Hair follicle was a tiny but complex organ consisting of numerous types of cells and tissues. The process of follicle shutdown in New Zealand cashmere goats was reported by Nixon et al. (1991a). Ryder (1966) indicated that the follicle cycle of cashmere goat was composed of anagen, catagen, and telogen stage. Paus et al. (1999) extended these pioneering studies, and reported a comprehensive guide for the recognition classification of distinct stages of hair follicle morphogenesis. Morphology research of the secondary follicle cycling of Inner Mongolia cashmere goat was described by Li et al. (2008). Research on the molecular biological aspects of hair follicle development, especially the signaling pathways was involved, has advanced in recent years (Fuchs and Horsley,2008; Michelet et al.,2008; Schneider et al.,2008). Identification of hair follicle characteristic and activity of one and two humped camels, Iranian cashmere goats, and Spanish goats was also reported by Ansari-Renani (2010a,b) and Wuliji et al. (2006), respectively. Although there is a long-lasting interest in the hair follicle of Cashmere goat, there are no descriptions to secondary follicles of Hexi Cashmere goat. What is more, there are no studies done what the ultrastructure changes of secondary follicle of Hexi Cashmere goat was under transmission electron microscope (TEM) within a hair follicle cycle.
Hexi cashmere goat, which was famous for excellent white down fiber, is one of the important breeds of china living in high mountain grassland at an altitude of 8530 feets, with extreme cool temperature and low oxygen content. The fleece of cashmere goats grows from specialized follicle in the skin. Primary follicles bear guard hair (>30 μm), and secondary follicles which are more numerous than primary follicle produce fine cashmere fiber (<24 μm), (Ryder,1966; Nixon et al.,1991a; Petrie,1995). The average diameter of fine cashmere fiber of Hexi cashmere goat, named soft golden, is <15.76 μm (Ma,2011). It would be interesting to know if the secondary follicle of Hexi cashmere goat had its own characteristics which were different from others. The present study was undertaken to observe the periodic changes of secondary follicle of Hexi cashmere goat within a year, in particular, the ultrastructural changes under TEM. Such information would also associate with harvesting down fiber at the appropriate time of the year to achieve maximum harvest.
MATERIALS AND METHODS
Hexi Cashmere Goat
Ten Hexi cashmere goats (five females, five males), which were 1-year old, were included in the investigation. All animals were kept under the same conditions of natural photoperiod and natural temperatures. They were principled by small-holders in Tianzhu County of Gansu Province. These Hexi cashmere goats were grazed at different regions during 1 year. During winter, they were housed and received supplementary forage.
Biopsy samples of skin of Hexi cashmere goats were taken from left midside under local anesthesia with 1% lignocaine (Jewin Pharmaceutical, Shangdong, China) each month within a year. The dose was 2 mg kg−1. To facilitate skin sampling, goats were restrained in a lateral position. The skin samples of flank regions were studied. All aspects of animal research have been conducted in according with guideline set by the Institution Animal Care.
Whole samples which were laid flat on a manila card to maintain their shape during fixation fixed overnight in a solution of 3.7% paraformaldehyde and 1% glutaraldehyde in 50 mM sodium phosphate buffer saline at pH 7.2 (PBS) at 4°C. For TEM, cut two blocks (1 cm3 each) with a razor blade from fixed samples for transverse and longitudinal ultrasections, and fixed in 3% glutaraldehyde at 4°C. Then the samples were postfixed with 1% OSO4 in PBS for 2 hr at 4°C, dehydrated in an increasing ethanol series and embedded in SPION 812. Ultrathin sections were prepared with an ultramicrotome (Leica EM UC6), stained with uranyl acetate and lead citrate, and examined under TEM (JEM 1230). Other fixed samples of skin for light microscope were dehydrated by an ascending series of alcohol, embedded in paraffin wax and stained with hematoxylin and eosin (HE). Transverse and longitudinal 7–8 μm sections of hair follicles were cut on a rotary microtome.
Secondary Follicle Cycle Within a Year Under Light Microscope
In January and February, the inner root sheath (IRS) disappeared. Hair germ nearby the old secondary follicle could be found (Fig. 1A). It can be identified by their compact, small, and ring arrangement (Fig. 1B). And the bulb or proliferative zone consisted of germinal epithelium enclosing the dermal papilla. The hair germ became finger cell group in March and April (Fig. 1C). Those developing hair germs had a common connective tissue sheath (CTS) with old secondary follicle (Fig. 1D). The elongation zone contained undifferentiated keratinocytes. It was the follicle rudiment.
In May and June, the secondary follicle became tubular structure, which grew down into dermis (Fig. 1E). At this level, the cells of the secondary follicles outer root sheath (ORS) regenerated. Secondary follicle started reconstructing. Hair bulb became visible but did not differentiate obviously in longitudinal section (Fig. 1F). IRS and hair shaft (HF) were absent in cross section (Fig. 1G). The secondary follicle which had the complete structure finished the reconstruction from July to August (Fig. 1H). At this level, hair bulb presented complete division; the cells of the dermal papilla (DP), with the shape of a pear, had the high density granules (Fig. 1I); HF surrounded by a red IRS, which was most longest and widest. Almost all secondary follicles were single (Fig. 1J). In the keratogenous zone, the cells of ORS have a large weakly staining cytoplasm.
The division speed of bulbar became slower, and the cell of hair bulbar became larger and less compact in September and October. IRS moved to the middle or upper of secondary follicle, and shorter than before. The cells of ORS adjacent to hair shaft became larger, and had a radicalized arrangement, whole secondary follicle became curve, and DP atrophied (Fig. 1K). ORS and CTS divided from each other and separated from surrounding connective tissue (Fig. 1L). From November, CTS of secondary follicle presented goffer (Fig. 1M), the bulbar epithelial cell's differentiation ceased, and papilla shrinks accompanied with massive epithelial cells apoptosis in secondary follicle (Fig. 1N). Gradually, the secondary follicle moved upward, till it reached the region nearby sebaceous gland in December and no longer varies.
Secondary Follicle Cycle Within a Year Under Transmission Electron Microscope
In January, the layers of the connective tissue sheath (CTS) of secondary follicle were not clearly visible. And no capillary and glass layer of CTS were found. The hemidesmosomes between ORS and basement membrane were rare or disappear. The cells of ORS in the lower or middle part were single and long-strip type (Fig. 2A), while in the upper part of secondary follicle often appeared some cells, which have some tubers and radiantly arranged, and no desmosome were found between them (Fig. 2B). In February, the cells in the upper of secondary follicle were fragmented, even died.
In March, the root of secondary follicle had risen to the level of the sebaceous gland. Almost hair shaft of secondary follicle had shed, leaving a hair shaft with a collapsed empty lumen (Fig. 2C). The remnant of CTS may still be observable surrounding the secondary follicle. In some cases, there may be an appreciable number of fiber tips which was surrounded by the cells of ORS (Fig. 2D). The new fiber which had the different cells from ORS was usually located adjacent to an existing brush end. In April, the thickness of CTS which presented layering increased. ORS which had the rounded shape like mitochondrion in the cytoplasm gathered to the end and was stretched out like fingers (Fig. 2E).
From May to June, CTS had the layering obviously, especially the glass layer. IRS consisted of three or four concentric cells which were not cornification completely (Fig. 2F). The cells of outmost in which the trichohyalin granules were found were Huxley's layer (Hx). Numerous desmosomes were found between the cells of Hx (Fig. 2G). The boundaries between ORS and Hx cells, as well as between Hx cells themselves, were extensive desmosomes (Fig. 2H). In July and August, the thickness of ORS increased and the volume of ORS cells increased. The IRS consisted of three concentric layers, each of which was known to contain special keratins. Among those, the outermost was henle's layer (He) which keratinized earlier than any other layer of IRS. The layers of Hx Cells were two or three. The structures of secondary follicles were most integral at this stage (Fig. 2I). Keratin bundles occurred randomly in the cytoplasm of hair cortex cells when observed in cross section (Fig. 2J), whereas their orientation was vertical in longitudinal sections (Fig. 2K). The tuber of hair cortex became longer and thinner than before so that the link between the cells of hair cortex viewed loose, but the abundant desmosomes between the tuber of hair cortex were found (Fig. 2L).
In September, the changes of IRS were most obvious. One of the earliest signs of the differentiation of Hx in IRS was the formation of Keratin bundles (Fig. 2M), which were not found before September. The area rich in Keratin bundles contained some trichohyalin granules. The cells of Hx degenerated gradually when keratin bundles and trichohyalin granules appeared in cytoplasm. The cell membranes of Hx became thinner or disappeared. In October, the cells of hair bulb of secondary follicle enlarged. The cells of hair matrix became bigger and moved upward. At a certain stage of catagen, numerous pseudopodia are formed on the surface of cells of ORS, so that the apparent gaps between them were often found (Fig. 2N). IRS moved upward and the layers of Hx cells decreased even disappear. A monolayer of He, which had the complete keratinization was observed. The structure of CTS was not distinct. Whole secondary follicle separated from the surrounding connective tissue almost.
From November to December, whole secondary follicle separated from surrounding connective tissue (Fig. 2O). After the complete keratinization, the projections of ORS cells decreased, desmosomes remain to form a structure like the corneodesmosome found in skin cornification. Hx layer of IRS disappeared. ORS and IRS also separated (Fig. 2P).
The cells of He, Hx, and the sheath cuticle, which together form IRS, had their own peculiarities. However, it was easy to identify them together because the type of intracellular product was the same in each. Birbeck (Birbeck and Mercer,1957a) has indicated that the intracellular product was trichohyaline, and that trichohyaline was certainly a precursor of the fibrous component of the sheath. Justyna et al. (2009) has reported that cell keratinization begun in IRS in the He layer. The first signal of keratinization is the appearance of trichohyalin granules in keratinocytes (Birbeck and Mercer,1957a; Morioka,2005). In this study we also found that the He layer cells were nearly completely differentiated, while keratinization of Hx cells was still incomplete. Trichohyaline droplets appeared in the cells of Hx, they were dense, rounded, apparently without an enclosing membrane. In addition, we found that the He layer was absent in the proanagen stage, and was present in late anagen and catagen. On the contrary, Hx layer appeared in anagen stage and disappeared in catagen stage. Therefore, it would be interesting to know whether the He layer was the result of the further keratinization of Hx layer.
The fleece of double-coated animals of Hexi cashmere goat of two major fiber types guard hair and down fiber (Millar,1986). Down fiber which is finer than the guard hair grows from secondary follicle (Mitchell et al.,1991; Nixon et al.,1991b). During the development and regeneration of secondary follicle, the amount and location of these follicle show both spatial and temporal changes in pattern. Transmission electron microscope research, beginning in the middle of the last century, has allowed us to gain a detailed understanding of the secondary follicle (Birbeck and Mercer,1957a,b,c; Morioka,2005; Shin et al.,2009). In the present study we found that the combination of the method using light microscope with transmission electron microscope would provide a satisfactory way for both microscopic and submicroscopic observations.
Our understanding of the molecular biological aspect of hair follicle development, especially the signaling pathways involved, has advanced considerably in recent years (Morioka,2009). The actions of signaling molecules are entwined with the complex structure of the hair follicle. Problems associated with harvesting fleece at the appropriate time of the year to achieve maximum harvest were emphasized, because the value of down fiber is determined by weight and diameter. Definition of follicle characteristics are required to enable a better understanding of the variability of fiber types and growth dynamics of down and hair and to provide information on the fiber harvest from Hexi cashmere goat.
Scientists have found that the follicle cycle is composed of anagen, catagen, and telogen phases. Nixon (1993) who used the Sacpic method summarized that the phase of hair follicle through a cycle can be divided into five levels, they were proanagen, anagen, early catagen, late catagen, and telogen, respectively. The occurrence of each level was grouped into active and quiescent classes. According to the results under light and transmission electron microscope, five levels were also partitioned in the course of the hair cycle in present study. Distinguished from Nixon, they were proanagen, anagen, procatagen, catagen, and telogen. Camel follicle cycle was described by Ansari-Renani et al. (2010b). Their study indicated that most secondary follicle began at telogen stage from February and March, and remained for 3–4 months before returning to anagen. Li et al. (2008) found that the secondary follicle of Inner Mongolia cashmere goat started growing from April, finished the reconstruction from August to September, entered into catagen in October, and at the telogen phase from December. In the present study, the different levels of developing secondary follicle in a cycle also were defined. Different from that in camels and Inner Mongolia cashmere goat, the stage of telogen, proanagen, anagen, procatagen, and catagen of Hexi cashmere goat was in January and February, March and April, May to August, September and October, and November and December, respectively. The contradiction between the present results and those of other researches may be because of differences in the breeds or the climate where the animals live, because the growth of down fiber had the intimate relationship with sunshine duration. Sunshine shortened, growth of secondary follicle accelerated; sunshine lengthened, growth became slow and stopped.
The study described above has been aimed at providing information on secondary follicle characteristics in Hexi cashmere goats, which were needed for the development of Hexi cashmere industry. It was indicated that the stage of telogen, proanagen, anagen, procatagen, and catagen was in January and February, March and April, May to August, September and October, and November and December, respectively. The key change observed in secondary follicle was IRS.