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.

During mouse embryo implantation, trophoblast invasion is controlled in part by a balance of trophoblast-derived proteinases and uterine decidual proteinase inhibitors. Our work has focused on cystatin C, the secreted inhibitor of cathepsins B and L. We have previously shown that cystatin C is synthesized by the uterine decidua and localized to the cells in close contact with the trophoblast during implantation in vivo. In the work reported here we have established that decidualizing cultures show a similar upregulation of cystatin C. Using Northern and Western blotting and immunolocalization techniques both cystatin C mRNA and secreted protein increased with the morphological differentiation of stromal or decidual capsule cultures. In an effort to understand the regulation of cystatin C expression, decidual cells were analyzed under various culture conditions. Cystatin C expression was upregulated by increased cell density and by the presence of serum in the media. The growth factors TGF-beta(1) and EGF were found to induce cystatin C to levels comparable to serum stimulation. Co-culture with ectoplacental cones (EPCs) likewise induced expression and resulted in the localization of cystatin at the decidua:trophoblast interface. This work shows that decidualizing cultures are a good system to study cystatin C expression and that the expression is controlled in part by TGF-beta(1) and EGF signaling.

The implantation of the mouse embryo requires the controlled invasion of the uterine stroma by the embryonic trophoblast. This event is dependent, in part, on the secretion of matrix metalloproteinases and serine proteinases for the extracellular degradation of the uterine matrix. Proteinase activity is controlled by stromal decidualization and specific proteinase inhibitors. This work adds to our understanding of implantation and placentation by reporting the expression and function of another class of proteinases/inhibitors closely related to invasive cell behavior. We focused on the cysteine proteinases, cathepsins B and L, and their inhibitor cystatin C. Northern blots showed that trophoblast expressed cathepsin B throughout the invasive period (days 5.5-10.5). Both cathepsin B message and cathepsin L protein were localized to the mature, invasive trophoblast giant cells. Substrate gel electrophoresis showed an increase in giant cell cathepsin activity with enzyme profiles changing at the end of the invasive period. Northern and western blotting showed that cystatin C, the main inhibitor of cathepsins, was a major product of the decidualizing stroma. Message levels first increased in peripheral decidualizing cells, with the protein localizing close to the embryo during implantation (days 5.5-8.5). With the regression of the decidua beginning on day 9.5, a coordinated upregulation of both cathepsin B and cystatin C was observed implying a role for controlled cathepsin expression during apoptosis. E-64, a synthetic inhibitor of cathepsins B and L, was injected into pregnant females at the stage of blastocyst attachment (days 4.5-5.5). High doses resulted in the complete failure of implantation while lower doses resulted in stunted embryos and a reduced decidual reaction. These results suggested that cathepsins B and L are necessary for normal embryo development and uterine decidualization, and that decidua contributes to their control by a coordinated expression of cystatin C within the implantation site.

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 extracellular fibronectin (FN). Using decidual cell cultures prepared from day 7 implantation sites, the synthesis, extracellular organization, and details of decidual celkFN 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 expresssion 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 a5 and PI 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.

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