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. 2010 May 11;217(1):57–66. doi: 10.1111/j.1469-7580.2010.01243.x

The differences between the localizations of MUC1, MUC5AC, MUC6 and osteopontin in quail proventriculus and gizzard may be a reflection of functional differences of stomach parts

Narin Liman 1, Emel Alan 1, Güner Küçük Bayram 1
PMCID: PMC2913012  PMID: 20492430

Abstract

Mucins are high molecular weight glycoproteins which constitute the major component of the mucus layer and are produce by many epithelial tissues in vertebrates. Osteopontin (OPN) is an adhesive phosphorylated glycoprotein that is expressed by a broad range of tissues and cells. Although gastric mucins MUC1, MUC5AC, MUC6 and OPN have been widely used in histological studies and in diagnostic pathology in order to diagnose gastric carcinomas, their localizations in the stomach of quail have not yet been studied. In this study, the localizations of MUC1, MUC5AC, MUC6 and OPN in the proventriculus and gizzard of Japanese quail during the post-hatching period were compared at light microscope levels by applying immunohistochemical methods. In all ages studied, the immunoreactivity of MUC5AC was present in the lining epithelium of both folds and superficial proventricular glands in the proventriculus, whereas MUC1, MUC6 and OPN reactivity was found in the oxynticopeptic cells of profound proventricular glands. In addition, some cells in the fold epithelium of the proventriculus showed a positive reaction to OPN. The immunoreactivity of MUC1 in gizzard was different from that of MUC5AC. Although MUC5AC was expressed in the cells of both the surface epithelium and profound glands of the gizzard, MUC1 was only localized in the profound glands of the gizzard. However, MUC6 and OPN immunoreactivity was absent in the gizzard. The results indicated that the differences between the localizations of MUC1, MUC5AC, MUC6 and OPN in quail proventriculus and gizzard may be a reflection of functional differences of stomach parts. Although the biological significances of the expressions of MUC1, MUC5AC, MUC6 and OPN in the quail stomach remains unknown, these notable glycoproteins may be associated with barrier function, host defence, and/or secretion.

Keywords: gizzard, MUC1, MUC5AC, MUC6, osteopontin, proventriculus, quail

Introduction

The avian stomach is peculiar in that it consists of two morphologically and physiologically distinct parts: the glandular portion, or proventriculus, and the muscular portion, or gizzard (Hodges, 1974; King & McLelland, 1984). Both of them are lined by tall columnar mucous cells. The proventriculus has two types of glands: the superficial simple tubular glands and the profound proventricular glands, which are lined by oxynticopeptic cells. The oxynticopeptic cells secrete both hydrochloric acid and the enzyme precursor pepsinogen, hence combining the functions of the mammalian peptic and parietal cells (Hodges, 1974). The interior surface of the gizzard is lined with a cuticle, sometimes called koilin, which is produced by the mucosal gland (Hodges, 1974; King & McLelland, 1984: Lumeij, 1994; Whittow, 2000). The proventriculus is the site of acid secretion, whereas the gizzard functions in mechanical digestion and is the site of gastric proteolysis. Hydrochloric acid, mucus and a digestive enzyme called pepsin are secreted in the proventriculus and start the process of breaking down the structure of the food material the bird has eaten. The food then passes to the second part of the stomach, the gizzard. The gizzard performs the same function as mammalian teeth, grinding and disassembling the food, making it easier for the digestive enzymes to break down the food. In most birds the gizzard contains sand grains or small rocks to aid the grinding process.

Mucins, high molecular weight glycoproteins which constitute the major component of the mucus layer, are secreted in large quantities by the mucosal epithelia of the gastrointestinal, respiratory, reproductive and urinary tracts, and the surface of the eye (Perez-Vilar, 2007; Linden et al. 2008). From a biochemical standpoint, mucins are composed of approximately 15–20% protein and up to 80% carbohydrate, present largely in the form of O-linked glycans (Strous & Dekker, 1992; Gendler & Spicer, 1995; Hideyuki, 2005). Epithelial mucins can be subdivided into secretory and membrane-associated forms. The secretory forms are synthesized, stored, and then released by exocytosis from the apical surface of secretory cells to help form the overlying mucus gel. Moreover, the secreted mucins are divided into two subgroups, gel-forming and soluble mucins. In contrast, the membrane associated forms have a hydrophobic membrane-spanning domain that serves to anchor them to the plasma membrane (Hattrup & Gendler, 2008; Thornton et al. 2008). Mucins are made up of core proteins coded by mucin genes specific to their types and chains of numerous oligosaccharides binding to the core proteins (Gendler & Spicer, 1995). To date, 21 human mucin genes, named MUC1, 2, 3A, 3B, 4, 5AC, 5B, 6–9, 11–13 and 15–21, have been distinguished by cDNA cloning (Porchet et al. 1999; Dekker et al. 2002; Rose & Voynov, 2006; Hattrup & Gendler, 2008; Itoh et al. 2008). Mucin genes are expressed in a regulated cell- and tissue-specific manner. The stomach provides a good example of such differential expression of mucin genes. The normal gastric mucosa shows cell type-specific expression of MUC1, MUC5AC and MUC6, with the first two mucins found in the superficial epithelium and MUC6 in the deep glands (Audie et al. 1993; Ho et al. 1993, 1995a,b; De Bolós et al. 1995; Reis et al. 1997; Bartman et al. 1998; Pinto de Sousa et al. 2002; McAuley et al. 2007; Lacunza et al. 2009a,b;). MUC1 is of the membrane-bound type, whereas MUC5AC and MUC6 are of the secreted, gel-forming type (Ho et al. 1995a; Corfield et al. 2000; Lee et al. 2001; Hattrup & Gendler, 2008).

Osteopontin (OPN) is a secreted, highly acidic, calcium-binding arginine-glycine-aspartate (RGD) containing, integrin-binding phosphorylated glycoprotein originally isolated from bone matrix (Franzén & Heinegård, 1985; Oldberg et al. 1986; Butler, 1989; Denhardt et al. 1995; Sodek et al. 2000). Luminal surfaces of epithelia that interface with the environment and contain a wide variety of secretory cells (i.e. gastrointestinal tract, gallbladder, pancreas, urinary tract, reproductive tract, lung, breast, salivary glands and sweat glands) commonly contain OPN (Brown et al. 1992). Sodek et al. (2005) have reported that OPN has the potential to regulate the specific function of epithelial cells involved in the barrier defence process (Sodek et al. 2005). Qu-Hong et al. (1997) have suggested that OPN in human stomach play an important role in barrier function, host defence, and/or secretion generally associated with epithelia. Stainings for gastric mucin MUC5AC (Ho et al. 1995b; Buisine et al. 2000; Nordman et al. 2002; Pinto de Sousa et al. 2002), MUC6, MUC1 (Lacunza et al. 2009a,b;) and OPN (Qu-Hong et al. 1997; Ue et al. 1998; Corfield et al. 2000; Sun et al. 2005; Higashiyama et al. 2007) have been used widely in histological studies and in diagnostic pathology to diagnose gastric carcinomas. Furthermore, mucin-type glycoproteins are widely distributed in nature, but our knowledge on this is limited by the scarcity of data on record in the literature for non-mammalian species. As OPN, MUC1, MUC5AC and MUC6 were found to be present in the human stomach, our attention has been focused on the localization of these proteins in quail stomach, which consists of two morphologically and physiologically distinct parts. Our aim was to evaluate, using immunohistochemistry, the pattern of expression of MUC1, MUC5AC and MUC6 and OPN in different regions of the gastric mucosa of quail during the post-hatching period.

Materials and methods

Animals and tissue preparation

The quails used in the study were obtained from a farm raising quails, which did not have any problem related to flock health (Safiye Cikrikcioglu Vocational College, University of Erciyes, Kayseri).

A total of 25 Japanese quails (Coturnix coturnix japonica) were used, starting from the first day of hatching. The animals were housed in individual cages, with free access to water and food. The photoperiod was 16 h of light and 8 h of dark. In the first post-hatching week, the temperature where the animals were housed was maintained at 35 °C. Subsequently, the pen temperature was reduced 5 °C per week until 22–24 °C was reached. The humidity in the pens was measured with a thermo-hygrometer, and the relative humidity of the environment was maintained at 50–60%. The pens were ventilated naturally, with the entrance of fresh air through windows, panels and low gate wings. This study was approved by the Ethics Committee of the Faculty of Veterinary Medicine, University of Erciyes (approval number 2003-28-32).

Five males each at ages 1, 5, 10, 30, and 125 days were sacrificed under ether anaesthesia and their stomachs were totally removed and separated into proventriculus and gizzard parts. The stomach parts were fixed in a 10% formol-alcohol solution for 24 h, dehydrated, cleared, and embedded in Paraplast.

Immunohistochemistry

Immunohistochemistry was performed at room temperature according to the previously reported immunostaining protocol of Thermo Fisher Scientific (Lab Vision Corporation, Fremont, CA, USA). Four slides were prepared from each sample, and each slide contained a minimum of two sections cut at 5 μm thickness. The tissue sections were mounted on glass slides coated with 3-aminopropyl-ethoxy-silane (APES) (Sigma-Aldrich Chemicals, St. Louis, MO, USA) and dried at 37 °C. Briefly, sections were de-paraffinized in xylene and rehydrated through a graded series of ethanol. To block any endogenous peroxidase activity/or non-specific staining, the sections were treated with 3% H2O2 in methanol for 15 min at room temperature and washed with phosphate-buffered saline (PBS; pH 7.4) two times. Antigen retrieval was performed in citrate buffer (pH 6) for 30 min at 95 °C with cooling for 30 min before immunostaining. After that, the sections were washed in PBS (pH 7.4), incubated in blocking serum (Ultra V Block; TA-125UB; Thermo Fisher Scientific Lab Vision Corporation) for 5 min at room temperature to block non-specific binding, and then left in the corresponding antibody for 60 min.

One series of slides was incubated with a rabbit polyclonal antibody against OPN (Epitope Specific Rabbit PAb IgG; Thermo Fisher Scientific Lab Vision Corporation) at a dilution of 1 : 50. The second series of slides was incubated with a mouse monoclonal antibody against MUC5AC [Mucin 5AC Ab-1 (Clone 45M1); Thermo Fisher Scientific Lab Vision Corporation] at a dilution of 1 : 100. The third series of slides was incubated with a mouse monoclonal antibody against MUC6 [Mucin 6 Ab-1 (Clone CLH5); Thermo Fisher Scientific Lab Vision Corporation] at a dilution of 1 : 100. The fourth series of slides was incubated with a hamster monoclonal antibody against MUC1 (Ab-5, Clone MH1; same as CT2; Thermo Fisher Scientific Lab Vision Corporation) at a dilution of 1 : 100. Thereafter, sections were washed in PBS, incubated with biotinylated anti-mouse antiserum (when primary antibody recognized MUC5AC and MUC6) or biotinylated anti-rabbit antiserum (when primary antibody recognized OPN) (Thermo Fisher Scientific Lab Vision Corporation) for 20 min at room temperature, and washed in PBS. Sections were incubated with streptavidin peroxidase for 20 min at room temperature, and washed with PBS. The peroxidase activity was visualized with 3,3-diaminobenzidine (DAB; Thermo Fisher Scientific Lab Vision Corporation) producing a brown color. Sections were counterstained with Gill’s haematoxylin, dehydrated through an alcohol series, cleared in xylene and finally embedded in Entellan® under a coverslip.

The specificity of immunohistochemical procedures was checked using positive and negative control sections. As positive controls, sections of human stomach and normal chicken proventriculus were incubated with primary antibodies. As negative control, sections were incubated with PBS alone, without the primary antibody. All samples were treated with exactly the same protocol.

Tissue sections from different post-hatching days were examined by conventional light microscopy (BX51; Olympus, Tokyo, Japan) and were evaluated for MUC1, MUC5AC, MUC6 and OPN localizations.

Results

In all ages studied, we observed that the walls of proventriculus and gizzard in the quails consisted of three tunics: the tunica mucosae, the tunica muscularis and the tunica serosae. The transverse section of mucosa of proventriculus was characterized by the presence of folds that are finger-like projections. The proventriculus had two types of proventricular glands. The surface epithelium, which is a simple prismatic epithelium, invaginated into the lamina propria to form the superficial, simple tubular glands. The profound proventricular glands, which are the principal component of the proventriculus, are located within the submucosa. The glands are composed of numerous rounded or polymorphic lobules. The each lobule is composed of numerous secretory tubules lined by oxynticopeptic cells. The secretory tubules of each lobule join together to form a short common tertiary secretory duct opening into the the secondary ducts. The secondary ducts of several lobules join together to form a primary secretory duct. The primary, secondary and tertiary ducts are lined with a simple prismatic epithelium similar to that of the mucosal surface.

The mucosa of gizzard was also characterized by the presence of low folds, which lined the simple prismatic epithelium. Over the epithelium of gizzard, a cuticle was found. The lamina propria was occupied by numerous simple tubular profound glands which expanded in the base of folds, located in parallel between them. Those glands are lined by a simple epithelium, which is lower in the base of the glands and higher in their upper portion.

In all ages studied, MUC5AC was highly expressed in the lining epithelium of both folds and superficial proventricular glands of the mucosa. In the fold epithelium of proventriculus, the MUC5AC immunoreactivity was localized throughout the entire supranuclear and apical cytoplasm of cells. MUC5AC was not detected in the oxynticopeptic cells of profound proventricular glands, except for an occasional staining of the apical cytoplasm of duct epithelial cells (Fig. 1A).

Fig. 1.

Fig. 1

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Immunohistochemical staining of quail proventriculus with anti-MUC5AC (A), anti-MUC1 (B), anti-MUC6 (C) and anti-OPN (D). (A) MUC5AC was highly expressed in the lining epithelium of both folds and superficial proventricular glands of the mucosa. (B,C,D) MUC1, MUC6 and OPN were detected in the cytoplasm of oxynticopeptic cells of profound proventricular glands. L, lumen; F, folds; SG, superficial proventricular glands; PG, profound proventricular glands; D, ducts, Lp, lamina propria. Scale bars: 200 μm.

In the proventriculus studied from the first day of hatching until day 125, expressions of MUC1 (Fig. 1B), MUC6 (Fig. 1C) and OPN (Fig. 1D) were detected in the cytoplasm of oxynticopeptic cells of profound proventricular glands. In addition, weak MUC1 immunoreactivity was found in the deep region of superficial proventricular glands. In 1-day-old quails, MUC1, MUC6 and OPN reactivities were usually visualized in basal compartment of oxynticopeptic cells, whereas the nucleus was localized in the apical pole of these cells (Fig. 2A,C,E). After day 10, the immunoreactivities for MUC1, MUC6 and OPN were mainly located in a para-nuclear position and throughout the entire apical cytoplasm of oxynticopeptic cells (Fig. 2B,D,F). Microscopic examination at ×400 or ×1000 (objective: ×40 or ×100, eye piece: ×10) and oil immersion revealed that the immunostainings for MUC1, MUC6, OPN are present in the cytoplasm in the form of granules of various sizes (Fig. 2A–F). It was also observed that OPN immunoreactivity in oxynticopeptic cells increased with the advance of age, and was weaker in 1-day-old quail than those of the other age groups (Fig. 2E,F). Following day 10 after hatching, although a few cells in the epithelium of the folds exhibited a positive reaction to OPN antibody, the superficial tubular glands showed a negative reaction to OPN antibody. It was also determined that the amount of cells of fold epithelium that exhibited OPN immunoreactivity increased with the advance of age and organ growth. OPN immunoreactivity was localized in the para-nuclear cytoplasm and the baso-lateral membrane (basal two-thirds of lateral surface of cells) in the cells of fold epithelium (Fig. 3). Cross-sections of fold epithelium showed that OPN in the baso-lateral membrane of the epithelial cells was outside the cell. In addition, OPN immunoreactivity was visualized in the cytoplasm of a few duct epithelium cells.

Fig. 2.

Fig. 2

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Immunohistochemical staining of quail proventriculus with anti-MUC1 (A,B), anti- anti-MUC6 (C,D) and anti-OPN (E,F). (A,C,E) In 1-day-old quails, MUC1, MUC6 and OPN reactivities were usually visualized in the basal compartment of oxynticopeptic cells, whereas the nucleus was localized in the apical pole of these cells. (B,D,F) After day 10, the immunoreactivities for MUC1, MUC6 and OPN were mainly located in a para-nuclear position and throughout the entire apical cytoplasm of oxynticopeptic cells. PG, profound proventricular glands; D, ducts. Scale bars: 25 μm (C,E), 50 μm (A,B,D,F).

Fig. 3.

Fig. 3

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OPN immunoreactivity was localized in the para-nuclear cytoplasm and the baso-lateral membrane (basal two-thirds of lateral surface of cells) in the cells of fold epithelium (F). L, lumen. Scale bars: 100 μm.

In the gizzard studied from the days 1–125 post-hatching, the cells of both surface epithelium and simple tubular profound glands and koilin exhibited a positive reaction for the MUC5AC (Fig. 4A). Although the surface epithelium of gizzard did not react with MUC1, the cells of simple tubular profound glands were immunopositive (Fig. 4B). Moreover, MUC1 and MUC5AC were highly expressed in the secretions filling the lumen of glands (Fig. 4A,B). However, MUC6 and OPN were not detected in gizzard mucosa.

Fig. 4.

Fig. 4

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The cells of both surface epithelium and simple tubular profound glands and koilin exhibited a positive reaction for the MUC5AC (A). Although the surface epithelium of gizzard did not react with MUC1, the cells of simple tubular profound glands were immunopositive. Immunohistochemical staining of quail gizzard with anti-MUC1 (B). L, lumen; C, koilin; F, folds; Ptg, profound simple tubular glands. Scale bars: 100 μm (A), 50 μm (B).

Discussion

The gastric mucosae of mammals and birds are covered by a continuous and adherent layer of viscoelastic mucus gel that provides a physical barrier with low permeability for pepsin and other macromolecules between the lumen and the apical cell surfaces. The viscous and gel-forming properties of this mucus gel are derived from mucin glycoproteins (mucins), which are made up of core proteins coded by mucin genes specific to their types and chains of numerous oligosaccharides binding to the core proteins (Gendler & Spicer, 1995). Mucins are characterized by a tandem repeat region rich in threonine and/or serine, which are O-glycosylation sites. MUC1, MUC5AC and MUC6 are the main constituents of the mucus barrier in the stomach (Audie et al. 1993; De Bolós et al. 1995; Ho et al. 1995a; Jia et al. 2010; Reis et al. 1997).

Previous histochemical studies on the stomach in birds have investigated the localization of the mucin carbohydrates, which are chains of numerous oligosaccharides bound to the mucin core proteins (Mogil’naia & Bogatyr’, 1977, 1983; Altamirano et al. 1984; Inforzato de Lima & Sasso Wda, 1985; Pastor et al. 1988; Pinheiro et al. 1989; Imai et al. 1991; Gheri et al. 1994, 1995; Sgambati et al. 1995, 1996). Moreover, early studies of changes in mucin expression during the development of the proventriculus and gizzard focused on alterations in the carbohydrate chain. When reviewing the literature, no information was found about the developmental changes and the localizations of mucin core protein MUC1, MUC5AC and MUC6 in the stomach of an avian species, the Japanese quail.

This study appears to be the first description of the presence and localization of mucin core proteins MUC1, MUC5AC and MUC6 in the stomach parts of quail during the post-hatching period. In this study, we used anti-M1 monoclonal antibody (Mabs) directed against the peptidic core of gastric M1 mucin. M1 immunoreactivity was recently described as encoded by the MUC5AC gene (Bara et al. 1998). To investigate the expressions of MUC1 and MUC6 mucins in quail stomach, we used two monoclonal antibodies, MH1 and CLH5, respectively.

Our findings, which showed the immunoreactivity of MUC1, MUC5AC and MUC6 in stomach parts of quails at hatching, support information in the literature (Altamirano et al. 1984; Avila et al. 1986; Gheri et al. 1994, 1995; Sgambati et al. 1995) that the mucin production in proventriculus and gizzard starts in the pre-hatching period, and suggest the hypothesis that the mucosal and glandular cells of stomach are functionally mature and secrete mucins at hatching.

Immunohistological studies have shown that the localizations of MUC1, MUC5AC and MUC6 are different in normal human gastric mucosa. Our results also indicated that the localizations of MUC1, MUC5AC and MUC6 in the mucosa of both the proventriculus and gizzard differed in the lining epithelium, the superficial glands and profound glands.

Studies in the stomach (Ho et al. 1993, 1995b; McAuley et al. 2007; Lacunza et al. 2009a,b;) showed that MUC1 is widely expressed in the apical membrane of cells of the surface epithelium and neck region of the gastric antrum, and is focally expressed in oxyntic glands of the fundus and pyloric glands of the antrum. Our immunohistochemical data demonstrated that MUC1 immunoreactivity is highly expressed in the oxynticopeptic cells of the proventricular profound glands and is moderately expressed in the lumen and the cell cytoplasm of the profound glands of gizzard. The oxynticopeptic cells secrete both hydrochloric acid and the enzyme precursor pepsinogen, hence combining the functions of the mammalian peptic and parietal cells. In birds, the proventriculus is lined by fundic glands, whereas the gizzard is lined by pyloric glands (Hodges, 1974). Therefore, our finding concerning the localization MUC1 in the oxynticopeptic cells of proventriculus and in the profound glands of gizzard is in agreement with previous reports in human stomach (Ho et al. 1993, 1995b). In addition, although the weak MUC1 immunoreactivity was detected in the deep region of superficial proventricular glands, no reaction was found in surface epithelium of either the proventriculus or the gizzard, contrary to previous studies (Ho et al. 1993, 1995b; McAuley et al. 2007; Lacunza et al. 2009a,b;).

The present study corroborated the presence of MUC5AC in the lining epithelia of the folds and in the superficial glands of the proventriculus, as well as in the cells of surface epithelium and profound glands of the gizzard. This is in agreement with previous reports from Lacunza et al. (2009a), who have observed the MUC5AC particularly stained the surface gastric epithelium from rat, rabbit, cat, pig and cow, as well as reports from other authors (Audie et al. 1993; De Bolós et al. 1995; Ho et al. 1995a; Reis et al.,1997), who have reported MUC5AC is highly expressed in the surface mucous cells of the cardia, fundus and antrum in human stomach. In agreement with Lacunza et al. (2009a), who did not find reactivity with MUC5AC in rat, rabbit, cat, pig or cow stomach glands, no reaction was found in the profound proventricular glands of quails. However, we observed that the cellular localization of MUC5AC in the profound glands of gizzard is restricted to the cell cytoplasm and the apical surface facing the lumen of glands.

The inner surface of the gizzard of granivorous birds is lined by a carbohydrate–protein complex called the koilin layer or cuticule (Hodges, 1974; King & McLelland, 1984; Lumeij, 1994). Previous histochemical studies reported that koilin contained both acid and neutral mucins (Pinheiro et al. 1989; Selvan et al. 2008). In the present study, the koilin also showed MUC5AC immunoreactivity, suggesting that MUC5AC is finally released into extracellular spaces together with mucin carbohydrates such as acid and neutral mucins. Our findings, which showed the immunoreactivity of MUC5AC in the koilin layer and in the mucosa of the gizzard in quails, support reports that koilin is formed by secretions originating from both the surface epithelium and the mucosal glands of gizzard (Hodges, 1974; King & McLelland, 1984; Lumeij, 1994).

Previous studies have shown that the MUC6 is expressed in the glands of the antrum and in mucopeptic cells of the neck zone of the fundus region of the normal human stomach (De Bolós et al. 1995; Ho et al. 1995a; Bartman et al. 1998). Reis et al. (2000) found that, in the human stomach, MAb CLH5 stained mucopeptic cells, which are characterized by particular histochemical and ultrastructural features of the mucin granules and include the glands of the antrum and the mucous cells of the neck zone of the fundus region. In the present study, we observed MUC6 expression in the oxynticopeptic cells of the profound glands of proventriculus. Because the oxynticopeptic cells in birds are analogues of the peptic and parietal cells in mammals, it can be assumed that this finding is similar to those of peptic cells in human stomach (De Bolós et al. 1995; Ho et al. 1995a; Bartman et al. 1998). In agreement with Reis et al. (2000), who did not detect reaction with MAb CLH5 in the foveolar cells of both antrum and fundus regions as well as the chief and parietal cells of oxyntic glands, no reaction was found in gizzard mucosa of quails.

This study indicates that the immunoreactivities for MUC1 and MUC6 are mainly located in a para-nuclear position and throughout the cytoplasm of oxynticopeptic cells. These immunostainings are in the form of granules of various sizes. This finding is in agreement with previous studies showing that the immunocytolocalization patterns of MUC6 in peptic cells of stomach were mainly diffuse cytoplasmic, and perinuclear or supranuclear (Reis et al. 2000) and that oxynticopeptic cells possess a very eosinophilic and somewhat granular cytoplasm. Although Lumeij (1994) reported that the oxynticopeptic cells do not contain PAS-positive mucin granules, our findings showed immunoreactivities of MUC1 and MUC6 in the oxynticopeptic cells of proventriculus, corroborating the presence of mucin core proteins of MUC1 and MUC6 in the oxynticopeptic cells.

Previous studies have reported that the histochemical characteristics and the trend of production of the mucous material in the gizzard during embryonic development are different when compared with those found in the proventriculus. These studies have further suggested that these differences are probably related to the different function of the mucins in the two parts of the stomach (Sgambati et al. 1995, 1996). Similarly, we may hypothesize that the functional difference in the two parts of the stomach is related to the presence of MUC1, MUC5AC and MUC6. We also suggest that MUC5AC is produced by the surface epithelium of both proventriculus and gizzard of quail. Furthermore, we suggest that MUC6 is only produced by the profound glands of proventriculus, whereas MUC1 is expressed in the profound glands of both proventriculus and gizzard of quail during the post-hatching period. In conclusion, it may be said that the expression of MUC1, MUC5AC and MUC6 was associated with stomach parts of quail. Nam et al. (2005) found that geranylgeranylacetone (GGA), a promising cytoprotective agent protected ethanol-induced gastric damage by upregulating MUC5AC and MUC6 rather than MUC1. Our findings, which showed immunoreactivity of MUC5AC in the surface epithelium of both the proventriculus and gizzard, and information in the literature prompt us to ask whether the function of mucin core protein MUC5AC in both stomach parts is to protect the epithelium against acid and pepsin in gastric juice and against exogenous damaging agents (e.g. pathogens, drugs), and against mechanical damage (Allen & Carrol, 1985; Allen et al. 1988).

OPN is an adhesive phosphorylated glycoprotein (Denhardt & Guo, 1993; Senger et al. 1994) that is expressed by a broad range of tissues and cells (Brown et al. 1992). OPN has been recognized as an important luminal regulator, due to its expression by epithelial cells covering luminal cavities capable of active secretion and absorption of nutrients or gases (Brown et al. 1992). Indeed, earlier studies, showing that epithelial cells secrete OPN first, indicated that OPN is involved in controlling the permeability of the epithelial barrier (Butler, 1989). In recent studies, it has been suggested that the constitutive expression of OPN by epithelial cells is required for maintaining the epithelial barrier in the gastrointestinal tract (Gassler et al. 2002) and OPN also has the potential to regulate the specific function of epithelial cells involved in the barrier defence process (Sodek et al. 2005). Furthermore, recent studies have reported that the increased expression of OPN is associated with increased cell mobilization, survival and activity, and is reflected in elevated concentrations of OPN in tissue fluids and plasma, which has potential diagnostic and prognostic value for gastric cancer progression (Wu et al. 2007). However, there has only been one study that reported the localization of OPN in normal gastric mucosa of human (Qu-Hong et al. 1997), consequently the exact function of OPN in gastric mucosa is unknown.

To the best of our knowledge, this is the first study to investigate the presence of OPN in avian stomach. This study revealed that OPN immunostaining was present only in the epithelial cells of the proventricular folds and oxynticopeptic cells of proventricular profound glands. Two OPN immunostaining patterns in the cells of proventricular folds were observed: cytoplasmic and cell membrane, and cytoplasmic staining was determined in the oxynticopeptic cells. OPN serves both a cell attachment function and a cell-signalling function via a number of receptors including several integrins and CD44 variants (Miyauchi et al. 1991; Ross et al. 1993). The integrins are expressed in the apical, lateral and basal surfaces of epithelial cells, whereas CD44 was generally restricted to the baso-lateral plasma membrane of polarized epithelial cells. Therefore, the localization of OPN outside the baso-lateral membrane of epithelial cells of the proventricular folds was similar to that of CD44 in polarized epithelial cells (Alho & Underhill, 1989; Neame & Isacke, 1993; Sheikh & Isacke, 1996). Sheikh & Isacke (1996) suggested that CD44, restricted to the baso-lateral domain of polarized epithelial cells, plays a role in interepithelial interactions (Sheikh & Isacke, 1996). The CD44 family of surface receptors regulates adhesion, movement and activation of normal and neoplastic cells (Weber et al. 1996). OPN, which is a protein ligand of CD44, regulates similar cellular functions (Denhardt & Guo, 1993; Denhardt et al. 1995, 2001). The observations of the present study and information in the literature prompted us to ask whether the function of OPN in the epithelial cells of the folds in the quail proventriculus is to regulate interepithelial interactions and cell–cell attachment.

In quails, the presence of OPN immunoreactivity in the oxynticopeptic cells of proventriculus was a feature common to all ages. However, OPN immunoreactivity in 1-day-old quails was weaker than that of the other age groups. Previous studies have shown that within 24 h after hatching, the peptic activity of the chick proventriculus was about 10 times that of the embryonic proventriculus of the 20-day embryo and increased at the same rate in the subsequent 24 h. The activity reached a plateau on the 3rd day after hatching. The specific activity of the adult proventriculus was only about three times that of the 2.5-day proventriculus (Yasugi et al. 1979; Yasugi & Mizuno, 1981). This may explain the weak OPN immunoreactivity at hatching. Based on these findings, we speculate that, in quails, the OPN production starts before hatching or at hatching, and that the oxynticopeptic cells of proventriculus are functionally mature at hatching. When reviewing the literature, no information was encountered about when OPN immunoreactivity began to appear and at which stages of life it was encountered. In this study, we also observed that at hatching, OPN immunoreactivity is present in basal cytoplasm of the oxynticopeptic cells, whereas the nucleus is located in the apical pole of these cells. In advanced age, although OPN immunoreactivity is located in the para-nuclear and or apical cytoplasm of these cells, the nucleus is displaced toward the central cytoplasm. Similarly, Vial et al. (1979) demonstrated that the oxynticopeptic cells undergo great changes in the shape of the apical pole in relation to secretory activity. Therefore, we suggest that with the onset of gastric secretion, OPN localization undergoes changes in relation to secretory activity.

In human stomach, Qu-Hong et al. (1997) have reported that both mucous and chief (peptic) cells of the epithelial layer and macrophages in the lamina propria stain for OPN protein, whereas parietal and endocrine cells of the epithelial layer and mast cells and plasma cells in the lamina propria did not contain this adhesive glycoprotein. These authors have also demonstrated that peptic cells contained pepsinogen-II positive granules and granules with mixtures of pepsinogen II and OPN. Our results, which showed the immunoreactivity of MUC1, MUC6 and OPN in oxynticopeptic cells, confirmed the findings of Qu-Hong et al. (1997). Our data are limited to an immunohistochemical study; as a result, we could not determine the exact role of OPN, but we suggest that OPN may be involved in the regulation of pepsinogen secretion or in the activation/or inactivation mechanism of this gastric proteinase, based on information in the literature (Qu-Hong et al. 1997) and our evidence, which showed a granular-like pattern of immunostaining of OPN and age-related changes of OPN localization in the oxynticopeptic cells. To test this or other hypotheses on the biological roles of OPN in oxynticopeptic cells, additional experimental laboratory studies are required. Furthermore, our findings, which revealed the different localization of OPN in the epithelial cells of proventricular folds and oxynticopeptic cells, support the opinion that the functions of OPN in this tissues are different and that OPN is a multi-faceted molecule (Chabas, 2005).

In conclusion, the expressions of MUC1, MUC5AC, MUC6 and OPN in quail proventriculus and gizzard were different. These differences may be a reflection of functional differences of stomach parts. In addition, the localization of MUC5AC in proventriculus was different from those of MUC1, MUC6 and OPN. The immunoreactivity of MUC5AC was present in the lining epithelium of both folds and superficial proventricular glands in the proventriculus, whereas MUC1, MUC6 and OPN reactivity was found in the oxynticopeptic cells of profound proventricular glands. Furthermore, the immunoreactivity of MUC1 in gizzard was different from that of MUC5AC. Although MUC5AC was expressed in the cells of surface epithelium and profound glands of the gizzard, MUC1 was localized in the simple tubular profound glands of the gizzard. However, MUC6 and OPN immunoreactivity was absent in the gizzard. These differences may also be a reflection of the functional differences between the surface epithelial cells and glandular cells of both the proventriculus and gizzard.

Acknowledgments

Osteopontin antibody used in this study belongs to a research project which is financially supported by the Scientific Research Council of Erciyes University (Project No. EUBAP-VA-07-06).

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