Kruppel-like factor 5 (KLF5) is critical for conferring
uterine receptivity to implantation
Xiaofei Suna, Liqian Zhangb, Huirong Xiea, Huajing Wanb,1, Bliss Magellaa, Jeffrey A. Whitsettb, and Sudhansu K. Deya,2
aDivision of Reproductive Sciences andbDivision of Pulmonary Biology, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of
Cincinnati College of Medicine, Cincinnati, OH 45229
Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved December 15, 2011 (received for review November 8, 2011)
A blastocyst will implant only when the uterus becomes receptive.
Following attachment, luminal epithelial cells undergo degenera-
tion at the site of the blastocyst. Although many genes critical for
uterine receptivity are primarily regulated by ovarian hormones,
Kruppel-like factor 5 (KLF5), a zinc finger-containing transcription
factor, is persistently expressed in epithelial cells independently of
ovarian hormones. Loss of uterine Klf5 causes female infertility due
to defective implantation. Cox2 is normally expressed in the lumi-
nal epithelium and stroma at the site of blastocyst attachment, but
luminal epithelial COX2 expression is absent with loss of Klf5. This
is associated with the retention of the epithelium around the im-
plantation chamber with arrested embryonic growth. These results
suggest that Klf5 is indispensable for normal implantation.
prereceptive on days 1–3, assumes receptivity on day 4 of preg-
nancy or pseudopregnancy (day 1 = vaginal plug), and becomes
refractory to implantation by day 5 (1). The implantation process,
which is initiated by blastocyst attachment to the receptive
uterine luminal epithelium, occurs in the evening of day 4 and
becomes more prominent on the morning of day 5. This coincides
with an increased uterine vascular permeability at the site of the
blastocyst. The event can be visualized as discrete blue bands
(sites of increased vascular permeability) by an i.v. injection of
a blue dye solution before sacrifice (2, 3). By day 6, epithelial cells
lining the implantation chamber disintegrate, presumably due to
apoptosis in response to the invading blastocyst (4). The blasto-
cyst is in direct contact with underlying stromal cells that are then
undergoing proliferation and differentiation into decidual cells.
A previous report indicated that trophoblasts do not directly
partake in epithelial cell degeneration (5), but the epithelium is
programmed to apoptosis at the site of the blastocyst following
the attachment reaction (6). However, the epithelium away from
the attachment site remains intact, suggesting that the blastocyst
signals trigger epithelial cell death (5).
In mice, uterine receptivity is achieved when the uterus is ex-
posed to estrogen after 24–48 h of P4priming (7). However, the
gene network that confers uterine receptivity is complex, making
it difficult to manipulate the window of receptivity by changing
the expression of several critical genes (8). Genetic studies in
mice have shown that certain genes, including Lif (9), Ptgs2 (10),
Ihh (11), Msx (12), and Fkbp52 (13), are critical to uterine re-
ceptivity. However, it is still unclear whether the signaling path-
ways work independently, in parallel, or converge onto a common
pathway to confer uterine receptivity.
The luminal epithelium plays a critical role in implantation: it
transmits embryonic signals to underlying stromal cells to initiate
and progress the implantation process. In mice, the luminal ep-
ithelium around the embryo is still intact on the morning of day
5, when stromal cells have already initiated decidualization. This
suggests that embryonic signals are transmitted to the stroma
via epithelial cells. In fact, removal of the epithelium prevents
decidualization (14). Although the exact nature of this signal has
varian estrogen and progesterone (P4) direct phases of
uterine sensitivity to implantation. In mice, the uterus is
not been clearly identified, prostaglandins (PGs) are considered
to play a role in triggering decidualization (10), and COX2-de-
ficient females have decidualization failure (10). However, it is
not clearly understood how uterine COX2 is regulated during
implantation and decidualization.
The Kruppel-like factors (KLFs) belong to a family of zinc
finger-containing transcription factors that regulate diverse cel-
lular processes, including development, differentiation, prolif-
eration, and apoptosis. Similar to the other 16 members in the
family, KLF5 contains three zinc-finger domains that function in
DNA binding (15). KLF5 is expressed in the human and mouse
digestive tract, pancreas, placenta, testis, prostate, skeletal mus-
cle, and lung (15). Studies in cell culture and animal models show
that KLF5 has essential roles in cell proliferation, apoptosis,
migration, differentiation, and stemness in a context-dependent
manner. Klf5 expression is regulated by estrogen and P4in breast
cancer cells: although estrogen facilitates KLF5 degradation
(16), P4induces its expression, and KLF5 mediates P4’s effects on
breast epithelial cell proliferation and dedifferentiation (17).
However, it is not known whether Klf5 is expressed in the uterus
or whether it has a role in pregnancy events.
Klf5 null (Klf5−/−) mice show embryonic lethality due to defects
in preimplantation embryo development (18). In its absence,
trophectoderm development is defective, resulting in develop-
mental arrest at the blastocyst stage. In addition, the expression
of Oct4 and Nanog, two pluripotency markers, is down-regulated
in the inner cell mass, whereas Sox17 expression is increased in
the primitive endoderm. These results suggest that Klf5 is a key
regulator of all three lineages in preimplantation embryos (19).
The generation of a conditional deletion mouse model has hel-
ped to study Klf5’s function in adulthood (20). To study KLF5’s
role in female fertility, we established a mouse line with uterine
deletion of Klf5 (Klf5d/d) using floxed Klf5 mice (Klf5f/f) crossed
with mice carrying a Cre gene driven by the progesterone re-
ceptor (Pgrcre/+). We found that Klf5d/dfemales are mostly in-
fertile due to defective implantation and decidualization.
KLF5 Is Expressed in a Spatiotemporal Manner in the Periimplantation
Uterus. Embryonic expression of Klf5 and developmental arrest
of Klf5−/−blastocysts provide evidence that Klf5 has a critical role
in stem cell differentiation and development of various organs.
Conditional deletions in mice show that Klf5 is required for
perinatal lung morphogenesis (20), formation and differentiation
Author contributions: X.S., L.Z., H.X., H.W., J.A.W., and S.K.D. designed research; X.S., L.Z.,
H.X., and B.M. performed research; H.W. and J.A.W. contributed new reagents/analytic
tools; X.S., H.W., J.A.W., and S.K.D. analyzed data; and X.S., J.A.W., and S.K.D. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1Present address: Huaxi Second University Hospital, Sichuan University, Chengdu, Sichuan
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 24, 2012
| vol. 109
| no. 4
of the bladder urothelium (21), epithelial proliferation and dif-
ferentiation of the gastrointestinal tract (22), and normal eyelid
development (23). However, its role in female fertility was not
known. To address this issue, we first examined KLF5 expression
in the mouse ovary, oviduct, and uterus. Immunohistochemistry
results show that KLF5-positive cells (if any) are very sparse in
the ovary (Fig. 1). In the oviduct, epithelial cell nuclei show
In the uterus, KLF5 expression is spatiotemporal. On day 1 of
pregnancy, KLF5 is present in the luminal and glandular epi-
thelia when the uterus is primarily under the influence of pre-
ovulatory estrogen secretion. On day 4, its expression is also
limited to the epithelium when the uterus is under the influence
of rising P4levels from newly formed corpora lutea superim-
posed with a small amount of estrogen secreted from the ovary.
With blastocyst attachment in progress on the morning of day 5,
stromal cells at the attachment site proliferate and differentiate
into decidual cells. Later on day 5, stromal cells immediately
surrounding the implantation chamber undergo differentiation
to decidual cells, giving rise to the primary decidual zone (PDZ).
At this time, proliferating stromal cells express KLF5, but the
expression declines in stromal cells that had already differenti-
ated into the PDZ (Fig. 1 and Fig. S1). In parallel, KLF5 ex-
pression is also down-regulated in the epithelium, especially the
epithelium surrounding the blastocyst. On day 8, little epithelium
is left at the implantation site, and stromal cells next to the PDZ
have undergone decidualization, forming the secondary decidual
zone. KLF5 expression persists in proliferating cells, but dis-
appears once the cells complete terminal differentiation. The
dynamic KLF5 expression in epithelial and decidual cells sug-
gests that it contributes to uterine receptivity to implantation and
Ovarian Hormones Modestly Influence KLF5 Expression. To address
whether estrogen and P4regulate uterine KLF5 expression, WT
females were ovariectomized and rested for 10 d. They were then
given a s.c. injection of oil (0.1 mL/mouse), estradiol-17β (E2, 100
ng/mouse), or P4(2 mg/mouse) and killed 2, 6, or 12 h later. The
presence of KLF5 in the epithelium of oil-injected ovariectomized
mice indicates that E2and P4are dispensable for KLF5 expres-
sion. However, E2or P4treatment increased the staining intensity
in the epithelium at 2 h, and both hormones modestly decreased
the staining at 12 h, specifically in the glandular epithelium (Fig.
S2). These results suggest that uterine KLF5 expression is not
greatly influenced by ovarian hormones.
Uterine Deletion of Klf5 Drastically Impairs Fertility. To examine the
role of Klf5 in uterine biology and pregnancy, we conditionally
deleted the floxed Klf5 gene via Cre expression driven by a pro-
gesterone receptor (Pgr) promoter (Pgr-Cre). Because KLF5 is
expressed in neonatal uteri when Pgr-Cre is active (Fig. S3), it is
possible that uterine loss of Klf5 from the neonatal stage to pu-
berty would affect normal uterine development and maturation.
We found that the uterus develops normally in Klf5d/dfemales,
precluding its role in uterine growth and maturation after birth. To
confirm efficient deletion of Klf5 in Klf5d/dfemales, Klf5d/dfemales
were mated with fertile WT males. In situ hybridization and im-
munohistochemistry performed on sections of implantation sites
on day 5 show that Klf5 is expressed in epithelial and stromal cells
in Klf5f/ffemales, but is absent in Klf5d/duteri except for Klf5flox/+
blastocysts that are positive for Klf5 (Fig. 2A). To test KLF5’s role
in female fertility, Klf5f/fand Klf5d/dfemales were mated with WT
males. Females in both genotypes showed normal mating with the
formation of vaginal plugs. However, only 2 of 10 Klf5d/dfemales
gave birth to only two and three pups, whereas all Klf5f/ffemales
delivered normal numbers of pups (approximately eight pups/
mouse) (Fig. 2 B and C). The results show that Klf5d/dfemales are
Klf5 Is Critical for Normal Implantation. Appropriate ovulation,
fertilization, preimplantation embryo development, oviductal
embryo transport, implantation, and decidualization all con-
tribute to a successful pregnancy. To ascertain the stage-specific
failure of pregnancy in Klf5d/dfemales, Klf5f/fand Klf5d/dfemales
were mated with WT males and pregnancy events were examined
on different days. On day 2, the number of two-cell embryos
retrieved from oviducts of both Klf5f/fand Klf5d/dfemales was
comparable, suggesting normal ovulation and fertilization in
Klf5d/dfemales (Fig. 3A). A comparable number of blastocysts
was recovered from day 4 uteri from these two groups, suggesting
that Klf5d/doviducts and uteri support normal preimplantation
embryo development (Fig. S4).
The expression of Hbegf at the site of blastocyst apposition
heralds the onset of attachment reaction (24). Hbegf is normally
in a spatiotemporal fashion. Immunohistochemistry of KLF5 in WT tissues.
Nuclear KLF5 staining is shown as dark-brown deposits on tissue sections
counterstained with eosin. KLF5 is present in the epithelia of day 1 and day 4
uteri and oviducts. On days 5 and 8, KLF5 is also expressed in decidual cells.
Positive signals were not detected in the ovary. le, luminal epithelium; s,
stroma; ge, glandular epithelium; em, embryo; sdz, secondary decidual zone;
cl, corpus luteum; f, follicle.
KLF5 is expressed in the oviduct and periimplantation mouse uterus
ization of Klf5 and immunohistochemistry of KLF5 in day 5 implantation
sites of Klf5f/fand Klf5d/dfemales. White and black arrowheads indicate the
locations of blastocysts. (B) Pregnancy outcomes in Klf5f/fand Klf5d/dmice.
(C) Litter sizes in Klf5f/fand Klf5d/dmice. Unpaired t test, *P < 0.05.
Uterine deletion of Klf5 impairs female fertility. (A) In situ hybrid-
| www.pnas.org/cgi/doi/10.1073/pnas.1118411109Sun et al.
expressed in the luminal epithelium surrounding the blastocyst
before and at the time of attachment reaction. In situ hybridiza-
tion results show that Hbegf expression is comparable between
Klf5f/fand Klf5d/dmice (Fig. 3D), suggesting that initiation of the
embryo–uterine interplay was not delayed in Klf5d/dfemales. We
then examined the status of implantation in these mice on day 5.
After an i.v. injection of a blue dye solution, the implantation sites
are demarcated as discrete blue bands along the uterus (2). We
found that the number of Klf5d/dmice with implantation sites was
lower than that of Klf5f/ffemales (Fig. 3B), but the most dramatic
effect seen in Klf5d/dfemales was the remarkably reduced number
of implantation sites (Fig. 3C). In addition, blue bands in Klf5d/d
uteri were not as distinct as those in the Klf5f/fgroup (Fig. 3E).
When flushed with saline, Klf5d/duteri with weak or no blue bands
yielded many blastocysts (Fig. 3E). These results suggest that,
although embryos develop to blastocysts in Klf5d/duteri, most fail
to implant or show defective implantation. We then sought to
explore the reason for implantation failure in Klf5d/dfemales.
Luminal Epithelium Surrounding the Blastocyst in Klf5d/dFemales Is
Retained Past the Timing of Implantation. The above observations
led us to examine the implantation status at later stages of
pregnancy in Klf5d/dfemales. On day 6, the epithelium around the
implantation chamber degenerates in Klf5f/ffemales, as is evident
from the disappearance of E-cadherin, an established marker of
epithelial cells. However, blastocysts remain entrapped within the
lumen with the intact epithelium in Klf5d/dfemales, and the ep-
ithelium shows distinct expression of E-cadherin (Fig. 4A).
Interestingly, the persistent epithelial barrier is associated with
retarded embryonic growth in Klf5d/dfemales. This is further ev-
ident from H&E staining of sections of day 7 implantation sites;
embryos in Klf5d/dfemales are either very small or resorbed (Fig.
4B). Ki67 immunostaining further confirmed our histological ob-
servations. Some embryos in Klf5d/dfemales were still surrounded
by epithelial cells, but showed signs of degeneration (Fig. 4C and
Fig. S5). Collectively, these data show that an intact epithelium
surrounding the implantation chamber in Klf5d/dfemales past the
normal window of implantation is not conducive to blastocyst
implantation and growth.
Klf5d/dFemales Show Compromised Decidualization. We found that
thenumber of implantation sites on day 6 is similar to that seen on
day 5 in Klf5d/dmice (Fig. 5A), suggesting that those implantation
sites on day 5 with weak blue bands continued to grow in size. This
increase in size is attributed to stromal growth (Fig. S6) because
embryonic growth is arrested at the implantation sites in Klf5d/d
females. Actin cytoskeleton reorganization is critical for normal
decidualization (25). On day 4 before decidualization, actin is
distributed around individual stromal cells without any obvious
pattern (Fig. S7). In day 6 implantation sites, actin expression is
up-regulated and becomes more organized, forming a network
around the embryo (Fig. 4A). A similar pattern of cytoskeletal re-
and Klf5d/dfemales (Fig. 4A), suggesting that Klf5d/dstromal cells
were proliferating and differentiating, despite the presence of the
intact luminal epithelium. Most Klf5d/dimplantation sites showed
continued growth until day 8, and the number of sites was similar
on day 8 (Fig. 5B) compared with those on days 5 and 6 (Figs. 3C
and 5A). However, decidual response in Klf5d/dimplantation sties
was dramatically compromised, and implantation sites were
smaller compared with those in Klf5f/fmice (Fig. 5 C and D). Al-
though KLF5 is expressed in decidual cells (Fig. 1), the results
suggestthat stromalcells are abletoinitiate decidualization in the
absence of KLF5. However, the decidual response of Klf5d/dstro-
mal cells is greatly attenuated in the absence of trophoblast pen-
etration through the luminal epithelium.
To further delineate the decidualization process in these mice,
we examined the expression of Bmp2 and Hoxa10, which are
ization in Klf5f/fand Klf5d/dmice. (B and C) Implantation rate and number of
implantation sites in Klf5f/fand Klf5d/dmice on day 5. (D) In situ hybridiza-
tion of Hbegf at the implantation site at 2400 hours on day 4. Arrowheads
indicate the position of embryos. (E) Representative uteri from Klf5f/fand
Klf5d/dfemales on day 5 after blue dye injection and embryos recovered
from Klf5d/duteri. Arrowheads indicate the implantation sites. IS, implan-
Klf5 is critical for normal implantation. (A) Ovulation and fertil-
the window of implantation in Klf5d/dfemales. (A) Confocal images of E-
cadherin and actin colocalization. Although the luminal epithelium (le) in
Klf5d/dmice at the site of the blastocyst on day 6 of pregnancy is retained, it
disintegrates in Klf5f/fmice with the loss of E-cadherin. Nuclei are shown in
blue color. s, stroma; em, embryo. (B) H&E staining of Klf5f/fand Klf5d/dim-
plantation sites on day 7. (C) Immunostaining of Ki67 in Klf5
dimplantation sites on day 7. M, mesometrial pole; AM, antimesometrial
pole. Arrowheads indicate the location of blastocysts. Arrows indicate the
degenerating embryos or residue of embryo debris, and the blue arrow-
heads indicate the luminal epithelium demarcation in which a blastocyst is
Blastocysts remain entrapped within the luminal epithelium past
Sun et al.PNAS
| January 24, 2012
| vol. 109
| no. 4
critical for decidualization (1). In situ hybridization results show
that these genes are expressed in stromal cells at implantation
sites on days 5 and 8 in Klf5d/dfemales, encompassing a smaller
domain of the stromal bed correlating with smaller decidual
sizes. Notably, viable embryos were not present in the implan-
tation chambers of Klf5d/dfemales on day 8 (Fig. 5E). Increased
vascular permeability and angiogenesis are also critical to suc-
cessful decidualization (6). Because Klf5 was shown to play a role
in angiogenesis, we asked whether angiogenesis is defective at
implantation sites in Klf5d/dfemales. In situ hybridization of Flk1
(an endothelial cell marker) in sections of day 6 implantation
sites showed that Klf5f/fand Klf5d/dfemales have comparable
density of microvasculature in decidual beds (Fig. 5F). This
suggests that changes in angiogenesis are apparently not a cause
for smaller decidual size.
The implanting blastocyst is the normal stimulus for decidual-
ization. However, it can also be experimentally induced by intra-
luminal oil infusion in pseudopregnant mice. Klf5d/dand Klf5f/f
females were mated with vasectomized males to induce pseudo-
pregnancy. On day 4, sesame oil (10 μL/horn) was intraluminally
infused into one uterine horn and the noninfused contralateral
horn was severed as a control. Mice were killed on day 8 (day of
maximal decidualization) to assess the extent of decidualization by
recording fold increases in uterine weights of infused-vs.-non-
infused horns. Klf5d/dfemales showed a remarkably reduced de-
cidual response (Fig. 5G). Collectively, these results suggest that
signals emanating from the blastocyst entrapped within the Klf5d/d
luminal epithelium contributed to decidual response in Klf5d/d
stroma, albeit at a reduced scale.
Klf5d/dMice Have Normal Levels of E2and P4and Uterine Respon-
siveness to These Hormones. Uterine functions are primarily di-
rected by estrogen and P4,and most of their actions are mediated
by estrogen receptor-α (ERα) and progesterone receptor (PR),
respectively (26, 27). Because KLF5 can interact with these
hormones in other systems (17, 28), observed infertility in Klf5d/d
females could be due to abnormal uterine responsiveness to es-
trogen and P4. We found that serum levels of E2and P4in Klf5d/d
females on day 4 are comparable to those in Klf5f/ffemales (Fig.
6A). Immunohistochemistry results show that Klf5d/duteri have a
normal expression pattern of ERα and PR on day 4 (Fig. 6B and
Fig. S8). These results suggest that infertility in Klf5d/dfemales is
not due to altered steroid hormone levels or reduced uterine
responsiveness to these hormones. Normal expression of Lif and
Ihh is indispensable to implantation (9, 11, 29). Whereas Lif is
regulated by E2, P4regulates Ihh expression. In situ hybridization
showed that Klf5d/duteri have normal expression of Lif and Ihh
on day 4 and day 5 (Fig. 6 C and D), suggesting that Klf5d/duteri
can appropriately respond to these genes.
Klf5 Regulates COX2 Expression in Luminal Epithelial Cells During
Implantation. COX2 is expressed at the implantation site, and
Ptgs2−/−females show defective implantation and decidualization
(10). The luminal epithelial and stromal COX2 expression is
differently regulated in some genetically altered mouse models.
For example, COX2 is expressed in the luminal epithelium, but
not in stromal cells, around the implantation chambers in Lif−/−
females that are infertile (29). Thus, we examined COX2 ex-
pression at the implantation site on day 5. In Klf5f/fimplantation
sites, COX2 was localized in both the epithelium and the stroma
(Fig. 7A). In contrast, COX2 was absent in the epithelium, but
present in stromal cells at the blastocyst site in Klf5d/dfemales.
These results suggest that poor attachment reaction in Klf5d/d
females could be due to aberrant COX2 expression. To com-
implantation sites on day 6 in Klf5f/fand Klf5d/dmice. Numerals above bars
indicate numbers of dams with implantation sites over total number of dams
examined. (B) Number of implantation sites on day 8 pregnancy in Klf5f/fand
Klf5d/dmice. (C) Representative uteri from Klf5f/fand Klf5d/dfemales on day
8. (D) Weight of implantation sites on day 8 in Klf5f/fand Klf5d/dmice. (E) In
situ hybridization of Bmp2 and Hoxa10 in implantation sites on days 5 and 8
in Klf5f/fand Klf5d/dfemales. (F) In situ hybridization of Flk1 in sections of
day 6 implantation sites of Klf5f/fand Klf5d/dfemales. (G) Fold changes in
uterine weights of oil-infused horns over noninfused horns indicate the
extent of decidualization in Klf5f/fand Klf5d/dfemales on day 8 of pseudo-
pregnancy. Unpaired t test, *P < 0.05.
Klf5d/dfemales show compromised decidualization. (A) Number of
hormones and uterine responsiveness to these hormones. (A) Serum levels of
E2and P4on day 4 of pregnancy. (B) Immunohistochemistry of ERα and PR on
day 4 uteri. (C) In situ hybridization of Lif in day 4 and day 5 uteri. (D) In situ
hybridization of Ihh in day 4 uteri. myo, myometrium. Arrowheads indicate
the position of embryos.
Klf5f/fand Klf5d/dmice have comparable levels of ovarian steroid
| www.pnas.org/cgi/doi/10.1073/pnas.1118411109Sun et al.
plement our in vivo observation, we examined whether KLF5 can
regulate Ptgs2 expression in cells in vitro. We transfected a human
endometrial epithelial cell line (Ishikawa cells) with Ptgs2 pro-
moter constructs fused to a luciferase gene together with a full-
length construct of the Klf5-coding region. We found that KLF5
up-regulated luciferase activity under the control of Ptgs2 pro-
moters (Fig. 7B). These results suggest that COX2 expression in
luminal epithelial cells requires KLF5 activity.
Successful implantation requires an intimate cross-talk between
the blastocyst and the receptive uterus. However, the molecular
basis for the initiation of uterine receptivity is not fully un-
derstood. The uterine luminal epithelium is the first cell type to
interact with the blastocyst trophectoderm to initiate the at-
tachment reaction. Thus, a receptive and functional epithelium is
essential for implantation. In WT females, the demise of the lu-
minal epithelium surrounding the blastocyst with the progression
of the attachment reaction helps trophoblast invasion into the
stroma for continued embryonic growth. We found that Klf5 is
persistently expressed in the Klf5f/futerine epithelium throughout
the preimplantation period and that deletion of uterine Klf5
confers female infertility due to defective and/or failed implan-
tation. The epithelium at the site of blastocysts fails to undergo
degeneration in most Klf5d/dfemales, thus preventing further
progression of the implantation process and embryonic growth.
The data suggest that Klf5 is critical to making the uterine lu-
minal epithelium conducive to blastocyst implantation and growth.
Klf5 is unique compared with other genes critical for implantation.
For example, Ihh and Lif are transiently expressed before and
during implantation and are regulated by P4or E2(9, 11, 29).
However, Klf5 is present in the uterine epithelium even in the
absence of ovaries (Fig. S1), suggesting that Klf5 signaling may be
parallel to E2and P4signaling. This raises the possibility that there
are other unidentified genes that are independent of E2and/or P4
regulation, but critical for implantation.
Klf5 is expressed mainly in the epithelia of various tissues (15,
30), but is also expressed in cardiovascular smooth muscle cells
(31), lymphoid cells (32), and neuronal cells (33). Our present
study shows that Klf5 expression is not limited to the epithelium,
but also occurs in stromal cells undergoing decidualization,
suggesting that Klf5 has a role in stromal cell transformation.
However, stromal decidualization, albeit at a reduced level, with
the expression of decidualization markers in Klf5d/duteri suggests
that Klf5 has a limited role in decidualization. Because Klf5d/d
epithelium seems to compromise decidualization, the definitive
role of Klf5 in decidualization warrants further investigation
using a mouse model with stromal cell-specific deletion of Klf5.
We speculate that Klf5 regulates uterine receptivity via luminal
epithelial expression of COX2, which is an inducible proin-
flammatory gene. A recent study also shows that Klf5 expression
in the renal collecting duct is essential for inflammatory responses
to unilateral ureteral obstruction (34). The apoptotic cell frac-
tions and glomerular sclerosis and tubular injury scores were all
reduced in the absence of Klf5. Implantation is also considered
a proinflammatory response, and COX2-derived PGs are im-
portant for implantation (10). Our study suggests that the absence
of luminal epithelial COX2 in Klf5d/dfemales may contribute
to implantation failure. Because KLF5 is known to exert an array
of context-dependent functions, COX2 signaling may not be the
only pathway that is dysregulated in the absence of Klf5. Fur-
thermore, pregnancy phenotype in Ptgs2−/−mice is genetic-
background-dependent, and COX1 can compensate for the loss
of COX2, partially rescuing pregnancy failure in Ptgs2−/−females
on certain backgrounds. These studies and COX2’s conserved
role in implantation in various species, including humans, have
previously been discussed (35). Future studies will determine
which PG ligands and corresponding receptors interact with
KLF5 in regulating pregnancy events.
In Lif mutant females or mice with uterine deletion of Msx1/
Msx2 , epithelial COX2 expression persists around the embryo on
d 5 of pregnancy, but stromal COX2 is lost (12, 29). In the present
study, only epithelial COX2 expression was affected, although
Klf5 was deleted in both epithelial and stromal cells. Collectively,
the findings suggest that COX2 in the epithelium and stroma is
differently regulated and affects implantation in diverse ways.
It is interesting to note that Klf5d/duteri show an initial re-
sponse to decidualization in the presence of a blastocyst, but at
a reduced rate. In contrast, oil-induced decidual response is very
poor in Klf5d/duteri. Similar phenotype is observed in several
other knockout mouse models in which implantation is aberrant
(12, 13). It is possible that the blastocyst stimulates stromal cell
decidualization indirectly via its interaction with the epithelium
in addition to sending paracrine signals to stromal cells. We
speculate that stromal cells respond to a paracrine signaling from
the blastocyst, although the stimulus arising from the Klf5-de-
ficient epithelium is disturbed. Collectively, the results suggest
that a circuitry connecting a viable blastocyst with the functional
epithelium and stroma is essential for successful decidualization
and establishment of pregnancy. Because KLF5 is expressed in
both the epithelium and the stroma, it would be exciting to de-
fine the relative contribution of epithelial vs. stromal KLF5 in
female fertility. By deleting floxed Klf5 by Cre driven by the
Müllerian inhibiting substance type II receptor (36, 37) or Wnt7a
promoter (38) in the stroma or the epithelium, future experi-
ments may help in dissecting the roles of epithelial vs. stromal
KLF5 in implantation and decidualization.
Nearly 1 in 12 US married couples under 45 y of age are in-
fertile; 40% of such infertility results from female reproductive
disorders. Despite significant improvement in in vitro fertiliza-
tion technology, implantation failure is still high. There is a need
to better understand the mechanism of uterine receptivity to
develop the means to extend uterine receptivity. Uteri in Klf5d/d
females are mostly unfavorable for normal implantation, pro-
viding evidence that Klf5 is a key player in uterine receptivity.
Klf5 is unique in that it is crucial for implantation, but not
influenced by ovarian hormones. It would be interesting to see
whether KLF5 is expressed in the human endometrium similarly
to that in mice. If so, this may have an implication in regulating
the window of implantation in humans by manipulating Klf5
expression independently of ovarian hormones.
implantation. (A) Immunohistochemistry of COX2 in day 5 implantation sites
of Klf5f/fand Klf5d/dfemales. Immunoreactive COX2 is absent in Klf5d/dlu-
minal epithelial cells. (B) Luciferase assay as evident from the luciferase gene
activity driven by a short and long form of the Ptgs2 promoter. Ishikawa cells
were transfected with a construct spanning the full-length coding region of
Klf5 or control plasmid together with luciferase reporter constructs. A
CMV–β-galactosidase plasmid was severed as a control for transfection ef-
Klf5 differentially regulates COX2 expression in the uterus during
Sun et al.PNAS
| January 24, 2012
| vol. 109
| no. 4
Materials and Methods Download full-text
Animals and Treatments. To generate mice with uterine deletion of Klf5,
Klf5loxP/loxPmice (20) were crossed with PgrCre/+mice (39). Klf5f/fand Klf5d/d
mice were housed in the animal care facility of the Cincinnati Children’s
Hospital Medical Center according to National Institutes of Health and in-
stitutional guidelines for laboratory animals. All protocols of the present
study were approved by the Cincinnati Children’s Hospital Research Foun-
dation Institutional Animal Care and Use Committee.
Immunostaining. Immunohistochemistry was performed in paraffin-embed-
ded sections using specific antibodies as indicated. Tissues fixed in 4% cold
paraformaldehyde were used for KLF5 immunostaining; tissues fixed in 10%
neutral buffered formalin were used for other stainings. Antigen retrieval
using microwave heating is required for KLF5 staining. A Histostain-Plus kit
(Invitrogen) was used to visualize specific antigens.
In Situ Hybridization. In situ hybridization was performed as previously de-
scribed (40) and is detailed in SI Text.
Cell Transfection and Luciferase Assay. IshikawacellsmaintainedinDMEMplus
10% FBS were transfected with specific plasmids (0.3 μg) and CMV–β-galac-
tosidase (0.06 μg), as transfection efficiency control (20), using poly-
ethylenimine (2.4 μg). KLF5 overexpressing and control plasmids have been
described (19). Plasmids containing two regions of the Ptgs2 promoter fused
with the luciferase gene were used to measure Ptgs2 transcriptional activity.
respectively, upstream of the translation start site were used. The promoter
activity was determined by luciferase activity normalized to β-galactosidase
Statistical Analysis. Comparison of means was performed by using Student’s t
test. Data are shown as mean ± SEM.
ACKNOWLEDGMENTS. We thank Serenity Curtis for editing the manuscript
and John Lydon and Francesco DeMayo (Baylor College of Medicine) for
providing us with PgrCre/+mice. This work was supported in part by National
Institutes of Health Grants HD12304 and HD068524 (to S.K.D.) and HL090156
and HL095580 (to J.A.W.). X.S. is supported by a Lalor Foundation postdoctoral
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| www.pnas.org/cgi/doi/10.1073/pnas.1118411109Sun et al.