Wool Follicle Bulbs Research Articles

Transcript variability in keratine 33 (K33) gene of magra sheep

In a study, single nucleotide polymorphisms (SNPs) were analysed in open reading frame (ORF) of keratin 33 (K33) gene from lustrous Magra sheep. Blood (120) and wool follicle bulb (20) samples were collected from November 2017 to April 2019. Tajima's D value was -0.212492 and was non-significantly (p>0.05) different from 0, which suggested that the neutral mutation hypothesis can explain the nucleotide polymorphism of K33 in lustrous Magra sheep. Seventeen mis-sense variations were observed in Magra K33 protein, out of which 12 variations were novel and were not reported earlier. In phylogenetic tree analysis, all sheep sequences were clustered nearest to goat and farthest from horse. Changes in amino acids viz., serine (S), threonine (T) and tyrosine (Y) were observed above threshold phosphorylation potential which warrant further investigation for their plausible role in regulation of K33 structure and function in lustrous wool.

MicroRNA-148b promotes proliferation of hair follicle cells by targeting NFAT5

MicroRNAs (miRNAs), small non-coding RNAs, are involved in many aspects of biological processes. Previous studies have indicated that miRNAs are important for hair follicle development and growth. In our study, we found by qRT-PCR that miR-148b was significantly upregulated in sheep wool follicle bulbs in anagen phase compared with the telogen phase of the hair follicle cycle. Overexpression of miR-148b promoted proliferation of both HHDPC and HHGMC. By using the TOPFlash system we demonstrated that miR-148b could activate Wnt/β-catenin pathway and b-catenin, cycD, c-jun and PPARD were consistently upregulated accordingly. Furthermore, transcript factor nuclear factor of activated T cells type 5 (NFAT5) and Wnt10b were predicted to be the target of miR-148b and this was substantiated using a Dual-Luciferase reporter system. Subsequently NFAT5 was further identified as the target of miR-148b using western blotting. These results were considered to indicate that miR-148b could activate the Wnt/β-catenin signal pathway by targeting NFAT5 to promote the proliferation of human hair follicle cells.

Expression profiling reveals genes involved in the regulation of wool follicle bulb regression and regeneration in sheep.

Wool is an important material in textile manufacturing. In order to investigate the intrinsic factors that regulate wool follicle cycling and wool fiber properties, Illumina sequencing was performed on wool follicle bulb samples from the middle anagen, catagen and late telogen/early anagen phases. In total, 13,898 genes were identified. KRTs and KRTAPs are the most highly expressed gene families in wool follicle bulb. In addition, 438 and 203 genes were identified to be differentially expressed in wool follicle bulb samples from the middle anagen phase compared to the catagen phase and the samples from the catagen phase compared to the late telogen/early anagen phase, respectively. Finally, our data revealed that two groups of genes presenting distinct expression patterns during the phase transformation may have important roles for wool follicle bulb regression and regeneration. In conclusion, our results demonstrated the gene expression patterns in the wool follicle bulb and add new data towards an understanding of the mechanisms involved in wool fiber growth in sheep.

Tunicamycin reduces wool growth by slowing the mitotic activity of wool follicles

Reductions in wool growth have previously been observed in ewes exposed to low levels of corynetoxins, the causal agents of annual ryegrass toxicity. In this experiment, tunicamycin, a commercially available and closely related toxin, was infused into an isolated area of skin on the abdominal flank. Eleven sheep were continuously infused for 5 days with saline on one side and a total dose of either 35 or 350 µg tunicamycin/kg affected skin on the other side. Both fibre length (P < 0·05) and fibre diameter (P < 0·01) were reduced by tunicamycin treatment. Cell division in the wool follicle bulb was also reduced by tunicamycin (P < 0·005), indicating that the toxin is able to have a direct effect on the follicles and their ability to produce wool. The permeability of the vascular system increased in the skin tissue treated with tunicamycin, but only at the highest toxin dose (P < 0·05); therefore, poor nutrient supply to the follicle may be a minor contributor to reduced wool growth. The direct effect of tunicamycin on the wool follicle explains why wool growth is reduced by low levels of corynetoxins independently of, and prior to, effects on the whole animal.

Differential lectin binding to presumptive cortical cells of the wool follicle bulb.

An alpha-D-galactoside-specific lectin from Bandeiraea simplicifolia (BSLI) showed differential binding to cortical cells of the wool follicle bulb. The lectin bound to cells on one side only of the bulb and was completely blocked by alpha-D-galactose. The region of lectin binding extended from presumptive cortical cells at the base of the bulb to cortical cells at the top of the bulb, disappearing as cortical cells entered the fibre cortex. An orthocortex-specific monoclonal antibody was used to show that cortical cells recognised by the lectin lie directly below the fibre orthocortex and presumably give rise to the orthocortex. The results suggest that two distinct populations of presumptive cortical cells are present only two to three cell layers from the base of the bulb in a region where no morphological differences are detectable. The lectin-bound pre-cortical cells appear to give rise to orthocortical cells while cells not bound by lectin presumably give rise to paracortical cells. Electron microscopy showed that the lectin bound to sites on the plasma membrane, probably on the extracellular surface. This suggests that the lectin target may be a membrane glycoprotein or glycolipid. Two polypeptides recognised by BSLI were separated from wool follicle extracts by SDS-gel electrophoresis. These polypeptides migrated at approximately 69 kDa and 17 kDa. However, only the 69 kDa molecule showed the expected loss of binding by BSLI in the presence of alpha-D-galactose. Further work will be required to determine if this glycoprotein is the bulb cell molecule recognised by BSLI.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the kinetics of in vivo labelling of proliferating wool follicle bulb cells with 5-Bromo-2′-deoxyuridine (BrdU): Intracutaneous labelling with BrdU and pharmacokinetics of free BrdU in the skin tissue of sheep

After establishing an immunocytochemical technique for the identification of BrdU labeled S-phase cells in the follicle bulb of wool fibres, the usefulness of a facilitated intracutaneous injection method for BrdU was investigated. This method enables easy and quick administration of the labelling agent and thus permits in vivo examination of cellular processes in the wool follicle bulb under field conditions. Because of very low dose rates, the local i.c. injection decreases the risk of toxic side effects of BrdU in the skin and more generally in the whole body, that accompany intravenous infusion. The accumulation of BrdU labelled cells over time in the follicle bulb was measured with immunocytochemical detection techniques. Intracutaneously injected BrdU was incorporated immediately into nuclear DNA of proliferating cells, and the labelling effect was confined to an area within 20 mm from the injection site. This allows sequential injections to be made on the same midside area of the sheep without interference of adjacent BrdU pools. Labelled S-phase nuclei accumulated in a linear fashion from 5 min to 24 h. Intradermally administered BrdU has a slow tissue clearance rate, with levels up to 10% of the initial peak still present after 24 h. The BrdU labelling effect showed detectable immunoreactivity at total injection loads as low as 5 8g. The free pool clearance rate in association with strong immunoreactivity suggests that a single intradermal injection can be regarded as comparable to a continuous labelling technique, if cell cycle parameters and growth fractions are to be determined. The method of administering BrdU intradermally opens a new perspective for cell population studies of wool follicles. It allows serial as well as concurrent estimates of cell activity to be made in the same animal, while eliminating difficulties and potentially toxic side effects associated with whole body infusion techniques.

Perturbation of wool fibre cell differentiation by a mixture of bromodeoxyuridine and fluorodeoxyuridine

As part of investigations into the pattern of cell division and differentiation in the wool follicle bulb, the thymidine analogue 5-bromo-2'-deoxyuridine was used to label cells in S phase. A mixture of 5-bromo-2deoxyuridine and 5-fluoro-2'-deoxyuridine was injected into sheep held at a constant plane of nutrition. fibre length growth rates were measured by injecting the animals with either 35S-labelled methionine or cysteine, followed by autoradiography of individual fibres. Fibres from 5-bromo-2'-deoxyuridine/5-fluoro-2'-deoxyuridine treated animals exhibited both abnormal cuticle scale patterns and disorganized ortho- and paracortex as determined by staining with methylene blue. Orthocortex differentiation in wool follicle bulb keratogenous zones was visualized using a monoclonal antibody specific for orthocortical cells (HiT 96). We determined that the normal pattern of orthocortical cell differentiation was reversibly inhibited by 5-bromo-2'-deoxyuridine/5-fluoro-2'-deoxyuridine treatment. Fibre length growth rate was reversibly decreased very slightly as a result of 5-bromo-2'-deoxyuridine/5-fluoro- 2'-deoxyuridine administration. We conclude that 5-bromo-2'-deoxyuridine/5-fluoro-2'-deoxyuridine can grossly perturb aspects of wool follicle bulb cell differentiation without profoundly affecting fibre length growth rate. Although 5-bromo-2'-deoxyuridine/5-fluoro-2~-deoxyuridine labelling is an inexpensive, nonradioactive method of visualizing S phase nuclei, the possibility of artefacts resulting from such treatment should be borne in mind.

An immunocytochemical method for studying patterns of cell proliferation in the wool follicle

The identification of cell proliferation sites in the wool follicle bulbs of the skin of New Zealand Romney sheep was investigated with two immunocytochemical techniques. These methods were based on the in vivo labelling of DNA synthesising follicle matrix cells with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and a surgical preparation of the skin on the lateral abdominal flank of the sheep. Using a monoclonal antibody to BrdU, an indirect immunoenzyme method and a biotin-streptavidin method were compared for specificity and sensitivity in detecting replicating bulb matrix cells which had incorporated infused BrdU during the S-phase of the cell cycle. The immunocytochemical results for both methods showed a black-brown staining reaction of cell nuclei entering mitosis. The biotin-streptavidin method proved to be more highly specific and sensitive than the immunoenzyme technique. The immunocytochemical detection of cell cycle S-phase is highly suited for studying cell proliferation sites and cytokinetics in wool follicle bulbs and in other mitotically active tissues. Immunocytochemical detection of mitotically active cells has the advantages of high specificity, cost efficiency and rapidity and may be an alternative to methods employing metaphase arresting agents like colchicine or autoradiography.

The use of high‐energy electrons to depilate the breech of sheep

The use of high-energy electrons for permanently depilating areas of sheep skin was evaluated. The most effective dose of electrons for depilation was 17.5Gy. Histological changes in skin treated at this dose were examined in one sheep over 18 months and in 5 sheep over 89 d. Effects of treatment on bodyweight gains and fleece growth were examined by comparing the productivity of a further 5 sheep treated on the breech with high-energy electrons, with that of conventionally mulesed sheep (n = 5) and untreated controls (n = 5). Electron treatment resulted in immediate death of cells in the germinative region of the wool follicle bulbs. Within 10 d of treatment the treated areas were completely depilated. Wool follicle shafts in the treated areas regressed rapidly towards the epidermis and remained quiescent for the whole trial (89 d). A sheep treated 18 months previously has remained largely depilated, although a few sparse fibres are present. Epidermal acanthosis and orthokeratosis were present at 26 d after treatment. The thickened stratum corneum then sloughed off, but the epidermis remained acanthotic for the entire trial. Sweat glands and most sebaceous glands were destroyed by the treatment and were replaced by fibrotic, avascular tissue in the dermis. In all other respects the external appearance of the electron-treated breech was similar to that of mulesed sheep. There were no apparent side-effects of treatment. Neither mulesing nor electron treatment altered weight gains or fleece growth rates.

Estimation of Cell Birth Rate in the Wool Follicle Bulb using Colchicine Metaphase Arrest or DNA Labelling with Bromodeoxyuridine

The birth rate of cells in the wool follicle cells (CBR) was measured in two experiments on sheep differing widely in wool growth rate as a consequence of differences in nutritional regime. In Experiment 1, CBR was determined in one sheep on a high and one on a low plane of nutrition by two methods: the colchicine-metaphase arrest method, or from the product of total cell number and cell cycle time (the latter determined by DNA labelling with 5-bromo-2'-deoxyuridine (BrdU) and analysis of the fraction of mitoses labelled curve). CBR by colchicine was 0.73 and 0.56 of that determined using BrdU for the low- and high-plane sheep respectively. Cell cycle times were 26.5 h (low nutrition) to 18.6 h (high nutrition). In Experiment 2, CBR was again measured by the colchicine method, and from the rate of increase in labelled cells following a pulse dose of BrdU, in sheep on two levels of nutrition (n = 4llevel) and with a 3.7-fold difference in wool growth rate. Again, CBR by colchicine was only 0.41 (high nutrition) to 0.74 (low nutrition) of estimates made using the BrdU method, this difference being statistically significant only for sheep on the high plane of nutrition. Nutrition significantly altered CBR determined by colchicine (Pc0.001) and by BrdU (Pc0.034). The rate of fibre growth per follicle measured on two separate occasions was closely related to CBR measured using colchicine (r= 0.83, P< 0.01 and 0.8 1, P c 0.02) or BrdU (r= 0.73, P c 0.04 and 0.64, P< 0.08). It is concluded that CBR is a major determinant of fibre growth, but that colchicine depresses CBR. Previous estimates of mitotic activity in wool follicle bulbs have been made using colchicine and are thus probably underestimated. The technique using BrdU labelling and the subsequent rate of increase in labelled cells appears to be a useful one for estimating follicle cell kinetics.

Mitotic activity in cells of the wool follicle bulb.

Mitotic activity in the cells of the germinative region of wool follicle bulbs was quantified by using small (0.1-0.5 ml) intradermal doses of colchicine and selective staining of the metaphase-blocked nuclei using either crystal violet, iodine and eosin or haematoxylin and eosin. The number of metaphase nuclei present 3 h after colchicine administration increased with colchicine dose from 0 to 1 microgram and thereafter remained relatively constant up to 200 micrograms colchicine. The accumulation of metaphase nuclei was linear for up to 6 h after intradermal colchicine. The metaphase-blocking effect of intradermal colchicine was confined to a radius of less than 5 cm from the injection site, allowing a number of estimates of mitotic rates to be made over a small area of skin. Such estimates revealed little variation in mitotic activity over the midside region of the sheep, although there were substantial differences in follicle activity at different sites over the body. The technique is simple, allows serial or concurrent estimates of mitotic activity to be made in the same animal, and eliminates problems associated with intravenous colchicine administration. It was used to derive the relationship between follicle activity and fibre production after nutritional changes, and to define the time course of mitotic events after administration of the antimitotic defleecing agent cyclophosphamide.

Inhibition of DNA synthesis in dermal tissue of merino sheep treated with depilatory doses of mouse epidermal growth factor.

Two groups of three Merino wethers were infused intravenously with either 0.12 mg mouse epidermal growth factor (mEGF)/kg fleece-free body weight or 0.9% (w/v) NaCl over 24 h. Sheep treated with mEGF rejected food during treatment but feed intake was kept equal for both groups. Wool growth and plasma concentrations of mEGF were measured during the experiment. Pieces of skin taken from the wool-growing regions of the body were incubated with radioactive thymidine in order to measure its rate of incorporation into DNA. The skin was then divided at about the level of the sebaceous glands into sections that contained the upper dermis and epidermis (E sections) and those containing the generative wool-follicle bulbs (D sections). No mEGF was detected in the controls whereas mean levels of about 35 micrograms mEGF/1 plasma were detected during the last 4 h of infusion in the protein-treated group. After infusion, wool growth was reduced by about 20% of the mean pretreatment level in the controls and no shedding of wool fibre was evident. In the mEGF-treated sheep, on the other hand, wool growth was depressed by 75-95% of the mean pretreatment level and the fleeces were almost completely cast in all three of the animals, leaving them nude on the wool-growing regions of the body. Wool growth was restored to its pretreatment level in this group about 1 month after infusion. The D sections of skin contributed 50-60% of skin wet weight in controls throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of internally administered N-[5-(4-aminophenoxy)pentyl]phthalimide on wool follicles and skin of sheep.

When N-[5-(4-aminophenoxy)pentyl]phthalimide was administered to sheep in sufficient quantities to permit manual removal of the fleece (400 mg/kg body weight orally, or 75 mg/kg body weight intravenously during a period of 48 h), cell division ceased in the wool follicle bulbs within 1 day. Dark-staining bodies (autophagic vacuoles) developed concomitantly in the cytoplasm of the bulb cells. The nuclei of cells in the keratogenous zones of the fibres became pycnotic 2 days after dosing and subsequent keratinization of these portions of the fibres was impaired. All the follicles retrogressed prior to day 7 after dosing, and the root ends which formed on the fibres moved towards the skin surface, reaching the level of the sebaceous glands by day 7. At this time mitotic activity recommenced around the dermal papillae in about 50% of the follicles. A small number of tips of new fibres emerged from the skin surface of some of the depilated sheep by day 14. The root ends on the fibres in the fleeces harvested at days 7-15 were fragmented with various degrees of taper. By 21 days, most follicles were growing emergent fibres. Thickening of the epidermis, increase in sebaceous gland size and decrease in skin thickness occurred in some of the depilated sheep. Smaller doses of the compound (200 mg/kg body weight orally or 40 mg/kg body weight intravenously during 24 h) produced weakness in the wool. Fewer autophagic vacuoles were present in follicle bulbs 1 day after dosing and not all follicles regressed. The weakened region of the fleece contained a mixture of shed fibre ends and continuously growing fibres with thin regions proximal to poorly keratinized lengths of fibre. No change was observed in other components of the skin of the sheep with weakened wool. The follicular changes produced by this compound are similar in some respects to those produced by other depilatory compounds or that occur during natural cyclic hair growth.

Cytoplasmic Degeneration in the Wool Follicle Bulb Following Administration of Cyclophosphamide1

Cytoplasmic Degeneration in the Wool Follicle Bulb Following Administration of Cyclophosphamide1

Mitotic Activity in Wool Follicle Bulbs

The quantitatIve significance of mitosis in wool follicle bulbs in relation to the rate of fibre growth was investigated in 10 major fleece phenotypes (fine .Merino, medium Merino mosaic, strong Merino, Merino lustre mutant, Corriedale carpet-wool, Corriedale mosaic, Southdown, and Border Leicester). Mitotic activity in the follicle bulbs was estimated with the aid of colchicine and the volume rate of fibre growth was inferred from fibre diameter.