Reichert's Membrane Research Articles

In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth—Holm—Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.

Characterization of a novel calcium‐binding 90‐kDa glycoprotein (BM‐90) shared by basement membranes and serum

The protein BM-90 was solubilized from the mouse Engelbreth-Holm-Swarm tumor with neutral buffers in molar yields lower (15-30%) than found for other basement membrane proteins (e.g. laminin, BM-40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84-kDa position to a 95-kDa one upon reduction. BM-90 was also shown to be a calcium-binding protein. The N-terminal sequence of BM-90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross-reactions with other known basement membrane proteins. Immunological assays demonstrated BM-90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20-40 micrograms/ml) and in the medium of various cultured cells (0.1-1 microgram/ml). The protein in these samples was identical in size to BM-90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM-90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM-90 appears to be a novel basement membrane protein whose functions remain to be studied.

Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues.

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.

A monoclonal antibody, 4H12, recognizes a surface antigen found on granulated metrial gland cells in the murine decidua

A monoclonal antibody (MAb), designated 4H12, was selected for reactivity to a surface antigen on PYS-2 teratocarcinoma cells. 4H12 was the product of a fusion of lymphoid cells of a non-immunized pregnant C57BL/6 mouse to NS-1 myeloma cells. Initial studies utilizing immunohistochemistry revealed that MAb 4H12 bound to an antigen found on cells in the decidua basalis of 7-, 8- and 10-day pregnant mice. Antigen-positive cells of 11–19-day pregnant mice were also found predominantly in the decidua. A few antigen-positive cells were found in the labyrinth of the placenta and up against Reichert's membrane. Antigen-positive cells were morphologically and spatially distinct, oval to round with large periodic acid Schiff positive granules. Indirect immunofluorescent (IIF) labeling of decidual cultures showed antigen on the surface of cells that were small, oval to round and adherent. The antigen recognized by MAb 4H12 was removed from tissue sections with trypsin and protease and therefore is suggested to be a protein. We conclude that MAb 4H12 recognizes a surface antigen found on cells historically described as granulated metrial gland (GMG) cells. This MAb should greatly facilitate the further analysis of the life history and function of GMG cells during pregnancy.

Blastocyst implantation in the chinese hamster (Cricetulus griseus)

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan.

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.

Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine), a Ca++-binding glycoprotein also known as osteonectin, is produced in significant amounts by injured or proliferating cells in vitro. To elucidate the possible function of SPARC in growth and remodeling, we examined its distribution in embryonic and adult murine tissues. Immunohistochemistry on adult mouse tissues revealed a preferential association of SPARC protein with epithelia exhibiting high rates of turnover (gut, skin, and glandular tissue). Fetal tissues containing high levels of SPARC included heart, thymus, lung, and gut. In the 14-18-day developing fetus, SPARC expression was particularly enhanced in areas undergoing chondrogenesis, osteogenesis, and somitogenesis, whereas 10-day embryos exhibited selective staining for this protein in Reichert's membrane, maternal sinuses, and trophoblastic giant cells. SPARC displayed a Ca++-dependent affinity for hydrophobic surfaces and was not incorporated into the extracellular matrix produced by cells in vitro. We propose that in some tissues SPARC associates with cell surfaces to facilitate proliferation during embryonic morphogenesis and normal cell turnover in the adult.

Heparan sulfate proteoglycan is present in basement membrane as a double-tracked structure.

Basement membranes contain 4.5-nm wide sets of two parallel lines, along which short prongs called "spikes" occur at regular intervals. The nature of this structure, referred to as "double tracks," was investigated in Lowicryl sections of mouse kidney and rat Reichert's membrane immunolabeled for basement membrane components using secondary antibodies conjugated to 5-nm gold particles. When the mouse glomerular basement membrane and rat Reichert's membrane were exposed to antibodies directed to the core protein of heparan sulfate proteoglycan, 95% or more of the gold particles were over double tracks, whereas after exposure of Reichert's membrane to antisera against laminin, collagen IV, or entactin, labeling of the double tracks remained at the random level. When heparan sulfate proteoglycan was incubated in Tris buffer, pH 7.4, at 35 degrees C for 1 hr, a precipitate resulted which, on electron microscopic examination, was found to consist of 5- to 6-nm wide sets of two parallel lines along which densities were observed. Immunolabeling confirmed the presence of the proteoglycan's core protein in the sets. Since double tracks were closely similar to this structure and were labeled with the same antibodies, they were likely to be also composed of heparan sulfate proteoglycan.

Ultrastructure of a model basement membrane lacking type IV collagen.

Basement membranes (BMs) are specialized extracellular matrices which have important roles in cell attachment, migration, growth, and differentiation. The major components of these matrices include type IV collagen, laminin, entactin, and heparan sulfate proteoglycan. The framework or scaffold of BMs has been proposed to be type IV collagen (Yurchenco et al., 1986, J. Histochem. Cytochem., 34:93-102). However, a murine teratocarcinoma cell-line, M1536-B3, has been described which produces an extracellular matrix (ECM) composed of some of the known components of BM, e.g., laminin, entactin, and sulfated proteoglycan, but lacking type IV collagen (Chung et al., 1979, Cell, 16:277-287). With the use of morphological techniques, we have found that the ECM assembled by these cells is composed of multiple layers of electron-dense cords arranged in an interweaving meshwork with short 2-4 nm-diameter cylindrical rods embedded throughout. This organization closely resembles that reported for naturally occurring BMs, e.g., Reichert's membrane (Inoué et al., 1983, J. Cell Biol., 97: 1524-1537). The previous identification of known in vivo BM components in M1536-B3 ECM and the correspondence in morphological appearance of M1536-B3 ECM with that present in naturally occurring BMs suggests that a BM-type of ECM can be assembled without a type IV collagen framework, thus indicating that other components of BMs have a critical role in BM organization and assembly.

Ultrastructure of trophoblast giant cell transformation during the invasive stage of implantation of the mouse embryo.

The fine structure of abembryonic and mural trophoblast cells of mouse embryos was analyzed during the initial stages of invasion of the endometrial stroma by the embryo (days 6-8 of pregnancy). On day 6 of pregnancy, most trophoblastic cells are flat and have spindle-shaped nuclei. A few large, round trophoblastic cells (giant cells) are present at the abembryonic pole. As pregnancy proceeds through days 7 and 8, the area occupied by the trophoblast becomes larger because of an increase in the trophoblastic cell population, growth of giant cells, and rearrangement of the latter cells into a network containing maternal blood. As flat cells transform into giant cells, their content of ribosomes, granular endoplasmic reticulum, Golgi complexes, lysosomelike bodies, and heterophagosomes increases. Reichert's membrane is always lined by cell bodies or by laminar processes of trophoblastic cells that are provided with small pores. Transformation of flat cells into giant cells is associated with an activation of the giant cells and their acquisition of invasive behavior.

Three-dimensional network of cords: the main component of basement membranes.

Basement membranes were divided into two types: 1) thin basement membranes, such as those of the epidermis, trachea, jejunum, seminiferous tubule, and vas deferens of the rat, the ciliary process of the mouse, and the seminiferous tubule of the monkey, and 2) thick basement membranes, such as the lens capsule of the mouse and Reichert's membrane of the rat. High-magnification electron microscopy was used to examine both types after fixation either in glutaraldehyde followed by postosmication or in potassium permanganate. The basic structure of thin and thick basement membranes was found to be a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the diameter of these cords was variable, but averaged 4 nm in all cases examined. The spaces separating the cords differed, however. In the lamina densa of thin basement membranes, the diameter of these spaces averaged about 14 nm in every case, whereas in the lamina lucida it ranged up to more than 40 nm. Intermediate values were recorded in thick basement membranes. Finally, the third, inconstant layer of thin basement membranes, pars fibroreticularis, was composed of discontinuous elements bound to the lamina densa: i.e., anchoring fibrils, microfibrils, or collagen fibrils. In particular, collagen fibrils were often surrounded by processes continuous with the lamina densa and likewise composed of a typical cord network. Finally, two features were encountered in every basement membrane: 1) a few cords were in continuity with a 1.4- to 3.2-nm thick filament or showed such a filament within them; the filaments became numerous after treatment of the seminiferous tubule basement membrane with the proteolytic enzyme, plasmin, since cords decreased in thickness and could be reduced to a filament, and 2) at the cord surface, it was occasionally possible to see 4.5-nm-wide sets of two parallel lines, referred to as "double tracks." On the basis of evidence that the filaments are type IV collagen molecules and the double tracks are polymerized heparan sulfate proteoglycan, it is proposed that cords are composed of an axial filament of type IV collagen to which are associated glycoprotein components (laminin, entactin, fibronectin) and the double tracks of the proteoglycan.

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

Differential expression of MHC class I antigens on the placenta of the rat. A mechanism for the survival of the fetal allograft.

In some mating combinations in rats, there is a maternal antibody response to the maternal antigenic components of the placenta without any previous immunization of the mother. The highest response occurs in the WF (u) female mated to the DA (a) male, and it is against a unique MHC-encoded class I antigen, the Pa antigen, and not against the major allele-specific transplantation antigen of the DA strain, RT1.Aa. The development of mAbs to the Pa and Aa antigens allowed us to localize these antigens on the placenta and to explore the reason for the differential antibody response to them using immunohistochemical and biochemical techniques. Both antibodies reacted with the WF X DA placenta and stained the endovascular and interstitial trophoblast of the decidua, the basal trophoblast, Reichert's membrane, and the yolk sac epithelium, but they did not stain the labyrinthine trophoblast. Blocking studies showed that each antibody reacted with a separate molecule in the placenta. Anti-class II mAbs reactive with the a or u haplotype did not stain the WF X DA, DA X DA, or WF X WF placenta; hence, there are no class II antigens in the placenta. Electron microscopic studies of the semiallogeneic WF X DA placenta using the immunogold technique with both single- and double-labeling showed that only the Pa antigen was expressed on the surface of the basal trophoblast, but that both the Pa and Aa antigens were in the cytoplasm of these cells; neither antigen was found in the labyrinthine trophoblast. By contrast, the placenta from the syngeneic DA X DA mating expressed both the Pa and Aa antigens on the surface of the basal trophoblast as well as in the cytoplasm; neither antigen was found in the labyrinthine trophoblast. These observations were quantified morphometrically using electron photomicrographs of single-labeled tissues. Both the Pa and Aa antigens isolated from the plasma membrane of lymphocytes have heavy chains of 46 kD, but those antigens isolated from the plasma membrane of basal trophoblast cells have heavy chains of 43 kD. Based on densitometric measurements of autoradiographs, the Pa/Aa ratio in the basal trophoblast membrane is 23.5, whereas it is 0.46 in lymphocyte membranes. These studies show that there is differential regulation of the expression of class I antigens on basal trophoblast cells in semiallogeneic pregnancies, but not in syngeneic pregnancies, such that the major allele-specific transplantation antigen is scarcely expressed on the surface of the basal trophoblast.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth and proliferation in mouse parietal yolk sac during whole embryo culture.

Use of the culture techniques for postimplantation rodent embryos, modified by explanting Day 9 or Day 10 embryos with the trophoblast cells removed but the remainder of the parietal yolk sac left intact, resulted in significant expansion of Reichert's membrane, accompanied by a marked increase in the numbers of the adherent parietal endoderm cells which synthesize the membrane. The surface area of the parietal endoderm/Reichert's membrane complex roughly doubled during culture, and the combined protein content of the cells and their basement membrane was also raised after incubation. Parietal endoderm cell numbers, calculated from area and cell density measurements on flattened membranes, increased by 54-190%, depending on the age of the embryo.

Radioautographic tracing of 3H-proline in the endodermal cells of the parietal yolk sac as an indicator of the biogenesis of basement membrane components.

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.

Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans.

Metabolically 35S- or 3H-labeled heparan sulfate was isolated from murine Reichert's membrane, an extraembryonic basement membrane produced by parietal endoderm cells, and from the basement membrane-producing Engelbreth-Holm-Swarm mouse tumor. The polysaccharides were subjected to structural analysis involving identification of products formed on deamination of the polysaccharides with nitrous acid. The polysaccharide from Reichert's membrane contained N- and O-sulfate groups in approximately equal proportions. It bound almost quantitatively and with high affinity to antithrombin. A high proportion of antithrombin-binding sequence was also indicated by the finding that 3-O-sulfated glucosamine residues accounted for about 10% of the total O-sulfate groups. In contrast, at least 80% of the sulfate residues in the heparan sulfate isolated from the mouse tumor were N-substituents. Only a minor proportion of this polysaccharide bound with high affinity to antithrombin, and no 3-O-sulfated glucosamine residues were detected. These results are discussed in relation to the possible functional role of heparan sulfate in basement membranes.

Trophoblast-decidual cell interactions and establishment of maternal blood circulation in the parietal yolk sac placenta of the rat.

Implantation sites from rats were studied on days 6, 7, and 8 of pregnancy to determine the sequence of events in the formation of blood spaces in the trophoblast that is part of the parietal wall of the yolk sac placenta and to determine how trophoblast gains access to maternal blood. The maternal blood flowing through these spaces is the source of nutrients that reach the embryo via the visceral endoderm. Tissues were prepared for light microscopy, scanning electron microscopy, and transmission electron microscopy. Trophoblast blood spaces are derived from the lateral intercellular spaces of trophoblast cells and are present in a collapsed condition until day 8, when maternal vessels are tapped by trophoblast. These spaces then contain circulating maternal blood, and trophoblast cells reflect adaptations for metabolic exchange including thinning of trophoblast covering Reichert's membrane and the appearance of numerous fenestrations, with and without diaphragms, in the areas where trophoblast is attenuated. Between days 6 and 7 decidual cells appear to form a barrier between the maternal circulation and trophoblast. On day 7, however, decidual cell processes penetrate the residual uterine luminal epithelial basal lamina, and then the decidual cells that are juxtaposed to trophoblast undergo degradative changes that resemble apoptosis. There is condensation of cytoplasmic contents, fragmentation of the cells, and phagocytosis of the fragments by trophoblast. Some decidual cells are interposed between endothelial cells in the walls of maternal vessels as early as day 7. Trophoblast may gain access to the maternal vessels by replacing decidual cells or by direct imposition of trophoblast cell processes between endothelial cells.

The role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3H-proline as a precursor of type IV collagen.

Biosynthesis of type IV collagen in the parietal endodermal cells of 12 day gestant Sherman rats was examined following intraconceptal injection of 3H-proline. The concepti were removed at times varying from 2 minutes to 24 hours after the injection. The parietal wall of the yolk sac, including endodermal cells and the associated basement membrane known as Reichert's membrane were processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Radioactivity was high in endodermal cells during the first 2 hr after 3H-proline injection and later dropped to some extent, while radioactivity rose in Reichert's membrane. Examination of endodermal cell organelles showed some early labeling over rER and Golgi apparatus without a clear-cut trend, except for a drop in Golgi label at late times after 3H-proline injection. The density of silver grains over secretory granules rose significantly by 40 min, reached a high peak by 4 hr and then declined at the time when radioactivity increased over Reichert's membrane. Furthermore, the radioactively-labeled secretory granules were localized mainly at the trans Golgi face soon after injection and near the cell surface adjacent to Reichert's membrane at later times. Biochemical reports indicate that a substantial amount of the proline taken up by the 12-14.5 day rat embryo endodermal cells is incorporated into type IV collagen. Since there is high labeling of the secretory granules from 40 min to 4 hr and the labeled granules are associated with the Golgi apparatus at early times, it is proposed that collagen precursors are processed through rER and Golgi apparatus, packaged into secretory granules and then transported to the cell surface where type IV collagen or its precursors are released and subsequently deposited into Reichert's membrane.

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac.

In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20-30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal antibodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40-100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.

Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor.

A novel acidic glycoprotein, BM-40, with Mr = 40,000, was purified from the basement-membrane-producing mouse EHS tumor and characterized with regard to its unique chemical and antigenic properties. It was obtained from the tumor in a neutral salt-soluble form or as a component requiring extraction with 6M guanidine X HCl. This protein could also be identified in many other tissue extracts and cell and tissue cultures. The most intact form of BM-40 consists of a single polypeptide chain which undergoes limited proteolysis during extraction and purification. BM-40 exists in most tissues in stoichiometric amounts compared to other basement membrane proteins (laminin, nidogen) and is secreted by various teratocarcinoma and epithelial cells. It can be visualized by immunofluorescence in the extracellular matrix of the EHS tumor and Reichert's membrane. Other tissues which contain extractable BM-40 were negative in immunofluorescence.