WGA-binding Sites Research Articles

Peritoneal fluid from women with endometriosis impairs human spermatozoa functionality

The success of mammalian fertilization depends largely on spermatozoa physiological events. However, the manner in which endometriosis influence morpho-functional spermatozoa biomarkers is poorly defined. Here, we studied in vitro the effect of peritoneal fluid (PF) from women with endometriosis (PFE) and non-endometriosis (PFNE) on spermatozoa parameters. This research was prospective and double-blind. A total of 45 PF samples were collected from women with (n = 25) and without endometriosis (n = 20). Semen samples were obtained from normozoospermic donors (n = 15) and cultured with 20 % (v/v) of PF or commercial culture medium (controls) during 0, 24, and 48 h. The outcome measures were spermatozoa/viability, motility, tyrosine phosphorylation (TP) and spontaneous acrosomal reaction. In addition, plasma membrane sugars were characterized by lectins [Aleuria aurantia agglutinin (AAA), Concanavalin A (ConA), Peanut agglutinin (PNA), and Wheat germ agglutinin (WGA)]. After a 24 -h culture, results reported a significant decrease in motility in cells cultured with PFE compared to the control, together with differences in the AAA and WGA-binding sites. Moreover, spermatozoa in contact with PFNE presented a significantly lower level of acrosome spontaneous reaction. At 48 h, no differences were observed in the biomarkers studied between the PFNE and the control, excluding ConA-binding sites. On the other hand, cells cultured with PFE exhibited significantly less motility, TP, and differences in the relocation of spermatozoa surface sugars. Viability was not affected in any culture condition. Overall, the effects of PFE could negatively affect spermatozoa quality, contributing to explain and diagnose the infertility associated to male partners of women with endometriosis.

Lectin binding in normal donkey eyeball

In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA), galactose (labeled by PNA, GSAI, ECA), GalNAc (labeled by SBA, VVA), and GlcNAc (labeled by WGA) residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA) and GlcNAc (WGA) binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues), and it lacks fucosyl residues.

Lectin binding in normal donkey eyeball

In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA), galactose (labeled by PNA, GSAI, ECA), GalNAc (labeled by SBA, VVA), and GlcNAc (labeled by WGA) residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA) and GlcNAc (WGA) binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues), and it lacks fucosyl residues.

Characterization of glycoconjugates in the pharyngeal cavity and the oesophagus of Odontesthes bonariensis by lectins

Seven types of biotinylated lectins were used to examine glycoconjugates (GCs) expression patterns in the mucosa of the pharyngeal cavity and the oesophagus of Odontesthes bonariensis. Con-A, RCA-I, and WGA binding sites were positive for glycocalyx, epithelial cells, and mucous cells in both organs with similar reaction patterns. PNA just stained glycocalyx and epithelial cells in the mucosa in both organs. DBA, SBA, and UEA-I stained only the oesophagus, and a greater affinity to glycocalyx and the epithelial cells was observed. The results of this study were discussed and their possible functional implications were compared.

Histological and Lectin Histochemical Characterization of the Epididymal Duct in the Dromedary (Camelus dromedarius)

Study of the regional microscopic anatomy of the epididymal duct of the dromedary camel revealed that the initial segment was lined with high pseudostratified columnar epi- thelium bearing stereocilia; varying height levels from place to placegive the duct a “festooned” appearance. The nuclei of both principal and basal epithelial cells were located in the basal one-third. The duct was surrounded by a layer of circularly oriented smooth muscle cells; this peritubular smooth muscle thickened toward the termination of the epididymal duct. The middle and terminal segments of the epididymal duct were lined with similar epithelium, but with no noticeable variations in height. The nuclei in these regions were located in the basal one-half of the cells and spermato- zoa were more evident in the lumen of the middle and terminal segments. Epithelial cells were stained with six lectins: Con-A, DBA, MAA, SBA, SNA-1, and WGA. Binding sites for Con-A, WGA, and SNA-1 were seen in the initial segment. No binding sites for SBA, DBA, or MAA were detected in the initial segment. Sug- ar moieties for all lectin studied were detected in the middle and terminal segments, except for DBA and MAA in the middle segment, or WGA and MAA in the terminal segment. These lectins showed selective regional binding to different portions of the epididymal duct and varying degrees of binding to different cellular compartments. These regional var- iations in carbohydrate composition imply the probability of functional compartmentalization in the epididymal duct.

WGA-QD probe-based AFM detects WGA-binding sites on cell surface and WGA-induced rigidity alternation

A strategy involving the conjugation of fluorescent quantum dot (QD) with wheat germ agglutinin (WGA) acting as fluorescent and topographic probes prior to cell surface staining is developed for fluorescence microscopy and atomic force microscopy (AFM). This strategy provided at least two advantages: (a) an amplified fluorescence of WGA-QD aggregates, strongly resistant to photobleaching, ensures repeated/real-time observations of the probe-labeled cells by fluorescence microscopy; (b) the enlarged size of WGA-QD probe makes it possible for labeled WGA to be distinguished from other membrane proteins by AFM. Here, the random distribution of WGA-binding sites on non-crosslinked cells and the uneven or polarized reorganization due to WGA-induced crosslinking on cell surfaces were studied using AFM-detectable WGA-QD probe. Moreover, we developed a method to rapidly detect the WGA-induced rigidity alternation of the whole cells, which is efficient and has the potentiality of being developed to a useful tool in clinical diagnosis.

Histochemical mapping of glycoconjugates in the testis of the one humped camel ( Camelus dromedarius) during rutting and non-rutting seasons

In the present study, the distribution of various sugar residues in the testicular cells of sexually mature camels during rutting and non-rutting seasons was examined employing 10 fluorescein isothiocyanate- (FITC) conjugated lectins. Lectin labeling was restricted to the germ cell lines and interstitial Leydig cells, while the Sertoli cells remained completely unlabeled. Our results revealed the presence of mannose (labeled by lectins PSA, LCA), galactose (labeled by PNA), GalNAc (labeled by HPA), and GlcNAc (labeled by WGA) residues in the camel spermatogonia. However, spermatocytes were only labeled with mannose (PSA, LCA) and GlcNAc (WGA) binding lectins. Binding sites for PSA, LCA and WGA in spermatogonia and spermatocytes were only evident during the rutting season. Although spermatids were exclusively labeled with PNA in the non-rutting seasons, other lectins (PSA, GSA-I, WGA) additionally bound to camel spermatids during the rutting period. Leydig cells and basal lamina of the seminiferous tubules of camel testis were consistently labeled with the mannose- (PSA, LCA) and GlcNAc- (WGA) binding lectins in both seasons, while DBA-labeling was seen in the Leydig cells during rutting period only. In conclusion, the findings of the present study clearly indicate that the camel testis contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosyl residues), and they lack fucosyl residues, both in the active sexual period and in the non-breeding season. The topographical distribution of the sugar moieties in the camel testis may indicate that specific carbohydrate structures are required for spermatogenesis during periods of sexual activity.

Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

Mammalian spermatozoa must become ;capacitated' in the female reproductive tract before they gain the ability to fertilize the oocyte. The attainment of a capacitated state has been correlated with a number of biochemical changes, the most notable of which is a dramatic increase in the tyrosine phosphorylation status of these cells. Despite its biological importance, the mechanisms responsible for initiating this tyrosine phosphorylation cascade in vivo are unknown. Here, we report that this signalling pathway can be elicited in a rapid, dose-dependent and lectin-specific manner by wheat germ agglutinin (WGA), but none of 18 other lectins assessed. This response was abrogated by prior enzymatic cleavage of either sialic acid or GlcNAc residues from the sperm surface and by treatment with a range of pharmacological inhibitors directed against protein kinase A, protein tyrosine kinases and intermediates including Src. Proteomic analysis of the WGA-binding sites on the sperm surface identified the putative cognate receptor as platelet cell adhesion molecule 1 (PECAM-1/CD31). This conclusion was supported by the following evidence: (i) anti-PECAM-1 antibodies identified a molecule of the correct molecular mass in human spermatozoa, (ii) PECAM-1 could be isolated from a pool of sperm surface proteins using WGA immobilized on a solid phase support, (iii) PECAM-1 and WGA co-localized to the sperm surface and (iv) anti-PECAM-1 antibodies could completely block the ability of WGA to stimulate tyrosine phosphorylation in these cells. Collectively, these data provide the first evidence that a receptor-mediated signal transduction pathway triggers human sperm capacitation and identifies PECAM-1 as the probable initiator of this second messenger cascade.

Probing lectin binding sites on isolated viable generative and sperm cells of tobacco

Generative cells and sperm cells of tobacco were isolated and labeled by fluorescein isothiocyanate conjugated concanavalin agglutinin (FITC–Con A), wheat germ agglutinin (FITC–WGA) and soybean agglutinin (FITC–SBA) to compare lectin binding site (LBS) distributive patterns between generative cells and sperm cells, and between a pair of sperm cells from the same pollen tube. With the help of cooled charge couple device (CCD) and confocal microscope, Con A binding sites were found on the surface of 96% generative cells and 91% sperm cells. WGA binding sites were also detected on plasma membrane of generative cells and inner plasma membrane of vegetative cells and the crosswall of male germ unit (MGU), but it was hardly found on most of sperm cell surface. No soybean agglutinin (SBA) binding sites were detected on the plasma membrane of both generative cells and sperm cells. The experiment revealed that LBSs on the membrane surface of germ cell were reorganized or modified during the development from generative cell to sperm cell, indicating sperm formation might couple with its function specialization. The possible dimorphism on surface glycoprotein distribution of sperm cells was not confirmed in tobacco in this experiment since sperm cells derived from the same generative cell could be both labeled or not labeled at all by FITC–Con A. Based on the finding in present work and our former reports, we proposed that there might be functional difference of sperm in the population of living sperms of higher plants.

Lectin-binding epitopes at the surface of Escherichia coli K-12: examination by electron microscopy, with special reference to the presence of a colanic acid-like polymer.

The presence and distribution of lectin-binding epitopes at the surface of Escherichia coli K-12, strain W1655, was studied by electron microscopy after lectin-gold labeling and negative staining. A comparison was made between the lectin-binding capacity of cells cultivated at 20 degrees C and 37 degrees C (in broth or on agar). A variety of pre-treatment protocols were applied prior to labeling. The gold-conjugated lectins used were wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Ulex europaeus lectin (UEA-I). For all culture conditions, the bacteria had moderate exposure of WGA-binding sites, and this was not changed after pre-treatment. Cells cultivated at 37 degrees C had exposed SBA- and UEA-I-binding epitopes apparently associated with the cell surface. These significantly increased in number after boiling the cells for 10 min. With bacteria cultivated at 20 degrees C these two lectins recognized sites situated on exopolysaccharide filaments. Affino dot-blot experiments with isolated polysaccharides of the strain identified the K-12 lipooligosaccharide as the source of WGA-binding epitopes, and the exopolysaccharide, colanic acid (CA) as the source of SBA- and UEA-I-binding sites. The interaction with these two lectins of bacteria cultivated at 37 degrees C could be due to altered translocation of CA from the cytoplasm to the environment. This suggestion was supported by the demonstration by electron microscopy of SBA and UEA-I binding at the surface of hot phenol-water extracted cell walls.

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.

Changes in the ultrastructure and glycoproteins of the cyst wall of Colpoda cucullus during resting encystment

Summary Our previous studies on long-term resting encystment of Colpoda inflata showed transcriptional activity of the macronucleus and the occurrence of protein synthesis. In this work we have investigated the structure, the glycoprotein composition, and the lectin-binding site localization of the cyst wall of C. cucullus at increasing cyst ages, by means of scanning and transmission electron microscopy, confocal microscopy, and cytochemical and lectin blotting techniques. During an 18-day resting encystment, the cyst wall undergoes structural modifications resulting in the compaction of its previously-formed layers and the production of new material that is placed on the inner side of the cyst wall. Changes in the localization of ConA-/WGA-binding sites have been checked by comparing 7-day-old cysts to 1-day-old ones. Moreover, differences in the electrophoretic and ConA-/WGA-positive patterns have been found in 18-day-old cysts when compared to 1-day-old ones. The results of in vivo ConA and WGA assays suggest that ConA promotes encystment and delays excystment.

IDENTIFICATION OF MATURATION-RELATED WHEAT-GERM LECTIN-BINDING PROTEINS IN THE CULTURE OF HUMAN CORPUS EPIDIDYMAL EPITHELIAL CELLS

This study is designed to investigate the synthesis of maturation-related wheat germ agglutinin (WGA) binding glycoproteins in the human corpus epididymal epithelial cells by in vitro culture. Epithelial cells were isolated from the corpus of human epididymides and cultured with RPMI 1640 medium supplemented with 10% fetal calf serum in type IV collagen-coated dishes at 37 degrees C. The epithelial nature, presence of fibroblasts, WGA-binding sites, and existence of GP-83 were determined by an indirect immunocytochemical and histochemical staining technique. Proteins in the cultured cells were analyzed by SDS-PAGE and autoradiography. After culturing for 10 days, the cells were shown to be positive with epithelial cell-specific keratins but devoid of fibroblasts. WGA-binding granules and positive binding sites of GP-83 were also detected in the cytoplasm. Immunoblots of cell extracts probed with the anti-GP-83 antibody from seminal fluid revealed the sperm maturation-related glycoprotein GP-83. The results indicate that WGA-binding proteins may be synthesized by the corpus epididymal epithelial cells of human and GP-83 may play an important role in sperm maturation. This culture model may be suitable for the investigation on the biosynthesis and physiology of human epididymal principal cells in vitro.

A Cytoplasmic Factor Required for Contraction of the Cleavage Furrow inafMutant Eggs of Xenopus laevis

The abnormal furrow (af) gene was identified through a maternal effect mutation that causes a defect in cell division in Xenopus embryos. In eggs obtained from mutant females (af eggs), polar body formation and cytokinesis are completely inhibited while nuclear division continues uninterrupted. Cleavage furrows are recognized as unpigmented narrow bands on the egg surface but they do not contract. Transfer of cytoplasm from wild-type eggs into af eggs partially rescues contraction of the cleavage furrow. The factor responsible for contraction promoting activity was present in wild-type eggs throughout the first cell division cycle and induced contraction in a dose dependent manner. The factor was characterized as a high molecu- lar weight protein complex that was associated with particulate fraction in the extract. The recovery of con- traction was associated with accumulation of filamentous actin and WGA-binding sites within the cleavage furrow of the af eggs which were normally not enriched for these components. Formation and contraction of a filamentous actin ring during cortical wound healing in af eggs was indistinguishable from that of wild-type eggs. From these results, the af gene product may be specifically required for reorganization of the actin filaments and WGA-binding sites in the cleavage furrow and contraction through these structures during cytokinesis.

Ultrastructural localization of lectin binding sites in the developing brain microvasculature.

The temporo-spatial patterning of lectin-binding sites was examined by lectin histochemistry and quantitative methods in the microvasculature of the optic tectum of 9-, 14-, 20-day-old embryos and 30-day-old chickens. Horseradish peroxidase and colloidal-gold-labelled lectins were used for detection of beta-D-galactose (RCA-I, Ricinus communis agglutinin-I) and of N-acetylglucosamine and sialic residues (WGA, Wheat germ agglutinin) at light and electron microscopical levels. At the light microscopical level, RCA-I and WGA binding sites were detectable in the early embryonic capillaries in a diffuse staining pattern; in later embryonic stages and in adult animals, RCA-I labelling became located on the abluminal surface of the vessels, while WGA staining was detected on the luminal surface. Ultrastructurally, gold labelling for RCA-I was seen intracytoplasmically in endothelial cells in 9-day-old embryos. In 14-to 20-day-old embryos and in chickens, binding sites for RCA-I were detected in endothelial tight junctions and basement membranes. In contrast, labelling of the gold-coupled WGA lectin was distributed almost exclusively on the luminal endothelial surface already in early embryos. The results indicate that the endothelial cells of the optic tectum acquire functional polarity early in their development and that glycoconjugates containing beta-D-galactose residues are involved in the biochemical composition of the tight junctions and basement membrane, which are considered to be key structures in blood-brain barrier (BBB) differentiation.

The wheat germ agglutinin binding sites and development of the mitochondria-rich cells in gills of tilapia ( Oreochromis mossambicus)

By labelling with gold-colloid-conjugated wheat germ agglutinin (WGA), the WGA-binding sites were identified in the apical region of certain mitochondria-rich (MR) cells in gills of freshwater-adapted tilapia (Oreochromis mossambicus). Bromo-deoxyuridine (BrdU) injection was used to discern the differentiation of the WGA binding sites during development of MR cells. Double labeling of gill sections of fish 2, 4, 6, and 8 days after a single BrdU injection showed that the BrdU labeled MR cells 2 and 4 days after injection were mostly WGA negative, whereas 6 and 8 days after injection, WGA and BrdU-labeled MR cell increased gradually in number. Furthermore, the ratio of the WGA-positive to total MR-cells was higher in gills of tilapia adapted to low calcium ([Ca] = 0.015 mm) freshwater than in hard freshwater ([Ca] = 1.898 mm). The results indicate that WGA binding site may be a marker expressed late during differentiation of MR cells, and physiologically functional in assisting uptake of calcium when environmental calcium is low. According to their WGA binding and also characteristics their likely function in calcium absorption, the WGA positive MR cells shown in this study are considered to be similar to the β cells described by Pisam et al. (1995).

Glycoconjugate expression during early mouse oculogenesis.

The carbohydrate side-chain of glycoconjugates can show a developmentally regulated expression pattern. In order to analyse these changes during the development of the eye, 13 lectins were used to reveal glycoconjugates histochemically in 8.5- to 14-day-old mouse embryos. During this period, eyes develop from the most immature vesiculation of the neural plate neuroepithelium into a primitive stage with all structures present, such as pigment epithelium, not yet differentiated neuroretina and lens. A striking diversity of carbohydrate side-chain expression was observed in the preocular somatoectoderm and neural plate of 8.5-day-old embryos, as indicated by the binding of nine different lectins. Binding sites at the apical poles of neuroepithelium of five of these lectins (PNA, LCA, SBA, LPA and GSA-II) disappeared completely during further development. The binding sites of four other lectins, WGA, MPA, Con A and BPA, remained expressed during the course of development, being indicative for the carbohydrate side-chains beta-GlcNAc(1-4)Gluc, alpha-Gal(1-3)GalNAc, alpha-D-Man/alpha-D-Gluc and alpha-GalNAc. In contrast, binding sites for GSA-I, RCA-I (alpha-D-Gal), UEA-I (alpha-L-Fuc) and DBA (alpha-GalNAc(1-3)GalNAc) were not present at any developmental stage. The time point of gross changes of lectin binding sites correlates well with the period of neural tube formation. During later development from neuroectoderm to the ocular pigment epithelium, a sharp reduction in all lectin binding sites at the apical cell poles, except for WGA and MPA, was observed. WGA binding sites were present until embryonic day 10, while those for MPA were present until day 9. At the basal cell poles of the pigment epithelium, all lectin binding sites except for WGA were lost after embryonic day 11.5. These results indicate that there are sophisticated kinetics of glycoconjugate expression during the course of early embryonic development of ectoderm into its descendent tissues.

Lectin-mediated bioadhesion: Proteolytic stability and binding-characteristics of wheat germ agglutinin and Solanum tuberosum lectin on Caco-2, HT-29 and human colonocytes

For the development of lectin-mediated drug delivery systems, the proteolytic stability of the non-toxic lectins from Arachis hypogea, Lens culinaris, Dolichus biflorus, Solanum tuberosum (STL) and Triticum vulgare was investigated by in vitro exposure to gastrointestine-located enzymes. No degradation products were observed within 24 h of incubation on sodium dodecyl sulfate polyacrylamide gels. Binding to human colon carcinoma cell lines was investigated by flow cytometry. The fluorescein-labelled derivatives of N-acetylglucosamine-specific wheat germ agglutinin (WGA) and STL exhibited the highest cell-associated fluorescence intensity. As determined by dilution experiments, the number of WGA-binding sites on Caco-2, HT-29 and human colonocytes exceeded those for STL by 5-, 1.7- and 1.4-fold, respectively. By a competitive flow cytometric assay using N,N′, N″-triacetylchitotriose for inhibition, WGA-affinity exceeded STL-affinity by ten-fold. The affinity of each lectin to Caco-2, HT-29 and human colonocytes was about the same, indicating that similar lectin receptors were involved. Preventing N-glycosylation of the carcinoma cells by pretreatment with 0.001% tunicamycin for 40 h resulted in 30% inhibition of WGA- and STL-binding. When WGA was covalently attached to Sepharose beads (250–350 μm), the interaction with HT-29 and Caco-2 cells showed stable and tight binding. Therefore, especially considering the comparable affinity of human colonocytes and monolayer-forming Caco-2 and HT-29 cells, this system is proposed as a model for the development of lectin-mediated particulate pharmaceutical devices.

Ultrastructural localization of binding sites for the lectins RCA I, WGA, and LTA in the preimplantation mouse embryo.

In biological tissues, specific carbohydrate moieties of the oligosaccharide chains of glycoproteins can be localized by lectin binding. Such carbohydrate moieties are among the factors that mediate cell-cell or cell-matrix interactions during pre- and postimplantation embryonic development. Binding sites for the lectins RCA I, WGA, and LTA were localized in preimplantation mouse embryos at the ultrastructural level with the help of postembedding lectin gold cytochemistry. WGA and RCA I binding sites, but no LTA binding sites, were present in the zona pellucida. WGA and RCA I binding sites were found at cell surfaces of morulae and in cells of the inner cell mass of blastocysts, suggesting that N-acetylglucosamine-, terminal beta-galactosyl-, and N-acetylgalactosamine-rich glycoproteins might be involved in cell-cell and cell-matrix interactions. WGA binding sites were found predominantly in electron lucid vesicles of the blastomeres, whereas RCA I was detected in electron dense vesicles of the compacted morula and later in the polar trophoblast cells. This allows early identification of blastomere cells that later differentiate into the polar trophoblast.

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization.

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction).