Fluorescein-labelled Lectins Research Articles

Semiautomated pipeline for quantitative analysis of heart histopathology

BackgroundHeart diseases are among the leading causes of death worldwide, many of which lead to pathological cardiomyocyte hypertrophy and capillary rarefaction in both patients and animal models, the quantification of which is both technically challenging and highly time-consuming. Here we developed a semiautomated pipeline for quantification of the size of cardiomyocytes and capillary density in cardiac histology, termed HeartJ, by generating macros in ImageJ, a broadly used, open-source, Java-based software.MethodsWe have used modified Gomori silver staining, which is easy to perform and digitize in high throughput, or Fluorescein-labeled lectin staining. The latter can be easily combined with other stainings, allowing additional quantitative analysis on the same section, e.g., the size of cardiomyocyte nuclei, capillary density, or single-cardiomyocyte protein expression. We validated the pipeline in a mouse model of cardiac hypertrophy induced by transverse aortic constriction, and in autopsy samples of patients with and without aortic stenosis.ResultsIn both animals and humans, HeartJ-based histology quantification revealed significant hypertrophy of cardiomyocytes reflecting other parameters of hypertrophy and rarefaction of microvasculature and enabling the analysis of protein expression in individual cardiomyocytes. The analysis also revealed that murine and human cardiomyocytes had similar diameters in health and extent of hypertrophy in disease confirming the translatability of our murine cardiac hypertrophy model. HeartJ enables a rapid analysis that would not be feasible by manual methods. The pipeline has little hardware requirements and is freely available.ConclusionsIn summary, our analysis pipeline can facilitate effective and objective quantitative histological analyses in preclinical and clinical heart samples.

A Novel Preparation Technique for Human Nasal Respiratory Mucosa to Disclose Its Glycosylation Pattern for Bioadhesive Drug Delivery

To shed some light on glycotargeting as a potential strategy for nasal drug delivery, a reliable preparation method for human nasal mucosa samples and a tool to investigate the carbohydrate building blocks of the glycocalyx of the respiratory epithelium are required. Applying a simple experimental setup in a 96-well plate format together with a panel of six fluorescein-labeled lectins with different carbohydrate specificities allowed for the detection and quantification of accessible carbohydrates in the mucosa. As confirmed by binding experiments at 4 °C, both quantitatively by fluorimetry and qualitatively by microscopy, the binding of wheat germ agglutinin exceeded that of the others by 150% on average, indicating a high content of N-acetyl-D-glucosamine and sialic acid. Providing energy by raising the temperature to 37 °C revealed uptake of the carbohydrate-bound lectin into the cell. Moreover, repeated washing steps during the assay gave a slight hint as to the influence of mucus renewal on bioadhesive drug delivery. All in all, the experimental setup reported here for the first time is not only a suitable approach to estimating the basics and potential of nasal lectin-mediated drug delivery but also meets the needs for answering a broad variety of scientific questions involving the use of ex vivo tissue samples.

The analysis of surface saccharide profiles through fluorescein-labelled lectins in a rat pancreatic tissue with established metabolic syndrome model

Abstract “Glycans”, which are generally referred as oligosaccharides and polysaccharides, are structures that are present on all cellular surfaces with proteins and lipids being attached to their basic chain structures. Many studies in the field of glycobiology have identified the various and complicated biological roles of these glycans which make them perfect molecules to use in labelling and selecting body cells specifically. This study aims at analyzing the modifications in saccharide units of glycans on a cell membrane surfaces of the pancreatic tissue of rats to which normal and metabolic syndrome (MetS) are established. To this end, a MetS model was created through a high fructose diet in Spraque Dawley breed of rats and the pancreatic tissue sections of the group with MetS and control group animals were evaluated comparatively. The targeted saccharide units were examined with Fluorescent Microscope by using two different Fluorescein (FITC) labelled lectins, namely Maackia amurensis-1 lectin [FITC-(MAL-I)] and the Wheat Germ Agglutinin (FITC-WGA). It was observed that FITC-MAL-1-labelled Galβ4GlcNAc units did not change much due to high- fructose diet. On the other hand, more GlcNAc, Neu5Ac and β-GlcNAc units which are labelled with FITC-WGA lectin increase in numbers in pancreatic sections of high fructose diet, compared to control group. Thus, a rapid and specific labelling method, which can identify surface saccharide sequences specifically, was developed. The method can be used in early diagnosis and/or treatment for metabolic diseases.

Identification of surface polysaccharides in akinetes, heterocysts and vegetative cells of Anabaena cylindrica using fluorescein-labeled lectins.

In response to environmental changes, Anabaena cylindrica differentiate three cell types: vegetative cells for photosynthesis, heterocysts for nitrogen fixation, and akinetes for stress survival. Cell-surface polysaccharides play important roles in cyanobacterial ecophysiology. In this study, specific cell-surface sugars were discovered in heterocysts, akinetes and vegetative cells of A. cylindrica using 20 fluorescein-labeled lectins. Both N-acetylglucosamine-binding lectins WGA and succinylated WGA bound specifically to the vegetative cells. Akinetes bound to three mannose-binding lectins (LCA, PSA, and ConA), and one of the galactose-binding lectins (GSL-I). Heterocyst also bound to ConA. However, the heterocysts in all4388 mutant of Anabaena sp. PCC 7120, in which the putative polysaccharide export protein gene all4388 was disrupted, exhibited diminished binding to ConA. Identification of distinct cell-surface sugar helped us to understand the role of polysaccharide for each cell type. Fluorescence-activated cell sorting may be applicable in isolating each cell type for comparative "omics" studies among the three cell types.

Lactoferrin Reduces Chorioretinal Damage in the Murine Laser Model of Choroidal Neovascularization

Purpose: To determine whether lactoferrin, specifically endogenous mouse lactoferrin and exogenous intraperitoneal lactoferrin treatment, plays a role in reducing the chorioretinal damage in the laser-induced model of choroidal neovascularization. Materials and methods: Four 532-nm argon laser spots were placed between the retinal vessels of each eye. At Day 7, Fluorescein Angiography was performed to grade the lesions. The mice were perfused with fluorescein-labeled tomato lectin and sacrificed. The retinal pigment epithelium-choroid-sclera complex was flat-mounted and analyzed with a confocal microscope to measure the volume of the lesions. The effect of endogenous lactoferrin was studied by comparing lactoferrin knockout and wild-type (WT) mice. The effect of exogenous lactoferrin treatment was studied by comparing lactoferrin knockout and WT mice treated with lactoferrin for seven days to their respective controls. Results: Lactoferrin knockout mice demonstrated 47% larger lesion volumes than WT mice (p < 0.001). Intraperitoneal treatment with Lactoferrin reduced the lesion volume in Lactoferrin knockout mice by 26% (p < 0.04). Regarding the fluorescein angiography, lesions indicating the greatest damage (grade 2B) occurred more frequently in control lactoferrin knockout mice compared with control WT mice (16% versus 5%). Intraperitoneal treatment with Lactoferrin reduced the grade 2B lesions from16% to 2% in Lactoferrin knockout mice. Conclusion: The endogenous lactoferrin present in WT mice appears to reduce the choroidal neovascularization in the laser-induced choroidal neovascularization model in mice. Treatment with exogenous lactoferrin is capable of reducing the choroidal neovascularization in lactoferrin knockout mice but does not add a significant protective effect to WT.

Distribution patterns of carbohydrates in murine glycocalyx

Enterocytes are unique cells governing an array of processes. They are covered by the gut glycocalyx, which is an extraneous carbohydrate-rich coat and an integral part of the plasma membrane. The intestinal glycocalyx and secreted mucins constitute a glycosylated milieu which has a number of physiological and protective functions. One of the important functions of the glycocalyx is its barrier function against microbial adherence to different membrane glycolipids. Thus, the glycocalyx is an important part of the mucosal immune system in newborns. The aim of our study was to identify the carbohydrates in the small bowel glycocalyx of Balb/c mice at different ages. We used plant lectins with different sugar specificities. Fluorescein-labelled lectins binding different carbohydrate moieties were detected using confocal laser scanning microscopy. Biotinilated lectins were used for transmission electron microscopy observations of the constituents of the gut glycocalyx at different periods of postnatal development in mice. Different carbohydrate moieties that were identified in the murine intestinal glycocalyx followed different distribution patterns and characteristics. Carbohydrates present on the mucus surface depended on tissue localization, cell type and stage of development. The distribution and mucins glycosylation could be of interest in analysing the response of the mucosal barrier to intestinal pathogens causing infection or inflammation.

Mucin–lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50–80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC–MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.

Tissue-engineered pro-angiogenic fibroblast scaffold improves myocardial perfusion and function and limits ventricular remodeling after infarction

Microvascular malperfusion after myocardial infarction leads to infarct expansion, adverse remodeling, and functional impairment. Native reparative mechanisms exist but are inadequate to vascularize ischemic myocardium. We hypothesized that a 3-dimensional human fibroblast culture (3DFC) functions as a sustained source of angiogenic cytokines, thereby augmenting native angiogenesis and limiting adverse effects of myocardial ischemia. Lewis rats underwent ligation of the left anterior descending coronary artery to induce heart failure; experimental animals received a 3DFC scaffold to the ischemic region. Border-zone tissue was analyzed for the presence of human fibroblast surface protein, vascular endothelial growth factor, and hepatocyte growth factor. Cardiac function was assessed with echocardiography and pressure-volume conductance. Hearts underwent immunohistochemical analysis of angiogenesis by co-localization of platelet endothelial cell adhesion molecule and alpha smooth muscle actin and by digital analysis of ventricular geometry. Microvascular angiography was performed with fluorescein-labeled lectin to assess perfusion. Immunoblotting confirmed the presence of human fibroblast surface protein in rats receiving 3DFC, indicating survival of transplanted cells. Increased expression of vascular endothelial growth factor and hepatocyte growth factor in experimental rats confirmed elution by the 3DFC. Microvasculature expressing platelet endothelial cell adhesion molecule/alpha smooth muscle actin was increased in infarct and border-zone regions of rats receiving 3DFC. Microvascular perfusion was also improved in infarct and border-zone regions in these rats. Rats receiving 3DFC had increased wall thickness, smaller infarct area, and smaller infarct fraction. Echocardiography and pressure-volume measurements showed that cardiac function was preserved in these rats. Application of a bioengineered 3DFC augments native angiogenesis through delivery of angiogenic cytokines to ischemic myocardium. This yields improved microvascular perfusion, limits infarct progression and adverse remodeling, and improves ventricular function.

Characterization of two blood–brain barrier mimicking cell lines: Distribution of lectin-binding sites and perspectives for drug delivery

In the present study plant lectins with distinct sugar specificities were applied to two blood–brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1–2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1–2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1–2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

Differences in the Glycocalyx of 5637 and SV-HUC-1 Cells and their Impact on Lectin Targeting

Background: Bladder cancer is one of the most common malignancies. Advances have been made in diagnosis, therapy, and surveillance resulting in increasing survival rates of patients, but the search for more effective therapies is imperative to be continued. Among others, lectin-mediated targeting might be a promising concept. Thus, the potential of fluorescein-labeled plant lectins was investigated using 5637 cells as a model for human urinary carcinoma [1] and SV-HUC-1 cells as a model for non-tumorigenic human uroepithelial cells.

Patterned Immobilization of Unprotected Carbohydrates on an Aminooxy Polymer-Grafted Solid Surface

Abstract We describe a graft-type modification of a solid surface to be able to immobilize unprotected carbohydrates with a desired pattern. Water soluble carbohydrate-trapping polymers were grown from a solid surface through surface-initiated polymerization. The polymerization of an N-tert-butonyloxycarbonyl (N-Boc)-protected aminooxy monomer 3 was carried out from the immobilized initiator on silicon or glass surfaces. The N-Boc protecting group was then removed in an acidic solution to generate an aminooxy group, which reacts with free carbohydrates through oxime bonds on immersion of the plate in a carbohydrate solution. Sugar attachment on the surface polymer was confirmed by using infrared reflection absorption spectroscopy (IR-RAS) and fluorescent images after sugar-specific binding of fluorescein-labeled lectin proteins. The polymer was also grafted in a desired pattern by partial UV-irradiation over a photomask, giving a new material to create carbohydrates arrays.

Red blood cell (RBC) age at collection and storage influences RBC membrane‐associated carbohydrates and lectin binding

Membrane-associated carbohydrate changes act as signals for removal of senescent and damaged red blood cells (RBCs) from the circulation and could play a role in the RBC storage lesion and RBC survival after transfusion. In this study, a panel of lectins was used to investigate the expression of carbohydrates on RBCs that had been separated before storage into young and old RBCs. Leukodepleted RBCs were separated before storage into young and old RBCs (n = 9 paired units) by centrifugation and sampled at nominated time points during 42 days of storage. Changes to carbohydrate expression at the RBC membrane during storage were determined by flow cytometry with a panel of fluorescein-labeled lectins. Old RBCs showed lower fluorescence intensity throughout storage, suggesting reduced binding of lectins compared to young RBCs. Progressively increased binding of lectins specific for galactose and N-acetylglucosamine residues was observed during storage of young and old RBCs. Changes to lectin binding during storage of RBCs suggest that significant changes occur to the carbohydrate structures at the RBC membrane. These findings provide further insight into the mechanisms of the RBC storage lesion and potential influence on RBC survival after transfusion.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin–cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha2,3- and alpha2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha1,6-linked l-fucosyl, and beta-D-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha1,2-linked l-fucosyl, terminal nonreducing alpha-D-galactosyl, N-acetyl-beta-D-glucosaminyl, nor N-acetyl-alpha-D-galactosaminyl groups were found by this approach. We also established the presence of Neu5Acalpha2,3/2,6-Galbeta1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha2,3-linked Neu5Ac as well as a change to exclusively alpha1,6-linked l-fucose upon differentiation.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses

Laser-capture microdissection (LCM) is a technique that enables selective extraction of desired cells from heterogeneous tissues compatible with subsequent molecular analyses. The specific visualization of desired cell types prior to LCM is essential for achieving selective capture. We have developed rapid and selective staining protocols for LCM extraction of microvessels from human and rat brain. Vessels in human and rat brain sections were visualized by a 2 min exposure to fluorescein-labeled lectins Ulex Europeaus Agglutinin I (UEA I) and Ricinus Communis Agglutinin I (RCA I), respectively. Immunohistochemical staining for the endothelial-specific marker, Factor VIII-related antigen (FVIII-rAg), co-localized with that for either UEA I or RCA I, confirming the selective staining of vascular structures with these lectins. Both brain vessels and perivascular parenchyma were captured using LCM, followed by RNA isolation. RT-PCR analyses demonstrated the enrichment of LCM-captured vessels and parenchyma in FVIII-rAg and GFAP mRNA, respectively. LCM-captured human vessels also expressed the tight junction-specific gene, zonula occludens 1 (ZO-1). LCM extraction of vessels from brain sections can be used to perform molecular fingerprinting of neurovascular unit in various brain pathologies.

Vimentin and heat shock protein expression are induced in the kidney by angiotensin and by nitric oxide inhibition

Angiotensin II (Ang II) infusion and nitric oxide synthesis (NOS) inhibition with Nomega-nitro-l-arginine-methyl-ester (l-NAME) are experimental models of hypertension associated with renal inflammation and oxidative stress. To gain insight into the nature of the tubulointerstitial injury induced in these models, we studied lectin-binding specificities, vimentin expression, and heat shock protein (HSP) 60 and 70 in these experimental models. Sprague-Dawley rats received Ang II infusion (435 ng/kg/min) for 2 weeks by subcutaneous minipumps (Ang II group, N = 5) or l-NAME in the drinking water (70 mg/100 mL) for 3 weeks (l-NAME group N = 7). The control group consisted of 10 rats. Systolic blood pressure (tail-cuff plethysmography), serum creatinine, and proteinuria were determined weekly. At the end of the treatment period, rats were sacrificed and kidneys studied. Binding specificities of fluorescein-labeled lectins were examined in frozen sections, and cellular infiltrates were identified by immunohistology and expression of vimentin and HSP 60 and 70 with immunohistochemistry and computer image analysis. Tubulointerstitial accumulation of macrophages, lymphocytes, and Ang II-positive cells were present in the Ang II group and l-NAME group. Vimentin, HSP 60, and HSP 70 were increased 8 to 20 times in the cortex of the rats of the Ang II group and the l-NAME groups. Neoexpression of vimentin and HSPs was found primarily in proximal tubular cells. Ang II infusion and NOS inhibition induce tubular injury with epithelial cell transdifferentiation and expression of stress proteins. The role of these changes in the accumulation and activation of the interstitial inflammatory infiltrate merits further investigation.

Fabrication of carbohydrate chips for studying protein-carbohydrate interactions.

A new tool for the high-throughput study of carbohydrate–protein interactions: Maleimide-linked carbohydrates (e.g. 1) were immobilized on thiol-derivatized glass slides, and the microspots were probed with fluorescein-labeled lectins (see picture). The binding of lectins to the carbohydrates on the slide depends on the concentration of the immobilized carbohydrates and on the length of the tethers.

Glycoconjugates on the surface of epididymal spermatozoa in a marsupial, the brushtail possum, Trichosurus vulpecula

Variation in localization and distribution of saccharides on the sperm surface of a marsupial, the brushtail possum, Trichosurus vulpecula, was compared between spermatozoa from the caput and cauda epididymides. Spermatozoa were subjected to the following treatments: (i) unfixed and fixed spermatozoa were stained with fluorescein-labelled lectins; (ii) unfixed spermatozoa were incubated with lectins for determination of agglutination; and (iii) spermatozoa were incubated with detergent to remove the plasmalemma, the glycoproteins were separated on SDS-PAGE and western blots were stained with biotinylated lectins. Many of the fluorescein isothiocyanate (FITC)-labelled lectins bound selectively to the sperm surface, and marked differences were found in lectin staining affinity between caput and cauda epididymal spermatozoa. Incubation of spermatozoa from the cauda epididymidis with neuraminidase reversed many of the differences in staining of the cauda epididymal spermatozoa, indicating masking of some terminal saccharides by sialic acid. Agglutination of spermatozoa from the caput epididymidis occurred after incubation with Concanavalin A (ConA) and soybean agglutinin (SBA), but agglutination was less extensive for spermatozoa from the cauda epididymidis. Western blot analysis indicated several ConA-positive bands in caput sperm extracts, but fewer positive bands in the cauda sperm extracts, whereas SBA stained four bands from caput but none from the cauda epididymal spermatozoa. These results demonstrate extensive glycosylation of the surface proteins of spermatozoa from the caput epididymidis and significant differences in spermatozoa from the cauda epididymidis. In general, the findings indicate similar glycosylation of the surface of marsupial spermatozoa to those from eutherian mammals despite marked differences in their morphology and early divergence of marsupials from eutherian mammals. It would appear that this situation differs markedly from that in sub-mammalian vertebrates.