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First published online 2 July 2003
doi: 10.1242/jcs.00648

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Involvement of connexin 43 in human trophoblast cell fusion and differentiation

¹ Institut National de la Santé et de la Recherche Médicale, U427, Paris, France
⁴ Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris, France
² Faculté des Sciences, Poitiers, France
³ Service d'Hormonologie, Hôpital Robert Debré, Paris, France

View larger version (54K): [in a new window]   Fig. 1. Effects of Cx43 antisense on Cx43 protein production in human trophoblasts. (A) Oligonucleotide uptake by primary cytotrophoblasts. After 4 hours of culture, cytotrophoblasts were incubated with FITC-labeled scrambled oligonucleotide for 1 hours, 2 hours, 24 hours and 48 hours. At each time-point, cells were washed three times in PBS, fixed and analyzed by fluorescence microscopy. Nuclei were stained in blue by DAPI. Intensely fluorescent green cells have internalized the scrambled antisense oligonucleotide (FITC) (x600). (B) Immunodetection of Cx43 in cytotrophoblast cells isolated from normal placentas. Cells were treated for 48 hours with a scrambled (control) or a specific Cx43 antisense (Cx43 antisense). Cell nuclei were labeled with DAPI (blue immunofluorescence). In the control, Cx43 punctuate immunofluorescence (IF) can be observed around nuclei and at the borders with neighboring trophoblastic cells. In cells treated with Cx43 antisense, the level of IF is largely decreased (x1000). (C) Cx43 protein levels in trophoblast cells after treatment with a scrambled antisense (control) or with a specific Cx43 antisense (Cx43 AS) were determined by western blotting using a mouse anti-Cx43 monoclonal antibody. An anti-actin monoclonal antibody was used as a standard. Proteins obtained from rat brain lysate were used as a positive control. One representative experiment out of the three performed is shown.

View larger version (81K): [in a new window]   Fig. 2. Effects of Cx43 antisense on the morphological differentiation of trophoblasts. Cytotrophoblasts isolated from human placentas were cultured in the presence of a scrambled antisense (Control) or with a specific Cx43 antisense (Cx43 antisense). After 72 hours of culture, the cells were fixed, immunostained with anti-desmoplakin monoclonal antibody and counterstained with DAPI. Large syncytia were observed in control cells (a) as immunofluorescent staining disappeared when cells have fused to form the syncytiotrophoblast. By contrast, immunoflurescent staining can be observed at the boundaries between aggregated cytotrophoblasts in cells treated with a specific Cx43 antisense (b).

View larger version (18K): [in a new window]   Fig. 3. Cell fusion index. Human cytotrophoblasts were incubated with a scrambled antisense (dark columns) or with a specific Cx43 antisense (white columns). After 24 hours (upper panel), 48 hours (middle panel) and 72 hours (lower panel), the cells were fixed, immunostained with anti-desmoplakin monoclonal antibody and counterstained with DAPI. One hundred syncytia were scored after staining and the nuclei were counted in each syncytium. Data show the distribution of syncytia as a function of the number of nuclei per syncytium. The figure illustrates the mean±s.e.m. of three independent experiments.

View larger version (30K): [in a new window]   Fig. 4. Functional intercellular communication measured by the gap-FRAP method. Upper four panels: typical computer-generated images of fluorescence distribution in villous trophoblastic cells cultured in the presence of scrambled antisense for 48 hours measured during a gap-FRAP experiment. After a prebleach scan (A), the fluorescent dye was photobleached in some selected cells (polygons 1 and 2) by means of a strong laser illumination. Isolated cells (polygon 4) kept unbleached served as a control for the spontaneous fading of fluorescent emission (B). The evolution of fluorescence intensities was measured starting just after photobleaching for 12 minutes with a scanning period of 2 minutes. After 12 minutes (C), a fluorescence recovery had occured in area 1, whereas the fluorescence intensity remained weak in area 2, indicating that cell 2 is not coupled to neighboring cells. D represents curves of fluorescence evolution in selected cells: fluorescence recovery in cell 1 follows a closely exponential time-course, reflecting the presence of open gap junctional channels. Note the low decrease of fluorescence intensity in the control unbleached cell (4) due to repeated scanning. Lower panel: percentage of coupled cells between villous trophoblastic cells after 48 hours of culture in the presence of scrambled antisense (dark column) or Cx43 antisense (white column). Coupled cells were characterized by an exponential time-course of fluorescence recovery from neighboring cells into a photobleached test cell. Functional communication was measured between cytotrophoblastic cells, between cyto- and syncytiotrophoblasts and between syncytiotrophoblasts. The number of intercellular contacts analyzed is indicated on top of the bars.

View larger version (26K): [in a new window]   Fig. 5. hCG expression and secretion. (A) hCGß mRNA levels were determined by real-time quantitative RT-PCR in cytotrophoblasts treated with a scrambled antisense used as a control (dark columns) or with a specific Cx43 antisense (white columns). These assays were carried out 24 hours, 48 hours and 72 hours after plating. Values are the levels of hCGß mRNA normalized to the level of PPIA (cyclophilin A) mRNA. (B) Amounts of hCG secreted into the culture medium at 24 hours, 48 hours and 72 hours of culture in the presence of a scrambled antisense (dark columns) or of a specific Cx43 antisense (white columns). Values are means from three separate dishes±s.e.m. and the figure illustrates one representative experiment from the three performed. *P<0.05. Values of ßhCG mRNA and hCG secretion in the three independent experiments are shown in the tables. Graphs are representative of experiment 1.