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Porcupine-dependent Wnt activity within the uterine epithelium is essential
for fertility
Omar Farah1,2, Steffen Biechele4, Dr. Janet Rossant5,6, Dr. Daniel Dufort1,2,3
1
Department of Obstetrics and Gynecology, McGill University Health Centre, Montreal, Quebec, Canada
2
Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada
3
Department of Biology, McGill University, Montreal, Quebec, Canada
4
Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, 35
Medical Center Way, University of California, San Francisco, CA 94143, USA
5
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
6
Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute,
Toronto, Ontario, Canada
Keywords: Porcupine, wnt, uterus, glands, decidualization, luminal closure
Corresponding Author:
Dr. Daniel Dufort
1001 Boul. Decarie
Room EM0.3211
Montreal, Quebec, Canada
H4A 3J1
Email: Daniel.dufort@mcgill.ca
Telephone: 514-934-1934 Ext. 34743
ABSTRACT
The secretion of mammalian Wnt ligands within the cell is dependent on the activity of Porcupine,
a gene located on the X-chromosome that encodes for a membrane bound o-acyl transferase. Here, we
report that post-natal ablation of Porcupine in the uterine luminal epithelium alone results in the decrease
in endometrial gland number. Despite having uterine glands, mutant females are completely infertile.
Epithelial ablation of Porcupine causes defects in timely apposition of the lumen, along with failure to
respond to artificial decidual induction. Interestingly, progesterone supplementation was able to rescue
the initiation of decidualization but the decidua was not maintained and subsequently resorbed.
Transcriptome analysis demonstrated that deletion of Porcupine in the epithelium resulted in the stromal
dysregulation of members of the Wnt signaling pathway (Lef1, Wnt4, Wnt16), dysregulation of receptors
and ligands in the Notch signaling pathway (Notch1, Notch4 and Dll4) as well as Hoxa10. Our results
demonstrate the crucial requirement of Wnt signaling in the epithelium for fertility and demonstrates that
epithelial Wnts regulate stromal Wnt gene expression as well as regulating the expression of essential
signaling factors and effectors required for successful embryo implantation.
INTRODUCTION
Proper development of the adult uterus is a prerequisite for successful implantation and fertility.
Embryo development, growth, and health are thus dependent on a uterine environment that is able to
provide both timely support and adequate nourishment. In murine species, the postnatal completion of
uterine development is marked by the establishment of endometrial glands (Bartol, Wiley et al. 1999,
Gray, Bartol et al. 2001). During early postnatal stages, the luminal epithelium invaginates towards the
anti-mesometrial (AM) side of the uterus forming the endometrial glands. Progesterone supplementation
during a critical postnatal window (PND3-9), results in the complete and permanent absence of uterine
glands, subsequently leading to infertility of those females during adulthood (Cooke, Ekman et al. 2012,
Filant, Zhou et al. 2012). Interestingly, progesterone supplementation with the slightest deviation from
this critical window (PND3-9) results only in a significant decrease, but not complete ablation of adult
uterine glands. In fact, females with a small number of glands are fertile and can produce viable offspring
(Stewart, Fisher et al. 2011). This observation suggests that pregnancy establishment and maintenance
requires only a small number of glands. Endometrial glands exclusively express several secreted factors
and transcription factors that are essential for early embryonic development, attachment and implantation;
these include Lif, Nodal, Foxa2, and Sox9. The dysregulation or deletion of these gland-derived factors
results in severe or complete sterility (Jeong, Kwak et al. 2010, Park and Dufort 2013, Salleh and
Giribabu 2014, Gonzalez, Mehra et al. 2016).
Recently, Wnt signaling has been shown to play essential roles in the coordinated and timely
orchestration of events that culminate in a successful reproductive cycle (Miller and Sassoon 1998,
Mohamed, Jonnaert et al. 2005, Boyer, Lapointe et al. 2010, Sonderegger, Pollheimer et al. 2010, Franco,
Dai et al. 2011). These include adenogenesis, embryo-uterine cross talk, implantation, decidualization,
and female reproductive tract patterning. Several members of the Wnt signaling pathway are known to be
expressed in the murine uterus; Wnt7a, Wnt7b, Wnt11, Fzd6 and Fzd10 are expressed in the luminal and
glandular epithelium, whereas the stroma expresses Wnt4, Wnt5a and Wnt16 (Hayashi, Erikson et al.
2009, Hayashi, Yoshioka et al. 2011). Wnt proteins from the different compartments have been shown to
interact with each other, as single gene deletions of several WNTs within either the epithelium or the
stroma have been shown to affect the expression level of WNTs within the other compartment of the
uterus (Lyons, Mueller et al. 2004, Hayashi, Erikson et al. 2009, Dunlap, Filant et al. 2011). Single Wnt7a
or Wnt4 conditional knockout models have demonstrated their essential role in the establishment of both
postnatal and post-pubertal uterine glands respectively (Dunlap, Filant et al. 2011, Franco, Dai et al.
2011). In addition, ablation of the canonical downstream effectors such, as β-catenin (Ctnnb1) or Lef1,
results in the disruption of neonatal and adult gland development (Jeong, Lee et al. 2009).
Secretion of all Wnt family members requires the same upstream secretory regulator, Porcupine.
Porcupine is a membrane bound O-acyl transferase that controls the release of WNT ligands from the
Wnt-secreting cells (van den Heuvel, Harryman-Samos et al. 1993, Kadowaki, Wilder et al. 1996, Proffitt
and Virshup 2012). Porcupine’s primary known function is the palmitoylation of WNT ligands in the
endoplasmic reticulum, allowing for their recognition by Wntless and subsequent export out of the cell
(Herr and Basler 2012). Interestingly, global Wnt ablation by means of Porcupine deletion within the
uterus results in normal initial development of glands post-natally, but causes subsequent loss of glands
during adulthood (Farah, Biechele et al. 2017). This phenotype is in sharp contrast to single Wnt gene
deletions that never develop glands post-natally (Dunlap, Filant et al. 2011), and revealed a more complex
mechanism in which the collective activity of all uterine Wnts is required for the post-pubertal
maintenance of glands as opposed to initial establishment. Thus, there seems to be a complex regulatory
network between different Wnts within the uterus regulating the adenogenic process.
In an effort to better understand the complex roles of Wnt-signaling in uterine development and
how it may affect fertility, we investigated the effects of ablating epithelial derived Wnt proteins by
conditionally deleting Porcupine in the uterine epithelium. Utilizing an estrogen-induced Crerecombinase driven under the Lactoferrin promoter (LtfiCre), we were able to ablate Porcupine specifically
in the luminal and glandular epithelium specifically (PorcnEpiΔ/Δ). We have previously reported the effects
of a global ablation of porcupine in uterus; including the luminal, stromal and myometrial tissues, where
we observed a lack of maintenance of post-pubertal glands after their initial formation during early
postnatal life (Farah, Biechele et al. 2017). The results presented here however, indicate that absence of
epithelial Wnt activity results in the decrease in endometrial gland number. Interestingly, PorcnEpiΔ/Δ
females are infertile despite being able to form uterine glands. Infertility in these females was attributed to
several defects in implantation and luminal closure, ultimately resulting in the arrest of embryo
development at the blastocyst stage. We also observed dysregulation of the Tcf-Lef/β-catenin signaling
pathway marking the future sites of implantation. Furthermore, we observed a lack of stromal cell
differentiation and decidualization that can be overcome by progesterone supplementation. Finally,
transcriptome analysis demonstrated that deletion of Porcn in the epithelium resulted in the stromal
dysregulation of members of the Wnt signaling pathway as well as the dysregulation of receptors and
ligands in the Notch signaling pathway in the uterus. Our results demonstrate the crucial requirement of
Wnt signaling in the epithelium for fertility and demonstrate that epithelial Wnt ligands regulate stromal
Wnt gene expression.
RESULTS
Loss of Porcn activity within the luminal and glandular epithelium reduces the number of uterine
glands
To determine the role of Porcupine (Porcn) within the luminal and glandular epithelium, we
crossed LtfiCre mice which expressed iCre in the luminal and glandular epithelium of the uterus at puberty
with Porcnflox/flox mice to generate LtfiCre/+ Porcnflox/flox which will be referred to as PorcnEpiΔ/Δ. In LtfiCre
mice, the iCre is followed by the IRES-EGFP so Ltf promoter activity can be visualized by EGFP activity.
As expected, EGFP activity was detected specifically in the luminal and glandular epithelium of 6 week
PorcnEpi Δ/Δ uterus (Supplemental Figure S1). Adult PorcnEpi Δ/Δ uteri appeared morphologically normal as
compared to control (Porcnflox/flox )(Figure 1A). Histological examination of the PorcnEpiΔ/Δ uteri revealed
the presence of endometrial glands at 9 weeks of age. Marker analysis, using the gland specific markers
FOXA2 and SOX9, confirmed the presence of uterine glands in the PorcnEpiΔ/Δ (Figure 1A, 1B).
Interestingly, this finding is in sharp contrast to the deletion of Porcn in both the stromal and epithelial
tissues using the progesterone receptor (Pgr)-Cre allele (PgrCre/+), from now on referred to as PorcnΔ/Δ,
where endometrial glands were absent in the adult uteri (Farah, Biechele et al. 2017). Quantification of
the gland number in PorcnEpiΔ/Δ however, revealed that there was a significant reduction in the number of
endometrial glands that form in the mutant uteri when compared to Porcnflox/flox control uteri (Figure 1C).
Although gland number was decreased in PorcnEpiΔ/Δ, the size of the glands that formed were unchanged
as compared to Porcnflox/flox control uteri (Figure 1D).
To determine if the presence of glands in PorcnEpiΔ/Δ uteri was either due to the later deletion of
Porcn from the LtfiCre/+ as compared to the PgrCre/+(1-2 months versus 3-4 days respectively) or due to the
remaining Porcn expression in the stroma, we induced Cre expression from the Ltf promoter by injecting
estrogen on post-natal day 2. Although Cre was expressed as early as post-natal day 4 as detected by
EGFP activity (results not shown), endometrial glands were still present at a reduced number in the 9
week-adult uteri (Supplemental Figure S2). This suggests that the presence of glands is not due to the late
excision of Porcn but may rather be due to the remaining stromal Porcn activity.
Porcn activity within the luminal epithelium does not affect the development of the stroma or
myometrium
We have previously shown that Porcn deletion in the entire female reproductive tract results in a
decrease in stromal size due to a decrease in proliferation accompanied by an increase in apoptosis
(Farah, Biechele et al. 2017). We therefore examined whether a similar defect would be present in the
PorcnEpiΔ/Δ mutant uteri. Immunofluorescence staining on both Porcnflox/flox and PorcnEpiΔ/Δ uteri was
performed using the stromal specific marker Vimentin and the myometrial marker smooth muscle actin
(Figure 2A, 2C). Quantification of stromal surface area, with the exclusion of the luminal and glandular
compartments, demonstrated that there was no difference in the size of the stroma in the PorcnEpiΔ/Δ as
compared to Porcnflox/flox controls (Figure 2B). Examination of the myometrial compartment also
demonstrated no visible difference in myometrial organization or myometrial size (results not shown).
These results suggest that Porcn activity within luminal and glandular epithelium does not affect the
growth, development or maintenance of the stroma.
Epithelial Porcn activity is essential for luminal closure and proper activation domain of the
canonical Wnt pathway.
We next assessed the fertility of PorcnEpi Δ/Δ females by performing a 5-month breeding trial. Five
Porcnflox/flox and five PorcnEpiΔ/Δ females were mated with proven wild type males. Porcnflox/flox controls
had on average three litters during this period with an average liter size of 13 pups. In contrast, all
PorcnEpi Δ/Δ females failed to become pregnant during this period (Table 1); PorcnEpiΔ/Δ females were thus
sterile. We next investigated whether these females were able to produce viable embryos. Similar
numbers of morphologically normal embryos were isolated from both Porcnflox/flox and five PorcnEpiΔ/Δ
females (Table 1, Supplemental Figure S3). Further, these embryos were able to produce pups when
transferred to wild type surrogates (data not shown). These results show that infertility in PorcnEpiΔ/Δ
females is due to a defective uterus.
In order to explain the reason for the infertility of the PorcnEpiΔ/Δ females, we examined TcfLef/β-catenin signaling during the implantation period since a deletion in Porcn would affect the Wnt
signalling pathway. We crossed a Tcf/Lef-LacZ reporter allele into the PorcnEpiΔ/Δ females. On the fourth
day of gestation (d3.5), Tcf/Lef signaling was restricted in activity to the anti-mesometrial side of the
luminal epithelium at the future site of embryo implantation the Porcnflox/flox control uterus as reported
previously. In contrast, Tcf/Lef signaling in the PorcnEpiΔ/Δ uterus appeared to be preferential activated on
the mesometrial side of the uterus (Figure 3A). Since activation of the Tcf/Lef signaling pathway in the
luminal epithelium is due to the presence of the embryo, the inversion of Tcf/Lef signaling from the anti-
mesometrial to the mesometrial side suggests that embryo homing to the anti-mesometrial side of the
uterus may be disrupted.
Luminal closure plays an important role in implantation and is necessary for normal crypt
formation allowing proper embryo implantation (Robinson and Fisher 2014, Zhang, Kong et al. 2014).
At the time of implantation, the opposing sides of the luminal epithelium become in close contact with
each other forming a slit-like structure with the long axis parallel to the uterine mesometrialantimesometrial (M-AM) axis. Whereas normal luminal closure was observed in day 5 in Porcnflox/flox
uteri, luminal closure failed to occur in most PorcnEpiΔ/Δ uteri; the lumen either remained open or formed
abnormal epithelial folds (Figure 3B). To quantitate luminal closure, the surface area of the lumen was
measured in both Porcnflox/flox and five PorcnEpiΔ/Δ uteri. The luminal surface area was significantly higher
in PorcnEpiΔ/Δ uteri confirming a luminal tube closure defect (Figure 3C). On rare occasions, luminal
closure did occur close to areas that harbored an embryo. Interestingly, trapped embryos at day 6 in
PorcnEpiΔ/Δ uteri appeared to be developmentally arrested as compared to embryos in day 6 Porcnflox/flox
uteri (Figure 3D). Interestingly, embryos could not be flushed out of day 5 or day 6 PorcnEpiΔ/Δ uteri
suggesting that the embryos potentially attach to the luminal epithelium (data not shown).
Porcn activity in the epithelium is essential for decidualization of stromal cells.
Shortly after implantation of the embryo, decidualization begins under the hormonal
control of progesterone; endometrial cells begin to differentiate, causing increased vasculature
and an increase in glycogen accumulation within the cytoplasm of the stromal cells. On day 6 of
gestation, deciduas were very clearly visible in the Porcnflox/flox uteri but were absent in the
PorcnEpiΔ/Δ uteri at the same gestational age (Figure 4A). To determine if decidualization was
defective in PorcnEpiΔ/Δ uteri, we artificially induced decidualization in Porcnflox/flox and
PorcnEpiΔ/Δ uteri as previously described (Behringer, Gertsenstein et al. 2014). While 4 of the 5
Porcnflox/flox uteri showed a decidual response as measured by the weight ratio the induced versus
the control horn, none of the PorcnEpiΔ/Δ uteri showed any decidual response (Figure 4B, 4C).
Thus, decidualization of stromal cells is impaired in the absence of porcupine activity in the
epithelium.
Since the process of decidualization is known to under the primary control of
progesterone (P4), we supplemented PorcnEpiΔ/Δ females with P4 by means of a subcutaneous
plug implanted on day 3 of gestation. After a 4-day period of supplementation, we observed a
partial rescue of the decidualization process in the mutant females that received the P4 plug
compared to mutant females that received a sham surgery treatment (Figure 4D). Decidualization
was further confirmed by alkaline phosphatase activity, which is restricted to decidual cells.
Alkaline phosphatase positive cells were present in P4 supplemented PorcnEpiΔ/Δ uteri but absent
in sham PorcnEpiΔ/Δ controls (Figure 4D). Although the decidual response was partially rescued
by P4 supplementation, the decidua appeared to be resorptive as large amounts of blood were
present in each decidua. This suggests that although P4 supplementation may partially restore
decidualization, it is not sufficient to support this process entirely during the early phase of
pregnancy. The ability of P4 supplementation to partially restore decidualization in PorcnEpiΔ/Δ
uteri suggests a potential interaction between Porcupine activity and the hormone dependent
decidual response.
Ablation of epithelial Porcn results in a change of the uterine transcriptome during
implantation.
To better understand the cause infertility of the PorcnEpiΔ/Δ females at the molecular level,
we examined levels of progesterone and estrogen as well as the expression levels of their
corresponding receptors by quantitative RT-PCR. On day 4 of gestation, no difference was
observed in the levels of both progesterone and estrogen in PorcnEpiΔ/Δ or Porcnflox/flox females
(Supplemental Figure S4A-B). At the level of steroid receptor expression, no difference was
observed in the progesterone receptor (Pgr) but a significant two-fold decrease in expression was
observed for the estrogen receptor (Esr1) in PorcnEpiΔ/Δ uteri (Supplemental Figure S4C, S4D
respectively).
In order to further investigate the changes in gene expression that may lead to infertility,
we compared the transcriptome of PorcnEpiΔ/Δ or Porcnflox/flox uteri on gestational day 4.
Microarray analysis identified a total of 257 genes whose expression showed a 1.5-fold
difference in levels of expression between PorcnEpiΔ/Δ or Porcnflox/flox (Figure 5A). Of these
genes, 195 genes were downregulated whereas 62 genes were upregulated (Supplementary Table
1). Interestingly, two important signalling pathways, Wnt and Notch, which have been shown to
be required for successful implantation, were deregulated. We therefore confirmed the
expression levels of Lef1 and the expression of the three Wnt genes expressed in the stroma,
Wnt4, Wnt16 and Wnt5a by qPCR (Figure 5B). Expression was significantly decreased for Lef1
and Wnt4, Wnt16 whereas the level of Wnt5a was not affected in PorcnEpiΔ/Δ uteri. Similarly, we
examined the expression of Dll4, Perp, Notch1 and Notch4. The expression of both receptors
Notch1 and Notch4, as well as the ligand Dll4 were significantly downregulated in the
PorcnEpiΔ/Δ uteri. Thus, deletion of Porcn in the epithelium affects Wnts expressed in the stroma
as well as affecting the expression of Notch receptors and ligands (Figure 5C).
In addition to the differential expression of genes involved in Wnt and Notch signalling,
several other genes known to be important for implantation were also deregulated. Interestingly,
Ihh and Cxcl15 were significantly upregulated, whereas Cdh5 and Pdgf-d were downregulated in
the absence of Porcn in the epithelium. We also examined the expression of candidate genes
whose dysregulation could contribute to the infertility phenotype and found that HoxA10 was
significantly downregulated (Figure 5D), whereas no change in expression were found for Lif,
Hgf, Hb-Egf, Nodal, Muc1 or Msx1 (results not shown). Taken together, our results demonstrate
that the absence of Porcn function in the epithelium leads to the dysregulation of the Wnt and
Notch pathways as well as several genes known to be required for successful implantation.
DISCUSSION
Our results clearly demonstrate that Porcn activity in the uterine epithelium is essential for
fertility as PorcnEpiΔ/Δ females are sterile. Interestingly, both histological and marker analysis
demonstrated that uterine glands are present in the adult uteri although a decrease in their number was
observed. This is in sharp contrast to our previous findings where ablation of Porcupine in the entire
female reproductive tract, using a progesterone receptor driven Cre-recombinase (PgrCre/+), results in the
initial formation of postnatal glands that are subsequently lost during the adult life of the female (Farah,
Biechele et al. 2017). Deleting Porcn activity in the epithelium would block the secretion of epithelial
expressed Wnts, namely Wnt7a, Wnt7b and Wnt11. It has been demonstrated that a deletion of Wnt7a in
the female reproductive tract results in the post-natal and post-pubertal disturbance of uterine gland
formation (Dunlap, Filant et al. 2011). These results at first glance would appear to contradict our
findings. However, considering the fact that a deletion of Wnt11 in the uterus results in a significant
increase in post-natal gland number (Hayashi, Yoshioka et al. 2011), there appear to be Wnts that
promote adenogenesis such as Wnt7a, and others (such as Wnt11) that repress gland formation. Thus in a
single gene deletion of Wnt7a, Wnt11 may be sufficient to repress adenogenesis. In our experimental
design, however, the activity of both Wnt7a and Wnt11 is eliminated. Since endometrial glands developed
in the PorcnEpiΔ/Δ females, Wnt genes within the stroma must be able to promote adenogenesis at least to
some degree. The most likely candidate is the stromal-expressed Wnt4. A conditional deletion of Wnt4
was shown to significantly reduce endometrial gland number (Franco, Dai et al. 2011), demonstrating that
it is also implicated in adenogenesis.
Deleting Porcn activity within the epithelium did not affect the development of the stroma or
myometrium. We had previously shown that in PorcnΔ/Δ mutants, where Porcn is deleted in the entire
female reproductive tract, exhibit a decrease in stromal size. This was due to a decrease in stromal
proliferation, accompanied by an increase in luminal apoptosis (Farah, Biechele et al. 2017). This stromal
defect could be rescued by injection of recombinant WNT5a in the luminal epithelium but not with
recombinant WNT7a, demonstrating the essential requirement for Wnt5a in stromal proliferation. We
have demonstrated that Wnt5a expression is unaffected in our PorcnEpiΔ/Δ mutants. The absence of stromal
proliferation defects in PorcnEpiΔ/Δ mutants provides further evidence that Wnt5a activity in the stroma is
needed for normal stromal development and proliferation. We cannot exclude however the involvement
of Wnt4 and Wnt16 activity to this end, although no stromal defects have been reported in the single
deletion of either gene (Franco, Dai et al. 2011, Hayashi, Yoshioka et al. 2011).
Although a decrease in uterine gland number was observed in the PorcnEpiΔ/Δ mutants, it is highly
unlikely that this would result in infertility of these mice, as a small number of uterine glands has
previously been reported to be sufficient to establish and maintain pregnancy to term (Stewart, Fisher et
al. 2011). We therefore shifted our focus to the molecular basis of pregnancy establishment in order to
explain the infertility phenotype observed in PorcnEpiΔ/Δ mutant females. The Tcf/Lef signaling pathway
has been shown previously to be activated in the most antimesometrial region of the luminal epithelium
prior to implantation, and that the activation of this pathway requires the presence of the embryo. It is
interesting to note that activation of the Tcf/Lef signaling pathway in the PorcnEpiΔ/Δ uteri was detected
more towards the mesometrial side and very rarely on the antimesometrial side. Since activation of this
pathway requires the presence of the embryo, this suggests that the embryo does not properly home
towards the antimesometrial side of the uterus. This also suggests that luminal epithelial Wnt regulation
may be necessary for proper homing of the embryo to the antimesometrial side. In the absence of luminal-
epithelial Wnts, as in the PorcnEpiΔ/Δ mutant uteri, embryos seem to preferentially remain in the
mesometrial side of the uterus. The process by which epithelial Wnts regulate this phenomenon remains
unknown.
In PorcnEpiΔ/Δ mutant uteri, the lumen fails to properly close and form a slit-like structure along a
line parallel to the uterine mesometrial-antimesometrial (M-AM) axis. The luminal epithelium either
remained open or formed abnormal epithelial folds. Little is known about the molecular cues that govern
luminal closure but its degree of precision in both timing and orientation remains remarkable in murine
species. Luminal closure defects have been reported in the mouse models with a uterine deletion of Rbpj,
a transcription factor residing downstream of the Notch signaling. In Rbpj mutants, luminal closure fails
to occur properly and also affects proper embryo implantation along the M-AM axis (Robinson and
Fisher 2014, Zhang, Kong et al. 2014). We have demonstrated that in the PorcnEpiΔ/Δ uteri, several
components of Notch signaling, including Notch1, Notch4 and Dll4 are all downregulated. Our results
suggest that epithelial-expressed Wnts regulate the expression of Notch signalling family members in the
uterus. Furthermore, defective Notch signaling in the PorcnEpiΔ/Δ uteri may contribute to the failure in
proper luminal closure in these mutants.
Embryo implantation in the mouse is known to induce the process of decidualization, whereby
stromal cell undergo differentiation in order to support the growing embryo until the development of the
placenta. We have demonstrated that in the PorcnEpiΔ/Δ, decidualization fails to occur and cannot be
induced artificially. This is especially interesting, as epithelial expressed Wnts have not been previously
linked to decidualization. Only stromal Wnt4 activity was shown to be essential for decidualization in a
progesterone dependent manner (Abrahamsohn and Zorn 1993, Ramathal, Bagchi et al. 2010, Franco, Dai
et al. 2011). Furthermore, Hoxa10 has also been shown to be required for decidualization by regulating
stromal cell responsiveness to P4 (Benson, Lim et al. 1996, Lim, Ma et al. 1999). In the PorcnEpiΔ/Δ uteri,
Wnt4 and Hoxa10 expression was decreased, which may be responsible for the decidualization defects
seen in these mutants. Interestingly, P4 supplementation was sufficient to partially rescue the initiation of
decidualization, although not sufficient to maintain the decidualization process. It is possible that P4
supplementation may lead to an increase in the expression of P4 target genes such as Wnt4 and/or
overcome the decrease in P4 responsiveness of the stromal cells due to a decrease in Hoxa10 expression.
In conclusion, our results shed new light on the involvement and importance of epithelial
Porcupine-dependent Wnt signaling in several facets of early pregnancy. Deletion of Porcn in the
epithelium of the adult mouse uterus leads to infertility due to defects in proper embryo implantation,
luminal closure, and decidualization. Transcriptome analysis has demonstrated that epithelial Wnts
regulate stromal Wnt4 and Wnt16 gene expression as well as members of the Notch signaling pathway.
The process of establishing and maintaining pregnancy is highly dependent on timely and intricate
crosstalk between the epithelial and stromal cells of the uterus as well as embryo-uterine communication.
The dysregulation of epithelial Wnt target genes is a probable cause for a defective epithelial-stromal and
embryo-uterine crosstalk, which ultimately results in infertility of mutant females.
MATERIALS AND METHODS
Generation and Maintenance of PorcnEpi Δ/Δ Mice:
All experimental protocols were approved by the Animal Care Committee of the McGill University
Health Centre and were in accordance with regulations established by the Canadian Council on Animal
Care. Mice with loxP sites flanking exon 3 of the Porcn gene (Porcnfloxed/floxed) on a CD1 background,
previously described (Biechele, 2013), were mated with Lactoferrin-iCre mice (LactoferriniCre/+) on a
C57BL/6J background (Daikoku 2014, obtained from Jackson labs). Both strains have previously been
reported to demonstrate normal fertility. Homozygous Porcnfloxed/floxed females were crossed with
homozygous LactoferriniCre/+ males and the offspring were genotyped by tail snip digestion and PCR.
The Porcn
min
floxed
each)
(248 bp) and Porcn wildtype (138 bp) alleles were amplified by PCR (94°C, 59°C, 72°C (1
for
30
cycles;
5’-CTGTTAAACCAAGACATGACCTTCA-3’;
5’-
TAACTAGGACGCTTTGGGATAGGAT-3’). The first generation offspring hemizygous (Porcnfloxed,
LactoferriniCre/+) males were crossed with homozygous (Porcnfloxed/floxed) females in order to obtain
(Porcnfloxed/floxed, LactoferriniCre/+) females (simplified from here on as PorcnEpiΔ/Δ or Porcn K.OEpi
females). Early excision of the Porcupine locus was achieved by 3 daily injections of 100 ng of 17βEstradiol into PND2 mice. For assessment of β-Catenin activity, a mouse strain containing a Tcf/LefLacZ reporter allele (Mohamed, Jonnaert et al. 2005) was crossed into the Porcn strain. Vaginal smears
were taken from females before tissue collection and processing in order to stage the females into the
correct estrous cycle stage as needed by experiments described. The various stages were determined
based on the presence and proportion of leukocytes (diestrus), nucleated cells (proestus) and cornified
cells (estrus).
Reverse Transcription and Real-time PCR:
RNA was collected using Trizol extraction and RNeasy Mini Kit [Qiagen Cat. No. 74104]. cDNA was
then synthesized using the QuantiTect Reverse Transcription Kit [Qiagen Cat. No. 205311]. Real-time
PCR was performed using the Rotor-Gene SYBR Green PCR Kit [Qiagen Cat. No. 204074] as per the
manufacturer’s protocol. The primers used are listed in Supplementary table 2.
Paraffin Embedding, Sectioning and Staining:
Uteri were dehydrated with increasing ethanol concentrations (25%, 50%, 75%, and 100%, 20 min each)
and submersed in xylenes (2×, 15 min). The tissue was placed in melted paraffin wax overnight and
embedded at room temperature, and the blocks were solidified at −80°C. Seven-micrometer sections were
cut with the Leica RM2145 microtome and dried overnight. Slides were then washed in xylenes,
rehydrated with a decreasing ethanol gradient (100%, 95%, 85%, 75%, 50%, and 20%, 2 min each).
Sections were then either used for immunofluorescence or stained with Hematoxylin and Eosin (H&E).
Briefly, sections were placed in Harris Modified Hematoxylin solution [Sigma] for 6 minutes and then
washed in running tap water for 20 minutes. Sections were then decolorized in 1% acidic alcohol (1
second), placed in a 1% sodium bicarbonate bluing agent (3 seconds), before being stained with Eosin
[Sigma] for 15 seconds. The slides were then dehydrated as previously described and mounted using
paramount.
Embryo flushing and collection:
Females were mated with fertile CD1 males, and the day of vaginal plug was assigned as Day 0.5.
Embryos were collected on Day 3.5 from the uterus in order to ensure that embryo transport to the uterus
was not compromised.
Immunofluorescence:
Whole uteri were dissected in PBS and fixed overnight at 4°C in 4% paraformaldehyde (PFA)/PBS.
Samples were dehydrated with increasing ethanol concentrations (25%, 50%, 75%, and 100%, 20 min
each) and submersed in xylenes (2×, 15 min). The tissue was placed in melted paraffin wax (TissueTek)
and xylenes (1:1) for 1 h at 60°C, followed by pure paraffin overnight under a vacuum. Samples were
embedded at room temperature and the blocks were solidified at −20°C. Seven-micrometer sections were
cut with a Leica RM2145 microtome and dried overnight. Slides were then washed in xylenes, rehydrated
with a decreasing ethanol gradient (100%, 95%, 85%, 75%, 50%, 20%; 2 min each). Samples were
permeabilized with PBT (0.2% BSA, 2.5% TritonX-100PBS for 10 minutes) and incubated with the
primary antibody: FoxA2 [Abcam ab108422; 1:500], Sox9 [Santa Cruz SC-20095; 1:50], Vimentin
[Abcam ab45939; 1:100], Smooth Muscle Actin [Abcam ab5694 1:500], at 4°C overnight. Slides were
washed with 0.1% PBS-Tween-20 three times before blocking with 5% heat-inactivated goat serum in
PBT for 1 hour at room temperature. Following several washes in with 0.1% PBS-Tween 20, slides were
incubated with a secondary antibody Alexa 488 [Life technologies 1:500] for 1 hour at room temperature.
Slides were then washed, counter stained with draq5 (1:5000) and mounted with Mowiol 4–88.
Surface Area Quantification:
Stromal surface area was calculated by measuring the area encompassed by Vimentin or smooth muscle
actin staining specific markers of the uterus. The ImageJ program was used to produce the values of the
surface areas; as designated by staining encompassed regions only. Quantification of the stromal surface
area excluded the uterine glands in the control uteri.
Detection of β-Galactosidase activity:
Uteri were collected on gestational days 3.5-3.75 from control and mutant females. Uteri were washed in
PBS and fixed in 4% Formaldehyde/PBS for 30 minutes. The uteri were subsequently washed 3 times for
15 minutes each in wash buffer (100 mM sodium phosphate, 2 mM MgCl2, 0.02% Nonidet P-40, 0.01%
sodium deoxycholate, pH 7.3). β-Galactosidase activity was then detected by incubating the uteri in
staining solutions (1 mg/mL X-Gal, 5mM K3Fe(CN)6, 5 mM K4Fe(CN)6) at 37°C overnight. The uteri
were then washed fixed overnight in 4% Formaldehyde/PBS at 4°C, before wholemount photography and
histological examination.
Artificial Decidualization
Control or mutant females were mated with vasectomized males. On gestational day 3.5, females were
sedated with isoflurane and an incision was made in the dorsal side, exposing the ovary and uterus on one
side. One uterine horn was subsequently scratched with a 22G needle. The uterus was then placed back
and the females were sutured and allowed to recover. On gestation day 7.5, the females were sacrificed
and the uterus was dissected out in order to assess the decidualization response.
Alkaline Phosphatase Staining
After tissue processing, paraffin embedding and sectioning, slides were deparaffinized twice in xylene for
5 min each. Slides were then rehydrated in a series of decreasing ethanol dilutions
(100%,100%,95%,75%,50%, dH2O; 2 min. each). Slides were pre-incubated overnight in 1% MgCl2/TrisMalate buffer (pH 9.2) at room temperature. Slides were incubated for 2 hours in AP-substrate solution
(NBT/BCIP) at room temperature. Slides were then washed, countered stained with nuclear fast red
dehydrated with series of increasing dilutions. The slides were then mounted with paramount and imaged.
Microarray
The microarray was performed by collecting RNA samples from pregnant (d3.5) whole uteri, as described
above. Samples were then sent to Genome Quebec for analysis, using the Affymetrix mouse Gene 2.0 ST
assay.
Statistics
Figure data is presented as the mean ± SEM of independent samples. Statistical analysis comparing
experimental groups was performed using a two-tailed Student t-test. Calculations were confirmed using
the GraphPad software. P-values less than 0.05 were considered statistically significant.
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Figure Legends:
Figure 1 – Epithelial ablation of Porcn results in the reduction of endometrial gland number but
not size. (A) Wholemount images and Immunohistochemistry staining on Porcnflox/flox and
PorcnEpi Δ/Δ uteri using the gland specific marker FOXA2 as indicated. (B) Immunofluorescence
staining on Porcnflox/flox and PorcnEpi Δ/Δ uteri using the gland specific marker SOX9 as indicated.
(C) Quantification of gland number in 10 week old Porcnflox/flox and PorcnEpi Δ/Δ uteri (N=6, 16
respectively). (D) Quantification of individual gland size in Porcnflox/flox and PorcnEpi
Δ/Δ
uteri
(N=69, 84 respectively).
Figure 2 - Ablation of Porcn in the uterine Epithelium does not affect the development of stroma
and the myometrium. (A) Immunofluorescence staining on Porcnflox/flox and PorcnEpi
Δ/Δ
uteri
using the stromal specific marker VIMENTIN as indicated. (B) Quantification of stroma surface
area, as marked by the Vimentin Immunofluorescence staining (N=5). (C) Immunofluorescence
staining on Porcnflox/flox and PorcnEpi
Δ/Δ
uteri using the myometrial specific marker Smooth
Muscle Actin as indicated
Figure 3 – Epithelial ablation of Porcn results in the dysregulation of Tcf-Lef Signaling and
luminal closure defects. (A) Wholemount and transverse of Tcf/Lef-LacZ stained Porcnflox/flox
and PorcnEpi
Δ/Δ
uteri on late gestational day 3.5. Several transverse sections, from different
females, of the stained uteri are shown to illustrate the dysregulation of signal in the mutant uteri.
Arrow indicates the M-AM axis (B) H&E staining Porcnflox/flox and PorcnEpi
Δ/Δ
uteri on
gestational day 5.5. Several transverse sections, from different females, are shown to illustrate
the variety of luminal closure phenotypes observed. (C) Quantification of luminal surface area of
control and mutant uteri on gestational day 5.5 (N=13, 27 respectively). (D) H&E staining
Porcnflox/flox and PorcnEpi Δ/Δ uteri at the implantation sites on gestational day 5.5.
Figure 4 – Epithelial Porcn activity is required for a decidual response, but can be rescued by
progesterone supplementation. (A) Wholemount images of pseudo-pregnant Porcnflox/flox and
PorcnEpi
Δ/Δ
uteri at gestational day 7.5 after artificial decidualization assays. (B) Graphical
representation of the percentage of uteri that responded to the artificial decidualization within
each respective genotype (N=5, 9 Respectively). (C) Weight ratio of the induced horn over the
contralateral horn for each respective genotype (N=5, 9 Respectively). (D) Wholemount, H&E
and alkaline phosphatase staining on mutant uteri, with and without Progesterone
supplementation, at gestational day 7.5.
Figure 5 – Pre-implantation transcriptome is altered after the ablation of Porcn activity within the
uterine epithelium. (A) Heat map a microarray performed on the control and mutant uteri at
gestational day 3.5. The Affymetrix Mouse gene ST 2.0 chip was used to perform the analysis
and the results were analyzed using the Affymetrix Expression Console software. Quantitative
real-time PCR analysis was conducted on various genes, including but not limited to, ones that
were shown to be misregulated in the microarray analysis. The genes were grouped into (B) Wnt
signaling related genes, (C) notch signaling genes, (D) gene showing a change or (E) no change
in expression between control and mutant. P-values are included in each panel; p<0.05 indicates
significance (N=5).
Table 1 - Porcn Epi Δ/Δ females exhibit server fertility issues.
Number of embryos
(E3.5) ± SEM
Average number of
Liters/5 month ± SEM
Average Liter Size
Porcn flox/flox
11.6 ± 1.35
3 ± 1.08
12.92 ± 2.68
Porcn Epi Δ/Δ
12.25 ± 0.75
0
0
p = 0.68123
p = 0.015928
p < 0.001
Genotype
± SEM
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