Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

138 Molecular Evolution of Glutamine Synthetase In Protists

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N-acetyl-D-galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus-specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi-step process and gamete binding is the initial step.     

137 Gamete Recognition and Sexual Isolation in a Red Alga,Aglaothamnion Byssoides (Rhdophyta)

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre‐incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N‐acetyl‐D‐galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus‐specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi‐step process and gamete binding is the initial step.     

Herpes simplex virus envelopment and maturation studied by fracture label.

Herpes simplex virus envelopment and maturation were investigated by thin-section fracture label. The distribution of glycoproteins B and D was analyzed by labeling with antibodies; the precursor and mature forms of the glycoproteins were differentiated by labeling with the lectins concanavalin A (ConA) and wheat germ agglutinin (WGA), respectively. We report that the two glycoproteins were readily detected in the intracellular virion, whether located between the inner and outer nuclear membranes or within cytoplasmic membrane-bound vesicles and in the inner and outer nuclear membranes themselves. The enveloped virion between the inner and outer nuclear membranes labeled with ConA but not with WGA. During the transit to the extracellular space the reactivity of the virion membranes with ConA decreased and that with WGA ensued. The results document that herpes simplex viruses acquire at the inner nuclear membrane an envelope carrying the immature forms of the glycoproteins and that during the transit to the extracellular space the envelope glycoproteins become of the fully processed type.     

Distribution and nature of saccharides of plasma membranes of rat ascites hepatoma cells as detected by ferritin-conjugated lectins and dialysed iron

Electron microscopic cytochemical studies were made on saccharides involved in the plasma membranes of rat ascites hepatoma cells (AH7974F) using ferritin-conjugated lectins and dialysed iron (DI). In the rat hepatoma cells, saccharide receptors for each of the three lectins used (concanavalin A (ConA), wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA)) were shown to be distributed homogeneously throughout the plasma membranes. When the cells were agglutinated, however, the saccharide receptors for each lectin appeared to form clusters on the plasma membranes. The cluster formation induced by one lectin was found to lead to a changed distribution of saccharide receptors for another lectin. None of the cluster formation types induced by lectins yield any noticeable effects upon the distribution of DI reactive acidic saccharides on the plasma membranes.     

Absence of cytochrome P-450 and presence of autolysosomal membrane antigens on the isolation membranes and autophagosomal membranes in rat hepatocytes

We wished to determine if phenobarbital (PB)-inducible cytochrome P-450 [P-450(PB)] and autolysosomal membrane antigens could be localized immunocytochemically on the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by the post-embedding protein A-gold method. P-450(PB) was maximally induced by PB treatment; then formation of autophagosomes and accumulation of autolysosomes were induced by cessation of PB treatment and by injection of leupeptin, respectively. P-450(PB) was detected neither on the isolation membranes nor on the limiting membranes of autophagosomes and autolysosomes. Autolysosomal membrane antigens, which were localized by the immunogold technique exclusively in post-Golgi compartments such as lysosomes, endosomes, and plasma membrane but were not found in pre-Golgi compartments such as endoplasmic reticulum (ER) and nuclear envelope, were detected in large amounts on the isolation membranes. These results suggest that the isolation membranes originate not from ER membranes but from post-Golgi membranes. We also present direct immunoelectron microscopic evidence that P-450(PB) is indeed degraded in the autolysosomes: when rats were treated with leupeptin, P-450(PB) was detected not only within the autophagosomes but also within the autolysosomes, whereas without leupeptin treatment, P-450(PB) was detectable only within the autophagosomes.     

Cellular heterogeneity in normal human urothelium: Quantitative studies of lectin binding

Summary A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II) was applied to cryosections of seven specimens of normal urothelium. Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium. Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types. Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties. Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation. Although there was some variation among individual cases, a general pattern emerged in this small series. WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding. MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.     

Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs

Receptors for Ricinus communis agglutinin I (RCAI), concanavalin A (Con A), and wheat germ agglutinin (WGA) were localized on the zonae pellucidae and plasma membranes of hamster, mouse, and rat eggs with ferritin-lectin conjugates. Intact eggs labeled with the ferritin conjugates showed dense concentrations of RCAI and WGA receptors in the outermost regions of their zonae pellucidae and sparse distributions of Con A receptors throughout the zonae. Ferritin-lectin labeling was specific, since inhibitory saccharides effectively blocked labeling. The asymmetric density of RCAI receptors across the zona was confirmed by ferritin-RCAI and fluorescein-RCAI labeling of mechanically isolated zonae pellucidae, indicating that the RCAI-binding sites are more densely distributed in the exterior zona regions. Plasma membranes of rodent eggs contained RCAI, WGA, and Con A receptors. These receptors were found to be more or less randomly distributed on surfaces of aldehyde-fixed eggs or on eggs labeled near 0 degrees C. However, eggs incubated at 25 degrees C showed aggregated WGA- and Con A-binding site distributions on their plasma membranes. This indicates that lectin- induced receptor redistribution occurs at this temperature. The possibility that plasma membrane receptor mobility is a requirement for sperm-egg fusion is discussed.     

Differentiated microdomains on the luminal surface of capillary endothelium: distribution of lectin receptors

Lectins conjugated with either peroxidase or ferritin were used to detect specific monosaccharide residues on the luminal front of he fenestrated endothelium in the capillaries of murine pancreas and intestinal mucosa. The lectins tested recognize, if accessible, the following residues: alpha-N-acetylgalactosaminyl (soybean lectin), beta- D-galactosyl (peanut agglutinin [PA] and Ricinus communis agglutinin- 120 [RCA]), beta-N-acetylglucosaminyl and sialyl residues (wheat germ agglutinin [WGA]), alpha-L-fucosyl (lotus tetragonolobus lectin), and alpha-D-glucosyl and beta-D-mannosyl (concanavalin A [ConA]). Thi labeled lectins were introduced by perfusion in situ after thoroughly flushing with phosphate-buffered saline the microvascular beds under investigation. Specimens were fixed by perfusion, and subsequently processed for peroxidase detection and electron microscopy. Control experiments included perfusion with: (a) unlabeled lectin before lectin conjugate; (b) labeled lectin together with the cognate hapten sugar, and (c) horseradish peroxidase or ferritin alone. Binding sites were found to be relatively homogeneously distributed on the plasmalemma proper, except for Lotus tetragonolobus lectin and Con A, which frequently bound in patches. Plasmalemmal vesicles, transendothelial channels, and their associated diaphragms were particularly rich in residues recognized by RCA and PA (beta-D-galactosyl residues) and by WGA (beta-N-acetylglucosaminyl residues). Receptors for all lectins tested appeared to be absent or considerably less concentrated on fenestral diaphragms. The results reported here extend and complement previous findings on the existence of microdomains generated by the preferential distribution of chemically different anionic sites (Simionescu et al., 1981, J. Cell Biol., 9:605-613 and 614-621).     

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.     

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.     

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.     

Effect of lectins on the invasion of Ichthyophthirius theront to channel catfish tissue.

This study determined the effects of lectin binding to theronts of Ichthyophthirius multifiliis on theront immobilization, invasion, trophont development and survival in channel catfish Ictalurus punctatus excised fins in vitro. Soybean agglutinin (SBA), lentil agglutinin (LCA), gorse agglutinin (UEA-I) and wheat germ agglutinin (WGA) were used to treat theronts. Percentages of theronts immobilized by 4 lectins ranged from 12.0 to 19.4% at a concentration of 1000 microg ml(-1). These lectins bound more than half of the theronts at a concentration of 50 microg ml(-1). More theronts were labeled by SBA and WGA than by lectin LCA at concentrations of 50 and 100 microg ml(-1), respectively. The binding of these lectins to theronts indicated that monosaccharides (D-galactose, L-fucose, D-mannose and D-glucose) and amino sugar derivatives (N-acetylgalactosamine and N-acetylglucosamine) were present on the surface of theronts. Invasion was reduced significantly for theronts treated with LCA, UEA-I and WGA. No difference in invasion was found between control and SBA bound theronts (p > 0.05). The binding of lectin LCA, UEA-I and WGA to theronts significantly reduced the development of trophonts (p < 0.05). The mean volumes of trophonts labeled with these 3 lectins were smaller than volumes in control trophonts from 8 to 48 h after exposure. Survival was lower in trophonts labeled with lectins than in control trophonts at 48 h after exposure.     

A survey of lectin binding in Paramecium

To better understand the general distribution of glycoproteins and the distribution of specific glycoprotein-bound sugar residues in Paramecium, a survey of the binding pattern of selected lectins was carried out in P. tetraurelia, P. caudatum, and P. multimicronucleatum. Lectins studied were concanavalin A (Con A), Griffonia simplicifolia agglutinins I and II (GS I and GS II), wheat germ agglutinin (WGA), Ulex europaeus (UEA I), peanut agglutinin (PNA), Ricinis communis toxin (RCA60) and agglutinin (RCA120), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), Dolichos biflorus agglutinin (DBA), and Maclura pomifera agglutinin (MPA). Those giving the most distinctive patterns were Con A, GS II, WGA, UEA I, and PNA. No significant differences were found between the three species. Concanavalin A, a mannose/glucose-binding lectin, diffusely labeled the cell surface and cytoplasm and, unexpectedly, the nuclear envelopes. Events of nuclear division, and nuclear size and number were thus revealed. Both WGA and GS II, which are N-acetylglucosamine-binding lectins, labeled trichocyst tips, the cell surface, and the oral region, revealing stages of stomatogenesis. The lectin WGA, in addition, labeled the compartments of the phagosome-lysosome system. The lectin PNA, an N-acetyl galactosamine/galactose-binding protein, was very specific for digestive vacuoles. Finally, UEA I, a fucose-binding lectin, brightly labeled trichocysts, both their tips and body outlines. We conclude that a judicious choice of lectins can be used to localize glycoproteins and specific sugar residues as well as to study certain events of nuclear division, cellular morphogenesis, trichocyst discharge, and events in the digestive cycle of Paramecium.     

Selective distribution of sugars on the tegumental surface of adult Bothriocephalus gregarius (Cestoda: Pseudophyllidea)

Whole specimens and histological and semi-thin sections of Bothriocephalus gregarius adults were exposed to lectins to identify carbohydrates present in the tegument and parenchyma. The sugars N-acetyl glucosamine, N-acetyl galactosamine, galactose, glucose (or mannose) and fucose were detected in the cestode using eight lectins: WGA (Wheat germ agglutinin), HPA (Helix pomatia agglutinin), SBA (Soy bean agglutinin), PHA (Phaseolus vulgaris agglutinin), RCA60 and RCA120 (Ricinus communis toxin and agglutinin), ConA (Concanavalin agglutinin) and UEA-I (Ulex europaeus agglutinin). Combined use of these methodological approaches (whole specimens, paraffin and semi-thin sections) revealed the presence of a gradient in the distribution of most of the sugars over the tegument, with the highest concentrations on the strobila (as shown by most of the lectins). Other sugars were specific for the scolex or strobila (as shown by UEA-I or HPA, respectively). The ultrastructural study showed that the distribution of glycoconjugates was associated with the presence of specific tegumental coats. The significance of this selective distribution and its relevance to cestode physiology and host-parasite relationships are discussed.     

Distribution of glycoconjugates on the plasma membrane and on membranes of the open-canalicular system in human platelets

Summary Distribution of carbohydrate moieties in the membrane system of the human blood platelet was studied by electron microscopy employing lectins as a probe. Glutaraldehyde-fixed platelets were treated with biotinylatedlectins (ConA, RCA, WGA, PNA, SBA, DBA and UEA-1) and labeled with horseradish peroxidase-conjugated avidin. Among the lectins used, ConA bound uniformly to the plasma membrane as well as to the membrane of the opencanalicular system (OCS). Other lectins showed more or less reduced binding on the OCS membrane compared with that on the plasma membrane, indicating that there exist regional differences in the distribution pattern of glycoconjugates in the membrane system of the platelet. The relationship of the distribution pattern of the glycoconjugates with the distribution of the major platelet glycoproteins GPIb and GPIIbIIIa is discussed.     

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.