Histochemical and Ultrastructural Observations of Respiratory Epithelium and Gland in Yak (Bos grunniens).код для вставкиСкачать
THE ANATOMICAL RECORD 293:1259–1269 (2010) Histochemical and Ultrastructural Observations of Respiratory Epithelium and Gland in Yak (Bos grunniens) BO YANG, SIJIU YU, YAN CUI,* JUNFENG HE, XINHUA JIN, AND RU WANG Faculty of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu, China ABSTRACT Submucous glands and epithelial mucous cells of yak (Bos grunniens) respiratory tract have been studied by a variety of histochemical methods and transmission electron microscopy for differentiating and characterizing serous and mucous cells. By light microscopy, the distribution, numbers of mucous cells, volume of mucous glands (Reid index), and the ratio of mucous cell to serous cell in the bronchial tree were measured with different staining. Histochemically, a majority of mucous cells, presented in the surface epithelium of bronchi and glands, secreted neutral and acid mucosubstances, only a few sulfated mucosubstances were present. No mucus-producing cells were observed from the terminal to respiratory bronchiolar level. Ultrastructurally, serous cells in glands of the lamina propria had two distinct forms: one type ﬁlled with many round dense secretory granules, plentiful RER and few other organelles, similar to other animals; the other type contained some oval mitochondrial and distended RER, the granules resembled the former. The mucous cells in gland were similar to that of epithelium, which containing abundant secretory granules with an eccentric core. The mucous cells of the surface epithelium differ from other animals in the structure and histochemistry of their secretory granules. Analysis of the size and distribution of the secretory granules and other organelles of serous cells suggested that differences represent different phases of a secretory cycle, not various populations of C 2010 Wiley-Liss, Inc. cell or granules. Anat Rec, 293:1259–1269, 2010. V Key words: respiratory epithelium; submucosal glands; mucous and Serous cells; histochemistry; ultrastructure; yak INTRODUCTION The surface of bronchial tree is coated with a layer of respiratory tract mucus containing electrolytes, proteins, and glycoproteins. The mucus is thought to be provided a mechanical barrier between airborne particles or gaseous products and the cilia, as well as the apical cell membrane for protecting the airways against inhaled irritant gases and particles, and changes in the temperature or humidity of inspired air. Comparative studies have shown that the abundance, distribution, and contents of secretory cells vary considerably among different kinds of species. It has been studied in several mammals, rodents (Dalen, 1983; McCarthy, 1964; Plopper et al., 1984), carnivores (Gatlagher et al., 1975; Robinson et al., 1986), primates C 2010 WILEY-LISS, INC. V (George et al., 1986; Plopper et al., 1989), domestic animals (Jones et al., 1975; Mariassay et al., 1988; Kahwa and Purton, 1996; Raji and Naserpour, 2007) and man (Lamb and Reid, 1972). Grant sponsor: The National Natural Science Foundation of China; Grant number: 30571342; Grant sponsor: Natural Science Foundation of Gansu Province; Grant number: 0802-02. *Correspondence to: Yan Cui, Faculty of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, Gansu, China. Fax: 86 931 7631220. E-mail: [email protected] Received 27 May 2009; Accepted 27 September 2009 DOI 10.1002/ar.21056 Published online 28 April 2010 in Wiley InterScience (www. interscience.wiley.com). 1260 YANG ET AL. Fig. 1. Bronchial wall of ﬁve-month-old yak stained with H.E. The mucous cells (Arrows) were stained blue. BV, blood vessels; Ca, cartilage; E, epithelium; G, glands; SM, smooth muscle. The yak originated in the Qinghai-Tibetan plateau of the People’s Republic of China. Approximately 14 million yaks are found in China, accounting for 90% of the total number of yaks in the world. Yaks play an important role in both livelihoods and natural ecosystems of the Qinghai-Tibetan plateau. But there is no related information available on Yak. The purpose of this study was to characterize the histochemical composition and the ultrastructure of bronchial surface epithelium and submucosal glands of the yak in order to make basic information for comparison with other mammalians. MATERIALS AND METHODS Five clinically normal, age between 5 and 6 months yaks of both sexes, from local farmer in Datong County of Qinghai Province, were used in this study. All animals were killed by exsanguinations via the abdominal aorta in slaughter house. The lungs were obtained immediately after the subjects were killed and ﬁxed by tracheal infusion of 4% neutral paraformaldehyde phosphate buffer (pH 7.3) at 30 cm water pressure for 2 hours, then stored in the same ﬁxative for one month. For light microscopy, the samples were taken randomly from the caudal lobe of the left lung, and then cut into pieces no larger than 1 cm3. Representive portions of each lung were dehydrated in graded alcohol and embedded in parafﬁn wax, serially sectioned at 4 lm. Every sixth section was mounted and stained with Alician blue (AB pH 2.5, 30 min) and periodic acid Schiff reaction (PAS). At intervals along the airway additional sections were also stained with either standard haematoxylin and eosin (HE) to examine the general morphology (Figs. 2A and 3A), or with AB at pH 1.0 to characterize further the histochemical nature of the epithelial cells, or even with Toluidine blue to examine the metachromasia of mucosubstances. The mucous cell density of airway epithelium was estimated by counting mucous cell nuclear proﬁles in 100 lm of basal lamina lining each airway (Mariassay and Plopper, 1983), and in submucosal glands the mucous and serous cell were also counted. The measurement of the gland to wall ratio was described by Reid (1960), which is generally referred to as the Reid index. The Reid index is expressed as ratio of the thickness of the gland to the distance between the basement and the inner aspect of cartilage (Reid, 1960; Shimura, 1990). RESPIRATORY EPITHELIUM AND GLAND IN YAK 1261 Fig. 2. Bronchial glands of ﬁve-month-old yak with H.E A: PAS/AB pH2.5 B: PAS C: AB pH2.5 D: AB pH1.0 E: and Toluidine blue F: staining. Ca, cartilage; M, mucous cells; S, serous cells. The epithelium was photographed and the length of the basal lamina of the counted regions was determined with Olympus DP71 microscopy (including DP control and Image-Pro Express), and expressed with mean SD. For transmission electron microscopy, tissue samples were taken randomly from the caudal lobe of the left lung immediately after slaughter, cut into pieces of approximately 1 mm3 immersed in 2.5% glutaraldehyde in neutral phosphate buffer (pH 7.3, at 4 C) and 1262 YANG ET AL. Fig. 3. Mucous cells of bronchial epithelium of ﬁve-month-old yak stained with H.E A: PAS/AB pH2.5 B: PAS C: AB pH2.5 D: AB pH1.0 E: and Toluidine blue F: C, ciliated cells; M, mucous cells. postﬁxed in 1% osmium tetroxide for 2 hours. The tissue was dehydrated in graded ethanol and embedded in Epon812. Orientation of the block was achieved by examining 1 lm sections stained with Toluidine blue. Thin sections (60–80 nm) were cut on a Leica EM-UC6 ultramicrotome, mounted on G200 grids, stained with alcoholic uranyl acetate and lead citrate and viewed with a JEOL 1230 electron microscope at 120 kV. 1263 RESPIRATORY EPITHELIUM AND GLAND IN YAK TABLE 1. Histochemical reaction of submucous glands and epithelium mucous cells Staining methods Submucous glands References PAS McManus, 1948 Red Red PAS/AB pH 2.5 Mowry, 1956 Bluish-purple, a few stained Red AB pH 2.5 Lev and Spicer, 1964 Lev and Spicer, 1964 Many cells red or reddish-purple, a few stained blue Mostly unstained, some blue More cells unstained compared to AB pH 2.5, a few blue Mostly unstained, some purple (metachromasia) AB pH 1.0 Toluidine blue Tock and Tan, 1969 Interpretation of staining reactions Mucous cells Blue Most cells light blue, a few unstained Purple (metachromasia) Periodate-reactive carbohydrates and/or glycogen To seperate acidic glycoconjugates (carboxylated and/or sulphated glycoconjugates) from neutral types Acidic glycoconjugates (carboxylated and sulphated types) Sulphated acidic glycoconjugates Sulphated acidic glycoconjugates TABLE 2. Number of mucous cell proﬁles in bronchial airways of yak Small Bronchi Bronchioles Gland (%) PAS n AB pH 2.5 n AB pH 1.0 n 7.3 2.3a 4.8 1.4 17.4 6.5b 65 50 54 6.9 2.2 4.4 2.2 16.5 7.5 68 53 58 7.3 1.8 4.1 2.0 17.4 6.9 58 52 63 Number of mucous cell proﬁles in bronchial epithelium of yak in per 100 lm. Proportion of mucous cell proﬁles in submucous glands in lung of yak. a b RESULTS The results of the histochemical reactions and number of mucous cells in bronchial airways obtained are summarized in Table 1. Submucosal Glands Light microscopy. The yak airways had extensive submucosal glands which lay between the muscle and the epithelium and/or the muscle and the cartilage, little or no penetration of the glands into the muscle layers (Fig. 1). The gland to wall ratio (Reid index) was 23.6 8.3 percent. These tubulo-alveolar glands, composed of two types of secretory cells, serous and mucous, were present throughout the tracheobronchial tree except in the terminal airway and respiratory bronchioles. They were numerous in the larger bronchi. The ratio of mucous to serous cells in gland acini with various staining is in Table 2. They were all stained red with PAS (Fig. 2C), with some whole acini or cells within an acinus staining with AB pH 2.5 as well (Fig. 2D). The majority of the cells in these glands were stained red or reddishpurple in the PAS-AB reaction. The staining was restricted to the apically localized granules, showing the presence of neutral mucin in them (Fig. 2B). There were however a smaller number of glandular cells in the bronchus, which contained acidic mucus. The AB pH 1.0 staining showed that few of cells contained sulphated mucin in submucosal glands (Fig. 2E). Electron microscopy. Two distinct granule types could be seen, electron-dense granules in serous cells and electron-dense granules with an eccentric core in mucous cells. The mean size of the serous granules for each cell was usually smaller than that of mucous granules. A cross section of a single acinus could contain either mucous or serous cells, or a mixture of cell types adjacent to each other (Fig. 4). Serous cell. In cross-sections, the serous cells were arranged around a small central lumen (Fig. 7). On the basis of differences in their cytoplasmic inclusions, two distinct forms of serous cells were recognized. The common serous cell (Fig. 5) had a round or elliptical nucleus, basal in position and with a prominent nucleolus. The serous cells contained numerous secretory granules, usually concentrated toward the apex of the cell. Although they were densely packed they did not distend the cell as in mucous cells. Individual granules were usually round and completely surrounded by a membrane. Granules were seen close to the unit membrane of the apical cell surface, whereas the mucous cell granules were often seen open and discharging. The mitochondria were round and ovoid; they were mainly concentrated in the base of the cell, but a few were found among the granules. Most of the rough endoplasmic reticulum (RER) was at the cell base, the cisternae being narrow and grouped in parallel arrays (Fig. 5). Free ribosomes were abundant through the cytoplasm. The Golgi apparatus was well developed and supranuclear, often with dilated lamellae and many associated vesicles. Multivesicular bodies were seen occasionally. The second type of serous cell was uncommon (Fig. 8), the nucleus was indented in shape, also basal in position and with a prominent nucleolus, but the cytoplasm was slightly dense than that of common one. The apical portion of the cell packed with abundant secretory granules, and many round mitochondria were observed among them. Plenty of dilated RER ﬁlled with the basal area. Golgi apparatus and multivesicular bodies were rarely observed. The cells of the epithelium were linked at their luminal surface by tight junctions, and throughout the depth 1264 YANG ET AL. Fig. 4. Yak bronchial gland with two types of cells A: mucous cell (M) and serous cell (S). The nuclear (N) of mucous cell was compressed at basal side B:. Numerous secretory granules (SG) located at the apical portion of cells, the sizes of dense granules of mucous cell with lucent core bigger than that of serous cell (B). The serous cells contained rough endoplasmic reticulum (RER) at basal portion and vacuoles (V) in perinuclear area b, C:. Smooth endoplasmic reticulum (SER), RER and mitochondrial (M) present among the granules of mucous cell D. GC, glandular cavity of the epithelium there was a degree of interdigitation between adjacent cells, with desmosomes occasionally being seen in Figure 6. lescing secretory granules (Fig. 4D). These granules contained an electron-dense granular matrix that surrounded a ﬁnely granular, with a round and eccentric electron-lucent core. In addition to granules, the cytoplasm contained mitochondria, small amounts of RER; occasionally a Golgi apparatus would be noted in the cytoplasm. The cytochemical reactivity of the granules was similar to the mucous cells of the epithelial surface. Mucous cell. The mucous cell had a columnar shape (Fig.4B), resembled that of the epithelial surface. Its nucleus was compressed at the basal side and was irregular in outline, with dense chromatin. The cytoplasm of the columnar cell was ﬁlled with large un-coa- RESPIRATORY EPITHELIUM AND GLAND IN YAK Fig. 5. Well-developed RER located at the basal portion of serous cell. BM, basement membrane; N, nuclear; SG, secretory granules; V, vacuoles. Fig. 6. Juction complex (JC) of serous cell and adjacent cells, intermediate junctions and desmosomes were present. 1265 Fig. 7. Another type of serous cell (Stars) in bronchial gland with plentiful distended RER and apical secretory granules. Fig. 8. Two types serous cell in bronchial gland. Numerous secretory granules (SG) and abundant mitochondrial present at supranuclear and distended RER (Triangle) surrounded the dentated nuclear (N) in serous cell (Top). The common serous cell with abundant RER, some mitochondria (M) and secretory granules (SG). Epithelial Mucous Cells Light microscopy. There were a large number of mucous cells of the surface epithelium in the bronchi and the bronchioles (Table 2), whereas no mucous cell was observed in terminal and respiratory bronchioles. Mucous cells were characterized by the presence of Alcian blue and/or PAS-positive granules. The majority of mucus-containing cells in the bronchi and bronchioles were stained blue with AB pH 2.5 showed that containing acidic mucosubstances (Fig. 3D), or red with the PAS showing the neutral mucosubstances in them (Fig. 3C). 1266 YANG ET AL. Fig. 9. Mucous cell in bronchial epithelium of yak contained basal nuclear and abundant dense secretory granules (SG) with a lucent core (A). Mitochondrial (M) and RER were also observed (B). C, ciliated cell; Ci, cilia; GL, globular leukocyte; N, nuclear. PAS decreased distally along the airways. Although no cells staining only with Alcian blue were seen, an alcianophilic layer was present at the epithelial-luminal interface at all levels of the airways, and appeared to lie over all types of cell. Electron Microscopy Serous sell. No serous cells were observed throughout the whole airways epithelium. Fig. 10. Mucous cell in bronchial epithelium had numerous vacuoles (V) among secretory granules (SG). C, ciliated cell; Ci, cilia; N, nuclear. In the combined AB-PAS staining these cells stain blue or bluish-purple (Fig. 3B). However, the AB pH 1.0 showed that the majority of the goblet cells had sulphated mucin. These cells stained blue or light blue (Fig. 3E). Cells staining only with Alcian blue were never seen, whilst the proportion of cells staining only with Mucous cell. Mucous cells were columnar cells with a microvillar surface, abundant cytoplasm, and a basal nucleus. The nucleus, looked oval in shape and contained electron-dense chromatin, was located immediately above the basal cell layer and below the level of the ciliated cell nuclei (Fig. 9). The cytoplasm was composed mostly of secretory granules. Mitochondria and RER were dispersed throughout the cytoplasm. Golgi apparatus were occasionally present. Mucous granules were roughly round membrane-bound structures. Some granules contained an eccentric lucent core (Fig. 9B). Some, less-frequent, mucous cells cytoplasm contained perinuclei Golgi apparatus, mitochondria, numerous speciﬁc vacuoles, amounts of distended RER, and few number of membrane-bound dense granules (Figs. 10 and 11). DISCUSSION Submucosal Glands Through extensive examination of tissue sections detected from lung of yak, we found there were substantial numbers of submucosal glands along tracheobronchial trees in this species of animals. Similar observations as previously reported in camel (Raji and Naserpour, 2007), sheep (Mariassay and Plopper, 1984), RESPIRATORY EPITHELIUM AND GLAND IN YAK 1267 Serous Cell Fig. 11. Mucous cell in bronchial epithelium had a basal dentate nuclear (N) with distended RER surrounded and many secretory granules (SG) at apical area. C, ciliated cell. goat (Kahwa and Purton, 1996), cat (Gatlagher et al., 1975), dog (Takenaka et al., 1996) and ferret (Robinson et al., 1986). There are variables between different species of animals. Submucosal glands in the treachea of the horse are known to be smaller than that of other mammalian species (Widdicombe and Pecson, 2002), and no similar gland had been reported in buffalo lung (Singh and Mariappa, 1981). In case of mice, some glands only had been discovered at the border between the trachea and the larynx (Choi et al., 2000), agreement with the ﬁndings of Pack et al (1980). For rat, the glands were present in the cranial third of the trachea, and the number of glandular tissues may increase near the carina (Steiger et al., 1995; Ohtsuka et al., 1997; Choi et al., 2000). In respect to guinea pig, there were some contradictions: some have reported that the glands of guinea pig were present in the trachea (Okamura et al., 1996; Widdicombe et al., 2001). Others believed that the tracheal submucosal glands in guinea pig were infrequent or even absent (Jeffery, 1983; Yeadon et al., 1995). Kennedy et al. (1978) claimed that they noted the distribution of glands in the hamster, which can be found in the trachea and the larynx, but very rare in the rest of the tracheobronchial tree. For small animals, such as rabbits, hamsters, rats and mice (Borthwick et al., 1999; Widdicombe et al., 2001) ﬁndings of submucosal gland were infrequent, and if they did appear, their appearances only occurred in the uppermost portion of the trachea. A signiﬁcant correlation has been found between airway diameter and gland volume per unit surface area of trachea in different of species, such as ox, goat, sheep, pig, monkey, dog, cat, rabbit, guinea pig, hamster, rat, mouse, and human, suggesting that the rate of deposition of inhaled particles may increase in large airways (Choi et al., 2000; Widdicombe et al., 2001). As aforementioned, under electron microscopic observations, two different types of serous cells in submucosal glands of yak lung are discernible. First type is termed as common serous cells, and second type as uncommon. Serous cells in yak lung were found only in submucosal glands resembled sheep (Mariassay and Plopper 1984). These cells ﬁlled with densely packaged granules, whereas in sheep it contained electron-dense granules with a more electron-dense core. In mice, submucosal gland usually contained only one form of secretory granulewhich produced either mucous or serous substance. It was conﬁrmed that the number of secretory granules varied possibly reﬂecting different states of synthesis or discharge, whereas a cell was almost devoid of granules, the cytoplasm was ﬁlled with a dense array of SER. In man bronchus tree, the volume of serous and mucous cells in glands was about 61 to 39% (Basbaum et al., 1990). As reported by some investigators, serous cells had electron-dense cytoplasm, more RER, in contrast with that of mucous cells, granules of which were described as discretely electron-dense and measured 300–1000 nm in diameter (Rogers et al., 1993; Jeffery and Li, 1997; Finkbeiner, 1999). Serous and mucous cells in the lung of yaks were calculated to be approximately 83% and 17% in the volume of the glands (Table 2). In addition, the second type serous resembled that of ferret (Basbaum, 1986), the common serous cells were similar to other animals. Mucous Cells At the TEM level, most mucus-producing cells contained a mixture of both serous (electron-dense) and mucous (electron-lucent) secretory vesicles. In the yaks, mucous cells, which are present in surface epithelia and submucosal glands, were found to include secretory granules. They appeared to be electron dense with lucent core and uncoalescing. The ﬁndings were agreement with the observation of the previous study (He et al., 2009). However, what is seen in the lung of yaks differs from granules in other species reported by others, such as sheep (Mariassay et al., 1988), monkeys (Wilson et al., 1984; Plopper et al., 1989), and rodents (Kennedy et al., 1978; Dalen, 1983). Mucous cells in sheep were classiﬁed into four types. Granules in mucous cells of types M1 and M2 had a less electron-dense meshwork, whereas granules in types M3 and M4 were electronlucent with electron dense cores. For Bonnet monkey, mucous granules were membrane-bound structures containing a centrally dense core, with occasional coalescing. In Rhesus monkey, mucous cells were ﬁlled with membrane-bound granules of variable electron densities. They were either biphasic or triphasic granule with a lucent rim and cores. Granules in guinea-pig and hamster mucous cells were all membrane-bound with a characteristic electron-dense cores, the former coalesced near the cell surface, but the latter did not coalesce. In mouse and rat, the goblet cells were rarely found in treacheobronchial tree, electron lucent incomplete membranebound secretory granules with an electron-dense core were often conﬂuent. Electron-dense cytoplasm containing conﬂuent granules of electron-lucent with about 800 nm in diameter were reported in mucous cells of 1268 YANG ET AL. man by Jeffery and Li (1997). The electron density and a series of different staining (PAS) of granules mucous cells in the glands and surface epithelium suggest that the cells are probable of the same phenotype. In the report by Staley and Trier (1965) who used AB/PAS staining a similar correlation between PAS reactivity and electron density for part of the ‘two-toned’ granule of the mouse Paneth cell, showed that the outer electron-lucent halo of the granule contained an acid mucosubstance (AB-positive), whereas the inner electrondense core contained a neutral protein (PAS-positive). Mucus Property The histochemical results shown here reveal three types of mucosubstances in yak tracheobronchial tree. These were neutral mucins, acidic mucins, and sulphated mucins. The nature of the mucosubstances of submucosal glands seen in this study was similar to those of many other species reported already. In yak, the mucosubstances of mucous cells in submucosal gland were strongly positive with PAS and/or AB and toluidine blue staining, whereas the serous cells were positive with PAS and negative with AB and toluidine blue staining. In goat, submucosal glands produced predominantly acidic mucosubstances with only a few producing a mixed reaction, neutral mucosubstances were rarely observed (Kahwa and Purton, 1996). Serous cells were found exclusively in submucosal glands in sheep, which stained light magenta with AB/PAS. The staining was restricted to the apically localized granules (Mariassay et al., 1988). In dog, a mixture of sulphomucin and sialomucin was found in the bronchial glands (Wheeldon et al., 1976). The ferret submucous glands contained predominantly neutral mucins, and scattered between these cells containing sulphated mucins and sialidase-labile and sialidaseresistant sialomucins (Robinson et al., 1986). The human tracheobronchial submucous glands contain various types of sulphomucins and sialomucins, also contain some neutral mucins (Lamb and Reid, 1972). In addition to mucins, tracheobronchial serous cells in glands of man secrete a number of antimicrobial and immunological products, and these are likely to play important roles in healthy, as well as diseased states (Thompson et al., 1995; Finkbeiner, 1999). In the yak bronchial tree, the mucosubstances produced by surface mucus-producing cells exhibited both acidic and mixed mucosubstances, and the present observation is in agreement with observations made in other mammalian species, including the camel (Raji and Naserpour, 2007), goat (Kahwa and Purton, 1996), sheep (Mariassay et al., 1988), Rhesus monkey (Plopper et al., 1984) and man (Spicer et al., 1983). However, in buffalo lung there were no goblet cells (Singh and Mariappa, 1981). The histochemistry of mucosubstances in the tracheobronchial tree of the yak similar to that observed in some mammalian species, such as sheep (Mariassay et al., 1988), pig (Jones et al., 1975) and one-humped camel (Raji and Naserpour, 2007) in which neutral mucosubstances were seen to predominate, but in adult Rhesus monkey (Plopper et al., 1989) and goat (Kahwa and Purton, 1996) the acidic mucosubstances were present in vast majority of surface goblet cells in trachea and proximal bronchi. But among the carnivores, Wheel- don et al. (1976) found sulphated and neutral mucin were the predominant mucosubstance in the epithelial goblet cells of dog with hardly any sialomucin. The cat bronchial tree contained mostly sulphated mucin with a very small amount of sialidase-resistant sialomucin (Gatlagher et al., 1975). In ferret tracheal goblet cells, as well as in the bronchi and larger bronchioles, contained sulphated mucins, only a smaller proportion of the goblet cells showed sialidase-labile and sialidase-resistant sialomucins (Robinson et al., 1986). Goblet cells in manhad been demonstrated to contain neutral, sialylated, and sulphated sugars (Kim and Jeffery, 1997; Kim et al., 1997). In the tissue sections of lung, mucous cell with granules were stained purple with toluidine blue (Figs.2F, 3F), Granules in serous cell would not be stained in such manner. This was probably an indication that two different types of granules in two different kinds of cells. However, other investigators have shown metachromatasia of toluidine blue or azur A in both mucous and serous cells, which they claimed distinguished between sialomucin and sulphomucin (Tock and Tan, 1969; Lamb and Reid, 1970). From this investigation, we concluded that histochemical nature and ultrastructural characteristics of mucous cells in the respiratory epithelia and submucosal glands of yak’s lungs were basically similar to those of other domestic animals. Though certain differences and variables were observed in each species, the discrepancies may be due to mutable environmental conditions, e.g. alpine, hypoxia, and cold climate, as well as dissimilarity in the use of histochemical techniques. ACKNOWLEDGMENTS The authors thank with deep and sincere appreciation for the editorial assistance provided by H. C. Dung, Ph.D., a retired professor of anatomy, University of Texas Health Science Center at San Antonio, San Antonio City, Texas, USA, also the farmers for the help with animal specimen and Yanyu He in Laboratory Center of Gansu Agricultural University for the technical support. LITERATURE CITED Basbaum CB. 1986. Regulation of airway secretory cells. Clin Chest Med 7:231–237. Basbaum CB, Jany B, Finkbeiner WE. 1990. The serous cell. Annu Rev Physiol 52:97–113. Borthwick DW, West JD, Keighren MA, Flockhart JH, Innes BA, Dorin JR. 1999. Murine submucosal glands are clonally derived and show a cystic ﬁbrosis gene-dependent distribution pattern. Am J Respir Cell Mol Biol 20:1181–1189. Choi HK, Finkbeiner WE, Widdicombe JH. 2000. A comparative study of mammalian tracheal mucous glands. J Anat 197(Part 3):361–372. Dalen H. 1983. An ultrastructural study of the tracheal epithelium of the guinea-pig with special reference to the ciliary structure. J Anat 136:47–67. Finkbeiner W. 1999. Physiology and pathology of tracheobronchial glands. Respir Physiol 118:77–83. Gatlagher JT, Kent PW, Passatore M, Phipps RJ, Richardson PS. 1975. Composition of tracheal mucus and its secretion in the cat. Proc R Soc Lond 192:49–56. George JA, Nishio SJ, Cranz DL, Plopper CG. 1986. Carbohydrate cytochemistry of rhesus monkey tracheal submucosal glands. Anat Rec 216:60–67. RESPIRATORY EPITHELIUM AND GLAND IN YAK He J-f, Yu SJ, Cui Y. 2009. Characteristics of lung structure in different age plateau yak. acta veterinaria et zootechnica sinica 40:748–755. Jeffery PK. 1983. Morphologic features of airway surface epithelial cells and glands. Am Rev Respir Dis 128:S14–S20. Jeffery PK, Li D. 1997. Airway mucosa: secretory cells, mucus and mucin genes. Eur Respir J 10:1655–1662. Jones R, Baskerville A, Reid L. 1975. Histochemical identiﬁcation of glycoproteins in pig bronchial epithelium J Pathol 98:213–229. Kahwa CKB, Purton M. 1996. Histological and histochemical study of epithelia respiratory tract in adult goat. Small Ruminant Res 20:181–186. Kennedy AR, Desrosiers A, Terzaghi M, Little JB. 1978. Morphometric and histological analysis of the lungs of Syrian golden hamsters. J Anat 125:527–553. Kim KC, Jeffery PK. 1997. Airway mucus. Eur Respir J 10:1438. Kim KC, McCracken K, Lee BCea. 1997. Airway goblet cell mucin: its structure and regulation of secretion. Eur Respir J 10: 2644–2649. Lamb D, Reid L. 1970. Histochemical and autoradiographic investigation of the serous cells of the human bronchial glands. J Pathol 100:127–138. Lamb D, Reid L. 1972. Quantitative distribution of various types of acid glycoprotein in mucous cells of human bronchi. Histochem J 4:91–102. Lev R, Spicer SS. 1964. Speciﬁc staining of sulphate groups with alcian blue at low Ph. J Histochem Cytochem 12:309. Mariassay AT, Plopper CG. 1983. Tracheobronchial epithelium of the sheep: I. Quantitative light-microscopic study of epithelial cell abundance, and distribution. Anat Rec 205:263–275. Mariassay AT, Plopper CG. 1984. Tracheobronchial epithelium of the sheep: II. Ultrastructural and morphometric analysis of the epithelial secretory cell types. Anat Rec 209:523–534. Mariassay AT, St George JA, Nishio SJ, Plopper CG. 1988. Tracheobronchial epithelium of the sheep: III. Carbohydrate histochemical and cytochemical characterization of secretory epithelial cells. Anat Rec 221:540–549. McCarthy C, Reid, L. 1964. Acid mucopolysaccharide in the bronchial tree in the mouse and rat. Qu J Exp Physiol 49:81–84. McManus JFA. 1948. Histological and histochemical uses of periodic acid. Stain Technol 23:99–108. Mowry RW. 1956. Alcian blue techniques for the histochemical study of acidic carbohydrates. J Histochem Cytochem 4:407–408. Ohtsuka R, Doi K, Itagaki S. 1997. Histological characteristics of respiratory system in Brown Norway rat. Exp Animals 46: 127–133. Okamura H, Sugai N, Kanno T, Shimizu T, Ohtani I. 1996. Histochemical localization of carbonic anhydrase in the trachea of the guinea pig. Histochem Cell Biol 106:257–260. Pack RJ, Al-Ugaily LH, Morris G, Widdicombe JG. 1980. The distribution and structure of cells in the tracheal epithelium of the mouse. Cell Tissue Res 208:65–84. Plopper CG, George JA, Nishio S, Etchison JR, Netfesheim P. 1984. Carbohydrate cytochemistry of tracheobronchial airway epithelium of the rabbit. J Histochem Cytochem 32:209–218. 1269 Plopper CG, Heidsiek JG, Weir AJ, George S, Hyde DM. 1989. Tracheobronchial epithelium in the adult Rhesus monkey: a quantitative histochemical and ultrastructural Study. Am J Anat 184:31–40. Raji AR, Naserpour M. 2007. Light and electron microscopic studies of the trachea in the One-Humped camel (Camelus dromedarius). Anat Histol Embryol 36:10–13. Reid L. 1960. Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis. Thorax 15:132–141. Robinson NP, Venning L, Kyle H, Widdicombe JG. 1986. Quantitation of the secretory cells of the ferret tracheobronchial tree. J Anat 145:173–188. Rogers AV, Dewar A, Corrin B, Jeffery PK. 1993. Identiﬁcation of serous-like cells in the surface epithelium of human bronchioles. Eur Respir J 6:498–504. Shimura S. 1990. Methods for the morphological study of tracheal and bronchial glands. In: Gil J, editor. Models of Lung Disease: Microscopy and Structural Methods. Florida: CRC Press. p 309–359. Singh K, Mariappa D. 1981. Histological studies of the conducting and the respiratory division of the buffalo lung. Indian Vet J 58:99–103. Spicer SS, Schulte BA, Chakrin LW. 1983. Ultrastructural and histochemical observations of respiratory epithelium and gland. Exp Lung Res 4:137–156. Staley MW, Trier JS. 1965. Morphologic heterogeneity of mouse Paneth cell granules before and after secretory stimulation. Am J Anat 117:365–384. Steiger D, Hotchkiss J, Bajaj L, Harkema J, Basbaum C. 1995. Concurrent increases in the storage and release of mucin-like molecules by rat airway epithelial cells in response to bacterial endotoxin. Am J Resp Cell Mol 12:307–314. Takenaka S, Heini A, Ritter B, Heyder J. 1996. Morphometric evaluation of bronchial glands of beagle dogs. Toxicol Lett 88: 279–285. Thompson AB, Robbins RA, Romberger DJea. 1995. Immunological functions of the pulmonary epithelium. Eur Respir J 8:127–149. Tock EP, Tan NT. 1969. A histochemical study of the mucins of the adult human nasopharynx. J Anat 104:81–92. Wheeldon EB, Pnue HM, Bassz RG. 1976. A histochemical study of the tracheobronchial epithelial mucosubstances in normal dogs and in dogs with chronic bronchitis. Folia veterinaria Latina 6:45–58. Widdicombe JH, Chen LL, Sporer H, Choi HK, Pecson IS, Bastacky SJ. 2001. Distribution of tracheal and laryngeal mucous glands in some rodents and the rabbit. J Anat 198:207–221. Widdicombe JH, Pecson IS. 2002. Distribution and numbers of mucous glands in the horse trachea. Equine Vet J 34:630–633. Wilson DW, Plopper CG, Hyde DM. 1984. The tracheobronchial epithelium of the bonnet monkey (macacaradiata): a quantitative ultrastructural study. Am J Anat 171:25–40. Yeadon M, Price RC, Payne AN. 1995. Allergen-induced glycoconjugate secretion in guinea pig trachea in vivo: modulation by indomethacin, BW B70C and ZD-2138. Pulm Pharmacol 8: 53–63.