Distribution of human endogenous retrovirus type W receptor in normal human villous placenta.

BACKGROUND: The fusion of trophoblast cells into the villous syncytiotrophoblast is crucial for appropriate placental function and fetal development. Fusion occurs following the interaction of syncytin-1, an envelope protein of the endogenous retrovirus HERV-W, and the RD114/mammalian type D retrovirus receptor (RDR/ASCT2) on adjacent cell membranes. This process must be tightly regulated in order to maintain the proliferative pool of cytotrophoblast cells as well as the function of the syncytia. AIM: We sought to investigate whether syncytial fusion of placental cytotrophoblast cells may be regulated via modulation of RDR/ASCT2 expression. METHODS: Expression of RDR/ASCT2 in term and first trimester villous placenta was assessed along with a number of molecular markers using immunofluorescent staining. In a complementary approach, Western blotting was used to investigate RDR/ASCT2 expression in a panel of choriocarcinoma cell lines before and after stimulation of fusion. RESULTS: Villous placental RDR/ASCT2 expression was found to be restricted to the cytotrophoblast compartment, being largely absent in the syncytiotrophoblast. Local variations in RDR/ASCT2 expression were not associated with the proliferative status of cytotrophoblast cells. RDR/ASCT2 expression was also shown to be down-regulated in BeWo choriocarcinoma cells after stimulation of syncytial fusion. CONCLUSION: This first report of the localisation and distribution of RDR/ASCT2 in human placental villi suggests that the fusion of placental trophoblast cells is not regulated by local or temporal variations of RDR/ASCT2 expression in villous cytotrophoblast cells.

Syncytin: the major regulator of trophoblast fusion? Recent developments and hypotheses on its action.

Syncytin is a membrane protein derived from the envelope gene of an endogenous retrovirus of the HERV-W family. The gene appears to be almost exclusively expressed in placenta; the protein was found in particular in syncytiotrophoblast. After transfection into various cell types it has proven to be a very fusogenic protein, inducing the formation of syncytia. Therefore, the question rises as to whether syncytin is responsible for the fusion process of villous cytotrophoblast into syncytiotrophoblast in vivo. If so, how is this fusion process regulated if syncytin is found all over the syncytiotrophoblast? Can this process be regulated through local or temporal changes in syncytin expression, or is syncytin merely one factor in a cascade of events leading to fusion limited at some other level? This review will try to summarize the published data on the regulation of fusion in trophoblast models as well as on the localization and regulation of syncytin expression and of its presumed receptors. Assuming that syncytin is the key factor inducing trophoblast fusion, a number of models will be presented by which syncytin and/or its receptors might regulate this process. In some of the hypotheses proposed, local coexpression of syncytin and receptor, leading to blocking of one factor by the other, is of functional relevance.

A two-colour fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines.

Syncytial fusion is a key event in implantation and placentation. Its regulation is only poorly understood. We present a cell-cell fusion assay based on staining of cells in two portions with a green and a red fluorescent cytoplasmic dye that become intracellularly mixed only after syncytial fusion. We quantified cell-cell fusion by fluorescence microscopy in choriocarcinoma cell lines BeWo, JAR and JEG3 and in some non-trophoblastic cell lines and found clear differences in fusion behaviour. Only BeWo cells fused with each other, while the other cell lines tested did not. BeWo cells also fused with all other cell lines tested. The efficiency of cell-cell fusion of BeWo cells was stimulated by forskolin. We tried to correlate messenger levels of syncytin and its receptor RDR with the fusion index of choriocarcinoma cells. BeWo and JAR cells contained readily detectable and forskolin-inducible levels of syncytin mRNA, whereas this messenger was barely detectable in JEG3 cells. RDR transcript levels were similar in all cell lines tested and were unaffected by forskolin treatment. The data suggests that the expression of syncytin and RDR messengers alone does not guarantee successful fusion. The fusion assay presented in this paper is a useful tool to study syncytial fusion in an accurate and quantitative way.

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity.

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.

Mechanisms of syncytial fusion: a review.

Syncytial fusion of trophoblast is a key process in placental morphogenesis and physiology. Disturbed syncytial fusion may lead to a number of pregnancy-associated pathologies. The mechanisms regulating syncytial fusion are only partly understood. This review tries to summarize the available knowledge on trophoblast fusion, originating from different scientific disciplines. Among the themes addressed in this paper are: morphogenesis and functions of syncytiotrophoblast; early apoptotic events and changes in plasmalemmal phospholipid orientation; proteins involved in membrane fusion: ADAMs and retrovirally-derived proteins and short-lived proteolipid intermediates in membrane fusion. Deeper understanding of syncytiotrophoblast fusion in future studies is only to be anticipated from collaborative studies focusing in parallel on physicochemical events in the participating plasmalemmas, early apoptotic/differentiation events preceding the fusion and role of the fusogenic membrane proteins.