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First published online 3 July 2008
doi: 10.1242/dev.019810
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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.
* Author for correspondence (e-mail: ibagchi{at}uiuc.edu)
Accepted 20 May 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Implantation, Endometrium, Neovascularization, Estrogen, Connexin 43, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Yoshinaga,
1988; Parr and Parr,
1989; Weitlauf,
1994). In the mouse and the human, as the embryo invades into the
stromal compartment, the fibroblastic stromal cells undergo differentiation
into a unique secretory tissue, known as the decidua. This transformation
process, known as decidualization, is crucial for executing the extensive
tissue remodeling that ensures proper maternal-fetal interactions, leading to
the establishment of pregnancy. Decidualization is also accompanied by the
creation of an extensive vascular network within the stromal bed that supports
embryonic and placental growth and maintains early pregnancy
(Cross et al., 1994;
Irwin and Giudice, 1999;
Carson et al., 2000).
During angiogenesis, new blood vessels are generated by the extension of
pre-existing vessels into avascular space. This process involves the local
degradation of the vascular basal membrane by proteases, proliferation and
migration of endothelial cells, and assembly of these cells into new vessels
(Folkman, 1995;
Hyder and Stancel, 1999). In
the female reproductive system, an active angiogenesis is required to support
the cyclic remodeling of the uterus. In the human and the non-human primates,
the spiral arteries that supply the functionalis layer of the endometrium
increase in length, branching and coiling during each menstrual cycle as the
endometrium is regenerated (Hyder and
Stancel, 1999). In rodents, a link between the steroid-driven
stromal differentiation program and active neovascularization within the
pregnant uterus has long been speculated, although the underlying mechanisms
are unknown. One of the earliest signs of a uterine response to an angiogenic
stimulus is an increase in microvascular permeability at the sites of
implantation (Chakraborty et al.,
1995; Rockwell et al.,
2002). E is recognized as a regulator of this phenomenon
(Chakraborty et al., 1995;
Rockwell et al., 2002).
However, the precise nature of the hormone-regulated pathways that influence
uterine angiogenesis remains unclear and controversial. Particularly
intriguing is a previous report that E is an inhibitor and P is a stimulator
of uterine angiogenesis (Ma et al.,
2001). As this study was performed using ovariectomized
non-pregnant mice following treatment with steroid hormones, the relevance of
these findings under normal pregnancy conditions is questionable. A major
challenge in reproductive medicine is, therefore, to gain a clear
understanding of the steroid hormone-regulated pathways that control
pregnancy-associated endometrial neovascularization in the stromal
compartment. The present study was undertaken to uncover and functionally
characterize these pathways.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to induce superovulation, 7- to 8-week-old female mice were injected intraperitoneally with 5 IU of pregnant mare serum gonadotrophin (PMSG) and 48 hours later with 5 IU of human chorionic gonadotropin (hCG). The mice were killed 16-18 hours post-hCG administration, and oocytes were flushed from the oviducts and counted.
To induce and maintain delayed implantation, mice were ovariectomized on day 4 (morning) of pregnancy and injected daily with P (2 mg) from days 5-7. To terminate delayed implantation and induce blastocyst attachment, the P-primed delayed implanting mice were given an injection of E (50 ng) on the fourth day of the delay (day 8). Mice were killed at different time points after E injection and uteri were collected.
Decidualization was experimentally induced in non-pregnant mice as
described previously (Cheon et al.,
2004). Mice were first ovariectomized. Two weeks following
ovariectomy, animals were injected with 100 ng of E in 0.1 ml of sesame oil
for 3 consecutive days. This was followed by daily injections of 1 mg of P for
3 consecutive days. Decidualization was then initiated in one horn by
injection of 50 µl oil. The other horn was left unstimulated. The animals
were treated with P for an additional 6 days post-stimulation and then killed
to collect the uterine tissue.
Immunohistochemistry
For Cx43, Pecam, Ki67 and Vegf immunostaining, uterine sections were
obtained and flash frozen. Samples were embedded in OCT, cryosections were
taken at 8 µm and subjected to immunostaining with antibodies against Cx43
(Zymed), Pecam1 (BD Biosciences), Ki67 (Santa Cruz Biotech) and Vegf (Santa
Cruz Biotech). For double immunostaining against Pecam1 and Cx43, uterine
tissues were collected from mice on day 8 of pregnancy and flash frozen in
liquid nitrogen. Incubations with primary antibody were carried out overnight
at 4°C (1:1000 dilution of rat anti-Pecam1 antibody, Pharmingen 557355;
1:500 dilution of a rabbit polyclonal antibody against Cx43, Zymed 71-0700),
using frozen sections. Secondary antibody incubations were carried out for 2
hours at room temperature with a 1:200 dilution of fluorescently conjugated
secondary antibodies: TRITC-conjugated goat anti-rabbit antibody (Sigma T6778)
and FITC-conjugated goat anti-rat antibody (Sigma F6258). Sections were washed
three times for 5 minutes each in PBS.
Human endometrial stromal cells
Primary human endometrial stromal cells were isolated from endometrial
biopsies of fertile women and immortalized by stable transfection of a gene
coding for an essential catalytic protein subunit of human telomerase reverse
transcriptase (Krikun et al.,
2004). These telomerase-expressing stromal cells (termed HESC-T)
were stably transfected with retroviral vectors expressing Cx43 small
interfering RNA (siRNA) and a control Cx43 non-silencing sequence
(Shao et al., 2005). The siRNA
insert-containing retroviral vectors were first transduced into PT67
retro-packaging cells (BD Biosciences) to generate infectious viral
particle-containing supernatant. Filtered supernatants were then used to
infect HESC-T cells and the infected cells selected in media containing 50
µg/ml hygromycin. Cells transfected with a control non-silencing siRNA and
Cx43 siRNA are designated as HESC-TC and HESC-T3, respectively. The cells were
grown in DMEM/F-12 medium containing 5% charcoal-stripped FBS. To induce in
vitro decidualization, the cells 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. Cell
culture supernatants were collected, and prolactin and VEGF were measured
using standardized ELISA kits. Three independent experiments were performed to
assure reproducibility and the data are presented as mean±s.d.
Comparisons between HESC-TC and HESC-T3 cells were made using two-tailed
Student's t-tests, with a significance threshold set at
P=0.05.
Dual label cell coupling assay
Donor cells were double labeled with the fluorescent dyes calcein (gap
junction permeable dye) and Dil (gap junction impermeable dye). These cells
were then placed in contact with unloaded cells in a monolayer. Dye transfer
was visualized after 2 hours. The cells that fluoresce both green (calcein)
and red (Dil) are the dual-loaded donor cells, whereas those fluorescing only
green were originally unlabeled in the monolayer and now demonstrate
functional coupling.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gidley-Baird,
1981). This hormonal profile mimics the transient preimplantation
surge of E that is essential for implantation in the rodents. Gene expression
profiling using Affymetrix mouse microarrays (430 2.0 Array) revealed that
connexin 43 (Cx43), also known as gap junction protein alpha 1 (Gja1), is one
of the many genes whose expression is altered in P-primed uteri in response to
E (Mantena et al., 2006)
(M.K.B. and I.C.B., unpublished). The connexins are a family of transmembrane
proteins that form hexameric assemblies in the plasma membrane to create gap
junctions, regulating intercellular communication. Cx43 is the principal and
most well-studied component of the gap junctions
(Kumar and Gilula, 1996;
Bruzzone et al., 1996).
We first confirmed the hormonal regulation of Cx43 expression in pregnant
uterus by performing immunohistochemical analysis. We observed that E
treatment dramatically enhances the expression of Cx43 protein in the uterine
stromal compartment during delayed implantation
(Fig. 1A). Our findings are
also in agreement with previous reports that Cx43 expression in the uterus is
primarily under E regulation (Grummer et
al., 2004). When we examined the profile of Cx43 protein
expression in mouse uterus during normal pregnancy, it was undetectable in
undifferentiated stromal cells during the preimplantation period
(Fig. 1B, parts a,b). However,
on day 5 of pregnancy, within 12 hours of the initiation of implantation, a
marked induction in Cx43 protein expression was observed in the stromal cells
in the primary decidual zone immediately surrounding the implanting embryo
(Fig. 1B, parts c,d). As
pregnancy progressed to day 7, Cx43 expression intensified and spread to the
secondary decidual zone (Fig.
1B, parts e,f). The close spatio-temporal relationship between
Cx43 expression and the progression of decidualization raised the possibility
that stromal gap junctions harboring this protein may play an important role
during the differentiation process.
To investigate the function of Cx43 during embryo implantation, we employed
a loss-of-function approach using genetically engineered mice.
Cx43-null mice exhibit a perinatal lethal phenotype due to impaired
cardiovascular development (Reaume et al.,
1995). To circumvent this problem, we created a conditional
knockout of the Cx43 gene in the uterus of adult mice by employing
the Cre-LoxP strategy. Transgenic mice expressing Cre under the control of
progesterone receptor (PR) promoter were used previously to ablate `floxed'
genes in the uterus (Lee et al.,
2006; Mukherjee et al.,
2006; Lee et al.,
2007). We crossed these PR-Cre mice with those harboring the
`floxed' Cx43 gene (Cx43fl/fl) to create the
Cx43d/d mice in which the Cx43 gene is deleted in
uterine cells expressing PR. The ablation of the Cx43 gene in the
uterine tissue of Cx43d/d mice during pregnancy was
confirmed when uterine sections obtained from these mice failed to show any
Cx43 protein expression in the stromal cells surrounding the implanted embryo
(Fig. 2).
An 8-month breeding study demonstrated that female
Cx43d/d mice exhibit severe fertility defects
(Table 1). Our study revealed
that 
50% of a cohort (n=13) of Cx43d/d
female mice analyzed during this breeding experiment never gave birth,
although they mated normally with wild-type males. Furthermore, the
Cx43d/d mice that did give birth exhibited a more than 60%
reduction in the number of pups per litter when compared with control
Cx43fl/fl mice. Overall, these results indicated that the
conditional excision of the Cx43 gene led to an 80% reduction in
the total number of pups born per Cx43d/d female compared
with a Cx43fl/fl female in the breeding program (35/13
versus 419/37). Superovulation experiments indicated that
Cx43d/d mice ovulate normally and release oocytes in
quantities comparable to those of Cx43fl/fl mice
(Table 1). In agreement with
normal ovarian activity, serum P levels were normal in the
Cx43d/d mice on day 8 of pregnancy
(Table 1). Collectively, these
data suggest that the observed fertility defect in Cx43d/d
mice is not likely to be due to an impairment of the
hypothalamic-pituitary-ovarian axis.
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The lack of endothelial cell proliferation in Cx43-deficient uteri was further ascertained by immunostaining for Ki67, a marker for cell proliferation (Fig. 3D). Whereas the uterine sections obtained from Cx43fl/fl mice on day 8 of pregnancy exhibited robust Ki67 immunostaining in endothelial cells, consistent with microvascular proliferation (Fig. 3D, part a), those obtained from mutant animals showed greatly reduced Ki67 staining (Fig. 3D, part b), confirming a severely compromised endothelial cell proliferation in the absence of stromal Cx43 expression.
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;
Rasmussen et al., 1997;
Croze et al., 1990;
Lin et al., 1997). As
expected, uterine sections from Cx43fl/fl mice exhibited
prominent PRP expression in both antimesometrial (AM) and mesometrial (M)
areas on day 7 of pregnancy (Fig.
4A). A similar pattern of PRP expression was observed in
Cx43-deficient pregnant uterus on this day
(Fig. 4B). On day 8 of
pregnancy in Cx43fl/fl mice, PRP expression was
drastically reduced in the decidual cells at both antimesometrial and
mesometrial regions. Only a few cells in the mesometrial region close to the
ectoplacental cone retained strong PRP expression
(Fig. 4C). By contrast, a
strikingly different spatial expression of PRP was seen in
Cx43-deficient uteri on day 8 of pregnancy
(Fig. 4D). In these mutant
uteri, expression of PRP, similar to that seen on day 7 of pregnancy,
persisted in the majority of the decidual cells in the antimesometrial and
mesometrial areas. A plausible explanation for the absence of timely
downregulation of PRP expression in these cells is that they fail to reach a
more advanced state of differentiation. When we analyzed PLP expression, it
was prominent in the antimesometrial region of uterine sections of
Cx43fl/fl mice on day 8 of pregnancy and was markedly
reduced in Cx43-deficient uteri
(Fig. 4E,F). Collectively, the
aberrant expression of both PRP and PLP indicated that the loss of Cx43
expression in the uterine stromal cells leads to an impairment in the proper
progression of the decidualization program.
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). Uteri of
ovariectomized Cx43fl/fl and Cx43d/d
mice were prepared by treating these animals with a well-established regimen
of E and P, and then decidualization was initiated in the left uterine horn by
injecting 50 µl oil while the right horn was left unstimulated. As shown in
Fig. 5A, widespread expression
of Cx43 is induced in stromal cells of Cx43fl/fl mice in
response to the deciduogenic stimulus. We then examined the gross anatomy of
the stimulated and unstimulated uterine horns of Cx43fl/fl
and Cx43d/d mice. As expected, the uterine horn of
Cx43fl/fl mice exhibited a robust decidual response within
48 hours of receiving the artificial stimulation
(Fig. 5B, upper left panel). By
contrast, the Cx43-deficient uteri under identical conditions showed
significantly reduced decidualization (Fig.
5B, upper right panel). When the decidual response was assessed by
measurement of uterine wet weight gain, the Cx43-deficient uteri
exhibited a markedly reduced weight gain relative to that seen in the
Cx43fl/fl uteri (Fig.
5B, middle panel). We further analyzed the decidualization
response of Cx43d/d uteri by monitoring the expression of
Hoxa10 and bone morphogenetic protein 2 (Bmp2), factors that are induced in
stromal cells during decidualization and that play important regulatory roles
during this process (Lim et al.,
1999; Lee et al.,
2007; Li et al.,
2007). As shown in the lower panel of
Fig. 5B, when
Cx43fl/fl and Cx43-deficient uteri were subjected
to artificial decidual stimulation, we observed a marked downregulation of
mRNAs corresponding to Hoxa10 and Bmp2 in the uteri lacking Cx43, consistent
with the other decidualization defects observed in the conditional mutant
mice. We next analyzed the angiogenic capacity of Cx43-deficient uteri during artificial decidualization. Immunohistochemical analysis of Cx43fl/fl and Cx43-deficient uteri with a Pecam antibody revealed that the artificially stimulated Cx43fl/fl horn displayed an extensive endothelial cell network spanning the endometrial bed (Fig. 5C, left panel). By contrast, similarly treated uterine horns of Cx43d/d mice displayed drastically reduced Pecam staining, indicating that only a rudimentary vasculature is formed (Fig. 5C, right panel). These studies clearly indicate that even in the absence of the conceptus, communication via Cx43 gap junctions plays a crucial role in stromal cell differentiation and angiogenesis in the steroid hormone-primed uterus.
We considered the possibility that the impaired decidualization of the
mutant stromal cells might affect the timely production of paracrine
regulators from these cells, thereby inhibiting endothelial proliferation or
angiogenesis. Ample evidence indicates that Vegf is a potent paracrine
stimulator of endothelial cell proliferation and is a crucial angiogenic
factor during decidualization (Ferrara et
al., 1996; Halder et al.,
2000). We, therefore, examined the pattern of Vegf protein
expression in Cx43fl/fl and Cx43d/d
uteri during the decidualization phase. Widespread expression of Vegf was
observed in Cx43fl/fl uteri on days 7
(Fig. 6A, part a) and 8
(Fig. 6A, part b) of pregnancy,
particularly in the mesometrial area, which is the primary source of the
growing implantation site vasculature. Its spatial expression pattern closely
overlapped with that of Pecam, which marked the endothelial network (compare
Fig. 3B and
Fig. 6A). By contrast, a
significant downregulation of Vegf expression, concomitant with the sharp
reduction in Pecam immunostaining, was seen in Cx43-deficient uteri
(Fig. 6A, part c,d; compare
with Fig. 3B, parts d-f). The
Vegf expression in Cx43d/d uteri was limited to only a few
layers of cells surrounding the implanted embryo, and was markedly reduced in
the mesometrial region. We also observed a marked downregulation of mRNAs
encoding angiopoietin 2 and angiopoietin 4 in Cx43-deficient uteri
during the decidualization phase (Fig.
6B). As these factors are known to play important regulatory roles
in endothelial cell proliferation, migration and new blood vessel formation,
our findings provide mechanistic insights into the pathways via which stromal
Cx43 gap junctions control angiogenesis during decidualization.
To further explore the relationship between Cx43 and Vegf expression, and
to test whether this important functional link is conserved among the species,
we extended our study to human endometrial stromal cells, which are known to
produce these proteins during decidualization
(Jahn et al., 1995;
Shifren et al., 1996;
Granot et al., 2000). We used
primary human endometrial stromal cells (HESC-T) that have been immortalized
by stable transfection of a gene encoding the catalytic subunit of human
telomerase (Krikun et al.,
2004). By using these cells, we established a low CX43-expressing
human stromal cell line HESC-T3 that is stably transduced with a retroviral
vector expressing small interfering RNA (siRNA) targeted to CX43
mRNA, generously provided by Dr Dale Laird
(Shao et al., 2005). A control
cell line, HESC-TC, containing the same retroviral vector expressing a
non-target sequence was also generated. Quantification by real-time RT-PCR
indicated that CX43 mRNA levels in HESC-T3 cells were drastically
reduced (>90%) relative to those in control HESC-TC cells (data not shown).
Correspondingly, western blot analysis demonstrated a marked reduction of CX43
protein in HESC-T3 cells (Fig.
7A). To examine whether the consequence of this forced suppression
of CX43 is an inhibition of gap junctions, we used a double dye-labeling
technique. As shown in Fig. 7B,
gap junction-permeable green calcein dye diffused from injected control
HESC-TC cells (yellow arrows, Fig.
7B) to adjacent cells, confirming that functional gap junctions
exist between stromal cells. By contrast, the injected dye failed to diffuse
from low CX43-expressing HESC-T3 stromal cells. The non-diffusible red DiI
marker identifies the microinjected cells. Interestingly, the HESC-T3 cells
also failed to undergo morphological decidualization in vitro following
treatment with a hormonal cocktail containing E, P and cAMP, whereas the
control HESC-TC cells, treated with this cocktail, exhibited distinct
epithelioid morphological characteristics
(Ryan et al., 1994) that were
indicative of their differentiated status
(Fig. 7C). These results
correlate with the impaired progression through decidualization displayed by
the uterine stromal cells of Cx43d/d mice.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yoshinaga, 1988). The
E-induced delayed implantation in rats and mice faithfully captures this
hormonal effect. Although the implantation-inducing action of E in rodents has
been known for a long time, the mechanism and mediators of this effect remain
largely unknown. Using gene expression profiling, we have identified Cx43 as a
target of E regulation in the uterus during delayed implantation. The
connexins constitute a large family of gap junction proteins that regulate
intercellular communications. Although other members of this family, including
Cx26 and Cx31, are expressed in the pregnant mouse uterus
(Grummer et al., 1996), our
microarray data indicated that Cx43 is the sole member of this family whose
uterine expression is induced by E during implantation. Our findings are in
agreement with previous reports that Cx43 expression in the uterus is
primarily under E regulation (Grummer et
al., 2004).
The expression of Cx43 in uterine stromal cells is intimately associated
with the decidualization phase of pregnancy. Following blastocyst attachment,
the underlying stromal cells undergo extensive proliferation and
differentiation that result in their transformation into the decidual cells.
In mice and a few other species, decidualization can also be experimentally
induced by a variety of artificial stimuli in steroid hormone-primed uteri. We
found that the expression of Cx43 is robustly induced in the decidual tissue
during both normal and artificial decidualization. This induction of Cx43 is
likely to be regulated by E acting via ER
(Esr1 - Mouse Genome
Informatics). In support of this view, our recent studies using conditional
knockout mice harboring a null mutation of the gene encoding ER in the
uterus have shown that E-induced expression of Cx43 is absent in these mutant
mice (M.J.L. and I.C.B., unpublished). A direct regulatory role for ER
in Cx43 expression, however, remains to be established, and would require a
detailed analysis of the 5'-flanking regulatory region of the
Cx43 gene for the presence of functional ER-binding sites.
Analysis of the Cx43-deficient uteri revealed an impaired decidual
response during both normal and artificial decidual reaction. A defect in
decidualization could arise from either compromised stromal cell proliferation
or an arrest in the differentiation program of these cells.
Immunohistochemical studies using the cell proliferation marker Ki67 indicated
that Cx43-deficiency did not significantly affect stromal cell
proliferation, which occurs predominantly during days 5 and 6 of pregnancy
(data not shown). However, in the absence of Cx43 gap junctions, the stromal
cells failed to progress properly in the differentiation program initiated in
response to a decidual stimulation. This was evidenced by a marked reduction
in uterine wet weight gain, which is considered a hallmark of decidualization.
Additionally, two well-known markers of uterine stromal differentiation, PRP
and PLP, exhibited impaired or aberrant expression in Cx43-deficient
uteri during days 7 and 8 of pregnancy. The altered expression of these
biomarkers indicated that the loss of Cx43 expression in the uterine stromal
cells leads to improper progression of the decidualization program. The defect
in stromal differentiation in the absence of Cx43 was further substantiated
when we analyzed the uterine expression of Hoxa10 and Bmp2, which are crucial
regulators of decidualization. Previous studies have shown that mice harboring
a targeted deletion of either the Hoxa10 or the Bmp2 gene
exhibit impaired uterine stromal differentiation and are infertile
(Lim et al., 1999;
Lee et al., 2007). The
expression of both of these factors was markedly reduced in
Cx43-deficient uteri. Furthermore, we observed that the loss of Cx43
expression in human endometrial stromal cells blocked their differentiation
into prolactin-producing decidual cells. Collectively, these results form the
basis of the important concept that the formation of Cx43 gap junctions
between stromal cells is critical for the efficient and timely progression of
the decidualization program.
Our study also suggests that stromal differentiation and angiogenesis are
intimately linked processes within the pregnant uterus. Paracrine factors
secreted by the decidualizing stromal cells might influence the proliferation
and function of uterine endothelial cells in the mesometrial region of the
pregnant uterus where neovascularization mostly occurs. Impaired
decidualization due to the loss of Cx43 gap junction communication might
result in reduced expression and secretion of angiogenic factors by the
uterine stromal cells, and might thereby lead to a concomitant impairment in
angiogenesis. In strong support of this concept, we observed a markedly
reduced expression of Vegf, a potent stimulator of endothelial cell
proliferation and a well-known angiogenic molecule, particularly in the
mesometrial region of the pregnant Cx43-deficient uterus. Previous
studies have shown that the morphogen Bmp2 mediates the enhancement of Vegf
expression and neovascularization in xenografts of breast tumors in mice
(Raida et al., 2005). It was
also demonstrated that Vegf expression in chondrocytes requires cooperative
interactions between the Bmp2 and β-catenin signaling pathways
(Chen et al., 2008). It is,
therefore, conceivable that Bmp2 produced by the decidualizing stromal cells
might regulate uterine Vegf expression during early pregnancy. Future studies
will evaluate the role of Bmp2 and other stroma-derived factors in the
regulation Vegf expression in the decidual uterus.
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; Lee et al.,
2004). The lack of expression of these crucial angiogenic factors
was predictably associated with a dramatic impairment in the formation of
neovasculature in pregnant Cx43-deficient uteri, and, consequently,
led to poor embryo development and early pregnancy loss. Most importantly, the
use of primary cultures of human endometrial stromal cells allowed us to
establish that the interrelationship between stromal CX43 expression, the
progression of decidualization, and the production of VEGF by the decidual
cells also exists in the human. Our study, therefore, uncovered a conserved,
fundamental role of Cx43 gap junctions in maintaining the stromal
differentiation program and supporting angiogenesis in the uterus during early
pregnancy. Another tantalizing possibility is that the Cx43 gap junctions
additionally play a role in the regulation of communication between the
maternal decidua and the conceptus. Further studies will be required to
carefully examine whether Cx43 expression is linked to proper functioning of
the extraembryonic tissues, such as the parietal endoderm, giant cells and the
ectoplacental cone.
Gap junction channels allow direct exchange of ions, second messengers,
metabolites, and other small molecules (up to 1200 Da) between the
cytoplasms of adjacent cells. This type of intercellular coupling is known to
regulate cell proliferation and differentiation
(Lo and Gilula, 1979;
Simon and Goodenough, 1998;
Evans and Martin, 2002). The
identity of the intercellular signal(s) being exchanged via the stromal gap
junction during decidualization needs to be established. Within the pregnant
uterus, stromal differentiation proceeds in a strictly coordinated manner,
spreading spatially from the antimesometrial to the mesometrial side as the
pregnancy advances. It is conceivable that stromal cells that are more
advanced in the differentiation program could facilitate the progression of
their less differentiated neighbors by establishing direct contact with these
cells. Once the gap junctions are formed, crucial signaling molecules may pass
from the donor to the recipient cells via these connections. These regulatory
molecules, which may include second messengers, such as cyclic nucleotides,
calcium ions or prostaglandins, are likely to impact the gene expression
program and differentiation fates of the recipient stromal cells, altering
their ability to produce Vegf, angiopoetin 2, angiopoetin 4, and possibly
other paracrine angiogenic effectors. In support of this concept, cAMP and
phorbol ester-dependent signaling pathways were found to enhance VEGF
expression by human endometrial stromal cells
(Popovici et al., 1999;
Fig. 7D). cAMP was also
implicated as the signaling molecule that passes through the gap junctions to
influence the adrenocorticotropin-stimulated growth and steroidogenic function
of adrenal cells (Murray and Fletcher,
1984; Shah and Murray,
2001). We postulate that a similar mode of intercellular coupling
via Cx43 gap junctions may underlie the unique spatiotemporal appearance of
the differentiation markers and the coordinated production of angiogenic
regulators in the uterus during early pregnancy.
In summary, the present study revealed a central role for Cx43-containing stromal gap junctions in the establishment of an elaborate vascular network within the endometrial bed that is essential for successful implantation and subsequent embryonic growth. The ovarian hormone dependence of Cx43 functionally regulates gap junction communication among stromal cells, advancing the process of decidualization and stimulating these cells to produce crucial paracrine effectors, such as Vegf. Most importantly, a novel, linear molecular pathway has emerged providing yet another link between nuclear actions of steroid hormones and cellular signaling at the level of the plasma membrane. Ultimately, these molecules control stromal differentiation and angiogenesis during early pregnancy.
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ACKNOWLEDGMENTS |
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