Histochemistry of glycoconjugates in the secretory epithelium of the goat bulbourethral gland

To investigate the histochemical nature of the secretory epithelium lining the goat bulbourethral gland, glycoconjugates contained in these secretory epithelial cells have been studied by means of light- and electron-microscopic histochemistry. The methods employed involved a series of conventional staining and peroxidase-labeled lectin diaminobenzidine procedures together with combined selective methods such as digestion with enzymes. According to the results obtained in the present work, the secretory cells of the goat bulbourethral gland can be grouped into two types: cells with glycoconjugate-rich granules and those with less amounts of glycoconjugates. The gland is at least dual in the nature of its secretory activities.     

Some histochemical studies on the prostate, urethral and bulbourethral glands of the one-humped camel (Camelus dromedarius)

Synopsis The histochemical localization of carbohydrates, ribonucleoproteins (RNA), lipids, some hydrolytic enzymes, succinate and lactate dehydrogenase and acetylcholinesterase were investigated in the prostate, urethral and bulbourethral glands of the camel. These glands probably secrete carbohydrate-protein complexes. In the bulbourethral glands, they are sulphated mucopolysaccharides. RNA was seen in the cytoplasm of the prostate and urethral glands. Neutral lipids were cytoplasmic and present in moderate amounts in the prostate and urethral glands and in traces, in the bulbourethral gland. Acid phosphatase-containing granules were abundant in the prostate, moderate in the urethral glands and in traces in the bulbourethral glands. Alkaline phosphatase was observed in the apical cytoplasm of the prostate and bulbourethral glands and in the ducts of the urethral glands. ATPase and adenosine 5-monophosphatase were seen in the basal laminae and interstitial tissue. In the urethral glands, adenosine 5-monophosphatase was distributed diffusely in the cytoplasm. Succinate dehydrogenase was seen in the urethral and bulbourethral glands. Varying degrees of lactate dehydrogenase activity was observed in all the glands. Acetylcholinesterase was confined to neural elements. The pars disseminata and the urethral glands were considered as two distinct glandular zones along the pelvic urethra. The significance of these histochemical results is discussed.     

Histology,ultrastructure, and seasonal variations in the bulbourethral gland of the African straw-colored fruit bat Eidolon helvum

We studied the morphological characteristics and seasonal changes of the bulbourethral gland of Eidolon helvum in a typical African tropical environment. Forty-eight bulbourethral glands were examined using gross anatomical, histological, histochemical, and ultrastructural techniques during the early rainy, late rainy, and peak dry seasons. The pear-shaped bilateral bulbourethral glands were located extra-abdominally in the inguinal region. Trabeculae from the capsule divided the parenchyma into numerous lobules of tubuloalveolar glandular acini. The mucosa was covered by a simple columnar epithelium consisting up of principal secretory cells, columnar dense cells and basal cells, which were progressively pronounced during the dry season. The principal cells contained eosinophilic granules, which were PAS positive while the dense cells did not show affinity for the stains. The mean gross weights, acini diameters, and epithelial heights were greater during the rainy season than the dry season. Ultrastructural evaluation showed that the cytoplasm of the principal cells contained well-developed Golgi complexes, rough endoplasmic reticulum, mitochondria, and secretory vesicles of varying electron densities and sizes. The secretory vesicles were numerous during the early rainy season, decreased during the late rainy season and were scanty during the peak dry season. The simple columnar epithelium observed during the rainy season was replaced by an undefined stratified epithelium during the dry season, and this was associated with cellular degenerations and regenerations. In conclusion, E. helvum has a typical mammalian bulbourethral gland, with a unique cell type, the dense cell whose functions are not well-understood. The gland exhibits cyclical seasonal variation in structure and secretory activity; being active during the early rainy season (breeding season), and showing the lowest activity during the dry season (non-breeding season). Glandular epithelial cell renewal occurs during the dry season in preparation for the next breeding season.     

Fluorescein-Conjugated Bandeiraea simplicifolia Lectin as a Marker of Endodermal, Yolk Sac, and Trophoblastic Differentiation in the Mouse Embryo

Abstract. The Bandeiraea simplicifolia lectin I (BSA-I) conjugated to fluorescein isothiocyanate was used as a histochemical reagent to study the mouse embryos from fertilization to early somitogenesis. No lectin binding could be detected on the embryonic cells in the preimplantation embryo. Lectin labeled intensely the zona pellucida. In the implanting embryos lectin binding was detected along the subtrophectodermal and Reichert's membrane, in the cytoplasm of the parietal and visceral endoderm, and the trophoblastic giant cells, but not in the ectodermal cells. Studies on explanted blastocyts cultured in vitro disclosed that the cytoplasmic BSA-I binding sites in trophoblastic cells develop gradually. In the 9-day somitic embryo BSA-I reacted with epithelial cells of the yolk sac, but not with the mesenchymal cells. A continuity between the lectin-reactive endoderm and the foregut epithelium could be demonstrated. These data indicated that BSA-I lectin can be used as a histochemical probe for endodermal (yolk sac) and trophoblastic differentiation in the peri-implantational mouse embryo.     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Binding of T-antigen-bearing neoglycoprotein and peanut agglutinin to cultured tumor cells and breast carcinomas

Chemical conjugation of appropriate carbohydrate ligands to an inert labeled carrier renders probes available to screen for the presence of respective binding sites. A set with a certain plant lectin and a suitable neoglycoprotein can thus determine complementary parts of a potentially relevant glycobiological interaction system. Owing to the interest in the peanut agglutinin-reactive T-antigen, we performed chemical synthesis of the respective disaccharide structure to serve as glycohistochemical ligand and established refinements of the synthetic patway. Coupling of the derivatized monomers had to be performed in the presence of sodium sulfate for optimal results. Complete removal of the protective groups from the p-nitrophenyl derivative of the N-acetylgalactosamine moiety was achieved under mild conditions with 2,3-dichloro-5,6-dicyanobenzoquinone without affecting any other functional groups. Specific binding sites for the synthetic neoglycoprotein as well as for the plant lectin were demonstrated in cell lines of human breast carcinoma colon adenocarcinoma, and erythroleukemia. ABC reagents in conjunction with DAB as peroxidase substrate were used to visualize specific binding sites. Binding complied with the accepted criteria for specificity. Moreover, carbohydrate-specific binding sites were detected in sections of nine out of 14 cases with malignant breast lesions. The percentage of positive tumor cells with both neoglycoprotein and lectin was similar in each of the individual sections, regardless of quantitative variations between cases, lectin staining intensity often being more pronounced. The reactivity pattern in sections of primary and metastatic lesions was not significantly correlated with the lymph node status. This study emphasized that custom synthesis of saccharides and histochemical application of the resulting neoglycoprotein has a remarkable potential for complementary assessment of endogenous binding sites for carbohydrate structures, localized by external tools such as plant lectins, as a step to elucidate the importance of a putative proteincarbohydrate interaction.     

Reference values for the genitalia of male dromedary before and after puberty using caliper and ultrasonography in subtropics

The aim of the present study was to characterize the dynamic changes of the testes and the pelvic genitalia in vivo in male dromedary. Eighty-one clinically healthy male dromedaries aged 1.5 to 12 years were assigned for the present study. Testicular length, breadth, and depth as well as epididymal head and tail were measured using caliper and ultrasonography. The pelvic genitalia, including bulbourethral gland, prostate, and pelvic urethra were examined using ultrasonography. The results revealed that the three dimensions of the testes and epididymal tail and head showed significant increase with age (P < 0.01). Concerning the epididymal measurements, differences between the pre- and peri-pubertal groups were not significant. Left testes tended to be larger than the right (not statistically significant) although only the breadth of the left testes in the prepubertal group was significantly larger (P < 0.05). The volume of both testes correlated positively with the age (r² = 0.91 for left and 1.00 for the right, P < 0.01). There were no significant correlations between the values measured using caliper and those by ultrasonography between groups, but the correlation was highly significant (P < 0.01) for the total number of the examined animals. There were significant and steady increases of the size of bulbourethral gland in all examined groups (P < 0.01). Pars disseminata of the prostate gland and pelvic urethra were significantly higher in sexually mature compared with prepubertal groups (P < 0.01). It was concluded that ultrasonography is a useful tool in studying the developmental changes of the testes and accessory glands of the male dromedary. The obtained data could provide a reference values for predicting camel puberty and future fertility.     

Comparative histochemical study of glycoconjugates of the major salivary glands and pancreas in humans

The human parotid, submandibular, sublingual salivary glands and pancreas have been studied with lectin--horseradish peroxidase conjugates (con A, PNA, SBA, WGA, LAL), aldehyde fuchsin and Bismark brown. Intercalated duct cells produce a specific aldehyde-fuchsin-reactive substance. These cells are found only in the submandibular and parotid, but not in the sublingual glands. Similar reactivity is found in B-insulocytes of the pancreas. Aldehyde-fuchsin marks cytoplasmic granularity of the striated duct cells of all large salivary glands. This specific granularity is also selectively stained with Bismark brown and con A. Using fucose-specific lectin from Laburum anagyroides bark (LAL), granularity in serocytes of the submandibular gland is demonstrated. Some individual variations are observed in PNA binding to serocytes of the submandibular gland. It reveals that thyroglobulin-peroxidase conjugate (previously reported as an available second-step reagent for indirect lectin histochemical methods) non-specifically binds to the striated duct cells of the submandibular gland. During control staining it is also found, that DAB-reaction for endogenous peroxidase can be used as a test-system for a selective histochemical exposure of nuclear regions of endotheliocytes, pericytes and striated duct epitheliocytes of the human salivary glands. Possible significance of the phenomena observed is discussed.     

Lectin histochemistry of the boar bulbourethral glands

The present study describes, for the first time, the glycosidic content of boar bulbourethral glands using lectin histochemistry. Fourteen horseradish peroxidase- or digoxigenin-labelled lectins with different carbohydrate specificities were used in samples obtained from 3 healthy Landrace boars. The results obtained indicate that endpiece and duct cells synthesize and secrete mainly O-glycoproteins with alpha- and beta-D-N-acetylgalactosamine, beta-D-galactose-beta(1-->3)-D-N-acetylgalactosamine, D-N-acetylglucosamine and neuraminic acid residues. Glycoproteins secreted by bulbourethral glands have a role in the protection and lubrication of the urethra. In addition, they may be also involved in the regulation of the sperm metabolic activity and in the maintenance of the structural integrity of acrosomal and plasma membranes.     

Histochemical reactivity of the Geodia cydonium agglutinin (GCA) in human tissues

We applied a peroxidase-antiperoxidase technique to study the distribution pattern and binding characteristics of the lectin from the marine sponge Geodia cydonium (Geodia cydonium agglutinin; GCA) in various human tissues. This lectin has been shown to possess a broad reactivity, but there was a distinct distribution of binding sites within the different organs. In the histochemical system GCA displayed no blood group specificity and labeled red blood cells, the vascular endothelium, and epithelial cells showing blood group antigen expression independent of the ABH blood group status. However, inhibition of GCA reactivity by simple sugars and complex carbohydrates demonstrated tissue-specific differences of lectin binding related to the ABH blood group status of the tissue and revealed information on the structural requirements of the histological lectin binding site. Tissues that totally lacked blood group antigens or that expressed only the H-antigen disclosed a GCA reactivity which was completely inhibited by lactose. In contrast, tissues that expressed blood group A- or blood group B-antigen exhibited a lactose-resistant lectin binding which was inhibited only by water-soluble blood group substance A from peptone A and by bovine glycophorin but not by other complex carbohydrates, including human glycophorin and human asialoglycophorin. Competitive inhibition studies in situ revealed that GCA binding was not inhibited by blood group type I/II carbohydrate sequence-specific lectins or by lectins with other sugar specificities. Inhibition by lactose of GCA binding to some histological sites indicates that the binding site consists of a beta-linked galactose-containing disaccharide. However, periodate oxidation of tissue sections had no effect on lectin binding, pointing to a subterminal location of the relevant sequence. The results obtained from inhibition studies with simple saccharides and complex carbohydrates in relation to the expression of ABH blood group antigens suggest a complex lectin combining site(s) in histological specimens. The lectin may possess either one binding site with a range of affinities for different carbohydrates (besides beta-linked disaccharides the GCA binding site accommodates to carbohydrate determinants carrying the blood group A or blood group B determinant), or may possess two different binding sites. Besides an acceptor site for beta-linked disaccharides, an additional binding site may exist accommodating to extended carbohydrate sequences related to A or B blood group structures. In conclusion, GCA represents a blood group-nonspecific lectin whose binding affinities are determined by the ABH blood group status of the tissue.     

In vivo effects of tunicamycin on chondrocytes of rat mandibular condyles as revealed by lectin cytochemistry

Summary The in vivo effects of tunicamycin on the glycosylation of proteoglycans and link protein in rat mandibular condylar chondrocytes were studied by ultrastructural lectin histochemistry. The binding of wheatgerm agglutinin was shown by using anti-lectin antibody followed by protein A-gold complex. In normal rats, wheat-germ agglutinin labeling was restricted to trans cisternae and vacuoles of the Golgi complex, whereas it was observed in neither the cis region of the Golgi complex nor in the rough endoplasmic reticulum. By 3 h after the drug administration, wheat-germ agglutinin binding sites on the disorganized Golgi vacuoles were dramatically reduced in number. At 6 h after the drug administration, the lectin binding sites on the Golgi vacuoles were restored. These results demonstrate that the in vivo use of tunicamycin in combination with histochemical analysis using lectin probes is of significant value for the study of protein glycosylation in chondrocytes of the rat mandibular condyle.     

A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]

Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids.     

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.     

Histochemical demonstration of prolactin binding sites

We have developed a new probe for histochemical demonstration of prolactin binding sites. Ovine prolactin (oPRL) was conjugated with the N-hydroxy-succinimide ester derivative of the fluorochrome 7-amino-4-methylcoumarin-3-acetic acid. Under mild reaction conditions the ester derivative reacted with available NH2 groups of the prolactin molecule to form stable bonds. The coupling reaction yielded products that co-migrated with oPRL but had slightly decreased isoelectric points. The receptor binding and bioactivity of the flurochrome-hormone derivatives were decreased essentially proportional to the extent of conjugation. The derivatives were further tested for their ability to label PRL binding sites, using frozen sections of mammary gland and brain tissue of lactating rats. The results presented in this report describe the validation of this probe with regard to labeling of PRL binding sites at the light microscopic level in known target organs (mammary gland and choroid plexus). In addition, this fluorescent probe was used to demonstrate the presence of PRL binding sites at a novel site, the ependymal lining of the third ventricle.     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.     

Lectin target cells in human central nervous system and the pituitary gland

Summary Peanut lectin (PNL), Concanavalin A (Con A) and Ulex europaeus lectin I (Ulex) were chosen to map their binding sites in different regions of formalin fixed and paraffin embedded human central nervous system tissue and pituitary gland tissues. An extended PaP method was used for PNL and Ulex, whereas a direct peroxidase technique was employed for Con A. In astrocytes, the cytoplasm as well as the delicate processes were stained by PNL and Con A; the most conspicious binding of PNL was seen in the ependymal cells and on the surface of plexus epithelial cells; in the anterior part of the pituitary gland a selective population was PNL positive. Intracytoplasmic Con A acceptors could be demonstrated in neurons, in ependymal cells, and in plexus epithelial cells. Intracytoplasmic Con A receptors were finely granular in astrocytes, oligodendrocytes, and in some cells in the pituitary gland. Ulex binding was restricted to the vascular endothelial cells and a selective population of cells in the pituitary gland. Our results suggest that lectins may be good tools for the evaluation of their respective target cells in the central nervous system and in the pituitary gland.     

Hormonal effects on the estrogen receptor system in the epididymis and accessory sex organs of sexually immature rabbits

The epididymis and male accessory sex organs (vesicular gland, prostate, and bulbourethral gland) of sexually immature rabbits contain a functional estrogen receptor system which is regulated in an organ-specific manner by various hormones. In both intact and castrated animals, acute estrogen challenge causes depletion of estrogen receptor from the cytosolic fraction and its appearance in the nuclear fraction of these tissues. A considerable amount of unoccupied nuclear receptor was detected both before and after estrogen challenge. An estrogen-activated, receptor-processing mechanism is operable in these organs since chronic treatment (daily for 14 days) with estradiol benzoate modified the levels of total estrogen receptor, and altered the relative amounts of occupied to unoccupied nuclear receptor present following estrogen challenge. Chronic treatment with estradiol benzoate, Tamoxifen, and testosterone propionate (alone and in combination) had differential, organ-specific effects on the ability of subsequent estrogen challenge to cause accumulation of nuclear receptor. The vesicular gland was the most responsive to estrogen treatment and the bulbourethral gland the least responsive.     

Lectin target cells in human central nervous system and the pituitary gland

Peanut lectin (PNL), Concanavalin A (Con A) and Ulex europaeus lectin I (Ulex) were chosen to map their binding sites in different regions of formalin fixed and paraffin embedded human central nervous system tissue and pituitary gland tissues. An extended PaP method was used for PNL and Ulex, whereas a direct peroxidase technique was employed for Con A. In astrocytes, the cytoplasm as well as the delicate processes were stained by PNL and Con A; the most conspicuous binding of PNL was seen in the ependymal cells and on the surface of plexus epithelial cells; in the anterior part of the pituitary gland a selective population was PNL positive. Intracytoplasmic Con A acceptors could be demonstrated in neurons, in ependymal cells, and in plexus epithelial cells. Intracytoplasmic Con A receptors were finely granular in astrocytes, oligodendrocytes, and in some cells in the pituitary gland. Ulex binding was restricted to the vascular endothelial cells and a selective population of cells in the pituitary gland. Our results suggest that lectins may be good tools for the evaluation of their respective target cells in the central nervous system and in the pituitary gland.