Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney

The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: implications for development

The interaction of transforming growth factor beta (TGF beta) with extracellular matrix macromolecules was examined by using radiolabeled TGF beta and various matrix macromolecules immobilized on nitrocellulose. TGF beta bound to collagen IV with greater affinity than to other extracellular matrix macromolecules tested. Neither laminin nor fibronectin, both of which bind type IV collagen, interfered with the binding of TGF beta to type IV collagen. TGF beta 2 competed effectively with TGF beta 1 for binding to type IV collagen. The biological effect of TGF beta was tested by an assay based on inhibition of proliferation of an osteoblast cell line, MC3T3-E1. The results demonstrated that the effect of TGF beta 1 was sustained when cells were grown on type IV collagen compared to cells grown on laminin, collagen type I, and plastic. These results demonstrate that extracellular matrix components may function as an affinity matrix for binding and immobilizing soluble growth and differentiation factors. In view of the demonstrated role of basement membranes in development the present results imply an important function for transforming growth factor beta bound to collagen IV in local regulation of cell proliferation and differentiation.     

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.     

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Type XVII collagen (BP180) can function as a cell−matrix adhesion molecule via binding to laminin 332

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knock-down of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix.     

Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix

This study examined the role of extracellular matrix in regulating matrix phenotype of hepatic lipocytes, the major source of matrix in liver. Lipocytes (Ito, stellate, or fat-storing cells) were purified from normal rat liver and established in primary culture on either uncoated plastic, plastic coated with individual matrix proteins, or a "complete" gel matrix, a basement membrane-like matrix derived from the Engelbreth-Holm-Swarm (EHS) murine tumor. The ultrastructure of lipocytes cultured on the gel matrix resembled that of cells in normal liver, whereas lipocytes on plastic had dispersed nuclear chromatin and expanded rough endoplasmic reticulum, consistent with active proliferation and secretion. Lipocytes on the gel matrix exhibited no proliferative activity; cells maintained on plastic proliferated and produced type I collagen predominantly. Total collagen secretion by lipocytes on the gel matrix was 29% of that of cells on plastic, and consisted of type III collagen only. This difference extended to proteoglycan production, which was less than 5% of the amount produced by cells in conventional culture on plastic. The effects of the EHS gel were not reproduced by the individual components of the gel (laminin, type IV collagen, and heparan sulfate proteoglycan) or by a type I collagen gel. They were also reversible upon transfer of the cells to conventional culture. In contrast to lipocytes, collagen synthesis by hepatocytes was similar whether cultured on EHS gel or on plastic. These results show that the extracellular matrix can modulate matrix protein production by lipocytes and imply that, in early hepatic inflammation, changes in the hepatic subendothelial matrix may underlie stimulation of lipocyte matrix production and progression of the fibrotic process.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE₂ stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc.     

Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells : Induction of ‘Assembly’ on fibrillar type I collagen substrata

Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable [3H]hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the all over rates of collagen synthesis and degradation. The proportion of the [3H]collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibres. Immunoprecipitation of the labelled extracts, electrophoresis, indirect immunofluorescence and immunoperoxidase techniques reveal the presence of type IV collagen, along with laminin and heparan sulfate proteoglycan in this layer, in excess over the amounts detectable on cells cultured on plastic. Transformed cells on collagen produce and accumulate more [3H]collagen, yet are less effective in basement membrane formation than normal cells, indicating that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface.     

Reconstituted basement-membrane matrix modulates fibroblast activities in vitro

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth-Holm-Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio of type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement-membrane matrix seems to influence the activities of fibroblasts significantly.     

Basal lamina formation by cultured microvascular endothelial cells

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

Immunomorphological research on the formation of the extracellular matrix in epithelial cell cultures

Formation of extracellular matrix structures in cultures of rat liver epithelial nontransformed cell line IAR2 was studied with antisera to fibronectin, laminin and type IV collagen by immunofluorescence and immunoelectron microscopy of platinum replicas. Fibronectin formed peripheral spots of variable size some of which outlined free cell edges, as well as fibrils located towards the center of single cells or of cellular islands. Similarly distributed structures were seen in isolated matrices. Codistribution of fibronectin and actin was observed only for the peripheral line of fibronectin spots and marginal circular actin bundle. Basement membrane components. laminin and type IV collagen, formed mainly spots of variable size predominantly beneath the cell or each cell in an island. Occasional fibrils were seen also. Essentially the same results were obtained by immunofluorescence and immunogold electron microscopy. Cytochalasin D treated cells displayed spots of both fibronectin and laminin. The relevance of previously postulated receptor-mediated assembly of extracellular matrix structures to the epithelial cells is discussed.     

Sertoli cell glycosylation patterns as affected by culture age and extracellular matrix

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.