Bruce Babiarz

Expression of cathepsin proteinases by mouse trophoblast in vivo and in vitro

Implantation and placentation in the mouse requires successful invasion of the uterine wall by primary and secondary trophoblast giant cells. Their invasive nature depends in part on the upregulation of proteinases for the phagocytosis and extracelluar digestion of maternal cells and matrix materials. The work reported here studies the expression of cathepsin proteinases during secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. Cathepsins B (CB), L (CL), and D (CD) were found to be upregulated during trophoblast differentiation in vivo at the message and protein level producing expression patterns equivalent to those of primary trophoblast. Invasive trophoblast cells expressed higher levels of the processed or active forms of the enzymes, coinciding with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials. Trophoblast differentiation in vitro showed a similar upregulation of cathepsin enzymes. The enzymes were localized to heterogeneous vesicles that resembled both lysosomes and heterophagic vesicles. The presence of a large lysosomal population within the giant cells was confirmed by vital staining with acridine orange. Analysis of trophoblast‐conditioned media also demonstrated secreted forms of CB and CL. The results suggest that cathepsin enzymes may contribute to trophoblast invasion not only through the intracellular breakdown of molecules phagocytosed by trophoblast cells, but also by the extracellular digestion of matrix molecules and activation of other pro‐enzymes. Dev Dyn 1999;216:374–384. ©1999 Wiley‐Liss, Inc.

The role of murine cell surface galactosyltransferase in trophoblast: Laminin interactions in vitro☆

Implantation of the mouse embryo involves the invasion of the secondary trophoblast giant cells of the ectoplacental cone (EPC) into the uterine decidua. The mechanisms of this event are poorly understood. The putative substrate molecules found in the decidua which could support trophoblast invasion include laminin, fibronectin, and collagen type IV. EPCs dissected from Day 7.5 embryos were cultured on all three matrices. Galactosyltransferase (GalTase) was localized by immunolabeling on trophoblast cell surfaces when grown on laminin but not the other matrices. Perturbations of the enzyme:substrate complex with alpha-lactalbumin, uridine diphosphogalactose, anti-GalTase, and pregalactosylation of the matrix did not affect rates of EPC attachment. However, decreased rates of migration or altered morphologies of spreading cells were observed. Laminin, and not fibronectin or collagen type IV, could be galactosylated with both exogenous GalTase or EPC outgrowths. Digests of galactosylated laminin produced a glycoconjugate substrate with a molecular weight of less than 10K. The results suggest that invasive secondary trophoblast cells possess a GalTase-mediated migration system that is functional on laminin.

Localization and expression of fibronectin during mouse decidualization in vitro: Mechanisms of cell:matrix interactions

During implantation, the embryonic trophoblast aggressively invades the uterine stroma. The resulting uterine reaction, decidualization, involves differentiation of new cell morphologies and remodeling of the extracellular matrix. This creates an environment that first permits invasion, then controls this invasion to allow the establishment of the placenta. The production, organization, and cellular interactions with the matrix are thought to underlie decidual functions. We have begun a reductional analysis of the components of the decidual matrix, focusing on extra‐cellular fibronectin (FN). Using decidual cell cultures prepared from day 7 implantation sites, the synthesis, extracellular organization, and details of decidual cell:FN interaction were studied. Employing immunofluorescence, immunoprecipitation, and dot blot analysis, decidualizing cultures showed a constitutive level of FN synthesis and deposition. The differentiating cells organized extracellular FN in patterns similar to that seen in vivo. The predominant, flattened dendritic decidual cells organized FN in long, thin fibrils. Large, rounded decidual cells, limited to the primary decidual zone in vivo, showed FN limited to punctate membrane patches and short, thick fibrils. Using double labeling techniques, FN expression was co‐localized with actin microfilament (MF) bundles during the cytoskeletal changes associated with the differentiation of both decidual cell types. The function of MFs in maintaining morphology was demonstrated by cytochalasin B perturbation. Attachment of decidual cells to FN was calcium dependent and gly‐arg‐gly‐asp‐ser‐pro (GRGDSP) sensitive, with dendritic decidual cells expressing the α5 and β1 integrin subunits. This suggests that an integrin system functions to attach decidual MF bundles to extracellular FN. This work shows that during decidual matrix remodeling, constitutive levels of FN are maintained to provide an extracellular framework to stabilize the decidual cytoskeleton and support morphological differentiation of decidual cells.

INTERACTION OF MOUSE ECTOPLACENTAL CONE TROPHOBLAST AND UTERINE DECIDUA IN VITRO

SummaryDuring the peri-implantation stages of mouse development, the secondary trophoblast invades into the uterine decidua. This uniquely controlled invasive process results in the formation of the placenta. We have analyzed this process in vitro using cultures of decidua and microdissected ectoplacental cones from Day 7 embryos. The results showed that the interaction between these two cell types is comparable to that seen in vivo. Morphologically, the decidua maintained close contact with the spreading trophoblast, limiting its invasion and producing a multilayered trophoblast outgrowth. Attachment to the decidua was not mediated through cell-matrix binding, but the subsequent invasion into the decidua was dependent on normal matrix interactions. Secretion of proteinases by the trophoblast also seemed to be a requirement for successful invasion, but not attachment.

Deletion mapping of the Tt complex: Evidence for a second region of critical embryonic genes☆

The developmental effects of three different deletion mutations of the T/t complex of the mouse have been studied. The three mutations, TOak Ridge (OR), TOrleans (TOrl), and THair pin (THp), each produce a unique homozygous lethal phenotype: THp homozygotes fail to develop normally past the morula stage, TOrl homozygotes past the blastocyst stage, and TOR homozygotes past the egg cylinder stage. In compound embryos (TX/TY), the lethal phenotype observed corresponds to the shared length of deleted chromosome. This interaction allows the regions of chromosome 17, containing genetic information critical to early mammalian development, to be mapped.

Expression of a glucose-regulated cell surface protein in early mouse embryos.

A 100,000-Da glucose-regulated surface protein (100K-GRP) has previously been isolated from the cell surface and culture medium of human fibroblasts. A rabbit antiserum directed against this protein reacts with the cell surface of both human and murine cultured cells and with a broad spectrum of mammalian tissues. It is shown, via indirect immunofluorescence, that this protein is also present on cells of the developing mouse embryo and can be detected as early as the 4-cell stage. The 8-cell embryo and morula show positive surface labeling; the inner cell masses of both the pre- and postimplantation blastocysts are also positive but the trophectoderm is not. At the 6-day egg cylinder stage, the embryonic and extra-embryonic ectoderm label intensely with the antiserum and visceral endoderm shows faint labeling. No labeling can be detected on parietal endoderm or on the trophoblastic giant cells invading the uterine decidua. However, the internal cells of the ectoplacental cone exhibit bright fluorescence. The same pattern is observed on 7- to 8.5-day embryos, except that at this stage no label is associated with the visceral endoderm. In addition, mesodermal cells emerging from the primitive streak are also labeled.

Distribution of a 100K Dalton glucose-regulated cell surface protein in mammalian cell cultures and sectioned tissues

Abstract Previously, we described a glucose-regulated cell surface glycoprotein (100K-GRP) which is present on the surfaces of non-transformed human fibroblasts. The 100K-GRP is released into the culture media in a soluble form. In this report, we utilize an antibody raised to the 100K-GRP and indirect immunofluorescence to localize the 100K-GRP on human and mouse cells in vitro, and on mouse tissues in vivo. On cultured cells, the 100K-GRP is associated with fibers in the pericellular matrix. Examination of sectioned mouse tissues reveals that the 100K-GRP is present in a wide variety of tissues. However, in contrast to fibronectin, it is not found in extracellular structures such as basement membranes. In vivo, the 100K-GRP is only found in close association with the surfaces of individual cells.

3T3 cell surface galactosyltransferase is a calcium-dependent adhesion molecule for collagen type IV.

Cell surface galactosyltransferase (GalTase) has been previously shown to mediate cell spreading or migration on laminin matrices. This work demonstrates that 3T3 cell surface GalTase also mediates cell attachment to collagen type IV. Attachment to collagen type IV was blocked by perturbations of GalTase or substrate pregalactosylation on cells possessing only calcium-dependent mechanisms of adhesion. Cells with both calcium-dependent and calcium-independent systems were not affected by GalTase perturbation. Collagen type IV was shown to possess GalTase substrates since matrices could be galactosylated by both soluble enzyme and 3T3 cells.

Developmental regulation of the monoclonally defined IIC3 antigen during primary and secondary trophoblast differentiation in vitro

The monoclonally defined IIC3 antigen has been found to be developmentally regulated during primary and secondary trophoblast differentiation in the mouse. Cell surface expression of the antigen was associated only with diploid and tetraploid trophoblast cell types. Endoreduplication to 8C DNA in differentiating trophoblast giant cells was associated with a loss of IIC3 cell surface expression and appearance of cytoplasmic expression. This developmental change was not temporally regulated, but dependent on the attachment and outgrowth of the trophoblast in vitro. The surface antigen was neither shed into the media nor masked by glycosylation, but was apparently internalized by the trophoblast giant cells.

Immunofluorescent localization of a monoclonally defined carbohydrate cell surface antigen (IIC3) during mouse development

A monoclonal antibody (anti-IIC3), raised against F9 embryonal carcinoma cells, detects an antigen which is first expressed at the compacted morula stage and segregates with the trophectoderm of the mouse blastocyst. We have further examined the expression of this antigen during embryonic development. Immunofluorescence experiments on sectioned embryos demonstrate that IIC3 expression is associated with the differentiation of extra-embryonic cell types. It is expressed at the cell surface of the trophectoderm of the attaching blastocyst and differentially by the two derivatives of this layer. The primary and secondary trophoblastic giant cells label intracellularly, whereas the cells of the ectoplacental cone and labyrinth placenta label at the cell surface. IIC3 is also expressed by the primitive endoderm of the blastocyst and subsequently by the visceral endoderm. The parietal endoderm does not express IIC3. Partial characterization of the IIC3 antigen with sugar hapten inhibition and glycosidase digestion experiments, suggests that the antigen is a lactosaminoglycan-like molecule, with galactose and N-acetylgalactosamine residues representing part of the antigenic determinant. Neuraminidase and fucosidase treatment exposed additional anti-IIC3-antigenic sites on the extra-embryonic ectoderm and chorion. A possible role for IIC3 in normal embryonic-uterine interactions is discussed.