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.

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.

Variations in lectin-binding on the zona pellucida during oocyte growth in some wild ungulates.

The aim of the present study was to examine the glycoconjugate modifications occurring in the zona pellucida during oocyte growth in fallow, red and roe deer using a battery of lectins combined with sialidase digestion and chemical treatments. This histochemical approach allowed us to sequence the oligosaccharidic side chains of the zona pellucida glycoproteins in these wild ungulates. The most effective lectins in the zona pellucida of these species were SBA, PNA, RCA-I GSA-IB4, and WGA, indicating the presence of beta-D-N-Acetylgalactosamine, beta-D-Galactose, alpha-D-Galactose and N-Acetylglucosamine residues. Additionally, sialic acid moieties were demonstrated. We also observed differences in the glycosidic residue content and in their spatial distribution, depending on the species and stage of follicle development.

Origin of enzootic intranasal tumor in the goat (Capra hircus): a glycohistochemical approach.

Enzootic intranasal tumor (EIT) appears glandular in type and has recently been classified as an adenocarcinoma of low malignancy. The aim of this study was to characterize the secretion of surface glycoconjugates (GCs) in EIT and in normal respiratory and olfactory mucosae of the goat by means of conventional and lectin histochemistry, in order to shed light on the histogenesis of EIT. Morphologic and ultrastructural investigations showed two growth types of EIT: i.e., tubular and papillary patterns. Conventional histochemistry revealed the presence of neutral and carboxylated GCs in the olfactory glands and in the tubular part of EIT, as well neutral and sulphated GCs in the respiratory glands and in the papillary part of EIT, suggesting that the papillary pattern tumor arises from the respiratory glands, whereas the tubular portion of EIT arises from the olfactory glands. Lectin histochemistry gave further information on the expressed GCs.

Glycoconjugate histochemistry in the duodenum of fetal and adult fallow deer.

This work aimed to characterize the complex carbohydrates in the duodenum of fetal and adult fallow deer. The proximal parts of the duodenum were removed from the intestine of 12- and 16-week-old fetuses and of adult fallow deer. Sections were assessed by conventional carbohydrate histochemistry combined with glycosidase digestions and treatment with KOH. During development, there was an increase in the acid carboxylated components of the glycoconjugates in goblet cells, while the duodenal glands expressed high quantities of sulphated glycoconjugates at the beginning of development but, in the adult. mainly secreted neutral and carboxylated glycoconjugates containing sialic acid. Sulphated components, probably represented by chondroitin sulphate B-like and heparan sulphate-like glycosaminoglycans may play a role in the morphofunctional differentiation of the duodenum.

Carbohydrate histochemistry of the alimentary canal of the shi drum, Umbrina cirrosa L.

Histochemical staining techniques, which differentiate the main categories of carbohydrates, were applied to sections from different segments of the alimentary canal of the shi drum Umbrina cirrosa L. to study patterns of distribution of epithelial glycoconjugates. In the oesophagus, mucous cells contained sulphomucins, together with a small amount of sialomucins. Stomach epithelial cells secreted neutral and acidic glycoconjugates, while gastric glands only produced small quantities of sialomucins. Goblet cells showed the presence of sialo and sulphomucins in the pyloric caeca, whereas intestinal mucous cells secreted sulphated glycoconjugates. This work serves as a baseline for further studies on carbohydrate composition of the mucosa of the shi drum digestive system.

Histochemical detection of the lectin-binding carbohydrates in the zona pellucida during oocyte growth in the wild boar (Sus scrofa scrofa).

The changes that occur in the carbohydrate composition of zona pellucida glycoproteins during oocyte maturation in the wild-boar were studied using periodic-acid Schiff (PAS), High Iron Diamine (HID) and Low Iron Diamine (LID). Lectin staining was performed with a panel of 11 HRP-lectin conjugates combined with neuraminidase digestion and chemical treatments. There were few internal glucidic residues, such as N-acetylglucosamine, in the wild boar zona pellucida but there were many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acid. In addition, beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine and beta-galactose-(1-4)-N-acetylglucosamine were detected in the sulphated form in the terminal and/or subterminal position. Some differences in the lectin reactive sites occurred in the zona pellucida, depending on the stage of oocyte maturation.

Localization of the lectin reactive sites in adult and prepubertal horse testes.

The testes of prepubertal and adult horses were investigated using 10 horseradish peroxidase conjugated lectins combined with sialidase digestion and potassium hydroxide treatment, to localise the oligosaccharide sequences of glycoconjugates during spermatid maturation. In adult animals, the lectins showed a variable affinity for spermatids and Sertoli cell apical extensions. Soybean agglutinin (SBA), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA-I) and wheat germ agglutinin (WGA) bound to the acrosomal structures of spermatids, whereas Griffonia simplicifolia agglutinin (GSA-II) labelled these structures only during Golgi and cap phases. These results suggested that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides and that these carbohydrate chains undergo modifications during spermiogenesis. Sialic acid residues were not detected throughout the acrosomal development. The lectin binding pattern of Sertoli cells was very similar to that of acrosome of spermatids during the maturation phase. In sexually immature horses, only the degenerated germinal cells and the Leydig cells showed reactivity towards lectins. The first cells reacted with SBA and Dolichos biflorus agglutinin (DBA), the latter with SBA, PNA, WGA, GSA-II, Canavalia ensiformis agglutinin (ConA), Lens culinaris agglutinin (LCA) and also with DBA after sialidase digestion.

Lectin histochemical detection of sulfoglycans in the zona pellucida of mammalian antral oocytes.

Sulphated esters are important to increase effectiveness of specific biological activities of carbohydrates. Biochemical studies revealed the presence of distinct sulphated glycoproteins in mammal zona pellucida (ZP) that bind proacrosin and thus participate in the sperm-egg fusion processes. In the present study, 6 lectin-horseradish peroxidase conjugates (SBA, PNA, RCA-I, GSA-IB4, GSA-II and DBA) were used in combination with desulphation and sialidase digestion to identify sulphocarbohydrates in the terminal and/or subterminal position of oligosaccharide side chains of glycoproteins in the ZP of bovine, ovine, caprine and porcine antral oocytes. In particular, we identified the following terminal sulphoglycans located in the outer layer of the ZP only: SO4-GalNAc in bovine ZP; SO4-Galbeta1,3GalNAc in bovine and ovine ZP; SO4-Galbeta1,4GlcNAc in bovine, ovine and caprine ZP; SO4-alpha-Gal in bovine, caprine and porcine ZP. Subterminal sulphoglycans linked to sialic acid residues were evenly distributed throughout the entire thickness of the ZP: Neu5Ac-SO4-Galbeta1,3GalNAc in bovine and porcine ZP; Neu5Ac-SO4-Galbeta1,4GlcNAc in caprine ZP; Neu5Ac-SO4-alpha-Gal in porcine ZP; Neu5AcSO4-GlcNAc in bovine ZP. The results demonstrate that the chemical composition of the ZP differs among species determining the species-specificity of gamete interactions.

Cytochemical identification of lymphocytes and other mononuclear cells in ovine and bovine hemal nodes.

Cytochemical and immunocytochemical studies were carried out with specific markers for B lymphocytes, dendritic reticulum cells (ATPase, 5'Nase, SIg) and T lymphocytes (ANAE, A.P.) in an attempt to identify the mononuclear cells present in bovine and ovine hemal nodes. The results show that primary nodes and mantle of secondary nodes are composed of B cells, whereas T cells are mainly localized in the interfollicular cords. Since such an arrangement resembles the picture in normal lymph nodes, but the direct contact between lymphatic tissue and blood is more reminiscent of the spleen, hemal nodes probably perform immunological functions similar to those of both normal lymph nodes and spleen.

Ultracytochemical and biochemical evidence for guanylate cyclase in guinea pig testis.

Guanylate cyclase activity has been studied biochemically and cytochemically in guinea pig testis. The results of the biochemical assays indicate an equal distribution of this enzyme between the soluble and particulate fractions, which have a different sensitivity to adenosine triphosphate. The cytochemical results demonstrate that the reaction product of guanylate cyclase is detectable in the interstitial capillary endothelial cells and, in the seminiferous epithelium, mainly at the level of the adjacent surfaces of Sertoli and germ cells of the intermediate and adluminal compartments. Guanylate cyclase activity appears at the level of pachytene spermatocytes and persists throughout subsequent stages of development. The distribution in the seminiferous epithelium seems to indicate that guanylate cyclase is involved in the interrelationships between Sertoli and germ cells during gamete differentiation.

Identification of lymphocytes extracted from bovine hemal nodes.

Lymphocyte populations extracted and separated from hemal nodes (HN) and peripheral blood of 20 male healthy bovines were characterized by surface markers and the rosette-forming test. After treatment with FITC- or peroxidase-conjugated antibovine IgG, about 22% of cells from HN and peripheral blood showed superficial fluorescence (B cells) and about 13% were able to form "E rosettes" (T cells) with sheep erythrocytes pre-treated with neuraminidase. Nearly equal percentages were obtained from peripheral blood lymphocytes. On the basis of the data shown, we can conclude that the function of HN is, at least in healthy animals, analogous with that of lymph nodes. In this study we also used the SEM to evaluate the possibility of classifying the lymphoid cells as B and T lymphocytes on the basis of their surface morphology. Some authors have stated that B and T lymphocytes have a rough and smooth surface, respectively. In our experience, however, because of cells with a moderate number of surface microvilli exist, this method used on its own is not suitable for lymphocyte identification.