Lectin histochemistry of human skeletal muscle

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.     

Lectin binding to rat spermatogenic cells: Effects of different fixation methods and proteolytic enzyme treatment

Summary The binding of a panel of eight different fluorescein-conjugated lectins to rat spermatogenic cells was investigated. Particular attention was paid to the effects of different fixation methods and proteolytic enzyme digestion on the staining pattern.Concanavalin A (Con A), wheatgerm agglutinin (WGA), succinylated WGA (s-WGA) and agglutinin from gorse (UEA I) stained the cytoplasm of most germ cells as well as the spermatid acrosome. In contrast, peanut agglutinin (PNA), castor bean agglutinin (RCAI) and soy bean agglutinin (SBA) mainly stained the acrosome. The staining pattern varied depending on the fixation method used. PNA was particularly sensitive to formalin fixation, while SBA, DBA and UEA I showed decreased binding and Con A, WGA, s-WGA and RCA I were insensitive to this type of fixation. Pepsin treatment of the sections before lectin staining caused marked changes in the staining pattern; staining with PNA in formalin-fixed tissue sections was particularly improved but there was also enhanced staining with SBA and horse gram agglutinin (DBA). On the other hand, in Bouin- and particularly in acetone-fixed tissue sections, pepsin treatment decreased the staining with several of the lectins, for example WGA and UEA I.     

Binding of fluorescein isothiocyanate conjugated lectins to rat spermatogenic cells in tissue sections

Summary The testes from three months old Spague-Dawley rats were fixed in Bouin's fluid or neutral buffered 10% formalin, embedded in paraffin, sectioned and after deparaffination stained with the following fluorescein isothiocyanate coupled lectins: PNA, WGA, Con A, RCA, SBA, DBA and UEA. The results show that there are considerable differences in the staining pattern of various spermatogenic cells between different lectins. The fixation in Bouin's fluid enhanced the staining of all the lectins compared to formalin fixation in which only a weak staining could be seen in the acrosomes of spermatids after WGA or PNA staining. PNA and WGA stained specifically the acrosome of the developing spermatids, which was seen from the beginning of the acrosome formation and lasted up to late spermiogenesis. However, the staining with PNA decreased in the late spermatids whereas the intensity of the staining remained unchanged with WGA. Con A did not stain the acrosome but stained unspecifically the cytoplasm of all spermatogenic cells. RCA stained faintly the acrosome throughout the spermatid differentiation. DBA and UEA stained specifically the chromosomes of B spermatogonia. DBA also faintly stained the cell membranes of early spermatids. SBA did not show any specific staining of the spermatogenic cells. Based on this it is suggested that the carbohydrates and glycoproteins which are known to be present in the acrosome are formed already in the beginning of the acrosome formation. The decrease in the PNA staining in late spermatids possibly reflects the fact that the receptor molecules are not synthesized in late spermatids but are formed in earlier developmental stages and are thereafter preserved in the acrosome. The enhancement of lectin binding caused by Bouin's fixative might also be applied to other tissues where previous experiments with formalin fixed tissue have failed to show any staining.     

Binding of fluorescein isothiocyanate conjugated lectins to rat spermatogenic cells in tissue sections. Enhancement of lectin fluorescence obtained by fixation in Bouin's fluid

The testes from three months old Sprague-Dawley rats were fixed in Bouin's fluid or neutral buffered 10% formalin, embedded in paraffin, sectioned and after deparaffination stained with the following fluorescein isothiocyanate coupled lectins: PNA, WGA, Con A, RCA, SBA, DBA and UEA. The results show that there are considerable differences in the staining pattern of various spermatogenic cells between different lectins. The fixation in Bouin's fluid enhanced the staining of all the lectins compared to formalin fixation in which only a weak staining could be seen in the acrosomes of spermatids after WGA or PNA staining. PNA and WGA stained specifically the acrosome of the developing spermatids, which was seen from the beginning of the acrosome formation and lasted up to late spermiogenesis. However, the staining with PNA decreased in the late spermatids whereas the intensity of the staining remained unchanged with WGA. Con A did not stain the acrosome but stained unspecifically the cytoplasm of all spermatogenic cells. RCA stained faintly the acrosome throughout the spermatid differentiation. DBA and UEA stained specifically the chromosomes of B spermatogonia. DBA also faintly stained the cell membranes of early spermatids. SBA did not show any specific staining of the spermatogenic cells. Based on this it is suggested that the carbohydrates and glycoproteins which are known to be present in the acrosome are formed already in the beginning of the acrosome formation. The decrease in the PNA staining in late spermatids possibly reflects the fact that the receptor molecules are not synthesized in late spermatids but are formed in earlier developmental stages and are thereafter preserved in the acrosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eosinophilic globule cells in mouse MFH-like sarcomas: lectin histochemistry.

Lectin binding patterns in ten mouse malignant fibrous histiocytoma (MFH)-like sarcomas containing eosinophilic globule (EG) cells and in granular metrial gland (GMG) cells of mouse placenta were stained with nine lectins (Con A, LCA, WGA, DBA, SBA, e-PHA, PNA, RCA-I and UEA-I) by an avidin-biotin-peroxidase-complex method. EG cells stained strongly with DBA, SBA and PNA which are specific for N-acetyl-D-galactosamine and/or D-galactose. DBA and SBA bound throughout the cytoplasm including the globules; PNA reacted preferentially at the cell surface. There was no evidence that these three lectins were reactive for immature EG cells. WGA, RCA-I and e-PHA also gave a slightly to moderately positive reaction to globules of EG cells. The results indicate that the globules contain abundant O-linked sequences of sugars, but also a few N-linked residues. MFH tumor cells showed a variable degree of binding with Con A, RCA-I, and WGA, but did not react with DBA, SBA and PNA. On the other hand, GMG cells exhibited specific affinities for DBA, SBA and PNA with staining patterns similar to those of EG cells. These findings suggest that EG and GMG cells may be of the same cellular lineage.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Lectin histochemical study on the infraorbital gland of the Japanese serow (Capricornis crispus)

Histology and lectin histochemistry were performed in the infraorbital gland of the Japanese serow. The gland is composed of glandular tissues and a pouch filled with the secretion. The tissues consist of an inner layer of sebaceous glands and an outer layer of apocrine glands. The male sebaceous layer is made up of the ordinary type, whereas the female's layer consists of the ordinary and modified types. In the apocrine gland stained with Arachis hypogaea (PNA), nine different patterns of glandular tubules were distinguished on the basis of staining of the cytoplasm, the Golgi area of secretory cells and secretion. Secretory modes of apocrine secretion and exocytosis were included in these stainings. Myoepithelial cells stained constantly with Glycine max (SBA) except when only the Golgi area of secretory cells was positive. The modified sebaceous gland was stained with PNA, SBA, Ricinus communis I (RCA), Triticum vulgaris (WGA), Canavalia ensiformis (Con A) and Ulex europaeus I (UEA), while the ordinary type was positive in PNA, RCA, SBA, WGA and Con A. The secretion in the pouch was stained with PNA, RCA, SBA, Dolichos biflorus (DBA), WGA and Con A. These findings suggest that the modified sebaceous gland contains large amounts of glycoconjugates and the apocrine gland shows a cyclic secretory process of apocrine secretion and exocytosis.     

A comparison of lectin binding in rat and human peripheral nerve

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.     

Lectin binding pattern in normal human labial mucosa

Summary The pattern of lectin binding in normal human labial mucosa was examined by light and electron microscopy using eight different lectins (ConA, LCA, WGA, UEA-1, RCA-1, SBA, DBA and PNA) and compared with the patterns in normal human skin and oesophageal mucosa. As seen by light microscopy, ConA, LCA, and WGA stained cell membranes in all layers of the mucosae. RCA-1 stained the plasma membrane of cells in the basal and middle layers, whereas cells in the superficial layers showed little positive staining. UEA-1, SBA, and PNA stained the cells in the middle layers weakly in some cases. No positive staining for DBA was seen. By electron microscopy, reaction product indicating ConA-binding sites was observed in the plasma membrane, cisternae of the endoplasmic reticulum, nuclear envelope and the Golgi apparatus. Binding of LCA, WGA, and RCA-1 was observed in the plasma membrane. These results show that the binding pattern of PNA, SBA, and RCA-1 in labial mucosa is different from that in the normal skin or oesophageal mucosa, although the labial mucosal epithelium, epidermis, and oesophageal epithelium are all stratified squamous epithelia. These differences in the cell-surface sugar residues are likely to be related to the possible functional differences in these tissues.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin binding to collagen strands in histologic tissue sections

Histologic sections from human skin and uterine ligaments were stained with the following FITC conjugated lectins: Con A, WGA, s-WGA, SBA, DBA, UEA I, PNA, RCA I, BPA, GSA I, GSA II, MPA and LPA. The staining of the connective tissue was similar in the dermis and the uterine ligaments and it was most intense in the extracellular matrix containing collagen strands whereas the fibrocytes remained unstained. The staining was clear with glucose or N-acetylglucosamine binding lectins like Con A, WGA, s-WGA and GSA II, which may be related to the presence of glucose residues in collagenous hydroxylysine. The staining with some of the galactose or N-acetylgalactosamine binding lectins like RCA I, DBA, and BPA was less intense. This may reflect the presence of terminal galactose sugars in the hydroxylysine of collagen. No staining was found with SBA, UEA I, PNA, GSAI, MPA or LPA. The results show that different particularly glucose specific lectins bind to the extracellular matrix and especially to collagenous strands in connective tissue. It is suggested that this might be used in histochemical studies of connective tissue and particularly concerning the changes that may occur in different disease states.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.     

Glycoconjugates in normal human kidney

Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

Changes in lectin-binding features of ram sperm surfaces associated with epididymal maturation and ejaculation

Ram sperm, isolated from the caput, corpus, and cauda epididymidis, plus ejaculated cells were washed free of loosely bound components and tested for their ability to bind fluorescein-conjugated lectins (Con A, SBA, RCA, PNA, ECA and WGA) as assessed by epiluminescent-fluorescence light microscopy and flow cytometry. Detailed preliminary studies established an appropriate lectin-to-sperm ratio and incubation conditions for quantitative comparisons of sperm cell types and permitted a detailed analysis of both the amount of lectin bound as well as its distribution on the various aspects of the cell surface. Con A (mannose positive) bound weakly over the entire surface, with little change associated with maturation in the male tract. SBA (N-acetylgalactosamine positive) bound moderately strongly to caput sperm, with an emphasis on the apical ridge portion of the cell; during epididymal transit this binding was greatly diminished and was regained upon ejaculation. RCA, PNA, and ECA (galactose positive) gave generally equivalent results, where initially strong binding to the entire sperm surface decreased (over all parts of the surface except the anterior head) during epididymal maturation, with no change associated with ejaculation. WGA (sialic acid positive) binding initially was weak, but increased with epididymal transit and ejaculation. In vitro incubations with beta-galactosidase and neuraminidase confirmed the assignments given above. These data, when coupled with previous reports describing the heterogeneous distribution of proteins and lipids and changes in their distribution associated with epididymal maturation, serve to quantitatively describe changes in those aspects of the cell surface that are probably responsible for the acquisition of the capacity of the sperm to bind successfully to the oocyte.     

Different binding to squamous and columnar epithelium of the uterine cervix as a marker of epithelial differentiation

Biotinylated lectins were used to investigate the expression of carbohydrate residues on columnar and squamous epithelium of the uterine cervix. Con A, WGA, RCA I, PNA, UEA I, DBA and SBA were used. In the native exocervical and in metaplastic squamous epithelium of the transformation zone, one group of lectins (Con A, WGA, RCA I and PNA) stained the cell periphery of all epithelial layers. A second group (UEA I, DBA and SBA) colored the cell periphery of the suprabasal cells. The basal layer was always negative. All lectins labeled the apical border and occasionally the cytoplasm of the endocervical columnar epithelium. Lectin-binding of metaplastic and native squamous epithelium could possibly be used as a marker of epithelial differentiation in normal and abnormal conditions.     

Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods

Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Acα2,3Galβ1,4GlcNAc, Neu5Acα2,6Gal/GalNAc, with Galβ1,3GalNAc, α/βGalNAc and glycans with terminal/internal αMan and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Galβ1,4GlcNAc (Ricinus communis₁₂₀ affinity) (RCA₁₂₀) and L-Fucα1,2Galβ1,4GlcNAcβ (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and post-acrosomal region did not display glycans terminating with GalNAc, GlcNAc, and αL-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Acα2,3Galβ1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with αGalNAc, GlcNAc, and L-Fucα1,2Galβ1,4GlcNAcβ (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and non-breeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.     

Glycoproteins of murine zona pellucida from unfertilized eggs and two-cell embryos: comparison of the reactivity to lectins

Glycoproteins of zona pellucida were fractionated by SDS-polyacrylamide gel electrophoresis, and their lectin binding was examined after blotting onto nitrocellulose. The specificity of lectin binding to the major zona glycoproteins was the same for zonae isolated from eggs or from two-cell embryos; RCA, WGA, SBA and DBA reacted with all of the glycoproteins, PNA with ZP-1 and ZP-3, and Con A with ZP-2. However, the reactivity of ZP-3 to SBA and DBA was decreased in zonae from two-cell embryos. In addition, two-cell embryo zona contained a band which had different binding specificity to lectins from the major glycoproteins.     

Secretory glycoproteins of major vestibular glands (Bartholin's glands) in female calves. Studies with usual histochemical methods and with lectin-horseradish peroxidase conjugates in normal animals and in animals implanted with anabolic drugs

The nature of mucins synthetized by the major vestibular glands of normal female calves and animals treated by anabolic drugs was investigated by usual histochemical methods and by lectin conjugate methods: Canavalia ensiformis (Con A), Limulus polyphemus (LPA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Glycine max (SBA) and Triticum vulgaris (WGA). Two main secretory cell types, i.e. acinar and tubular cells, could be distinguished. The former produced sialomucins, whereas the latter produced neutral mucins. WGA and PNA showed strong binding to all secretory cells while LTA, RCA I and SBA exhibited a weaker affinity. Treatment with anabolic drugs enhanced O-acetyl sialic acid synthesis and we noted de novo synthesis of sulfomucins. However, the staining intensity of LTA was stronger than in control calves. Tubular secretory cells of treated animals revealed an intense secretion of neutral mucins but, in contrast, all tested lectins were less intensively bound. The present study provides additional histochemical information on Bartholin's glands. A shortened procedure is proposed to detect animals treated with anabolic drugs when morphological changes are lacking.