Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.     

High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV

Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with a Kd value of 10(-9) M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10(-6) M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin 5 and collagen IV. We postulate that the ability of the NC1 domains to bind with high affinities to laminin 5 and collagen IV facilitates stabilization of the structure of the basement membrane itself and that the NC1-collagen I interaction may be less important for stabilization of the dermal-epidermal junction.     

Immuno-electron-microscopic localization of types III pN-collagen and IV collagen,laminin and tenascin in developing and adult human spleen

The distribution of the extracellular matrix proteins types III pN-collagen and IV collagen, laminin and tenascin was investigated in fetal, infant, and adult human spleens by using immuno-electron microscopy. The presence of type III pN-collagen was assessed by using an antibody against the aminoterminal propeptide of type III procollagen. All the proteins other than type III pN-collagen were found in reticular fibers throughout development. In the white pulp of the fetus aged 16 gestational weeks, only an occasional type III pN-collagen-containing fibril was present, although type III pN-collagen was abundant in the reticular fibers of the red pulp. Conversely, in adults, most of the reticular fibers of the white pulp, but not of the red pulp, were immunoreactive for type III pN-collagen. Ring fibers, the basement membranes of venous sinuses, were well developed in both infant and adult spleens. The first signs of their formation could be seen as a discontinuous basement membrane, which was immunoreactive for type IV collagen, laminin, and tenascin in the fetus aged 20 gestational weeks. Intracytoplasmic immunoreactivity for all the proteins studied was visible in the mesenchymal cells of the fetus aged 16 gestational weeks and in the reticular cells of the older fetuses, which also showed labeling for type IV collagen and laminin in the endothelial cells. The results suggest that proteins of the extracellular matrix are produced by these stationary cells.     

Localization of the 110 kDa receptor for laminin in brains of embryonic and postnatal mice

Laminin, a large glycoprotein of the basement membrane that promotes the growth of nerve cell processes in vitro has also been detected in the brains of developing embryos in situ where it is postulated to promote or guide neural outgrowth. We have investigated the histological and developmental patterns of a receptor to a specific pentapeptide sequence in the A chain of the laminin molecule (PA22-2 or IKVAV) that has been identified as a neuron growth-promoting sequence. Standard immunocytochemical procedures were used to localize the receptor by means of a polyclonal antibody to affinity-purified receptor (M_R=110 kDa) from mouse brains. Results for postnatal stages (P) stages (P 1,7,8,25,30,and adult) show that the 110 kDa receptor is localized in fibers in the cortex and hippocampus, in astroglial cells at the surface of the cortex, and in neuronal cell bodies in the hippocampus. In contrast, the A-chain ligand is localized in cell bodies in the same regions at P stages. For embryonic stages (E) (E 14 and E 16) the receptor is localized in bundles of fibers in the superficial and deep cortical layers, and in cell bodies in these regions at E 14 only. Staining for the A chain ligand of the receptor was first seen postnatally. We speculate that the inverse histological pattern of receptor and ligand with respect to cell bodies and fibers may reflect a role in controlling axon guidance during development or repair during regeneration.This research was supported by the AOA Grant #89-08-296 and by the Ohio University College of Osteopathic Medicine     

Distribution of laminin,type IV collagen,and fibronectin in the cell columns and trophoblastic shell of early macaque placentas

Summary The cytotrophoblastic cell columns and trophoblastic shell of macaque placentas accumulate progressively greater amounts of intercellular material during early gestation. We studied the composition of this material in placentas collected from 22–34 days of gestation by using immunoperoxidase techniques directed to the extracellular matrix molecules fibronectin, type IV collagen, and laminin. These antigens co-localized within the intercellular deposits at all stages studied. At day 22 the proximal cell columns were composed of cells with narrow interstices and which lacked immunoreactivity for the 3 antigens. Distally the cells were vacuolated and the intercellular spaces increased in size and contained dense matrix deposits. The trophoblastic shell consisted of closely packed, non-vacuolated cytotrophoblast cells with only a delicate meshwork of matrix. By day 27 the matrix deposits of the distal cell columns increased markedly in size. The trophoblastic shell contained larger numbers of vacuolated cells and was occupied by accumulations of matrix. By 34 days the matrix deposits of the cell columns expanded substantially along the longitudinal axes of the columns. These deposits were often continuous with a matrix-dense, cell-deficient layer in the trophoblastic shell. This matrix-rich zone lay between a cellular layer adjacent to the intervilous space and a similar, but discontinuous, cell layer that formed the junctional zone with the endometrium.     

Distribution of laminin and types IV and III collagen in fetal,infant and adult human spleens

Summary The immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroth's cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Short and long range order in basement membrane type IV collagen revealed by enzymic and chemical extraction

Oriented bovine lens capsules give X-ray diffraction patterns suggesting a considerable degree of order in the collagenous components, predominantly type IV collagen. Here we report the effects of preliminary treatment of lens capsules before orientation. Extraction with 4 guanidinium hydrochloride or with heparinase/hyaluronidase reveals the same collagenous diffraction patterns previously seen after extraction with 1 NaCl. There is a four-point pattern of d-spacing 3.9 nm, indicating liquid crystal cybotactic nematic organization, along with sharp streaked meridional reflections which index as orders of 21 nm. This suggests that the removal of basement membrane proteoglycans results in a reduction in diffuse scatter and clarification of the pattern. Extraction of the lens capsules with trypsin or dithiothreitol greatly reduces the intensity of the four-point pattern while leaving the meridional pattern unaffected. This strengthens the evidence that the 21 nm period has its origins in the collagen IV helix. Reduction in the four-point pattern could arise if disruption of non-helical NC1 domains or 7S overlap regions allows slippage of the collagen molecules on orientation, weakening the proposed 1 nm intermolecular stagger. Ultra-low angle diffraction patterns of extracted lens capsules show meridional reflections which index as a long-range axial repeat of approximately 95 nm. This is consistent with a model of microfibrils of type IV collagen in which the NC1 domains bind to the collagen helix at approximately 100 nm intervals, as has been previously suggested.     

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

Nerve growth factor receptor-like immunoreactivity in primary and permanent canine tooth pulps of the cat

Summary The distribution of nerve growth factor receptor (NGF receptor)-like immunoreactivity in pulps of developing primary and mature permanent cat canine teeth was examined, by use of a monoclonal antibody against NGF receptor detected by fluorescence immunohistochemistry and pre-embedding immunocytochemical light- and electron microscopy. Both primary and permanent pulps contained a vast number of NGF receptor-like immunoreactive nerves. Immunolabelling appeared to be localized both to axons and Schwann cells. In addition, many blood vessel walls in immature primary tooth pulps showed NGF receptor-like immunoreactivity, in contrast to permanent pulps where blood vessels rarely were NGF receptor-immunoreactive. Double-labelling immunofluorescence experiments revealed that in the permanent pulp a majority of the NGF receptor-positive nerves also showed calcitonin gene-related peptide (CGRP)-like immunoreactivity, and many showed substance P-like immunoreactivity. However, nerve fibers with neuropeptide Y-like immunoreactivity lacked NGF receptor-like immunoreactivity. In developing primary tooth pulps fewer NGF receptor-positive nerves were CGRP-like immunoreactive or substance P-like immunoreactive, as compared to the permanent pulp. Neuropeptide Y-like immunoreactive nerve fibers were not detected in the primary tooth pulp. The results suggest a role for nerve growth factor in both developing and mature sensory nerves of the tooth pulp.     

Determination of collagen type IV by Surface Plasmon Resonance Imaging using a specific biosensor

Serum collagen type IV (COLIV) is a promising tumor marker. High COLIV concentrations have been found in the serum of patients with colorectal, gastric, lung, liver and breast cancers. The aim of this work was to develop a biosensor for use with the Surface Plasmon Resonance Imaging (SPRI) technique for COLIV determination. The biosensor consists of glass covered with gold and immobilized monoclonal mouse anti-human collagen type IV antibody via cysteamine linker. The biosensor works selectively within a dynamic response range between 10 and 300 ng mL⁻¹, with LOD 2.4 ng mL⁻¹ and LOQ 8 ng mL⁻¹. The precision of determination is 4.7% at a 150 ng mL⁻¹ COLIV spike and 8.0% at a 20 ng mL⁻¹ spike, with recoveries of 101% and 106% respectively. A 100-fold excess of collagen I, albumin, laminin and fibronectin is tolerated. The average COLIV blood plasma concentration of healthy donors determined by the developed method was 69 ± 10 ng mL⁻¹, while the median of six results available in the literature was approximately 80 ng mL⁻¹. The average COLIV blood plasma concentration of breast cancer patients was 360 ± 68 ng mL⁻¹, showing the high potential of COLIV as a marker of this type of cancer.     

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.     

Laminin B1 and Collagen Type IV Gene Expression in Transected Peripheral Nerve: Reinnervation Compared to Denervation

The expression of B1 laminin and type IV collagen was followed in the microsurgically isolated endoneurium of transected rat sciatic nerves from 3 days until 8 weeks. Northern hybridizations revealed that after nerve transection the proximal stumps of denervated, as well as freely regenerating, nerves showed a markedly increased expression of laminin and type IV collagen which lasted from 3 days up to 8 weeks. In the distal stumps, close to the site of transection (2-7 mm), the expression of laminin, and to a certain extent that of type IV collagen, seemed to be enhanced if free axonal reinnervation was allowed. Further distally (10-15 mm), the patterns of B1 laminin and type IV collagen expression were similar in both experimental groups, so that an increased expression was noticed during the first 2 weeks. The present results suggest that laminin and type IV collagen gene expression is markedly different in different parts of transected rat sciatic nerve. During peripheral nerve regeneration, there is a long-lasting basement membrane gene expression in the proximal stump. In the distal part of the transected nerve, the axonal reinnervation possibly up-regulates, but is not essential for, the expression of B1 laminin and type IV collagen.     

Basement membrane components of bronchial epithelium in humans suffering from chronic nonspecific lung diseases

Collagen IV and laminin are important constituents of the basement membrane (BM). By use of immunocytochemistry we examined the occurrence and distribution of these two components in the BM beneath normal, mucoid and metaplastic epithelium of large bronchi in 22 adults suffering from chronic nonspecific lung diseases. Both collagen IV and laminin were expressed as a thin and continuous layer beneath the epithelium in most tissue specimens with normal epithelium. In a few specimens the layer showed interruptions with a patchy distribution of the immunoreactivity. Three patterns of distribution of BM components were found under the metaplastic epithelium. Total absence of immunoreactive collagen IV and laminin was the most common variant. Weak and scarce staining for both proteins in the BM characterized the second pattern. The third variant showed strong collagen IV immunoreactivity but lack of laminin. The BM beneath the mucoid epithelium was characterized by irregular distribution of collagen IV and laminin. We suggest that the occurrence and distributional pattern of the BM components are related to the type of overlying epithelium and connected with an altered synthesis of these components.     

Inhibition of parasympathetic axonal sprouting in the cat submandibular gland by sympathetic axons

Summary The sprouting of parasympathetic axons into the submandibular sympathetic nerve trunk following sympathetic denervation has been investigated. It was found that a permanent sympathetic denervation was necessary in order for the sprouting to develop and be maintained: if reinnervation by adrenergic nerves was delayed, the sprouting developed but was reduced at longer survival times when the original innervation was reestablished. The evidence for suppression of the cholinergic sprouting by the adrenergic axons is discussed, as is the evidence that these sprouts arise from the submandibular gland.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

Expression and localization of laminin 5, laminin 10, type IV collagen,and amelotin in adult murine gingiva

The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules—laminin 5 (γ2 chain), type IV collagen (α1 chain), and laminin 10 (α5 chain)—and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals.