Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles.

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.

Topographical localisation of glucidic residues and their variations in the canine zona pellucida during folliculogenesis.

In the present ultrastructural study, horseradish peroxidase-labelled lectins, in conjunction with antiperoxidase antibody and protein A-gold, were used to characterise and localise the oligosaccharide sequences of zona pellucida glycoproteins at different stages of follicular development in the canine ovary. Deacetylation and sialidase digestion were also performed before lectin cytochemistry. The zona pellucida of oocytes present in unilaminar primary follicles reacts with WGA- and RCA-I-lectins. The zona pellucida of oocytes present in bilaminar and trilaminar secondary follicles displays positivity to WGA, RCA-I, Con-A, UEA-I, and sialidase/SBA. This labelling pattern persists in the zona pellucida of oocytes present in antral tertiary follicles with the exception of WGA and RCA-I reactive sites which are differently distributed throughout the zona pellucida. The topographical distribution of these carbohydrates is not uniform throughout the zona pellucida, indicating the regionalization of oligosaccharide chains within three concentric bands of the zona matrix: an inner surface close to the oocyte plasma membrane, an intermediate portion and an outer layer in contact with the follicular cells. Our results demonstrated variations in the presence and distribution of the carbohydrate residues in the canine zona pellucida during different stages of follicular growth. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the zona pellucida.

Apoptotic cell death in canine hair follicle.

Apoptotic cell death is an essential homeostatic mechanism involved in the control of cellular turnover in a variety of adult tissues. Cytoplasmic and nuclear condensation morphologically define this process whose biochemical hallmark is extensive DNA fragmentation into discrete oligonucleosomic units. Hair follicle growth and regression has been shown to be correlated with apoptosis in humans, mice, rats and guinea pigs. The present study was carried out to evaluate its implication in canine hair biology in order to define the spatio-temporal relationship between apoptosis and the hair cycle in dogs. As assessed by terminal deoxy-nucleotidyl transferase-mediated d-UTP nick-end-labelling (TUNEL) and by basic histological and ultrastructural assays, apoptotic cells appeared both in the growing and in the regressing follicle epithelium showing the well characterized morphological features described in the previous relevant literature.

Complex carbohydrates occurring in the digestive apparatus of Umbrina cirrosa (L.) fry.

The glycosoaminoglycans in the digestive apparatus of immature fish have important biological functions and are involved in morphofunctional differentiation. The aim of this study was to investigate the glycoconjugate histochemistry in the different parts of the digestive apparatus (oesophagus, stomach, intestine) of Umbrina cirrosa (L.) fry using classical histochemical reactions (periodic acid-Schiff, Alcian blue pH 2.5, Alcian blue/periodic acid-Schiff, high iron diamine, low iron diamine) in conjunction with glycolytic digestions that degrade different classes of glycosoaminoglycans. No differences were observed in the reactivity to conventional histochemical staining of the oesophagus, stomach or intestine among 27-, 34- or 44-day-old fry. In the oesophagus, the mucopolysaccharides contained chondroitin sulphates B and A and/or C, heparan sulphate and chondroitin. In the stomach, only neutral glycoconjugates were revealed, whereas in the intestine there were only chondroitin sulphates. Some differences in the type and content of glycoconjugates were found in Umbrina cirrosa (L.) fry compared to those of adult subjects, probably related to different dietary habits and to changes in the environmental conditions.

Immunogold study on lectin binding in the porcine zona pellucida and granulosa cells.

An ultrastructural localization of lectin receptors on the zona pellucida (ZP) of porcine antral oocytes and on the granulosa cells was performed using a panel of horseradish peroxidase-labelled lectins in conjunction with antiperoxidase antibody and protein A-gold. In some cases, lectin incubation was preceded by sialidase digestion. WGA-, Con-A-, UEA-I-, RCA-I-, PNA- and SBA-reactive sites were distributed differently in the porcine ZP. Sialidase digestion increased the positivity obtained with RCA-I and it was necessary to promote PNA and SBA reactivity. These results indicated that the ZP contained N-acetylglucosamine, a-mannose, a-fucose, b-Gal-(1-4)GlcNAc, b-Gal- (1-3)GalNAc, b-GalNAc and sialic acid residues. We also observed the presence of vesicles in both the ooplasm and granulosa cells, showing a similar lectin binding pattern to that of the ZP, thus suggesting that the oocyte and granulosa cells are the site of synthesis of ZP glucidic determinants.

Retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults.

The morphometric characteristics and the ultramicroscopic findings of Cryptosporidium spp. at various stages of their life cycle in the intestinal and bursal epithelial cells of naturally infected 30-day-old commercial turkeys are reported. Small, sporulated oocysts, observed in the small intestinal content after flotation, were identified as Cryptosporidium meleagridis on the basis of morphometric characteristics (round in shape and 4.5-5.0 microm in size) and the small intestinal localization. Light section examinations revealed the presence of the protozoon in multiple organs, but its prevalence was highest in the intestinal and bursal epithelial cells. Ultramicroscopic studies on ileum and bursal samples showed the presence of all the life cycle stages in the microvillar brush epithelial cells in both the organs examined. On the basis of the comparison of the morphology and the sizes of the microorganisms parasitizing the ileum and the bursa, hypotheses are considered on the possible species involved.

Carbohydrate histochemistry of lamb duodenum.

In order to elucidate the carbohydrate profile of the mucosa of lamb duodenum, conventional histochemical methods and a panel of 7 labelled lectins were used. In some cases, treatment with sialidase preceeded lectin staining. Carbohydrate histochemistry revealed the presence of sugar residues in the brush border of enterocytes, goblet cells and duodenal glands. All sites contained neutral and acid glycoconjugates. The presence of sulphomucins in goblet and duodenal gland cells was age-dependent. Enterocytes and duodenal gland cells contained abundant amounts of oligosaccharides with terminal sialic acid-galactosyl(beta1 --> 3)N-acetylgalactosamine, whereas goblet cells contained the penultimate N-acetylgalactosamine residue linked to sialic acid. These findings were not age-dependent, whereas scarce amounts of fucose were found in all sites especially in young animals. The findings obtained in the present study serve as a basis for future pathological studies in lamb and sheep.

Characterisation of sugar residues in glycoconjugates of pig mandibular gland by traditional and lectin histochemistry.

Sugar residues are important components of salivary gland secretion. Traditional histochemical methods and lectin histochemistry were used to characterise glycoconjugates present in the mandibular gland of normal adult pigs. Acinar cells contained abundant quantities of glycoconjugates with the terminal trisaccharide sialic acid - (alpha 2-->3, 6) galactosyl (beta 1-->3) N -acetylgalactosamine. Mandibular acinar cells also contained alpha and beta N -acetylgalactosamine and N -acetylglucosamine residues, whereas the demilunar cells contained glycoconjugates with fucose, mannose and N -acetylglucosamine residues. In the duct system a range of sugar residues were localised throughout the cell cytoplasm or limited to the apical surface. These results provide new knowledge concerning the structure of salivary glycoconjugates in normal adult pig and a basis for future pathological studies.

Complex carbohydrate histochemistry and ultracytochemistry of the sheep lacrimal gland.

The chemical content of the secretion of the sheep lacrimal gland was analysed at the light and electron microscope levels by applying histochemical techniques and an ultrastructural histochemical method (periodic acid, thiocarbohydrazide and silver proteinate). Mucosubstance histochemistry demonstrated acidic glycoconjugates, mainly sulphated, in the mucous and seromucous glandular cells and in the apical portion of the cells lining the terminal ducts. Moreover, secretory granules, stained with PA-TCH-SP, showed a different localization of the reaction product. The presence of lysozyme was also found in the glandular serous cells. These histochemical studies demonstrate that the secretion of sheep lacrimal glands is mixed, having serous, mucous and seromucous components, and that an excellent correlation exists between the secretory granule substructure and glycoprotein localization.

Ultrastructural study of sheep lacrimal glands.

Sheep lacrimal glands are mixed glands, consisting of tubulo-acinar units succeeded by ducts of simple morphology. The secretory portions consist of three cell types: mucous, seromucous and serous, which may be intermingled in the same acinus or may form acini wholly made of only serous or mucous cells. Mucous cells show a rough endoplasmic reticulum that is reduced to a few cisternae located near the cell base and among the interstices of the secretory droplets. Mucous granules appear uniformly electron-lucent. Serous cells display a typical structure; serous granules can be uniformly electron-dense or composed of dense inclusions dispersed in an electron-lucent matrix. The seromucous granules have a bizonal substructure: a dense core is embedded in a highter matrix. Secretory acini are succeeded by intercalated ducts; the epithelium of these ducts gradually increases in height to form a kind of excretory duct, without the intervention of striated ducts.

Detection of glycosidic residues in carpal glands of wild and domestic pig revealed by basic and lectin histochemistry.

Carpal glands are compound tubuloalveolar glands, located on the medial surface of the carpus. This study was carried out on samples from carpal glands of adult wild and domestic pigs of both sexes. We elucidated the glycosidic composition of carpal gland secretion in situ using traditional histochemical methods and lectin histochemistry. Some secretory cells exhibited an intense reaction with PAS in both wild and domestic pigs. Lectin histochemistry showed differences in the localization and composition of glycoconjugates secreted by carpal glands. A cytoplasmic positivity was revealed in the wild pig by the sequence sialidase-PNA and WGA, whereas in the domestic pig the reactivity was localized at the apical surface of some cells. LTA positive cells were found only in the carpal glands of the domestic pig.

Presence and distribution of neuroendocrine cells in the gastroenteropancreatic endocrine system of fallow deer foetuses.

The gastroenteropancreatic endocrine system was studied in 11- and 17-week-old fallow deer foetuses using an immunocytochemical technique. In the gastrointestinal tract, gastrin-, serotonin-, somatostatin- and cholecystokinin-containing cells were found: their frequency and distribution were also determined. Anti-glucagon and anti-insulin antibodies did not stain any cells along the gut. In the pancreas, somatostatin-, pancreatic polypeptide-, insulin- and glucagon-immunoreactive cells were detected. The different distribution and number of neuroendocrine cells, in the two investigated stages of foetal life, are discussed. Data obtained in this study were compared with those published in a previous study on the gastrointestinal system of the adult fallow deer.

Immunohistochemical localization of endometrial oestrogen and progesterone receptors in the cow.

The presence and distribution of oestrogen and progesterone receptors have been investigated by means of immunohistochemical procedures in the uterus of two groups of cows: the first group underwent superovulatory hormonal treatment while the second group was used as a control group. After the immunohistochemical study no differences regarding the presence and distribution of hormone receptors seemed to be apparent between the two groups, so it was concluded that the topographical distribution and staining intensity of hormone receptors seem to be unaffected by hormonal superovulatory treatment.

Histology and ultrastructure of the gut of the tilapia (Tilapia spp.), a hybrid teleost.

The morphology of the intestine has been studied in a species of warm water fish, Tilapia spp., a hybrid teleost of notable economic importance. Light and electron microscope results show that the intestine is a relatively undifferentiated muscular tube lined with a simple columnar epithelium interspersed with goblet cells. The proximal region has a greater surface area, manifested by elongated mucosal ridges. The enterocytes are covered apically with uniform microvilli and exhibit the typical ultrastructural features of pinocytosis, namely extensive invaginations of the luminal plasma membrane and massive accumulation of vesicles in the apical cytoplasm. The distal intestine mucosa is thinner and less elaborately folded and consists of columnar cells with shorter and sparser microvilli. Their supranuclear cytoplasm contains abundant clear vacuoles. Numerous endocrine cells can also be seen. Regional cellular ultrastructural features are correlated with digestive functions.

Ultrastructural study on the stomach of Tilapia spp (Teleostei).

An ultrastructural study has been made of gastric mucosa of a teleostean fish, Tilapia spp. The cytological features of the surface mucous cells, mucous neck cells, glandular cells and endocrine cells are described. The surface mucous cells, identified by their superficial localization, are characterized by apical granules. The mucous neck cells are distinguished by the appearance of their mucous granules and their localization between surface mucous cells and glandular cells. The gastric glands contain only one form of cell whose fine structure is similar to cells that secrete hydrochloric acid. Physiological implications of some ultrastructural features are also discussed.

Basic and lectin histochemical characterization of bovine gustatory (von Ebner's) glands.

The Bovine tongue possesses numerous circumvallate papillae (8-16 each side). The troughs around the papillae are the openings of the ducts of the gustatory (von Ebner's) glands. In this study, we have characterized in situ the glycosidic composition of the secretion of bovine gustatory glands using traditional histochemical methods and lectin histochemistry with and without prior neuraminidase (sialidase) digestion. The lectin-horseradish peroxidase conjugates employed were: PNA, DBA, SBA, WGA, LTA, UEA I and ConA. Acinar cells show a diffuse positivity towards PAS and Alcian blue at pH 2.5 and the most intense and homogeneous lectin staining was obtained with PNA. This indicates that bovine gustatory glands secrete glycoproteins with 1,2-glycol containing hexoses and carboxyl-rich glycoconjugates and that galactosyl (beta 1-->3) Nacetylgalactosamine is the most frequent sugar residue present in these glycoproteins. Results were compared with data reported in the literature on the same glands of other species.

Histochemical study of lectin binding in the major salivary glands of adult fallow-deer (Dama dama L.).

The sugar residues in glycoconjugates present in the parotid and mandibular glands of the adult fallow-deer were detected and characterized by using a battery of eight different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. Parotid secretory cells produced glycoconjugates with N-acetylgalactosamine, N-acetylglucosamine and mannose residues. Mucous acinar cells were the most reactive sites of the mandibular gland and contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(beta 1-->3)N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Mandibular mucous cells also presented N-acetylglucosamine and sialylated components with the terminal dimer sialic acid-N-acetylgalactosamine. Demilunar cells contained glycoconjugates with fucose and mannose residues. The apical surface of duct cells was stained by all the lectins.

The presence of granular excretory ducts in the rabbit zygomatic gland.

The proximal and interlobular ducts of rabbit zygomatic glands show a particular morphological feature consisting of numerous secretory granules localized in the apical cytoplasm of the cells. It is suggested that there is a similarity between these ducts and the granular ducts of submandibular glands in rodents.