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BIOLOGY OF REPRODUCTION 77, 425–432 (2007)
Published online before print 30 May 2007.
DOI 10.1095/biolreprod.107.062414
Maternal Pentraxin 3 Deficiency Compromises Implantation in Mice
1
Susanne Tranguch,
4
Anindita Chakrabarty,
5
Yong Guo,
3,5
Haibin Wang,
5
and Sudhansu K. Dey
2,4,5,6
Departments of Cell & Developmental Biology,
4
Pediatrics,
5
and Pharmacology,
6
Vanderbilt University Medical Center,
Nashville, Tennessee 37232
ABSTRACT
Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have
been attributed to fertilization failure. However, our global gene
expression studies showed high uterine Ptx3 expression at the
implantation site in mice, suggesting its role in blastocyst
implantation. We initiated molecular and genetic studies in
mice to explore the importance of uterine Ptx3 in this process.
We found that Ptx3 is expressed in a unique and transient
fashion at implantation sites. With the initiation of implantation
on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively
in stromal cells at the site of blastocysts. On Day 5, its
expression is more intense in decidualizing stromal cells, but it
disappears on Day 6. The expression again becomes evident in
the deciduum on Day 7, followed by a more robust expression
on Day 8, particularly at the antimesometrial pole. From Day 9,
with the initiation of placentation, Ptx3 expression becomes
undetectable. These results suggest a role for PTX3 in implan-
tation and decidualization. Indeed, deletion of Ptx3 results in
both compromised implantation and decidualization. Interleu-
kin 1B (IL1B), a known inducer of Ptx3, is also transiently
expressed in stromal cells at the implantation site, suggesting
that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine
Ptx3 expression follows that of Il1b induced by lipopolysaccha-
ride treatment on Day 7 of pregnancy. Collectively, these
findings provide evidence for an important role for PTX3 in
implantation and decidualization. This study has clinical
implications, since PTX3 is expressed in the receptive endome-
trium, and trophoblast cells influence decidual Ptx3 expression
in humans.
embryo, implantation, pregnancy, uterus
INTRODUCTION
A reciprocal interaction between the blastocyst and uterus is
absolutely required for implantation [1]. The major events for
the success of this process involve epithelial-epithelial
interactions between the blastocyst trophectoderm and the
uterine luminal epithelium, regulated invasion of the tropho-
blast through the luminal epithelium and the underlying
basement membrane, transformation of stromal cells into
decidual cells surrounding the implanting blastocyst, and
protection of the semiallogenic embryo from the mother’s
immunologic responses. Unlike other tissues, the uterus is
considered an immunologically privileged site, because the
semiallogenic embryo, despite its genetic incompatibility, is
not rejected by maternal immunologic surveillance [2, 3]. The
mechanism by which the semiallogenic embryo is protected
during pregnancy from the maternal immunologic responses is
not fully understood.
Global gene expression studies in mice and humans have
shown that numerous immunologically relevant genes are
downregulated at the site of blastocyst implantation, with the
exception of a few genes, including pentraxin 3 (Ptx3) and
decay-accelerating factor 1 (Daf1) [4, 5]. The upregulation of
these genes implies their potential role in protecting the embryo
from maternal immunologic and/or other noxious stimuli.
Although Daf1-deficient female mice are fertile [6], most Ptx3-
null females show pregnancy failure, attributed primarily to
fertilization defects [7, 8]. However, a role for PTX3 in uterine
biology and implantation remains unknown and is therefore the
subject of the present investigation.
PTX3, also known as tumor necrosis factor (TNF)-
stimulated gene 14 (TSG14), is a member of the pentraxin
superfamily, a family characterized by a cyclic multimeric
structure and subdivided into two classes: short and long
pentraxins [9]. Pentraxins are acute-phase molecules whose
expression increases in response to inflammatory stimuli. They
are composed of monomers that usually assemble into
pentameric, noncovalently associated structures [10–12]. Pen-
traxin 3 is a long pentraxin, originally cloned as an interleukin
1 (IL1)-inducible and tumor necrosis factor a(TNFa)-
inducible gene in endothelial cells and fibroblasts, respectively
[13, 14]. Interestingly, PTX3 does not bind to classical
pentraxin ligands, such as phosphoethanolamine, phosphocho-
line, or fibronectin. Instead, PTX3 binds with high affinity to
the globular domain of complement component C1q [15, 16].
In vitro studies have shown that PTX3 recognizes membrane
moieties, including galactomannan, OmpA (a bacterial moiety
outer membrane protein A), histones, and matrix protein
TSG6 (reviewed in [17, 18]). There is also evidence that the
N-terminal of PTX3 binds to fibroblast growth factor 2 (FGF2)
to restrain FGF2’s proliferative and proangiogenic activity [19,
20]. PTX3 is also known to play roles in immunity and
apoptosis [9, 17, 18, 21].
Targeted deletion of Ptx3 results in reduced fertility, due to
an early loss of cumulus investment, leading to fertilization
failure [7, 8]. It is thought that PTX3 binding to TSG6
stimulates the assembly of a hyaluronic acid-rich extracellular
matrix, which is essential for cumulus expansion, and therefore
this event fails with deletion of Ptx3 [8]. In this investigation,
we studied the role of PTX3 in implantation and decidualiza-
tion. We observed that not only is Ptx3 uniquely expressed in
the mouse uterus during the periimplantation period in a
temporal and cell-specific manner, but implantation and
decidualization are also compromised in mice deficient for
1
Supported in part by National Institute of Child Health & Human
Development grants HD 12304 (to S.K.D.) and HD050315 (to H.W.).
S.T. is supported by a National Institutes of Health National Research
Service Award individual pre-doctoral fellowship from the National
Institute of Drug Abuse (F31 DA021062).
2
Correspondence: Sudhansu K. Dey, Department of Pediatrics, Division
of Reproductive and Developmental Biology, Vanderbilt University
Medical Center, MCN-D4100, Nashville, TN 37232-2678.
FAX: 615 322 4704; e-mail: sk.dey@vanderbilt.edu
3
Current address: Department of Animal Science and Technology,
Beijing University of Agriculture, Beijing, China.
Received: 21 April 2007.
First decision: 14 May 2007.
Accepted: 30 May 2007.
Ó2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
425
Ptx3. These results provide molecular and genetic evidence that
PTX3 is an important member of the signaling network
operative during implantation and decidualization. Recent
studies showing its upregulation in receptive human endometria
and its induction in decidua by trophoblast cells also suggest an
important role for PTX3 in human implantation [22, 23].
MATERIALS AND METHODS
Mice
Disruption of the Ptx3 gene was originally achieved in AB2.2 embryonic
stem cells by homologous recombination as described [8]. Breeding pairs of
Ptx3-null mice on a C57/129 mixed background were kindly provided by
Martin M. Matzuk (Baylor College of Medicine, Houston, TX) and are being
maintained on the same background in our animal care facility. Genotyping is
routinely performed by PCR analysis of genomic DNA. CD1 mice used for
expression studies were purchased from Charles River Laboratory (Raleigh,
NC). All mice were housed in the animal care facility at Vanderbilt University
Medical Center according to the National Institutes of Health and institutional
guidelines for laboratory animals.
Experimentally Induced Deciduoma and Delayed
Implantation
Adult virgin females (20–25 g) were mated with fertile males of the same
strain to induce pregnancy (Day 1 ¼vaginal plug). Implantation sites on Day 5
of pregnancy were visualized by an intravenous injection (0.1 ml per mouse) of
Chicago blue dye solution (1% in saline) as described previously [24] and were
collected separately from interimplantation sites. Implantation sites collected on
Days 7 and 8 of pregnancy are easily visualized, requiring no blue dye
injection, and were also collected separately from interimplantation sites.
Conditions of delayed implantation were induced by ovariectomizing females
on the morning (0900–0930 h) of Day 4 and were maintained with daily
subcutaneous injections of progesterone (P
4
, 2 mg per mouse) from Days 5 to
7. To initiate implantation with the activation of dormant blastocysts, P
4
-primed
mice were injected with estradiol-17b(E
2
, 25 ng per mouse) on Day 7 of
pregnancy and were killed 12 and 24 h later to examine implantation status by
the blue dye method. Wild-type and null females were mated with fertile males,
and uteri were collected on Days 1 and 4 of pregnancy to examine expression
of estrogen and progesterone target genes, respectively. For experimentally
induced decidualization, wild-type or Ptx3-null littermate females were mated
with vasectomized wild-type males to induce pseudopregnancy. Sesame oil (20
ll) was infused intraluminally in one uterine horn on Day 4 of pseudopreg-
nancy, and the noninfused contralateral horn served as a control. Wild-type
mice were killed on Days 5, 6, and 8 of pseudopregnancy to examine Ptx3
expression. Control and oil-infused uteri from wild-type and null mice were
weighed on Day 8 to assess the extent of decidualization.
Blastocyst Transfer
Pseudopregnant recipients were generated by mating wild-type or Ptx3
/
females with vasectomized wild-type males. Wild-type Day 4 blastocysts were
transferred into Day 3 or 4 uteri of wild-type or Ptx3
/
pseudopregnant
recipients. No differences were observed whether embryos were transferred on
Day 3 or 4 of pseudopregnancy, so results were pooled. Mice were examined
for implantation sites 24 h (Day 5) or 96 h (Day 8) later by the blue dye
method. All female mice used were 2–5 mo of age. Uteri devoid of
implantation sites were flushed with saline to recover unimplanted blastocysts.
LPS Treatment
Adult virgin female mice (20–25 g) were mated with fertile males of the
same strain to induce pregnancy. On Day 7 of pregnancy (0900 h), mice were
intraperitoneally injected with lipopolysaccharide (LPS; 100 lg/0.1 ml saline;
Sigma, St. Louis, MO). Liver and uterine tissues were collected at 0, 1, 2, and 6
h after injection for isolation of RNA.
Hybridization Probes
For in situ hybridization, sense and antisense
35
S-labeled cRNA probes for
Ptx3, Il1b, Ltf, Areg, and Hoxa10 were generated from cDNAs using
appropriate polymerases. For Northern hybridization, antisense
32
P-labeled
cRNA probes for Ptx3,Il1b, and Rpl7 (a housekeeping gene) were generated.
Probes had specific activities of ;2310
9
dpm/lg.
RNA Preparation and Northern Blot Hybridization
Total RNA was extracted from three independent tissue samples using
Trizol reagent (Invitrogen, Carlsbad, CA). RNA (6 lg) was denatured, separated
by formaldehyde-agarose gel electrophoresis, and transferred onto nylon
FIG. 1. Ptx3 is expressed in a spatiotem-
poral manner during the periimplantation
period. A) Northern blot hybridization of
Ptx3 in implantation sites (IS) and interim-
plantation sites (IIS) in the mouse uterus on
Days 5 and 8 of pregnancy. Rpl7 was used
as a housekeeping gene to confirm integrity
of RNA samples and equal loading. B)In
situ hybridization of Ptx3 in the mouse
uterus during early pregnancy. Hybridiza-
tion signals in representative darkfield
photomicrographs of uterine sections on
select days of pregnancy are shown. Arrows
indicate locations of embryos. Bar ¼200
lm. le, luminal epithelium; s, stroma; myo,
myometrium; pdz, primary decidual zone;
sdz, secondary decidual zone; D, day of
pregnancy.
426 TRANGUCH ET AL.
membranes. Cross-linked blots were prehybridized, hybridized, and washed as
previously described [25]. Hybrids were detected by autoradiography.
In Situ Hybridization
In situ hybridization was performed as previously described by us [25]. In
brief, sections were prehybridized and hybridized at 458C for 4 h in 50%
formamide hybridization buffer containing
35
S-labeled antisense or sense
cRNA probes. RNase A-resistant hybrids were detected by autoradiography.
Sections were poststained with hematoxylin-eosin. Sections hybridized with
sense probes did not exhibit any positive signals and served as negative
controls. Experiments were repeated at least two to three times using
independent samples.
RESULTS
Ptx3 Is Expressed at Higher Levels at the Implantation Site in
a Spatiotemporal Manner
Our gene profiling studies in mice showed that while most
immunologically related genes are downregulated at the site of
implantation, Ptx3 is one of the few genes that is upregulated
instead [5]. Later, it was found that Ptx3 expression is also
upregulated in human endometrium during the receptive phase
[4]. These results suggest that PTX3 is important for uterine
function relevant to implantation. To first confirm our micro-
array results [5], we compared the levels of Ptx3 mRNA
between implantation and interimplantation sites on Days 5 and
8 of pregnancy in mice by Northern hybridization. As noted in
Figure 1A, the levels of Ptx3 mRNA are considerably higher at
implantation sites compared with those at interimplantation
sites on Days 5 and 8, affirming our microarray results.
The uterus is comprised of heterogeneous cell types that
undergo dramatic changes during early pregnancy [26]. Thus,
we next examined the cell-specific and temporal expression of
Ptx3 in the periimplantation uterus using in situ hybridization.
We found an interesting expression pattern. Ptx3 is expressed
at low to undetectable levels in the uterus on Days 1 and 4 of
pregnancy (Fig. 1B). The Day 1 uterus is predominantly under
the influence of preovulatory estrogen, whereas the Day 4
uterine milieu is dominated by rising progesterone (P
4
) levels
from the newly formed corpora lutea [26]. These results
suggest that ovarian steroids estrogen and P
4
play a small, if
any, role in regulating uterine Ptx3 expression. In contrast, the
expression pattern of Ptx3 dramatically changed with the onset
of implantation. The initiation of blastocyst attachment
coincides with an increased endometrial vascular permeability
and increased stromal cell proliferation at the site of blastocyst
attachment. We found that Ptx3 is first expressed in stromal
cells surrounding the blastocyst with the initiation of its
attachment with the luminal epithelium on midnight of Day 4
of pregnancy. On Day 5, stromal cell proliferation is more
intense at this site, and so is the expression of Ptx3 (Fig. 1B).
The proliferating and differentiating stromal cells surrounding
the implanting blastocyst begin to form the primary decidual
zone (PDZ) late on Day 5 of pregnancy. The PDZ is avascular
and densely packed with decidual cells. By Day 6, the PDZ is
well established, and a secondary decidual zone (SDZ) is
formed around the PDZ. At this time, cell proliferation ceases
in the PDZ but still continues in the SDZ. On this day, Ptx3
TABLE 1. Implantation is compromised in Ptx3 null mice.
Genotypes
No. of blastocysts transferred No. of recipients Day of Sacrifice No. of IS (%) No. of blastocysts recoveredDonors Recipients
þ/þþ/þ87 5 5 46 (53%) N/A
þ/þ/121 8 5 21 (17%)* 26 (24%)
þ/þ/81 7 8 19 (23%)* 0
* The number of IS in null females on Day 5 or Day 8 of pregnancy is significantly different from those in wild-type females (P,0.001, chi-square
analysis).
FIG. 2. Estrogen- and progesterone-regulated genes are expressed
appropriately in Ptx3-null uteri. A) In situ hybridization of Ltf in wild-
type and Ptx3-null uteri on Day 1 of pregnancy. B) In situ hybridization of
Ltf,Areg, and Hoxa10 in wild-type and Ptx3-null uteri on Day 4 of
pregnancy. Bar ¼200 lm. le, luminal epithelium; ge, glandular
epithelium; s, stroma.
MATERNAL Ptx3 DEFICIENCY COMPROMISES IMPLANTATION 427
expression becomes very low to undetectable. Surprisingly, its
expression reappears on Day 7 of pregnancy at the border
between the PDZ and SDZ, and it becomes more intense on
Day 8. With the beginning of placentation and decidual
regression on Days 9 and 10, Ptx3 expression disappears
(Fig. 1B and data not shown).
Ptx3-null Mice Show Compromised Implantation and
Decidualization
The unique expression pattern of Ptx3 in the peri-
implantation uterus led us to ask whether uterine PTX3 plays
a role during early pregnancy. Targeted deletion of Ptx3 in
mice results in severely reduced fertility, primarily resulting
from failure of oocyte fertilization associated with defective
cumulus cell expansion [7, 8]. Although pregnancy can occur
in Ptx3-null females, litter sizes are drastically reduced [8].
These observations, together with the unique uterine expression
pattern of Ptx3, led us to explore whether the implantation
process is also defective in Ptx3-null females. Since these null
females have severely compromised fertilization [7, 8], we
employed reciprocal blastocyst transfer experiments to exam-
ine implantation status in these mice. Day 4 wild-type
blastocysts were transferred into the uteri of Day 4 pseudo-
pregnant wild-type or null recipients. Although 53% of the
transferred blastocysts implanted into all five wild-type
recipient uteri, only a small percentage (17%) of transferred
blastocysts showed signs of implantation in just four of eight
Ptx3-null uteri. Recovery of blastocysts from null recipients
confirmed efficacy of transfer. When examined on Day 8,
again, only three of seven Ptx3-null mice showed signs of
implantation, and the number of implantation sites was low
(23%; Table 1).
Estrogen- and Progesterone-Regulated Genes are Normally
Expressed in the Ptx3 Uterus Prior to Implantation
The results of blastocyst transfer experiments suggested that
uteri missing PTX3 are not fully competent for implantation.
One cause of this compromised implantation could be due to
inadequate preparation of the uterus to the receptive state in
response to ovarian estrogen and P
4
. To ensure that Ptx3-null
females attained appropriate uterine receptivity, we examined
estrogen- and P
4
-responsive genes that are differentially
regulated on Day 4 (the day of the receptive phase). Although
the estrogen-responsive gene lactoferrin (Ltf ) is highly
expressed in the uterine epithelium on Day 1 of pregnancy
under the preovulatory estrogen surge, this gene is dramatically
downregulated when the uterus undergoes P
4
dominance on
Day 4 [26]. We found that although Ltf is highly expressed
in the uterine epithelia of both wild-type and Ptx3
/
mice on
Day 1 (Fig. 2A), it is dramatically downregulated on Day 4 of
pregnancy in both null and wild-type mice (Fig. 2B). Hoxa10
and amphiregulin (Areg) are P
4
-regulated genes in mice and are
expressed in a cell-specific manner in the receptive uterus on
Day 4 of pregnancy [27, 28]. We found that the expression
pattern of these genes is comparable between wild-type and
null uteri on Day 4 (Fig. 2B), suggesting that the loss of Ptx3
does not interfere with uterine receptivity achieved under
coordinated P
4
and estrogen signaling.
Decidualization is Compromised in the Absence of PTX3
Since Ptx3 expression is biphasic, first showing its
expression in stromal cells with the onset of blastocyst
attachment and then in decidual cells following implantation,
we sought to examine whether PTX3 participates in decidu-
alization and whether embryonic signals are absolutely
FIG. 3. Decidualization is compromised
in Ptx3-null uteri. A) In situ hybridization of
Ptx3 in sections of oil-induced deciduoma
(oil) and in noninfused wild-type uteri on
Days 5, 6, and 8 of pseudopregnancy.
Representative darkfield photomicrographs
of uterine cross-sections are shown. Bar ¼
200 lm. B) Fold changes in uterine weights
(oil versus control) between þ/þand /
uteri. Data are presented as fold change 6
SEM. *P,0.05, unpaired t-test. C) Photo-
graphs of decidual responses in a represen-
tative wild-type (þ/þ) and a maximally
responsive Ptx3-null (/) uterus are
shown.
428 TRANGUCH ET AL.
required for decidual Ptx3 expression. We used the model of
experimentally induced decidualization to address these
questions. In the absence of fertilized embryos in pseudopreg-
nant mice, the steroid hormonal milieu within the uterus is
similar to normal pregnancy, and nonspecific stimuli, such as
mechanical trauma or intraluminal oil infusion, evoke many
aspects of decidual cell reactions similar to those induced by
living blastocysts [26]. On Day 4 of pseudopregnancy, 20 ll
sesame oil was injected intraluminally into one uterine horn of
each pseudopregnant wild-type mouse; the contralateral horn
served as a control. Mice were sacrificed on Days 5, 6, and 8,
and in situ hybridization was performed to localize Ptx3
expression in uterine sections (Fig. 3A). We found that the
Ptx3 expression pattern in oil-induced decidualizing stromal
cells closely resembles that observed during normal pregnancy,
suggesting that signals arising from the implanting blastocysts
are not an absolute requirement for decidual Ptx3 induction.
Since Ptx3 is expressed in the deciduoma induced by
intraluminal oil infusion, we next investigated this event in the
absence of PTX3. We found that intraluminal oil infusion on
Day 4 of pseudopregnancy in Ptx3-null mice resulted in
significantly diminished decidual responses compared with
those observed in oil-infused wild-type uteri (Fig. 3, B and C).
More specifically, while all of the wild-type mice showed
typical decidual responses averaging 8-fold change in uterine
weight between control and oil-infused horns, three of seven
null mice showed no decidual responses. The highest fold
change in weight between control and oil-infused uterine horns
observed in one Ptx3
/
female was 4.3; a photograph of this
uterus is shown (Fig. 3C). These results suggest that the ability
of the uterine stroma to respond to decidualization is
compromised in the absence of Ptx3, implying a role for
PTX3 in this event as well.
Il1b Induction Precedes Ptx3 Expression in the Pregnant
Mouse Uterus
Unlike classic pentraxins present in the liver, PTX3 is
produced by macrophages and a variety of tissues upon
exposure to inflammatory stimuli, one being IL1B [9]. LPS is a
known inducer of IL1B, and we have previously shown that an
injection of LPS induces Il1b expression in both the liver and
mouse decidua on Day 7 of pregnancy [29]. We therefore
asked whether Ptx3 induction follows that of Il1b in this
system. Our Northern blot analysis shows that LPS induces
Il1b expression in the liver and decidua maximally at 1 h after
injection (Fig. 4A). While Ptx3 expression, as expected, was
not induced in the liver, its expression was maximally elevated
in Day 7 implantation sites (IS) at 2 and 6 h following LPS
injection (Fig. 4A). These results suggest that IL1B is an
inducer of Ptx3 in the uterus during early pregnancy.
To address whether endogenous IL1B is involved in
inducing the unique expression pattern of Ptx3 in the uterus,
we examined the spatiotemporal expression of Il1b in the peri-
implantation uterus using in situ hybridization. Il1b is
expressed abundantly but in a punctate pattern on Day 1 of
pregnancy in the luminal epithelium (Fig. 4B), perhaps
participating in the acute inflammatory response known to
occur in the uterus after coitus [30]. Il1b expression is
undetectable on Day 4, the day of uterine receptivity. However,
its expression appears in stromal cells immediately underneath
the luminal epithelium at the site of blastocyst attachment on
midnight of Day 4, and this pattern persists on Day 5 of
pregnancy. Furthermore, its temporal expression pattern
mimics that of Ptx3 in that it is undetectable on Day 6 but
reappears on Days 7 and 8 of pregnancy, albeit in fewer cells
(Fig. 4B).
IL1B is classically referred to as a marker for monocytes
and macrophages. Such IL1B-expressing monocytes/macro-
phages are transiently found within the capillaries in the
FIG. 4. Il1b induction precedes Ptx3 expression in the pregnant mouse
uterus. A) Northern blot hybridization of Il1b and Ptx3 in the liver and
implantation sites (IS) from wild-type mice injected with LPS on Day 7 of
pregnancy. Rpl7 was used as a housekeeping gene. B) In situ hybridization
of Il1b in the mouse uterus during early pregnancy. Hybridization signals
in representative darkfield photomicrographs of uterine cross-sections on
indicated days of pregnancy (D) are shown. Arrowheads indicate the
location of embryos. Bar ¼200 lm. le, luminal epithelium; s, stroma.
MATERNAL Ptx3 DEFICIENCY COMPROMISES IMPLANTATION 429
stromal bed at the implantation site on Day 5 of pregnancy in
mice [31]. Our observed localization of Il1b in the subluminal
stromal cells suggests a different source for this cytokine.
Regardless of the source, however, the pattern of Il1b
expression on midnight of Day 4 and Day 5 of pregnancy
correlates with that of Ptx3, suggesting a role for IL1B in
inducing uterine Ptx3 expression in a paracrine manner.Their
spatial localization on Days 7 and 8, however, is somewhat
different. Il1b expression is evident in scattered cells at both the
mesometrial and antimesometrial poles (Fig. 4B), whereas
expression of Ptx3 is more localized to the antimesometrial
pole (Fig. 1B).
Uterine Il1b and Ptx3 Expressions are Associated with
Blastocyst Activation
Our observation of both Il1b and Ptx3 expression in the
stromal cells surrounding the implanting blastocyst led us to
question whether implanting blastocysts regulate Il1b, and
therefore Ptx3, expression locally in the uterus. This would
suggest a role for PTX3 during the onset of implantation,
supporting our observation of its higher expression at
implantation sites (Fig. 1A). We used the delayed implantation
model to identify whether Ptx3 is upregulated at the time of
blastocyst activation for implantation. In situ hybridization was
performed on serial uterine sections of dormant (P
4
only) and
activated (P
4
þE
2
) wild-type uteri at 12 and 24 h following an
injection of E
2
(Fig. 5). We observed that uterine Il1b and Ptx3
expressions are undetectable surrounding dormant blastocysts
and throughout the P
4
-primed uterus, whereas activation of
blastocysts with the initiation of implantation by estrogen
upregulates both Il1b and Ptx3 expression in stromal cells
adjacent to implanting blastocysts 12 h after injection (Fig. 5).
This finding is consistent with Ptx3 expression in stromal cells
surrounding the implanting blastocyst on Day 4 midnight and
Day 5 of pregnancy (Fig. 1B). Whereas Ptx3 expression in
stromal cells surrounding the implanting blastocyst is main-
tained 24 h after injection, Il1b expression disappeared.
Collectively, these results suggest that signaling arising from
the implantation-competent blastocyst and/or uterus at the time
of implantation switches on the IL1B-PTX3 signaling pathway.
DISCUSSION
PTX3’s role in the immune system is readily apparent, since
Ptx3 null mice are more susceptible to select pathogens [8], and
mice overexpressing Ptx3 have improved survival in response
to endotoxic shock and sepsis [21]. Studies in Ptx3-null mice
have also shown its important role in female fertility,
specifically in cumulus-oocyte interactions during ovulation,
impacting fertilization [7, 8]. Our present study showing Ptx3
expression in stromal cells at the time of blastocyst attachment
on Day 4 (2400 h) of pregnancy, its persistent expression
through Day 5 during the PDZ formation, and the observed
compromised implantation and decidualization in Ptx3-null
females suggests that PTX3 plays a crucial role in implantation
and decidualization. PTX3 could be involved in protecting the
embryo from maternal immunologic or other blood-borne
noxious stimuli before the establishment of the avascular PDZ
barrier. This is consistent with its disappearance on Day 6,
when the PDZ is fully established. The appearance of Ptx3 at
the border of the PDZ and SDZ on Days 7 and 8 also implies
an embryo protective role, since PDZ cells undergo demise
around this time to make room for the growing embryo.
Alternatively, PTX3 could also play a role in the formation
of the PDZ, which begins to form late on Day 5, with its full
establishment on Day 6 [26], the day that Ptx3 expression
disappears. Since PTX3 is known to play a role in clearing
apoptotic cells [21], it could serve to clear dying PDZ cells
during this time. It is also known that PTX3 inhibits FGF2-
dependent angiogenesis and proliferation [19, 20]. Since Fgf2
is also expressed in cells immediately surrounding the embryo
upon its attachment to the uterus [32], perhaps PTX3 acts to
FIG. 5. Uterine Il1b and Ptx3 expression
are associated with blastocyst activation
and implantation. In situ hybridization of
Il1b and Ptx3 in dormant (P
4
) versus
activated (E
2
þP
4
) uteri is shown. Arrow-
heads indicate locations of embryos in
serial sections. Bar ¼200 lm.
430 TRANGUCH ET AL.
counter FGF2-induced angiogenesis to contribute to the
avascular nature of the PDZ and differentiation of stromal
cells to form this zone [19, 26]. These putative roles of PTX3
could explain the reason for implantation and decidualization
failure in Ptx3-null mice.
The significance of Ptx3 expression on Days 7 and 8 of
pregnancy is not clearly understood. On these days, Ptx3 is
specifically expressed at the boundary between the PDZ and
SDZ. Perhaps PTX3 serves as a barrier between the
degenerating PDZ and proliferating SDZ on these days. It is
also possible that PTX3 present at the interface regulates cell
death in the PDZ and differentiation of decidualizing stromal
cells in the SDZ. This is consistent with findings showing
PTX3’s role in the clearance of apoptotic cells and activation of
the complement cascade [9, 17, 18, 20]. PTX3 has also been
characterized as a marker of human inflammatory conditions,
and studies suggest a protective role for PTX3 at sites of
inflammation, perhaps to prevent autoimmune reactions [17,
21]. The underlying cause and meaning of the abrupt
disappearance of Ptx3 expression from Day 9 onwards is not
clearly understood. It is possible that other immunologic or
physiologic fetoplacental factors are in place to protect the
developing embryo at this time.
Our finding that LPS stimulates Il1b expression to induce
Ptx3 in decidua of Day 7 pregnant mice implies that IL1B is an
inducer of PTX3 in vivo. In fact, Il1b appears in stromal cells
immediately surrounding the implanting blastocyst, with Ptx3
expressed in stromal cells a few cell layers away, suggesting
that IL1B induces Ptx3 expression in neighboring stromal cells.
Furthermore, similar to Ptx3 expression, Il1b expression is
concentrated at implantation sites compared with interim-
plantation sites, and both are expressed with the initiation of
the blastocyst attachment by estrogen in a delayed implantation
model. In the same vein, there is a recent report showing that
signals emanating from trophoblast cells upregulate decidual
Ptx3 expression in humans [23]. It is, however, possible that
other factors play a role in inducing PTX3 during decidualiza-
tion. We speculate that implantation-related stimuli arising
from the blastocyst and/or uterus cooperate to induce Ptx3
expression. This question is currently under investigation in
our laboratory.
Our present study illustrates for the first time an important
role for uterine PTX3 in implantation and decidualization.
These results have clinical implications, since PTX3 is
expressed in the receptive endometrium, and trophoblast cells
influence decidual PTX3 expression in humans [22, 23].
ACKNOWLEDGMENTS
We are grateful to Martin M. Matzuk (Baylor College of Medicine,
Houston, TX) for providing us with Ptx3-null mice, to Fuhua Xu for
assistance with statistical analysis, and to Sung Tae Kim for assistance with
cloning.
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