Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Characterization of collagen precursors found in rat skin and rat bone.

Two genetic types of collagenous proteins, type I and type III, were isolated by extraction and differential salt precipitation from rat skin. The yield of collagen precursors was increased by injecting animals with colchicine 30 min before sacrifice to inhibit secretion of collagen. DEAE-cellulose chromatography was used to separate collagen from collagen precursors. Although these preparations contained more type I collagen than type III collagen, there were always more type III than type I precursors. The precursor chains of type I fractions were separated on CM-cellulose chromatography after denaturation. Three precursor forms were found for each collagen alpha chain, a complete chain (proalpha chain), and a precursor chain with only an amino-terminal (pNalpha chain) and carboxy-terminal extension (pCalpha chain). Species differences were demonstrated between rat collagen precursors and other species using rat calvaria (frontal and parietal) bones extracted with either 0.5 N acetic acid or neutral salt buffers containing protease inhibitors. Native rat procollagen elutes earlier than chicken or human procollagen on DEAE-cellulose chromatography and does not separate significantly from the pC collagen form. The collagenase resistant amino terminal peptides of rat pNalpha1 and pNalpha2 were the same size (16 000) but could be separated by DEAE-cellulose chromatography.     

Binding of soluble type I collagen to fibroblasts: specificities for native collagen types, triple helical structure, telopeptides, propeptides, and cyanogen bromide-derived peptides

Unlabeled collagenous proteins were quantified as inhibitors of binding of native, soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V a minor cartilage isotype (1 alpha 2 alpha 3 alpha), and the collagenlike tail of acetylcholinesterase did not inhibit binding. Collagen types II and III behaved as competitive inhibitors of type I binding. Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the separated constituent alpha chains. Inhibition of binding by unlabeled type I collagen was not changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in cyanogen bromide-derived peptides from various regions of helical alpha 1 (I) and alpha 1(III) chains. The aminoterminal propeptide of chick pro alpha 1(I) was inhibitory for binding, whereas the carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The data are discussed in terms of the proposal that binding to surface receptors initiates the assembly of periodic collagen fibrils in vivo.     

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.     

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.     

Identification of binding partners interacting with the α1-N-propeptide of type V collagen

The predominant form of type V collagen is the [α1(V)]?α2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the α1(V)- and α2(V)-collagen chain, cause classic EDS (Ehlers-Danlos syndrome), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remain unexplained. Type V collagen controls collagen fibrillogenesis through its conserved α1(V)-N-propeptide domain. To gain an insight into the role of this domain, a yeast two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the N-propeptide as a bait. We identified 12 interacting proteins, including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or co-immunoprecipitation: α1(I)- and α2(I)-collagen chains, α1(VI)-, α2(VI)- and α3(VI)-collagen chains, tenascin-C, fibronectin, PCPE-1 (procollagen C-proteinase enhancer-1), TIMP-1 (tissue inhibitor of metalloproteinases-1), MMP-2 (matrix metalloproteinase 2) and TGF-β1 (transforming growth factor β1). Solid-phase binding assays confirmed the involvement of the α1(V)-N-propeptide in the interaction between native type V collagen and type VI collagen, suggesting a bridging function of this protein complex in the cell-matrix environment. Enzymatic studies showed that processing of the α1(V)-N-propeptide by BMP-1 (bone morphogenetic protein 1)/procollagen C-proteinase is enhanced by PCPE-1. These interactions are likely to be involved in extracellular matrix homoeostasis and their disruption could explain the pathogenetic mechanism in unresolved classic EDS cases.     

Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.     

The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes.

Previous studies have indicated that the morphology and behaviour of bovine retinal microvessel endothelial cells are influenced by culture conditions in vitro. Data are presented here concerning the biosynthesis of matrix macromolecules by bovine retinal endothelial cells cultured under conditions in which the cells display either the 'cobblestone' or the 'sprouting' phenotype. Newly synthesized matrix proteins were identified by their characteristic electrophoretic mobilities, immunoprecipitation with specific antibodies, susceptibilities to enzymic digestions and chromatographic behaviour. Type IV procollagen was the major collagenous species synthesized by early-passage cells forming a 'cobblestone' monolayer. In contrast, cells displaying the 'sprouting' morphology switched to the predominant synthesis of interstitial fibrillar collagens (types I and III). Fibronectin was synthesized by retinal endothelial cells under all the experimental conditions studied. A non-collagenous glycoprotein of Mr approx. 47,000 was also a major biosynthetic product of these cells. The synthesis of thrombospondin was very much dependent on the nature of the substratum on which the cells were cultured. This glycoprotein was synthesized in large amounts by 'cobblestone' endothelial cells cultured on gelatin-coated dishes, whereas its synthesis was markedly decreased by culturing the cells on collagen gels, and the protein appeared to be absent when the cells were plated within collagen gels ('sprouting' cells). Late-passage retinal cells synthesized predominantly type I procollagen, variable amounts of type III procollagen and only traces of type IV procollagen, irrespective of whether the cells displayed a 'cobblestone' or 'sprouting' morphology.     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Procollagen and collagen produced by a teratocarcinoma-derived cell line,TSD4: Evidence for a new molecular form of collagen

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of θ chains (150,000 daltons), pro α chains (130,000 daltons), and α chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (α1)₃, as did collagen isolated from the cell layer. Each α1 chain appears to be slightly larger than α1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type α1(X)₃, although similar to that of the α1 chain of type I collagen, [α1(I)]₂α2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [α1(I)]₃.     

Lysyl hydroxylase 2 is a specific telopeptide hydroxylase, while all three isoenzymes hydroxylate collagenous sequences.

Lysyl hydroxylase (LH), with three isoenzymes in vertebrates, catalyzes the formation of hydroxylysine by acting on -X-Lys-Gly- triplets in the collagenous domains of proteins of the collagen superfamily and also in -X-Lys-Ala- or -X-Lys-Ser- sequences in the telopeptides located at the ends of the polypeptide chains in some fibril-forming collagens. The hydroxylysine residues are essential for the stability of collagen crosslinks and act as carbohydrate attachment sites. The extent of lysine hydroxylation varies between collagen types, between tissues in the same collagen type and in certain diseases, suggesting that the LH isoenzymes may have different substrate specificities. We studied here the hydroxylation of synthetic peptides representing various hydroxylation sites in type I and IV collagens by purified recombinant LHs in vitro and of a recombinant full-length type I procollagen chain coexpressed with each LH in insect cells. All three LHs hydroxylated peptides representing collagenous sequences of type I and IV collagens, although with different K(m) and V(max) values. Furthermore, all three hydroxylated the collagenous domain of the coexpressed type I procollagen chain to a similar extent. None of the isoenzymes hydroxylated peptides representing the N and C telopeptides of type I collagen, but LH2, unlike the other two isoenzymes, hydroxylated the N telopeptide in the coexpressed procollagen chain. Hydroxylation of the telopeptide lysines by LH2 thus occurs only in the context of a long peptide. These data provide the first direct evidence that LH2 is a specific telopeptide hydroxylase, while all three LHs act on collagenous sequences.     

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.     

Processing of procollagen types III and I in cultured bovine smooth muscle cells

The processing of type III and type I procollagen molecules in cultured bovine aortic smooth muscle cells was investigated. The molecular identities of the processing intermediates of type III and type I procollagen were characterized by analysis of the radioactive collagenous components using mammalian collagenase and pepsin digestions and cyanogen bromide peptide mapping. The results indicate that the processed intermediates for procollagen type III and type I are their respective pC components. Although the processing pathways for both collagen types are the same, data from pulse-chase experiments suggest that the rates at which the processing occurs are different. Type I procollagen is processed more rapidly to its intermediate than is type III procollagen. The type I pC intermediate is almost completely processed to alpha-chains and a significant portion of these fully processed molecules remains in a soluble form even after 11 h. In the same time period, the type III pC intermediate is slowly converted to alpha-chains. Since beta-aminopropionitrile was not employed in these studies, significant accumulation of collagen chains into the insoluble extracellular matrix was observed during the chase period.     

Attaching human fibroblasts secrete a type I procollagen rich in 3-hydroxyproline.

The predominant collagenous protein secreted during the attachment of freshly trypsinized human foreskin fibroblasts was found to be Type I procollagen. Evidence is presented that both the α₁ and α₂ chains exhibit a 3-hydroxyproline/4-hydroxyproline ratio 4–5 fold higher than that of normal Type I collagen. These findings suggest that caution should be exercised in assigning an observed increase in the 3-hydroxyproline/4-hydroxyproline ratio to the synthesis of a basement membrane type collagen.     

Comparison of the collagenous products synthesized in culture by pig aortic endothelial and smooth-muscle cells. Variability in endothelial-cell cultures.

In contrast with smooth-muscle cells from the same tissue, endothelial cells from pig aorta were found to exhibit in culture considerable variability in the pattern of collagen synthesis between one isolation of cells and the next. Synthesis varied from largely collagen type I to virtually all type III in the absence of type I but with small amounts still of collagens types IV and V, to, in one instance, synthesis basically of only type V. Synthesis usually by these cells of collagen predominantly of the interstitial type (I and III) rather than, as might be expected, that from basement membrane (type IV) was not attributable to the influence of subculture. All four collagen types were deposited in the cell layer to an increased extent in primary compared with secondary cultures of either smooth muscle or endothelial origin. Endothelial cells appeared sometimes to synthesize a large-Mr collagenous entity that might conceivably be related to 'short-chain' collagen. In addition, small-Mr hydroxyproline-containing peptides were detected that might reflect rapid collagen(s) turnover in endothelial cultures.     

Biosynthesis and proteolytic processing of type XI collagen in embryonic chick sterna

The biosynthesis and proteolytic processing of type XI procollagen was examined using pulse-chase labelling of 17-day embryonic chick sterna in organ culture with [3H]proline. Products of biosynthesis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with and without prior reduction of disulfide bonds. Pro-alpha chains, intermediates, and matrix forms were identified by cyanogen bromide or Staphylococcus aureus V8 protease digestion. The results show that type XI pro-alpha chains assemble into trimeric molecules with interchain disulfide bonds. Proteolytic processing begins at least 40 min after the start of labeling which is later than that of type II procollagen (25 min). This first processing step involves the loss of the domain containing the interchain disulfide bonds which most likely is the carboxyl propeptide. In the case of the pro-alpha 3 chain, this generates the matrix form, m alpha 3, which retains its amino propeptide. For the pro-alpha 1 and pro-alpha 2 chains, this step generates intermediate forms, p alpha 1 and p alpha 2, which undergo a second proteolytic conversion to m alpha 1 and m alpha 2, and yet retain a pepsin-labile domain. The conversion of p alpha 2 to m alpha 2 is largely complete 2 h after labeling. p alpha 1 is converted to m alpha 1 very slowly and is 50% complete after 18 h of chase in organ culture. The apparent proteolytic processing within the amino propeptide, and the differential rate of processing between two chains in the same molecule are unusual and distinguish type XI from collagen types I, II, and III. It is possible that the extremely slow processing of p alpha 1 affects the formation of the heterotypic cartilage collagen fibrils and may be related to the function of type XI collagen.     

Interstitial collagen synthesis by somatic testicular cells in culture

The synthesis, distribution and types of collagen produced by somatic testicular cells in culture was studied. To investigate whether changes in collagen synthesis correlate with the age of the animal, cultures derived from immature and pubertal rats were established. Immature rats synthesize 40 per cent more collagen than pubertal rats. Both groups of animals synthesize procollagen types I and III. Pro-collagen type I is present in the culture medium as well as in the cell fraction, while type III is only detected in the culture medium. In the transition from immature to pubertal rat, the ratio of procollagen type III to procollagen type I diminishes from 5.7 to 1.7. These results indicate that the synthesis, distribution and molecular characteristics of interstitial collagens changes with the age of the animal. Since, the content of other extracellular matrix components such as proteoglycans and collagen type IV also varies with age, we postulate that the composition of the extracellular matrix in the testes is not constant but changes with sexual development.