First published online 2 July 2003
doi: 10.1242/jcs.00648
Involvement of connexin 43 in human trophoblast cell fusion and differentiation
Jean-Louis Frendo1,
Laurent Cronier2,
Gwladys Bertin1,
Jean Guibourdenche1,3,
Michel Vidaud4,
Danièle Evain-Brion1 and
André Malassiné1,*
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
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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.
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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).
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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.
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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.
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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.
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© The Company of Biologists Ltd 2003
