Structural components and characteristics of Reichert's membrane, an extra-embryonic basement membrane.

Rat parietal yolk sacs (PYS) at gestational ages 7.5, 9.5, 11.5, 13.5, 14.5, and 16.5 d were reacted with antibodies against laminin or plasma fibronectin. At all times studied, laminin consistently gave a positive reaction with Reichert's membrane and with the cytoplasm of PYS cells. In contrast, fibronectin gave a negative reaction with Reichert's membrane at day 7.5, was weakly positive at day 9.5, and from then on was increasingly positive with maximum reactivity at 14.5 d. By electron microscopic immunohistochemistry, antilaminin reacted strongly with 14.5-d Reichert's membrane and with the contents of the rough endoplasmic reticulum RER cisternae of the PYS cells. Antifibronectin had some spotty reactivity with Reichert's membrane, but the cytoplasm of the PYS cells was negative. The contents of the vitelline vessels and the interface between trophoblast and Reichert's membrane were strongly positive.Metabolic labeling of PYS cells in organ culture clearly demonstrated the presence of laminin, type IV procollagen, and entactin both in the medium and in tissues, but fibronectin was absent. No component in the medium bound to gelatin-Sepharose columns.These studies demonstrate that PYS cells, which actively synthesize and secrete basement membrane components, do not synthesize any detectable fibronectin. Furthermore, the antifibronectin staining pattern in the vitelline vessels and trophoblast-Reichert's membrane interface strongly suggests that the fibronectin present in Reichert's membrane is derived from the maternal circulation and is merely "trapped" in the membrane.The relationship between fibronectin and basement membranes has been controversial. Initial immunofluorescence studies describe the localization of fibronectin in the basement membranes of a variety of tissues (8,16,32,34,35,38), but more recent light and electron immunohistochemical studies fmd that fibronectin and basement membrane antigens have different tissue distributions (1, 21, 37). Because most of the prior studies were performed in tissues with a slow rate of basement membrane synthesis and turnover, they could provide only a relatively static picture. Therefore, we decided to address this question using a more dynamic system characterized by rapid basement membrane synthesis and deposition.The rodent parietal yolk sac (PYS) in vitro synthesizes, secretes, and deposits onto Reichert's membrane a number of high molecular weight glycoprotein components. These include basement membrane procollagen [pro-cd(IV) and proa2(IV)], laminin (PYS A and PYS B), and entactin (PYS C) (2,4,7,11,12,31). Biochemical experiments suggest that neither the PYS, the endodermal cells derived from the PYS, nor most PYS carcinoma cells in culture synthesize significant amounts offibronectin (t 1-13, 15, 29, 31, 38). In contrast, there are reports that the extracellular matrix associated with such cells and tissues reacts with antibodies directed against fibronectin (13, 34-36, 38, 39).Here we report that, even at embryonic stages when Reichert's me...

Section: Discussionsupporting

confidence: 78%

mentioning

confidence: 99%

Deposition of fibronectin and laminin in the basement membrane of the rat parietal yolk sac: immunohistochemical and biosynthetic studies.

Amenta¹,

Clark²,

Martínez-Hernández³

1983

“…Because our procedure could localize extraceUular as well as intraceUular substances (7,35,36), the lack of immunostaining in the lamina lucida was not due to lack of penetration; indeed the projections from the basal lamina across the lamina lucida were immunostained. In any case, it was likely that the antigenicity of the basal lamina was due to the presence of laminin itself, because laminin had been extracted from Reichert's membrane (37)(38)(39) Heparan sulfate proteoglycan had not previously been immunolocalized at the level of the electron microscope. On the basis of reactions with cationic markers and dyes, this substance had been assigned to the basal lamina (15,16), to its surface (17,18) or to the lamina lucida (19,20).…”

Section: Discussionmentioning

confidence: 99%

Localization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin to the basal lamina of basement membranes.

Laurie¹,

Leblond²,

Martin³

1982

435

180

Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-/~m thick slices of formaldehyde-fixed tissues.In the basement-membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries.The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.

“…The other components of the membrane are rigidlooking basotubules that may impart strength to the layers, and tiny particles referred to as double pegs whose role is unknown. By immunohistochemical staining, Reichert's membrane has been previously shown to contain not only laminin (14)(15)(16)(17)43) and type IV collagen (18)(19)(20), but also heparan sulfate proteoglycan (44), fibronectin (43), and entactin (unpublished results with D. Grant). Since these five substances have also been detected by immunostaining of the glomerular basement membrane in the rat kidney (laminin, reference 45; type IV collagen, reference 45; heparan sulfate proteoglycan, reference 46; fibronectin, reference 45; entactin, Laurie et al, in preparation), we have recently examined whether this basement membrane has an ultrastructure comparable to that of Reichert's membrane.…”

Section: O N C L U D I N G Remarksmentioning

confidence: 92%

“…genicity. Since Reichert's membrane was known to contain laminin (14)(15)(16)(17) and type IV collagen (18)(19)(20), it was hoped that treatment with plasmin could help in assigning laminin to some of the structural components. In a third stage, immunostaining for laminin and type IV collagen was applied to Reichert's membrane.…”

mentioning

confidence: 99%

Ultrastructure of Reichert's membrane, a multilayered basement membrane in the parietal wall of the rat yolk sac.

Inoué¹,

Leblond²,

Laurie³

1983

126

The ultrastructure of Reichert's membrane, a thick basement membrane in the parietal wall of the yolk sac, has been examined in 13-14-d pregnant rats. This membrane is composed of more or less distinct parallel layers, each one of which resembles a common basement membrane. After routine fixation in glutaraldehyde followed by osmium tetroxide, the layers appear to be mainly composed of 3-8-nm thick cords arranged in a threedimensional network. Loosely scattered among the cords are unbranched, straight tubular structures with a diameter of 7-10 nm, which mainly run parallel to the surface and to one another; they are referred to as basotubules. Permanganate fixation emphasizes the presence of a thick feltwork of irregular material around basotubules. Finally, minute dot-like structures measuring 3.5 nm and referred to as double pegs are present within the meshes of the cord network.Reichert's membranes have been treated for 2-48 h at 25°C with plasmin, a proteolytic enzyme known to rapidly digest laminin and fibronectin. After a 2-h treatment, most of the substance of the cords is digested away leaving a three-dimensional network of 1.5-2.0-nm thick filaments. The interpretation is that the cords are formed of a plasmin-resistant core filament and a plasmin-extractable sheath. When plasmin treatment is prolonged for 15 h or longer, the filaments are dissociated and disappear, while basotubules are maintained. Plasmin digestion also reveals that basotubules are composed of two parts: a ribbon-like helical wrapping and tubule proper. Further changes in the tubule under plasmin influence are interpreted as a dissociation into pentagonal units suggestive of the presence of the amyloid P component. After 48 h of plasmin treatment, basotubules are further disaggregated and dispersed, leaving only linearly arranged double pegs.Reichert's membranes with or without a 2-hr plasmin treatment have been immunostained by exposure to antibodies against either laminin or type IV collagen with the help of peroxidase markers. The results indicate that the sheath of the cords contains laminin antigenicity, while the core filament contains type IV collagen antigenicity.It is proposed that Reichert's membrane consists mainly of a three-dimensional network of cords composed of a type IV collagen filament enclosed within a laminin-containing sheath. Also present are basotubules--which may contain the amyloid P component--and double pegs whose nature is unknown.