Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins

Capacitated acrosome-intact mouse spermatozoa bind to the egg's extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor-ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars (N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94-101]. To further elucidate the specificity of sperm-ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm-bead assay was carried out at 37 degrees C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm-bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor(s) that recognize and bind to the sugar residues.     

FGF binding by extracellular matrix components of Wharton's jelly

Our earlier paper has reported that Wharton's jelly is a reservoir of several peptide growth factors, including acidic and basic fibroblast growth factors (aFGF and bFGF, respectively). Both can be extracted by buffered salts solutions in the form of high molecular mass complexes, probably with a component(s) of the extracellular matrix. Both aFGF and bFGF from such extracts hardly penetrate 10% polyacrylamide gels during electrophoresis. Pre-treatment of Wharton's jelly with hyaluronidase slightly increased the extractability of aFGF, but did not affect the extractability of bFGF. In contrast, the pre-treatment of tissue homogenate with bacterial collagenase (2000 U/ml, 37 degrees C, 18 h) increased the extractability of bFGF. The presence of beta-mercaptoethanol in the extracting solutions increased the extractability of both FGFs, but did not release FGFs in their free form, despite reducing the molecular mass of the FGF-containing complexes. We conclude that both aFGF and bFGF are bound through disulphide bonds to a protein component of Wharton's jelly. We propose that ground substance composed mainly of collagen fibrils and hyaluronate molecules, which surrounds the cells of Wharton's jelly, prevents the access of the extracting solution to aFGF and bFGF. Although hyaluronate and collagen do not bind aFGF or bFGF directly, they may constitute a barrier which prevents the dispersion of FGFs in Wharton's jelly. Thus, the high concentration of FGFs around the cells of Wharton's jelly may facilitate the interaction of these factors with membrane receptors, thereby resulting in stimulation of cell division and differentiation, as well as of the synthesis of extracellular matrix components.     

In vitro adhesion of mouse fetal germ cells to extracellular matrix components

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5–12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitinsulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5–14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.     

Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches

Summary The distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyer's patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyer's patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyer's patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.     

Production and utilization of extracellular matrix components by human melanocytes

Normal human melanocytes were separated from keratinocytes and maintained in culture using KGM medium supplemented with 12-O-tetradecanoylphorbol acetate and cholera toxin. The melanocytes were examined for the production of extracellular matrix molecules including fibronectin, laminin, and thrombospondin and for the utilization of these molecules in adhesion and motility assays. Melanocytes produced significant amounts of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). Fibronectin was expressed on the surface of these cells. Laminin was also produced by melanocytes and expressed on the cell surface. The amount of laminin produced was significantly less than the amount of fibronectin. In contrast, melanocytes did not produce measurable thrombospondin as indicated by biosynthetic labeling/immunoprecipitation. Only traces of thrombospondin were detected by ELISA and no surface fluorescence was observed. When examined in adhesion and motility assays, melanocytes were found to utilize fibronectin for both processes. Laminin also stimulated adhesion but it was much less effective than fibronectin. Thrombospondin did not stimulate either attachment and spreading or motility. The pattern of extracellular matrix molecule production and utilization by melanocytes is significantly different from that shown previously for human epidermal keratinocytes (J. Varani et al., 1988, J. Clin. Invest. 81, 1537). These differences may underlie the differences with which the two cell types interact with basement membranes in vivo.     

Interaction of Campylobacter jejuni with extracellular matrix components

The adhesion of three strains of Campylobacter jejuni to coverslips and microwells coated with isolated extracellular matrix components, fibronectin, laminin and types I, III, IV and V collagens was studied. Fibronectin mediated the adherence of C. jejuni, but there were differences in the binding capacities of the strains. Type I, III and V collagens mediated very strongly the attachment of two strains of C. jejuni. All three strains attached weakly to basement membrane-specific type IV collagen. Laminin was capable of mediating the adhesion only when present at a higher concentration. The observations indicate that extracellular matrix components may serve as anchor molecules for C. jejuni adhesion and that several attachment mechanisms occur simultaneously.     

Mechanism of human papillomavirus binding to human spermatozoa and fertilizing ability of infected spermatozoa

Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases in females and males. Precise data about the presence, mechanism of infection and clinical significance of HPV in the male reproductive tract and especially in sperm are not available. Here we show that HPV can infect human sperm, it localizes at the equatorial region of sperm head through interaction between the HPV capsid protein L1 and syndecan-1. Sperm transfected with HPV E6/E7 genes and sperm exposed to HPV L1 capsid protein are capable to penetrate the oocyte and transfer the virus into oocytes, in which viral genes are then activated and transcribed. These data show that sperm might function as vectors for HPV transfer into the oocytes, and open new perspectives on the role of HPV infection in males and are particularly intriguing in relation to assisted reproduction techniques.     

Bacterial proteins binding to the mammalian extracellular matrix

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best-characterized bacterial proteins active in these interactions are the mycobacterial fibronectin-binding proteins, the fibronectin- and the collagen-binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A-protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein-protein or on a protein-carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

Immunohistochemical localization of extracellular matrix components in human breast tumours with special reference to PG- M/versican

Immunohistochemical localization of the large proteoglycan, PG-M/versican, was studied in 36 breast tumours, including infiltrating ductal carcinomas, benign tumours and fibrocystic diseases. The relation between the proteoglycan and the other extracellular matrix components was also investigated. In the carcinoma tissues, the interstitial elements of the ‘specific stroma’, consisting of fibroblastic cells and fine fibrils, were reactive to antibody 2B1, which specifically recognizes the large proteoglycan, PG-M/versican. In the peripheral invasive areas of infiltrating ductal carcinoma, the most intense 2B1-positive reaction was visualized in mesenchymal tissues between carcinoma cell clumps and the surrounding tissues, where hyaluronic acid could be demonstrated histochemically. The 2B1-positive elements were not reactive to antibody 6B6, which specifically recognizes small proteoglycan. In the central sclerotic areas, where antibody 6B6 was reactive, a 2B1-positive reaction was detected only in elastosis masses, which also bound antibodies to type IV collagen and laminin, and to some extent antibody raised against chondroitin 6-sulphate proteoglycan. Elastic tissues of blood vessel walls and perivascular elements became reactive to antibody 2B1 when they were involved in carcinoma invasion. The present results have shown that PG-M/versican was localized in the proliferating interstitial tissues, in particular in hyaluronic acid-rich portions, in association with carcinoma cell growth, and also that PG-M/versican accumulated in vascular and perivascular elastic tissues involved in carcinoma invasion. The biological significance of PG-M/versican was briefly discussed     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

Quantitative characterization of binding of small molecules to extracellular matrix

Extracellular matrix (ECM) is a major tissue component that, besides its cell support function, is implicated in cell-cell signaling, wound repair, cell adhesion, and other cell and tissue functions. For small molecules acting in tissues, including chemicals, signaling peptides, effectors, inhibitors, and other man-made and physiological compounds, non-specific binding to ECM is a critical phenomenon affecting their disposition. We describe here a method for a quantitative characterization of the ECM binding, using a solidified ECM layer incubated with medium containing studied small molecules. Working conditions of Matrigel, a commercial basement membrane preparation, were optimized in terms of the protein concentration, surface area, gel layer thickness, solidification time, and mixing speed. The release of proteins from the solidified layer into the buffer was monitored and taken into account. Two major proteins, laminin and collagen IV, dissolve at different rates. The Matrigel stability data, obtained under varying incubation conditions and gentle mixing, can also be useful in other ECM-related research. The experimental binding data, averaged over all binding sites, were analyzed assuming a fast linear binding. The binding constants were determined for 10 small organic molecules for both dissolved proteins and the solidified layer. The binding constants tend to increase with lipophilicity of the compounds, as characterized by the 1-octanol/water partition coefficients.     

Epithelial shape change in mouse embryonic submandibular gland: modulation by extracellular matrix components

Early morphogenesis of mouse submandibular gland provides an excellent model for the formation of epithelial lobules as a consequence of epithelial-mesenchymal interactions. Both proteoglycans and a glycosaminoglycan, high molecular weight components which contain amino-sugars and hexuronic acids, seem to be important in maintaining the lobular structure through the formation of epithelial basal lamina. Collagen also appears to play a crucial role in this morphogenesis. By visualizing the distribution of collagen fibrils and by changing the concentration of collagen in the gland, we have developed a new hypothesis which emphasizes the mechanical role of mesenchyme in epithelial cleft formation. Precise mechanisms for the involvement of these molecules have not been elucidated, yet it is now clear that knowledge of the function of the extracellular matrix components is a prerequisite for understanding the epithelial-mesenchymal interactions.     

Expression of extracellular matrix components fibronectin and laminin in the human fetal heart.

It has been well documented that the extracellular matrix components fibronectin and laminin promote or regulate morphogenesis of the myocardial cells in mammalian heart. However, their chronological change of expression (or localization) in the human heart remains elusive. In this study, fibronectin and laminin in the left ventricle of forty-two human fetuses aged from 8 to 26 weeks gestation and left ventricular tissues obtained from a 2-week old infant and two adults were investigated by Western blot analyses and indirect immunofluorescence technique with monoclonal antibodies. In the fetal heart, fibronectins were present along the endocardium, epicardium, and linings of larger blood vessels. In 14-16 weeks gestation, fibronectin immunofluorescence became stronger but not evenly dispersed in the interstitium. After 24 weeks gestation, they were strongly positive only in the relatively larger blood vessels, as well as those in the infant and adult cardiac tissues. Laminins were strongly positive along the endocardium and basement membrane of the myocardial cells and fibroblasts during fetal life. After birth, laminins formed fine fibrillar network along the basement membrane in association with the transverse tubules of myocardial cell; these morphological characteristics remained in the adult cardiac tissues. These results indicate that fibronectin expression is relatively constant during fetal life but decreases after birth; in contrast, laminin expression is not age-dependent and constant throughout the life.     

Adherence of Candida albicans to components of the subendothelial extracellular matrix

Candida albicans yeasts adhered avidly to extracellular matrix (ECM) proteins, type IV collagen, laminin, and fibronectin immobilized on plastic. Type IV collagen showed an increase of adherence of 400% above control values; laminin, 300%; and fibronectin, 150%. In addition, all three (in quantities of 0.02-200 micrograms/well of a culture tray) bound yeasts in a dose-response fashion. Adherence was inhibited when the proteins were preincubated with specific antibody, except with type IV collagen. Soluble laminin or fibronectin inhibited yeast adherence to the same proteins by 36 and 94%, respectively. Soluble fibronectin bound to the yeast surface and in so doing inhibited subsequent yeast adherence to fibronectin by 66%. By comparison, Candida albicans yeasts adhered in smaller numbers to glycosaminoglycans (GAGs). Keratan sulfate, hyaluronic acid, chondroitin sulfate, Type B, and heparin actually decreased yeast adherence compared to control from 10% to 25%.     

Selective proteolysis by matrix metalloproteinases of photo-oxidised dermal extracellular matrix proteins

Photodamage in chronically sun-exposed skin manifests clinically as deep wrinkles and histologically as extensive remodelling of the dermal extracellular matrix (ECM) and in particular, the elastic fibre system. We have shown previously that loss of fibrillin microfibrils, a key elastic fibre component, is a hallmark of early photodamage and that these ECM assemblies are susceptible in vitro to physiologically attainable doses of ultraviolet radiation (UVR). Here, we test the hypotheses that UVR-mediated photo-oxidation is the primary driver of fibrillin microfibril and fibronectin degradation and that prior UVR exposure will enhance the subsequent proteolytic activity of UVR-upregulated matrix metalloproteinases (MMPs).We confirmed that UVB (280-315 nm) irradiation in vitro induced structural changes to both fibrillin microfibrils and fibronectin and these changes were largely reactive oxygen species (ROS)-driven, with increased ROS lifetime (D₂O) enhancing protein damage and depleted O₂ conditions abrogating it. Furthermore, we show that although exposure to UVR alone increased microfibril structural heterogeneity, exposure to purified MMPs (1, −3, −7 and − 9) alone had minimal effect on microfibril bead-to-bead periodicity; however, microfibril suspensions exposed to UVR and then MMPs were more structurally homogenous. In contrast, the susceptibly of fibronectin to proteases was unaffected by prior UVR exposure. These observations suggest that both direct photon absorption and indirect production of ROS are important mediators of ECM remodelling in photodamage. We also show that fibrillin microfibrils are relatively resistant to proteolysis by MMPs −1, −3, −7 and − 9 but that these MMPs may selectively remove damaged microfibril assemblies. These latter observations have implications for predicting the mechanisms of tissue remodelling and targeted repair.     

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca²⁺ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca²⁺ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca²⁺, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca²⁺-dependent binding reaction was rapid, reversed by EGTA, specific for Ca²⁺, and did not require the transport of Ca²⁺ into the cell. Sperm bound to the zona surface following preincubation with Ca²⁺ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.     

Use of extracellular matrix components for cell culture

Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior. The response observed is dependent on the type of cell and matrix used. Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo. More differentiated phenotypes are observed and cells generally survive longer on such matrices. In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors. As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the alpha(2), alpha(5) and beta(1) integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the beta(1) integrin subunit. Antibodies to alpha(3) integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with collagen. It seems that alpha(3) subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy.