth. The methods described form a unique experimental system to identify leads for new technologies that improve

Cells from the dermal papilla and dermal sheath of hair follicles exhibit pronounced plasticity in vitro, being capable of adopting fat, bone, hematopoietic, and nerve cell phenotypes. In this study, we show that bovine dermal papilla cells (DPC) are also capable of undergoing skeletal muscle differentiation. DiI labeled DPC incorporated into myotubes when co-cultured with differentiating C(2)C(12) myoblasts. Bovine-specific PCR assays showed that the muscle markers MyoD and myogenin were up-regulated, confirming that the DPC had adopted a myogenic gene expression program. Nine clonal lines of DPC underwent both adipogenic and myogenic differentiation, demonstrating the multipotency of individual cells. Primary populations of both DPC and extra-follicular dermal fibroblasts were also capable of both adipogenic and myogenic differentiation. However, on myogenic differentiation, cells derived from dermal papillae expressed higher levels of myogenin than primary fibroblasts derived from extra-follicular dermis, suggesting that papilla cells undergo myogenesis more efficiently. This result shows that populations of fibroblastic cells derived from different anatomical sites within the skin are not equivalent with respect to their plasticity. Cultured DPC and dermal fibroblasts both expressed Pax3, a marker for the dermomyotome which represents a common embryological origin of muscle and dermis. Quantitative PCR showed that Pax3 expression levels before myogenic induction correlated with myogenin expression levels after myogenesis. These results suggest that a degree of dedifferentiation may underlie the plasticity of dermal cells in vitro, and that this plasticity may be predicted, at least in part, by levels of Pax3 expression.

Seasonal patterns of hair growth are governed, at least in part, by levels of prolactin in circulation, and although receptors for prolactin (PRLR) have been demonstrated in hair follicles, little is known of their regulation in relation to follicular cycles. In this study, a photoperiod-generated increase in prolactin was used to induce a wool follicle cycle during which changes in PRLR expression in sheep skin were determined by ribonuclease protection assay and in situ hybridisation. mRNA for prolactin and both isoforms of PRLR were also detected in skin by reverse transcription and polymerase chain reaction. As circulating prolactin began to rise from low levels, PRLR mRNA in the skin initially fell. These changes immediately preceded the catagen (regressive) phase of the hair cycle. Further increase in prolactin resulted in up-regulation of PRLR during telogen (dormancy), particularly in the epithelial hair germ, to reach a peak during proanagen (reactivation). In anagen (when follicle growth was fully re-established), PRLR mRNA returned to levels similar to those observed before the induced cycle. Hence, this longer term rise and fall of PRLR expression followed that of plasma prolactin concentration with a lag of 12-14 days. PRLR mRNA was most abundant in the dermal papilla, outer root sheath, hair germ, skin glands and epidermis. Location of PRLR in the dermal papilla and outer root sheath indicates action of prolactin on the growth-controlling centres within wool follicles. These cycle-related patterns of PRLR expression suggest dynamic regulation of PRLR by prolactin, thereby modulating hormonal responsiveness of seasonally growing hair follicles.

Wool fibres are formed from the germinative epithelium, a population of keratinocytes at the base of wool follicles. In order to test the function of candidate wool growth genes and pharmaceuticals, we have developed methods to isolate and maintain these cells in culture. Pure, clonal lines of ovine keratinocytes were isolated from both vibrissae and wool follicles. Vibrissa-derived cells proliferated more extensively before senescing, but cells from both follicle types possessed ample proliferative potential for further experimentation. Standard assays have been established to quantify keratinocyte proliferation and apoptosis in culture. Differentiation of keratinocytes could be induced by maintaining them at high density for a week. Keratinocytes expressed differentiation markers for epidermis and inner root sheath. Expression of a chosen gene could be up-regulated by transfection with a liposome-borne plasmid. A transfected plasmid encoding a fluorescent reporter protein was expressed in 68-100% of cells. Similarly, expression of a gene of interest could be suppressed by RNA interference. The experimental tractability of these keratinocytes suggests that they will be a valuable model for investigating the cell biology and molecular genetics of wool growth. The methods described form a unique experimental system to identify leads for new technologies that improve

The relationships between circulating prolactin (PRL), wool follicle growth and daylength were investigated in 24 New Zealand Wiltshire ewes housed indoors from September 1989 to May 1991. Twelve control (C) ewes were maintained under natural photoperiod. Two other groups were held in short days (SD; 8 h light: 16 h darkness) commencing from the winter solstice (22 June 1990) for either three (group SD3, n = 7) or six (group SD6, n = 5) months before reversion to natural daylength. Skin was sampled at one- to four-week intervals for histological determination of percentages of growing primary and secondary follicles. Hourly blood samples over 24 h were collected via jugular cannulae from C sheep in March and July and then monthly from all animals until December 1990 for estimation of mean monthly PRL concentrations for each treatment group. Between autumn (March 1990) and winter (July) primary follicle activity (PFA) and secondary follicle activity (SFA) declined in C ewes (PFA: 97 to 43%, SFA: 100 to 57%). Follicle regrowth during July and August in eight C ewes preceded the initial rise in plasma PRL from the winter minimum (1.6 ng/ml). Across the three groups, four instances of decreased follicle activity were observed, closely following or concurrent with increases in plasma PRL concentrations. The resumption of spring growth in four C sheep was temporarily checked by falls in follicle activities during September and October as PRL concentrations began to increase (3.4 to 8.9 ng/ml). Follicle activity also declined in November and December in eight C sheep, coincident with the rapid rise in PRL to a seasonal maximum in late November (165.4 ng/ml). The increase in SD3 follicle activity over spring was not delayed by short days but during October, after release from treatment, PRL concentrations rose (1.8 to 12.0 ng/ml) and follicle activity declined (PFA: 65 to 38%, SFA: 68 to 43%). In SD6 ewes, PRL concentrations were suppressed (2.1 ng/ml) and relatively constant levels of follicle activity (PFA: 73%, SFA: 95%) were maintained throughout short-day treatment. Release of SD6 ewes into summer photoperiod in January 1991 temporarily interrupted follicle growth (PFA: 68 to 17%, SFA: 96 to 19%) and caused out-of-season shedding in March and April. Contemporary C follicle activities were high (PFA: 95%, SFA: 98%). These data suggest that natural and experimental increases in daylength have a short-term inhibitory effect on growing wool follicles which could be mediated through rising concentrations of plasma prolactin.

Exposure of NZ Wiltshire sheep to a long-day photoperiod during late winter/spring has previously been shown to alter wool follicle growth patterns. The present experiment was undertaken to evaluate the plasma prolactin and wool follicle response to treatment with either 1,2 or 3 months of long days. Fourgroups ofsix, NZ Wiltshire ewes were subjected to long-day photoperiod (16L:8D) for 1 month (LD I), 2 months (LD2), 3 months (LD3) or natural photoperiod (control), commencing on the 3 August, after which they were released from light treatment into natural (indoor) spring daylength. Weekly jugular blood samples were assayed for prolactin (PRL) and skin biopsies were histologically assessed for wool follicle activity. Circulating PRL concentrations were low in all treated and control sheep at the start of the experiment (19 It: 2 ng/ml). In treated sheep mean PRL concentration increased (P&OS) above control levels (8 f 1 @ml) within I week after exposure to long days (52 4 4,5 1 f 5 and 55 f 2 ng/ml for LDl, LD2 and LD3, respectively). PRL concentrations remained significantly higher than controls (PcO.05) for the duration of each treatment reaching maxima of 148 4 27 @ml, 180 f 43 @ml, and 199 f 33 @ml for LDI, LD2 and LD3 respectively. In LD2 and LD3 treatments, PRL concentrations declined to control levels by 3 and 2 weeks after treatment had ceased, respectively, but in the LDI treatment PRLremained significantly above (PcO.05) control levels until between 2 and 3 months aftertreatment. Primary follicle activity (PFA) decreased in response to long-day treatment (P < 0.01) reaching minima of 47 f 9%, 36 f 6%, and 33 + 11% in LDl, LD2, LD3 sheep, respectively. Secondary follicle activity (SFA) responses were most marked in theLD2 and LD3 treatments where they fell below (P c 0.05) control levels reaching minima at 2 months into treatment (66 + 15% and 58 f 13% for LD2 and LD3, respectively). Both PFA and SFA increased to over 90%, within 1 month after release into natural light, in all treatments groups (except PFA for the LD I group which took 2 months after release to reach over 90%). In support of previously reported trials an increasing level of plasma PRL was followed by a decline in follicle activity. The optimal timeframe toachieveaneffectivesynchronisedfollicleregression wasbetween2and3monthsoflong-day treatment.Thelong-day treatment system produced a complete follicle growth cycle in temporal synchrony and is therefore a potential model for the investigation of structural and biochemical processes surrounding transitions between active and resting stages of fibre growth.