Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and peri-implantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.     

Histamine enhances cytotrophoblast invasion by inducing intracellular calcium transients through the histamine type-1 receptor

Blastocyst implantation and placentation require molecular and cellular interactions between the uterine endometrium and blastocyst trophectoderm. Previous studies showed that histamine produced in the mouse uterine luminal epithelium interacts with trophoblast histamine type-2 receptors (H2) to initiate blastocyst implantation. However, it is unknown whether similar histamine activity is operative in humans. Using a human cell line (HTR-8/SVneo) derived from first-trimester cytotrophoblasts that expresses both histamine type-1 receptor (H1) and H2, we found that histamine promotes cytotrophoblast invasiveness specifically through activation of H1. Stimulation of H1 in human cytotrophoblasts by histamine induced intracellular Ca2+ (Ca(2+)i) transients by activating phospholipase C and the inositol trisphosphate pathway. The enhanced invasion induced by histamine was blocked by pretreatment with H1 antagonist or by chelation of Ca(2+)i. These findings suggest possible differences between rodents and humans in histamine signaling to the trophoblast.     

Trophoblast giant-cell differentiation involves changes in cytoskeleton and cell motility

Trophoblast giant-cell differentiation is well-characterized at the molecular level, yet very little is known about how molecular changes affect the cellular functions of trophoblast in embryo implantation. We have found, using both explanted E7.5 mouse embryo ectoplacental cone and the rat choriocarcinoma (Rcho-1) cell line, that trophoblast differentiation is distinguished by dramatic changes in cytoarchitecture and cell behavior. Undifferentiated trophoblast cells contain little organized actin and few small, peripheral focal complexes and exhibit high membrane protrusive activity, while differentiated trophoblast giant cells contain prominent stress fibers, large internal as well as peripheral focal adhesions, and become immotile. The dramatic changes in cell behavior and cytoskeletal organization of giant cells correlate with changes in the activities of the Rho family of small GTPases and a decrease in tyrosine phosphorylation of focal adhesion kinase. Together, these data provide detailed insight into the cellular properties of trophoblast giant cells and suggest that giant-cell differentiation is characterized by a transition from a motile to a specialized epithelial phenotype. Furthermore, our data support a phagocytic erosion, rather than a migratory infiltration, mechanism for trophoblast giant-cell invasion of the uterine stroma.     

Molecular mechanisms of trophoblast survival: from implantation to birth

Fetal development depends upon a coordinated series of events in both the embryo and in the supporting placenta. The initial event in placentation is appropriate lineage allocation of stem cells followed by the formation of a spheroidal trophoblastic shell surrounding the embryo, facilitating implantation into the uterine stroma and exclusion of oxygenated maternal blood. In mammals, cellular proliferation, differentiation, and death accompany early placental development. Programmed cell death is a critical driving force behind organ sculpturing and eliminating abnormal, misplaced, nonfunctional, or harmful cells in the embryo proper, although very little is known about its physiological function during placental development. This review summarizes current knowledge of the cell death patterns and molecular pathways governing the survival of cells within the blastocyst, with a focus on the trophoblast lineage prior to and after implantation. Particular emphasis is given to human placental development in the context of normal and pathological conditions. As molecular pathways in humans are poorly elucidated, we have also included an overview of pertinent genetic animal models displaying defects in trophoblast survival.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the initial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Mechanisms of trophoblast migration,endometrial angiogenesis in preeclampsia: The role of decorin

abstract

The objective of the present review is to synthesize the information on the cellular and molecular players responsible for maintaining a homeostatic balance between a naturally invasive human placenta and the maternal uterus in pregnancy; to review the roles of decorin (DCN) as a molecular player in this homeostasis; to list the common maladies associated with a break-down in this homeostasis, resulting from a hypo-invasive or hyper-invasive placenta, and their underlying mechanisms. We show that both the fetal components of the placenta, represented primarily by the extravillous trophoblast, and the maternal component represented primarily by the decidual tissue and the endometrial arterioles, participate actively in this balance. We discuss the process of uterine angiogenesis in the context of uterine arterial changes during normal pregnancy and preeclampsia. We compare and contrast trophoblast growth and invasion with the processes involved in tumorigenesis with special emphasis on the roles of DCN and raise important questions that remain to be addressed. Decorin (DCN) is a small leucine-rich proteoglycan produced by stromal cells, including dermal fibroblasts, chondrocytes, chorionic villus mesenchymal cells and decidual cells of the pregnant endometrium. It contains a 40 kDa protein core having 10 leucine-rich repeats covalently linked with a glycosaminoglycan chain. Biological functions of DCN include: collagen assembly, myogenesis, tissue repair and regulation of cell adhesion and migration by binding to ECM molecules or antagonising multiple tyrosine kinase receptors (TKR) including EGFR, IGF-IR, HGFR and VEGFR-2. DCN restrains angiogenesis by binding to thrombospondin-1, TGFβ, VEGFR-2 and possibly IGF-IR. DCN can halt tumor growth by antagonising oncogenic TKRs and restraining angiogenesis. DCN actions at the fetal-maternal interface include restraint of trophoblast migration, invasion and uterine angiogenesis. We demonstrate that DCN overexpression in the decidua is associated with preeclampsia (PE); this may have a causal role in PE by compromising endovascular differentiation of the trophoblast and uterine angiogenesis, resulting in poor arterial remodeling. Elevated DCN level in the maternal blood is suggested as a potential biomarker in PE.     

Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia

Maternal uteroplacental blood flow increases during pregnancy. Altered uteroplacental blood flow is a core predictor of abnormal pregnancy. Normally, the uteroplacental arteries are invaded by endovascular trophoblast and remodeled into dilated, inelastic tubes without maternal vasomotor control. Disturbed remodeling is associated with maintenance of high uteroplacental vascular resistance and intrauterine growth restriction (IUGR) and preeclampsia. Herein, we review routes, mechanisms, and control of endovascular trophoblast invasion. The reviewed data suggest that endovascular trophoblast invasion involves a side route of interstitial invasion. Failure of vascular invasion is preceded by impaired interstitial trophoblast invasion. Extravillous trophoblast synthesis of nitric oxide is discussed in relation to arterial dilation that paves the way for endovascular trophoblast. Moreover, molecular mimicry of invading trophoblast-expressing endothelial adhesion molecules is discussed in relation to replacement of endothelium by trophoblast. Also, maternal uterine endothelial cells actively prepare endovascular invasion by expression of selectins that enable trophoblast to adhere to maternal endothelium. Finally, the mother can prevent endovascular invasion by activated macrophage-induced apoptosis of trophoblast. These data are partially controversial because of methodological restrictions associated with limitations of human tissue investigations and animal studies. Animal models require special care when extrapolating data to the human due to extreme species variations regarding trophoblast invasion. Basal plates of delivered placentas or curettage specimens have been used to describe failure of trophoblast invasion associated with IUGR and preeclampsia; however, they are unsuitable for these kinds of studies, since they do not include the area of pathogenic events, i.e., the placental bed.     

Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo.

For implantation and placentation to occur, mouse embryo trophoblast cells must penetrate the uterine stroma to make contact with maternal blood vessels. A major component of the uterine epithelial basement membrane and underlying stromal matrix with which they interact is the extracellular matrix protein laminin. We have identified integrin alpha 7 beta 1 as a major receptor for trophoblast-laminin interactions during implantation and yolk sac placenta formation. It is first expressed by trophectoderm cells of the late blastocyst and by all trophectoderm descendants in the early postimplantation embryo through E8.5, then disappears except in cells at the interface between the allantois and the ectoplacental plate. Integrin alpha 7 expression is a general characteristic of the early differentiation stages of rodent trophoblast, given that two different cultured trophoblast cell lines also express this integrin. Trophoblast cells interact with at least three different laminin isoforms (laminins 1, 2/4, and 10/11) in the blastocyst and in the uterus at the time of implantation. Outgrowth assays using function-blocking antibodies show that alpha 7 beta 1 is the major trophoblast receptor for laminin 1 and a functional receptor for laminins 2/4 and 10/11. When trophoblast cells are cultured on substrates of these three laminins, they attach and spread on all three, but show decreased proliferation on laminin 1. These results show that the alpha 7 beta 1 integrin is expressed by trophoblast cells and acts as receptor for several isoforms of laminin during implantation. These interactions are not only important for trophoblast adhesion and spreading but may also play a role in regulating trophectoderm proliferation and differentiation.     

Conditional deletion of Msx homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs.     

The rat as an animal model for fetoplacental development: a reappraisal of the post-implantation period

Following implantation in rodents, the uterine stromal fibroblasts differentiate into densely packed decidual cells. This process, called decidualization, is well-orchestrated and progresses both antimesometrially and mesometrially, creating two regions with distinctive cellular morphologies. In addition, subsequent placental development is dependent on the invasion of the trophoblast, the process intimately linked to the endometrial tissue remodelling and depending largely on the environment created by the decidua; this phenomenon is crucial for the establishment and maintenance of pregnancy. The key mechanisms underlying the maternal tissue remodelling and trophoblast invasion remain poorly understood. The rat, just like human beings, exhibits a highly invasive type of placental development, the haemochorial placentation. For obvious ethical reasons, the studies of endometrial tissue remodelling throughout pregnancy in humans are greatly limited. Although the rat differs somewhat from humans with regards to the implantation process, it is an appropriate model for studying the mechanisms of decidualization as well as subsequent remodelling of the uterine tissues and fetoplacental development. As decidual remodelling is very closely linked to placentation and the maternal-fetal interactions in the rat show several important similarities to human placentation, the morphological alterations occurring during the post-implantation period in the rat have been addressed in the present review.     

Interaction of extravillous trophoblast galectin-1 and mucin(s)—Is there a functional relevance?

ABSTRACT

In the course of embryo implantation extensive interaction of the trophoblast with uterine tissue is crucial for adequate trophoblast invasion. This interaction is highly controlled, and it has been pointed out that a specific glycocode and changes in glycosylation may be important for successful implantation and maintenance of pregnancy. Both uterine and trophoblast cells have been shown to express cell surface glycoconjugates and sugar binding proteins, such as mucins (MUC) and galectins (gals). An increasing number of studies have investigated potential candidates interacting in this process. However, knowledge about the biochemical nature of the interactions and their importance for trophoblast cell function, and, consequently, for pregnancy outcome are still lacking. This review is aimed at deliberating the possibility that mucins, as heavily glycosylated proteins, might be among the functionally relevant galectin ligands in human trophoblast, based on both published data and our original research.     

Development of the implantation chamber in the pregnant bitch.

Uteri taken from 25 bitches at various times during the early stages of pregnancy were studies cytologically to determine how the implantation chamber developed and how fetal-maternal relations were established. On day 13 after the end of estrus, knobs of trophoblastic syncytium formed and became wedged between cells of the uterine luminal epithelium. The syncytium quickly spread along the uterine lumen and into the mouths of the glands, dislodging and surrounding maternal cells. As invasion continued trophoblastic villi, consisting of cores of cytotrophoblast covered by a continuous layer of syncytium, penetrated deeper into the endometrium. The syncytium spread to surround maternal vessels and decidual cells. By day 26 the trophoblast had extended down to the large lacunae. Here syncytial trophoblast covering tips of the villi degenerated, leaving cytotrophoblast exposed to the necrotic zone. These cells possessed characteristics of absorbing cells. Hematomas were formed by focal necrosis of fetal and endometrial tissue at the poles of the implantation sites. Large pools of extravasated blood accumulated and red blood cells were phagocytized by surrounding trophoblastic cells. Therefore, the endotheliochorial relationship in the canine placenta appeared to be established by syncytial trophoblast invading a cellular endometrium. In the necrotic zone and hematomas, cellular trophoblast may have lost its syncytial covering, but elsewhere maternal vessels and decidual cells in the placenta were in direct contact only with syncytial trophoblast.     

Expression and regulation of integrin receptors in human trophoblast cells: role of estradiol and cytokines

Embryo implantation and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-cell communication amongst the implanting blastocyst and the receptive endometrium through integrins, a large family of proteins involved in the attachment, migration, invasion and control of cellular functions. Integrins display dynamic temporal and spatial patterns of expression by the trophoblast cells during early pregnancy in humans. However, the precise mechanism of embryo implantation and the modulation of the integrin receptors during blastocyst attachment and further implantation remain elusive in the humans. The present study elucidates the expression and hormonal modulation of fibronectin, vitronectin and laminin integrin receptors by estradiol and IL-1alpha in human trophoblast cells. Human first trimester trophoblast cells showed the induction of the classical estrogen receptor (ER)-alpha by its own ligand, estradiol. Treatment with either estradiol or IL-1alpha induced the expressions of alpha4, alpha5, alpha6 and alpha(v) integrin receptor subunits at both the mRNA and protein levels, while expression of beta1 remained unaltered. Furthermore, estradiol upregulated the expression of IL-1alpha, thereby suggesting the possibility that estrogen may either directly or via the proinflammatory cytokine induces the expression of the cell surface integrin receptors. The findings delineate the role of hormones and the cytokines in modulating the adhesiveness and attachment of the trophoblast cells. This may reflect the in vivo scenario where the implanting embryo is surrounded by a hormone-cytokine rich uterine microenvironment that may precisely regulate the expression of integrins and thereby facilitate implantation.     

Adhesion of trophoblast to uterine epithelium as related to the state of trophoblast differentiation: in vitro studies using cell lines

At the initial phase of embryo implantation, the trophoblast must have acquired competence for adhesion to the uterine epithelium, a condition whose cell biological basis is far from understood. In the present study, trophoblast-type cells (BeWo, JAr, and Jeg-3 choriocarcinoma cell lines) were treated with retinoic acid, methotrexate, dibutyryl-cAMP, or phorbol-12-myristate-13-acetate in order to modulate their ability to adhere to uterine epithelial cells (RL95-2). In an established model, multicellular spheroids of choriocarcinoma cells were transferred onto the surface of monolayer cultures of RL95-2 cells followed by a centrifugal force-based adhesion assay. In controls, about 45% of BeWo and JAr cell spheroids and 75% of Jeg-3 spheroids adhered to uterine monolayers within 30 min. Pretreatment of spheroids with either of the agents stimulated differentiation as indicated by the rate of chorionic gonadotropin secretion, but consistently reduced the adhesion to the endometrial monolayer in all three choriocarcinoma cell lines. While previous investigations had shown that invasiveness of trophoblast cells (into extracellular matrix) does not seem to be linked to the differentiation program in a simple manner, the present data suggest that such an (inverse) link may indeed exist with respect to the ability to initiate an adhesive interaction with the uterine epithelium. These observations support the view that epithelial cell interactions as typical for the initial phase of embryo implantation are regulated in a way that is clearly different from cell-matrix interactions governing later phases of trophoblast invasion into the endometrial stroma.     

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

Summary In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the intial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.