To identify steroid-regulated gene networks with functional relevance in implantation, we used a delayed implantation mouse model in which implantation is induced by an acute administration of E to ovariectomized pregnant mice maintained in the presence of P (Yoshinaga and Adams, 1966
; 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) (M.K.B. and I.C.B., unpublished).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 (). 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 (, 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 (, parts c,d). As pregnancy progressed to day 7, Cx43 expression intensified and spread to the secondary decidual zone (, 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.
Induction of Cx43 expression in uterine stromal cells during implantation
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
) 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 ().
Loss of Cx43 expression in the uterus of the Cx43 conditional-knockout mouse
An 8-month breeding study demonstrated that female Cx43d/d mice exhibit severe fertility defects (). 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 (). In agreement with normal ovarian activity, serum P levels were normal in the Cx43d/d mice on day 8 of pregnancy (). 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.
Impact of Cx43 ablation on female fertility
Further analysis indicated that the Cx43d/d mice are able to initiate embryo implantation and support pregnancy up to day 7 of gestation. Implanted embryos embedded in the stroma were observed in both Cx43fl/fl and Cx43d/d mice (, parts a,c). However, starting on day 8 of pregnancy in Cx43d/d mice, we detected distinct signs of arrest of embryonic growth and noted embryo loss or resorption. Morphological analysis of uterine sections obtained from these mice on day 8 of pregnancy showed abnormally small embryos compared with those of Cx43fl/fl mice (compare parts b and d in ). An examination of the histological sections from day 8 pregnant uteri revealed a severe impairment in the development of an angiogenic network in the stromal bed of Cx43-deficient uteri (, parts e,f). When the uterine sections of pregnant Cx43fl/fl mice were subjected to immunohistochemistry using an antibody against platelet/endothelial cell adhesion molecule (Pecam), a marker of endothelial cells, they displayed a well-developed vascular network spanning the endometrial bed that surrounds the implanted embryo on day 7 or day 8 of pregnancy (, parts a–c). The Pecam immunostaining, however, was reduced drastically in uterine sections of Cx43d/d mice on day 7 or day 8 of pregnancy, indicating that only a rudimentary vasculature formed in the mutant uteri (, parts d–f). It is important to mention here that immunofluorescence experiments using differently labeled antibodies against Pecam and Cx43 indicated that during early pregnancy Cx43 expression occurs solely in the uterine stromal cells and not in the endothelial cells (). These results support the concept that the loss of Cx43 expression in the stromal gap junctions is responsible for the drastic reduction in the endothelial cell population in the pregnant uterus.
Impaired embryo development and lack of angiogenesis in uteri of Cx43 conditional knockout mice on day 8 of gestation
The lack of endothelial cell proliferation in Cx43-deficient uteri was further ascertained by immunostaining for Ki67, a marker for cell proliferation (). 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 (, part a), those obtained from mutant animals showed greatly reduced Ki67 staining (, part b), confirming a severely compromised endothelial cell proliferation in the absence of stromal Cx43 expression.
To gain further insight into the mechanisms underlying the phenotypical defects of Cx43d/d
uteri, we examined whether the loss of Cx43 expression affected the stromal differentiation program. We analyzed the decidual response in Cx43d/d
uteri by monitoring the expression of decidual prolactin-related protein (PRP) and prolactin-like protein (PLP), well-known biochemical markers of decidualization (Orwig et al., 1997
; 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 (). A similar pattern of PRP expression was observed in Cx43
-deficient pregnant uterus on this day (). 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 (). By contrast, a strikingly different spatial expression of PRP was seen in Cx43
-deficient uteri on day 8 of pregnancy (). 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 (). 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.
Cx43-deficient uteri exhibit impaired decidualization
It is important to address whether Cx43 controls stromal decidualization and angiogenesis independently of embryonic development. We, therefore, subjected non-pregnant mice to experimentally induced decidualization in which a mechanical perturbation of the steroid-primed uteri triggers a decidual response in the absence of the implanting embryo (Cheon et al., 2004
). Uteri of ovariectomized Cx43fl/fl
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 , 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
mice. As expected, the uterine horn of Cx43fl/fl
mice exhibited a robust decidual response within 48 hours of receiving the artificial stimulation (, upper left panel). By contrast, the Cx43
-deficient uteri under identical conditions showed significantly reduced decidualization (, 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 (, 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 , when Cx43fl/fl
-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.
Cx43 is essential for decidual response and angiogenesis
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 (, 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 (, 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
uteri during the decidualization phase. Widespread expression of Vegf was observed in Cx43fl/fl
uteri on days 7 (, part a) and 8 (, 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 and ). By contrast, a significant downregulation of Vegf expression, concomitant with the sharp reduction in Pecam immunostaining, was seen in Cx43
-deficient uteri (, part c,d; compare with , 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 (). 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.
Reduced expression of angiogenic factors in Cx43-deficient uteri
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 (). 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 , gap junction-permeable green calcein dye diffused from injected control HESC-TC cells (yellow arrows, ) 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 (). These results correlate with the impaired progression through decidualization displayed by the uterine stromal cells of Cx43d/d
Attenuation of CX43 expression in human endometrial stromal cells leads to impaired gap junction communication, decidualization and a reduction in VEGF production
We next examined whether this lack of stromal differentiation in the absence of CX43 resulted in differences in protein production from HESC-TC and HECS-T3 cells. Prolactin, a biomarker of decidualization in human endometrial stromal cells, similar to the expression of PRP in the mouse, was undetectable in untreated cells but was induced after 7–11 days incubation with E, P and cAMP. Prolactin production in hormone-treated HESC-T3 cells was reduced by 66±8% relative to control HESC-TC cells (n=3, P<0.02). In addition to a reduction in this classical marker of stromal differentiation, indicates that basal and phorbol ester (TPA)-induced production of VEGF also was reduced in low CX43 expressing HESC-T3 cells compared with control HESC-TC cells. Quantification of VEGF synthesis was performed in three independent E, P and cAMP-treated cell cultures. In control HESC-TC cells, hormone treatment stimulated VEGF production 11.4±5.7 fold, whereas the hormone-induced augmentation in HESC-T3 cells was only 1.4±0.1-fold (n=3, P<0.04). Our findings indicate that CX43 gap junctions are directly involved in the regulation of VEGF production in human endometrial stromal cells during in vitro decidualization. Furthermore, conservation of the important functional link between CX43 and VEGF expression in both mouse and human stromal cells supports a fundamental role of gap junction communication during uterine angiogenesis.