A Yolk-granule Component Acts as an Adhesive Material for Dissociated Gastrula Cells of the Newt, Cynops pyrrhogaster

Yolk granules were collected from full-grown ovarian oocytes of the newt, Cynops pyrrhogaster , and dissolved in 3% acetic acid or 8 M urea solution. Culture dishes were then coated with either of the yolk-granule solutions. The yolk-coated surfaces acted as adhesive substrata for dissociated gastrula cells, which showed active locomotion when spread on the surfaces. Divalent cation was required for cell adhesion and spreading on the yolk-coated surface. Trypsin and glycosidase digestions of dissociated cells or the yolk-coated surfaces inhibited cell adhesion and spreading. Addition of concanavalin A to the culture medium inhibited cell spreading on the yolk-coated surfaces, while high concentration (50 mM) of the saccharides, D-galactose, D-lactose and D-mannose, had a slightly inhibitory effect on cell spreading.
Two yolk components (30-kD and 108-kD proteins) were isolated from yolk granules and applied to the culture dish surfaces. Surfaces coated with the 30-kD protein showed strong adhesiveness for dissociated gastrula cells.     

Cell adhesion to extracellular matrix is different in marine hydrozoans compared with vertebrates

Extracellular matrices (ECMs) of phylogenetically very distant organisms were tested for their ability to support cell adhesion, spreading and DNA replication in reciprocal xenograft adhesion tests. Mechanically dissociated cells of the medusa Podocoryne carnea (Cnidaria, Hydrozoa) were seeded on ECMs of polyps and medusa, and on several ECM glycoproteins or entire ECMs from vertebrates. In reciprocal experiments, cells from different vertebrate cell-lines were seeded on ECMs of polyps, medusae and also on electrophoresed and blotted extracts of both types of ECMs. The results demonstrate that medusa cells adhere and spread on polyp and medusa ECMs but do not recognize vertebrate ECMs or purified ECM glycoproteins. Vertebrate cells in contrast adhere, spread and proliferate on ECMs of polyps and medusae. The number of attached cells depends on the cell type, the type of ECM and, in certain cases, on the stage of the cell cycle. Cell adhesion experiments with pretreated ECMs of polyps and medusae, e.g. oxidation of carbohydrate residues with sodium-metaperiodate, or blocking of certain carbohydrate moieties with the lectin wheat germ agglutinin or a carbohydrate-specific monoclonal antibody, demonstrate that ECM carbohydrates are more important for cell-ECM interactions of medusa cells than for vertebrate cells. Furthermore, the experiments indicate that polyp and medusa ECMs contain different components which strongly modulate adhesion, spreading and DNA replication of vertebrate cells.     

Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.     

Integrin-dependent cell behavior on ECM peptide-conjugated chitosan membranes

Extracellular matrix (ECM) plays an important role in tissue regeneration by promoting cell adhesion, migration, proliferation, and differentiation. ECM mimetics are of importance for tissue engineering because of their functions as scaffolds for cells. Previously, we developed bioactive laminin-derived peptide-conjugated chitosan membranes and demonstrated their cell- and peptide-type specific functions. Here, we conjugated twelve integrin-binding peptides derived from ECM proteins onto chitosan membranes and examined biological activity. Seven peptide-chitosan membranes promoted human foreskin fibroblast attachment. Additionally, FIB1 (YAVTGRGDSPAS; from fibronectin), A99 (AGTFALRGDNPQG; from laminin alpha1 chain), EF1zz (ATLQLQEGRLHFXFDLGKGR, X = Nle; from laminin alpha1 chain), and 531 (GEFYFDLRLKGDKY; from collagen alpha1 (IV) chain) conjugated chitosan membranes promoted integrin-dependent cell adhesion. Various integrins, including alphav, beta1, and beta3, were involved in the cell adhesion to the peptide-chitosan membranes. Further, only the FIB1- and A99-chitosan membranes promoted neurite outgrowth with PC12 rat pheochromocytoma cells. These data demonstrate that peptide-chitosan membranes can regulate specific integrin-mediated cell responses and are useful constructs as ECM mimetics.     

Locomotion and adhesion of amphibian gastrula and neurula cells cultured on substrata of varied surface charge

This paper reports experiments to determine the effects of electrostatic repulsion on cell adhesion and locomotion, and to demonstrate quantitative differences in adhesiveness among cell populations of the amphibian gastrula, where it may be a factor controlling morphogenesis. Rana pipiens gastrula presumptive germ layer cells and neural crest were cultured on substrata of known surface charge and composition; cell-substratum adhesion assayed, and locomotory rate measured. Because electrokinetic mobilities of R. pipiens gastrula cells are known and that of neural crest has been measured here, various cell-substratum combinations have been ranked in order of the electrostatic force between them. Experimental methods include time-lapse cinematography and single-cell electrophoresis. The results indicate differential cell-substratum adhesion for R. pipiens inner neural ectoderm (INE), chordamesoderm (CHM) and neural crest (NC). In addition, it was found that increase in electrostatic repulsion decreases cell-substratum adhesion, and at first increases rate of locomotion, but then, as repulsion continues to increase, locomotor rate decreases. Increase in the surface potential of presumptive pharyngeal endoderm and chordamesoderm may bring about a decrease in cohesiveness thought to be necessary for the beginning of amphibian gastrulation.     

The effect of substrate and adsorbed proteins on adhesion, growth and shape of CaCo-2 cells

Extracellular matrix (ECM) proteins play a critical role in many cellular functions, from spreading, migration and proliferation to apoptosis. This role can be altered when proteins of the native ECM are adsorbed to different substrates which cause structural modifications that can influence their biological function. The effects on CaCo-2 cells of laminin-1, fibronectin, collagen-1 and ECM gel adsorbed to glass and to tissue culture polystyrene (PS) were compared in terms of adhesion, proliferation, shapes and spreading of cells in culture. Significant differences between glass and PS surfaces were observed for proliferation and cell shape. Protein surfaces prepared on PS substrates had, in most cases, more pronounced effects on cells than uncoated PS, especially if coated by collagen-1. Adsorbed ECM gel was the most adhesive for cells, but its effect on cell proliferation was not notably different from the controls (glass or PS). These findings indicate that the choice of the substrate can have a significant effect on experimental results and should be taken into consideration when comparing results obtained on different surfaces.     

A Shared Mechanism of Adhesion Modulation for Tenascin-C and Fibulin-1

Adhesion modulatory proteins are important effectors of cell–matrix interactions during tissue remodeling and regeneration. They comprise a diverse group of matricellular proteins that confer antiadhesive properties to the extracellular matrix (ECM). We compared the inhibitory effects of two adhesion modulatory proteins, fibulin-1 and tenascin-C, both of which bind to the C-terminal heparin-binding (HepII) domain of fibronectin (FN) but are structurally distinct. Here, we report that, like tenascin-C, fibulin-1 inhibits fibroblast spreading and cell-mediated contraction of a fibrin–FN matrix. These proteins act by modulation of focal adhesion kinase and extracellular signal-regulated kinase signaling. The inhibitory effects were bypassed by lysophosphatidic acid, an activator of RhoA GTPase. Fibroblast response to fibulin-1, similar to tenascin-C, was dependent on expression of the heparan sulfate proteoglycan syndecan-4, which also binds to the HepII domain. Therefore, blockade of HepII-mediated signaling by competitive binding of fibulin-1 or tenascin-C represents a shared mechanism of adhesion modulation among disparate modulatory proteins.     

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.     

Extracellular Matrix Proteins in Hemostasis and Thrombosis

The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets.The balance between hemostasis and thrombosis relies on a finely tuned adhesive response of blood platelets. Inadequate adhesion leads to bleeding, whereas excessive or inappropriate adhesion leads to thrombosis. Resting platelets are nonadhesive anuclear discs and do not interact with the vessel wall, but they have a plethora of receptors that sense activating signals (agonists) of various sorts. The activating signals include soluble factors such as thrombin, adenosine diphosphate (ADP), and epinephrine, all of which act on G-protein-coupled receptors (GPCRs) on the platelets. In addition, certain receptors for extracellular matrix (ECM) proteins (e.g., GPIb, GPVI, and some integrins) can also act as activating receptors. These diverse receptors trigger intracellular signaling pathways that activate (1) actin assembly leading to cell shape change and extension of filopodia; (2) exocytosis of secretory granules that release additional platelet agonists as well as adhesive ECM proteins; and (3) activation of additional cell-surface receptors such as the major platelet-specific integrin, αIIbβ3, that contribute further to the adhesion and aggregation of activated platelets. Thus, the interactions of platelet-ECM adhesion receptors with ECM proteins from the vessel wall, from the plasma, and from the platelets themselves, are central to both the initial adhesion and the subsequent activation and aggregation of platelets (Varga-Szabo et al. 2008). These adhesive interactions, together with coagulation (to which platelets also contribute), generate the fibrin clot, essentially a facultative ECM that forms the initial occlusion of the damaged vessel but also serves as a subsequent ECM substrate for wound healing. In this article, we will review what is known about the roles of ECM proteins and their receptors in platelet adhesion and aggregation, summarize the roles of the clot and provisional ECM in subsequent wound healing, point out various unanswered questions, and discuss briefly the contributions of the relevant cell–ECM interactions to disease and the potential for therapeutic interventions.     

Dermatopontin promotes adhesion,spreading and migration of cardiac fibroblasts in vitro

Dermatopontin (DPT), an extracellular matrix (ECM) protein, has been previously shown to be upregulated in the infarct zone of experimentally induced myocardial infarction (MI) rats. However, the accurate role that DPT exerts in the ventricular remodeling process after MI remains poorly understood. In this study, we evaluated the expression pattern of DPT mRNA and protein as well as its secretion in cultured neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) under conditions of hypoxia and serum deprivation (hypoxia/SD). Further, we tested the possible roles of DPT in CFs adhesion, spreading, migration and proliferation, which greatly promote the ventricular remodeling process after MI. Results showed that hypoxia/SD stimulated DPT expression and secretion in CMs and CFs and that DPT promoted adhesion, spreading and migration of CFs whereas had no effect on CFs proliferation. In addition, functional blocking antibodies specific for integrin α3 and β1 significantly reduced CFs adhesion and migration that DPT induced, suggesting that integrin α3β1 is at least one receptor for CFs adhesion and migration to DPT. These results implicated that DPT participates in the ventricular remodeling process after MI and may act as a potential therapeutic target for ventricular remodeling.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

Isolation of plasma membranes from Xenopus embryos

Summary Plasma membranes were isolated in high yield from Xenopus gastrulae by repeated sedimentation in discontinuous sucrose gradients. Most of the yolk was separated by lowspeed sedimentation before centrifugation on the discontinuous sucrose gradients. The isolation of plasma membranes was followed by covalent labelling of the surface of dissociated gastrula cells with diazoniobenzene sulphonate, by electron microscopy and the distribution of enzymatic markers. The isolated plasma membranes have a low neural inducing activity as compared to other cell constituents.     

The Effect of Extracellular Matrix Molecules on the in Vitro Behavior of Bovine Endothelial Cells

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.     

Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF): I. Effects on short-term adhesiveness and on long-term organization of the culture

A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly. In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM). BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition. The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic. The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins. However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM. ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer. BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo. These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo.     

Towards Gene Banking Amphibian Maternal Germ Lines: Short-Term Incubation,Cryoprotectant Tolerance and Cryopreservation of Embryonic Cells of the Frog,Limnodynastes peronii

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40–60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca²⁺-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both—indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.     

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.     

Behaviour of dissociated hypoblast cells on the basal lamina and on extracellular fibrils in the gastrulating chicken embryo.

The spreading behaviour of dissociated hypoblast cells on and besides a band of aligned fibrils associated with the basal lamina of the epiblast was investigated by the use of scanning electron microscopy. A horse-shaped band of aligned fibrils, first demonstrated by Wakely and England (1979), is present during the gastrulation stages of chicken embryos on the ventral side of the epiblast at the cranial and lateral borders of the area pellucida. The basal lamina of the area pellucida situated inside the fibrillar band enables the spreading and probably the locomotion of dissociated cells, which appeared as polarized cells. Numerous cells were also found on the fibrillar band, and these cells lacked distinct lamellae and a polarized shape. Extensions of the cells contacted the extracellular fibrils and, at these sites of contact, the pattern of the fibrils was frequently deformed. From these observations and from previous results emerged the concept that spreading and locomotion of dissociated hypoblast cells, as well as single mesoblast cells and healing hypoblast epithelium, are inhibited by the band of extracellular fibrils, which acts as a physical barrier. The cell biological basis of the mechanism by which extracellular fibrils associated with the basal lamina arrest the migration of hypoblast and mesoblast cells, but guide the migration of primordial germ cells, is discussed.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.