First published online 3 July 2008
doi: 10.1242/dev.019810
Development 135, 2659-2668 (2008)
Published by The Company of Biologists 2008
Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival
Mary J. Laws1,
Robert N. Taylor3,
Neil Sidell3,
Francesco J. DeMayo4,
John P. Lydon4,
David E. Gutstein5,
Milan K. Bagchi2 and
Indrani C. Bagchi1,*
1 Department of Veterinary Biosciences, and University of Illinois
Urbana/Champaign, Urbana, IL 61802, USA.
2 Department of Molecular and Integrative Physiology,
3 Department of Gynecology and Obstetrics, Emory University School of Medicine,
Atlanta, GA 30322, USA.
4 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA.
5 New York University School of Medicine, NY 10016, USA.
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Fig. 1. Induction of Cx43 expression in uterine stromal cells during
implantation. (A) Mice were subjected to delayed implantation as
described in the Materials and methods. Uteri were collected 1 hour (b) and 24
hours (c,d) after E treatment and subjected to immunohistochemistry using
anti-Cx43 antibody. (d) Higher magnification view of the 24-hour sample. The
0-hour sample (a) represents pregnant uterus obtained from a mouse treated
with P alone for three days. Results are from two independent experiments.
(B) Uteri were collected in the morning of day 1 (a), day 4 (b), day 5
(c,d) and day 7 (e,f) of pregnancy. Higher magnifications of Cx43 expression
in stromal cells surrounding the implanted embryo on day 5 and day 7 are shown
in d and f, respectively. Results are from two independent experiments.
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Fig. 2. Loss of Cx43 expression in the uterus of the Cx43
conditional-knockout mouse. (A,B) Uterine sections from
Cx43fl/fl (control) mice show prominent Cx43 expression in
the stromal cells surrounding the embryo. (C,D) Uterine sections
from Cx43d/d (conditional knockout) mice show efficient
ablation of Cx43 in uterine stromal cells. E denotes implanted embryo.
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Fig. 3. Impaired embryo development and lack of angiogenesis in uteri of
Cx43 conditional knockout mice on day 8 of gestation. (A)
Hematoxylin and Eosin staining of uterine sections obtained from
Cx43fl/fl (a,b) and Cx43d/d (c,d) mice
on days 7 and 8 of pregnancy. Note that implanted embryos (E) develop poorly
in Cx43d/d mice compared with those in
Cx43fl/fl mice. Histological analysis by Hematoxylin and
Eosin staining of perfused uterine sections obtained from pregnant
Cx43fl/fl (e) and Cx43d/d (f) mice on
day 8 of pregnancy. E, implanted embryo; M, the mesometrial region. Note the
extensive vasculature in the uteri of Cx43fl/fl mice and
the lack of it in the uteri of Cx43d/d mice. (B)
Immunohistochemical staining (indicated by red deposits) for Pecam in pregnant
uteri. Uterine sections of Cx43fl/fl (a-c) and
Cx43d/d (d-f) mice on day 7 (a,b,d,e) and day 8 (c,f) of
pregnancy are shown (n=3). (C) Uterine sections collected from
mice on day 8 of pregnancy were subjected to immunoflouroscence employing
anti-connexin 43 (a) and anti-Pecam1 (b) antibodies. c represents a merge of
both Cx43 and Pecam staining indicating the expression of Cx43 in the stromal
cells but not in the endothelial cells (n=2). (D) Uterine
sections from Cx43fl/fl (a) and
Cx43d/d (b) mice on day 8 of gestation were subjected to
immunohistochemistry using an antibody against Ki67, a marker of cell
proliferation (n=2).
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Fig. 4. Cx43-deficient uteri exhibit impaired decidualization.
(A-D) Uterine sections from Cx43fl/fl and
Cx43d/d mice on day 7 (A,B) and day 8 (C,D) of pregnancy
were subjected to immunohistochemical staining using an antibody specific for
the prolactin-related protein (PRP). (E,F) Uterine sections from
Cx43fl/fl (E) and Cx43d/d (F) mice on
day 8 of pregnancy were subjected to immunohistochemical staining using an
antibody specific for the prolactin-like protein (PLP). AM and M denote
antimesometrial and mesometrial regions, respectively. Results are from two
independent experiments.
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Fig. 5. Cx43 is essential for decidual response and angiogenesis. (A)
Expression of Cx43 in artificially decidualized uterus. Mice were subjected to
artificial decidual stimulation. Uteri were collected at 0 (a) and 72 (b,c)
hours following the application of the stimulus. Uterine sections were
analyzed for Cx43 expression by immunohistochemistry. c is a higher
magnification of the 72-hour sample. (B, upper) Gross morphology.
Cx43fl/fl (left) and Cx43d/d (right)
mice were subjected to artificial decidual stimulation for 48 hours as
described in the Materials and methods. For each mouse, one uterine horn was
stimulated, while the other horn was left undisturbed. The stimulated horn is
indicated as `s' and the unstimulated one as `us'. (Middle) Comparative wet
weight gains in uteri of Cx43fl/fl and
Cx43d/d mice. Following artificial decidualization,
stimulated and unstimulated horns were assessed for wet weight gain. The
histogram shows the ratios of average weights of stimulated over unstimulated
horns from Cx43fl/fl and Cx43d/d mice.
The data are represented as means±s.e.m. (n=4). (Lower)
Expression of Hoxa10 and Bmp2 mRNA in the uteri of
Cx43fl/fl and Cx43d/d mice. Total RNA
was isolated from uteri collected 96 hours after the application of the
artificial decidual stimulus and qPCR analysis was performed using
gene-specific primers. (C) Immunohistochemical staining for Pecam in
artificially decidualized uteri. Uterine sections of
Cx43fl/fl (a) and Cx43d/d (b) mice are
shown. The results are representative of two independent experiments.
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Fig. 6. Reduced expression of angiogenic factors in Cx43-deficient
uteri. (A) Uterine sections from Cx43fl/fl mice
on day 7 (a) and day 8 (b) and those from Cx43d/d mice on
day 7 (c) and day 8 (d) were subjected to immunohistochemical analysis using
anti-Vegf antibody. E, implanted embryo; M, the mesometrial region. (B)
qPCR analysis was performed to monitor angiopoetin 2 and angiopoetin 4 mRNA
expression in the uteri of Cx43fl/fl and
Cx43d/d mice on day 8 of pregnancy (n=3).
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Fig. 7. Attenuation of CX43 expression in human endometrial stromal cells leads
to impaired gap junction communication, decidualization and a reduction in
VEGF production. (A) Western blotting was used to analyze the
expression of CX43 in HESC-T cells stably transfected with a CX43 siRNA
(HESC-T3) or a nonsilencing control vector (HESC-TC). Pronounced silencing of
CX43 protein was observed in HESC-T3 cells, whereas the levels of beta actin
protein remained unaltered. (B) Gap junction intercellular
communication was determined using a double-labeling technique. Donor cells
were double labeled with the fluorescent dyes calcein (gap junction permeable
dye, green fluorescence) and DiI (gap junction impermeable dye, red
fluorescence), and were then placed in contact with unloaded cells in the
monolayer. Dye transfer was visualized after 2 hours. HESC-T3 cells with
reduced CX43 expression exhibited lack of green dye diffusion. Arrows show
examples of functional coupling between HESC-TC cells. (C) HESC-TC and
HESC-T3 cells were grown in DMEM/F-12 medium containing 5% charcoal-stripped
FBS. To induce in vitro decidualization, they were treated with or without a
hormone cocktail containing 1 nM E, 1 mM P and 0.5 mM 8-bromo-cAMP for 7-11
days (Ryan et al., 1994 ). When
the cells were examined morphologically, a distinct transition from
fibroblastic to a plump, epitheloid phenotype, characteristic of decidual
cells, was observed in control HESC-TC cells. Low CX43-expressing HESC-T3
cells failed to show this morphological transformation. (D) HESC-TC and
HESC-T3 cells were grown for 24 hours in the absence (Con) or presence (TPA)
of 50 nM phorbol ester. The VEGF in supernatant was measured in duplicate by
ELISA.
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