Suzanne Afonso

ABSTRACT 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.

Trophoblast

Placentation

Decidualization

Cathepsin L

Cystatin

Decidua

Cathepsin E

10.1242/dev.124.17.3415

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Citations (192)

Control and expression of cystatin C by mouse decidual cultures

Molecular Reproduction and Development (2002)

Suzanne Afonso Chris Tovar Linda Romagnano Bruce Babiarz

Abstract 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‐β 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‐β 1 and EGF signaling. Mol. Reprod. Dev. 61: 155–163, 2002. © 2002 Wiley‐Liss, Inc.

Decidua

Trophoblast

Decidual cells

Cystatin

10.1002/mrd.1142

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Citations (30)

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

Developmental Dynamics (1996)

Bruce Babiarz Linda Romagnano Suzanne Afonso George Kurilla

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. © 1996 Wiley-Liss, Inc.

Decidual cells

Decidua

Decidualization

Trophoblast

10.1002/(sici)1097-0177(199607)206:3<330::aid-aja10>3.0.co;2-3

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Citations (31)

An in vitro system for the study of matrix metalloproteases during decidualization in the mouse

Biochemistry and Cell Biology (1996)

Linda Romagnano Suzanne Afonso Bruce Babiarz

Decidualization results in the remodeling of the extracellular matrix with the loss of collagen type I and the appearance of basement membrane matrix components. We have developed an in vitro assay system to study matrix metalloproteases during mouse decidualization. Uterine stroma, or decidua isolated from day 7.5 pregnant mice, were grown on a three-dimensional collagen type I matrix (Vitrogen). Gelatin zymography of conditioned media from these cultures showed constitutive secretion of processed forms of gelatinase A at 65, 62, and 59 kDa with 62 kDa predominating. Similar patterns of gelatinase A expression were obtained from tissue lysates of decidualizing uteri from days 5.5 to 7.5 of development. Cells cultured on Vitrogen, but not on plastic or matrix-coated dishes, were able to process the proenzyme to the 59 kDa form as observed in vivo. Only stroma cells cultured on a coating of collagen type I displayed the same increase in the 59 kDa zymogen. Decidua cells grown on Vitrogen attached and then migrated into aggregates that eventually penetrated the gel and spread as differentiated decidua on the underlying plastic. These preliminary results suggested that the in vitro assay system can be used to study the role of metalloproteases in matrix remodeling during decidualization.Key words: matrix metalloproteases, uterine stroma, decidua, gelatinase A, Vitrogen.

Decidualization

Matrix (chemical analysis)

Zymography

Decidua

10.1139/o96-096

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Citations (10)

Cellular Interactions and the Cysteine Proteinases in the Process of Mouse Implantation

Springer eBooks (1999)

Bruce Babiarz Suzanne Afonso Linda Romagnano

Placentation

Trophoblast

Decidualization

Decidua

10.1007/978-1-4612-1548-6_6

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Citations (0)

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

Developmental Dynamics (1996)

Bruce Babiarz Linda Romagnano Suzanne Afonso George Kurilla

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. © 1996 Wiley-Liss, Inc.

Decidual cells

Decidua

Decidualization

Trophoblast

10.1002/(sici)1097-0177(199607)206:3<330::aid-aja10>3.3.co;2-7

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Citations (2)

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

Developmental Dynamics (1999)

Suzanne Afonso Linda Romagnano Bruce Babiarz

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.

Trophoblast

Expression (computer science)

Cathepsin L

10.1002/(sici)1097-0177(199912)216:4/5<374::aid-dvdy6>3.0.co;2-n

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Citations (41)

Suzanne Afonso

Papers

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Papers

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