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Cytosolic phospholipase A2αdeficiency is crucial for ‘on-time’ embryo implantation that
directs subsequent development
Song, H., Lim, H., Paria, B. C., Matsumoto, H., Swift, L. L., Morrow, J., Bonventre, J. V. and Dey, S. K. Development 129, 2879-
2889.
In the printed version of this article, the title is incorrect. The correct title is given below.
Cytosolic phospholipase A2αis crucial for ‘on-time’ embryo implantation that directs
subsequent development
CORRIGENDUM 3761
INTRODUCTION
Embryo implantation in the uterus is one of the most crucial
steps in mammalian embryo development. It requires both
prior preparation of the receptive uterus and activation of the
blastocyst, and is initiated by the attachment of the blastocyst
trophectoderm to the uterine luminal epithelium between 22:00
and 24:00 hours on day 4 of pregnancy in mice (Das et al.,
1994). Concurrently with the attachment reaction, an increased
endometrial vascular permeability becomes evident at the
site of implantation. This event is followed by localized
endometrial decidualization with luminal epithelial apoptosis
and subsequent invasion of the trophoblast through the
basement membrane into the stroma (Dey, 1996). Coordinated
interactions between ovarian estrogen and progesterone (P4)
prime the uterus for implantation. For example, removal of
preimplantation ovarian estrogen secretion in mice by
ovariectomy results in blastocyst dormancy and prevents the
attachment reaction. Dormant blastocysts can be activated to
implant in the P4-primed uterus by a single injection of
estrogen (Paria et al., 1993a; Paria et al., 1993b). The
molecular signals that regulate implantation are of
considerable clinical relevance as understanding the nature of
these signals should lead to strategies for correcting
implantation failures and pregnancy losses.
Prostaglandins (PGs) are implicated in various female
reproductive functions (Lim et al., 1997; Lim et al., 1999a). PGs
are generated from arachidonic acid (AA) by phospholipase A2s
(PLA2s) followed by cyclooxygenases (COX). PLA2plays
crucial roles in diverse cellular functions, including
phospholipid metabolism, immune functions and signal
transduction by generating bioactive lipid mediators (Gijon and
Leslie, 1999; Valentin and Lambeau, 2000). Once activated by
a variety of stimuli, PLA2hydrolyzes the ester bonds of fatty
acids at the sn-2 position of phospholipids, producing free fatty
acids and lysophospholipids. The mammalian PLA2
superfamily consists of four major subfamilies that include
cytosolic (cPLA2), secretory (sPLA2), Ca2+-independent
(iPLA2) and platelet-activating factor (PAF) acetylhydrolase.
While cPLA2and sPLA2participate in various cellular
functions by generating free fatty acids, including AA, iPLA2
and PAF acetylhydrolase primarily contribute to membrane
remodeling and attenuation of PAF bioactivity, respectively
(Murakami et al., 2000). PLA2-derived AA gives rise to various
lipid mediators, including PGs, leukotrienes, thromboxanes and
endocannabinoids. These mediators via various signaling
2879
Development 129, 2879-2889 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV3654
Cytosolic phospholipase A2α(cPLA2α) is a major provider
of arachidonic acid (AA) for the cyclooxygenase (COX)
system for the biosynthesis of prostaglandins (PGs). Female
mice with the null mutation for Pla2g4a (cPLA2α) produce
small litters and often exhibit pregnancy failures, although
the cause(s) of these defects remains elusive. We show that
the initiation of implantation is temporarily deferred
in Pla2g4a–/– mice, shifting the normal ‘window’ of
implantation and leading to retarded feto-placental
development without apparent defects in decidual growth.
Furthermore, cPLA2αdeficiency results in aberrant
uterine spacing of embryos. The deferred implantation and
deranged gestational development in Pla2g4a–/– mice were
significantly improved by exogenous PG administration.
The results provide evidence that cPLA2α-derived AA
is important for PG synthesis required for on-time
implantation. This study in Pla2g4a–/– mice, together with
the results of differential blastocyst transfers in wild-type
mice provides the first evidence for a novel concept that a
short delay in the initial attachment reaction creates a
ripple effect propagating developmental anomalies during
the subsequent course of pregnancy.
Key words: cPLA2α, implantation, uterus, embryo, pregnancy
SUMMARY
Cytosolic phospholipase A2αdeficiency is crucial for ‘on-time’ embryo
implantation that directs subsequent development
Haengseok Song1, Hyunjung Lim2, Bibhash C. Paria1,3, Hiromichi Matsumoto1, Lany L. Swift4,
Jason Morrow4, Joseph V. Bonventre5and Sudhansu K. Dey1,*
1Departments of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160-7336, USA
2Departments of Obstetrics and Gynecology, Cell Biology and Physiology, Washington University School of Medicine, St. Louis,
MO 63110, USA
3Department of Pediatrics, University of Kansas Medical Center, Kansas City, KS 66160-7336, USA
4Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-2279, USA
5Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
*Author for correspondence (e-mail: sdey@kumc.edu)
Accepted 26 March 2002
2880
pathways exert a wide range of cellular functions (Clark et al.,
1995; Mechoulam et al., 1998; Serhan and Oliw, 2001). Among
the PLA2superfamily members, cPLA2is a key regulator of
eicosanoid biosynthesis, because it selectively releases AA
(Clark et al., 1995). It lacks sequence similarity to other
members of the PLA2superfamily, suggesting a unique role of
cPLA2. Recently, two other cPLA2isoforms, cPLA2βand
cPLA2γ, have been identified in the human EST database
(Underwood et al., 1998; Song et al., 1999), assigning a new
name for cPLA2as cPLA2α, although they, unlike cPLA2α, do
not show substrate preference for AA (Song et al., 1999).
As a major source of AA for PG synthesis, cPLA2α
undergoes Ca2+-dependent translocation to the perinuclear and
endoplasmic reticular (ER) membranes, the sites of COX
enzymes (Clark et al., 1995; Murakami et al., 2000). To date,
more than ten PLA2 and two COX isoforms (COX1 and COX2)
have been identified in mammals (Smith and DeWitt, 1996; Six
and Dennis, 2000; Valentin and Lambeau, 2000), indicating
their differential roles in distinct biological responses to
various stimuli. For example, in mast cells, sPLA2and cPLA2α
are functionally coupled to COX1 and COX2, and participate
in early and late PGD2synthesis, respectively (Reddy and
Herschman, 1997).
Owing to their vasoactive, mitogenic and differentiating
properties, PGs are implicated in ovulation and implantation
(Lim et al., 1997). Recent genetic evidence points towards
essential functions of enzymes responsible for PG biosynthesis
in reproduction. Consistent with our previous report of uterine
induction of COX-2 at the site of blastocyst implantation
(Chakraborty et al., 1996), mice with null mutation for Ptgs2,
which encodes COX2, show multiple female reproductive
failures, such as defective ovulation, fertilization, implantation
and decidualization (Lim et al., 1997). By contrast, female
mice with null mutation for Ptgs1, which encodes COX1, are
fertile with limited parturition defects (Langenbach et al.,
1995). Studies on Ptgs2–/– mice further showed that while
prostacyclin (PGI2) plays a major role in implantation, PGE2
plays a complementary role in this process (Lim et al., 1999a).
Of the many PLA2s, cPLA2(cPLA2α) is known to couple
functionally to COX2 in specific cell types (Reddy and
Herschman, 1997; Takano et al., 2000), and mice with null
mutation for Pla2g4a, which encodes cPLA2α, have small
litters with presumed parturition defects and often show
pregnancy failures (Bonventre et al., 1997; Uozumi et al.,
1997). However, the underlying cause of this reduced fertility
still remains unknown. Using Pla2g4a mutant mice and
differential blastocyst transfers in wild-type mice, we show
here that a short deferral of the normal ‘window’ of
implantation results in severe progressive developmental
anomalies during the course of pregnancy. In addition, cPLA2α
deficiency results in abnormal uterine spacing of embryos.
MATERIALS AND METHODS
Mice
The disruption of the Pla2g4a gene was originally achieved in J1 ES
cells by homologous recombination as described (Bonventre et al.,
1997). Genotyping was by PCR analysis of genomic DNA. All of the
mice used were housed in the Animal Care Facility at the University
of Kansas Medical Center, according to NIH and institutional
guidelines for laboratory animals.
Ovulation and fertilization
To examine ovulation and fertilization, wild-type or Pla2g4a–/– mice
were bred with fertile males with same genotypes, respectively. The
morning of finding a vaginal plug was designated day 1 of pregnancy.
Mice were killed on day 2 of pregnancy and oviducts were flushed
with Whitten’s medium to recovery eggs and embryos. Their
morphology was examined under a dissecting microscope. In vitro
maturation and fertilization were performed with partial zona
pellucida dissection as previously described by us (Matsumoto et al.,
2001).
Implantation and decidualization
Implantation sites on days 5 and 6 of pregnancy were visualized by
an intravenous injection of Chicago Blue dye solution (Paria et al.,
1993b). The uteri of mice with a few or without implantation site were
flushed with Whitten’s medium to recover unimplanted blastocysts.
Recovered embryos were observed under a dissecting microscope.
For reversal experiments, PGE2and cPGI (Cayman Chemical, Ann
Arbor, MI) were prepared in 10% ethanol/90% saline. Injections
(PGE2+ cPGI, 5 µg each/mouse, i.p.) were given at 10:00 and 18:00
hours on day 4 of pregnancy. The control mice received the vehicle.
Implantation sites were recorded on day 5 morning (10:00 hours) by
the blue dye method.
To induce artificial decidualization, pseudopregnant wild-type or
Pla2g4a–/– mice received intraluminal infusion of sesame oil (25 µl)
in one uterine horn on day 4 or day 5 (10:00 hours) and were killed
4 days later. Uterine weights of the infused and non-infused (control)
horns were recorded and the fold increases in uterine weights were
used as an index of decidualization (Lim et al., 1997).
Blastocyst transfer
Mating females with vasectomized males induced pseudopregnancy.
Day 4 wild-type or Pla2g4a–/– blastocysts were transferred into the
uteri of wild-type or Pla2g4a–/– pseudopregnant recipients on day 4
(Paria et al., 1993b). The number of implantation sites was recorded
by the blue dye method on day 5 morning (10:00 hours) (Paria et al.,
1993b). To examine pregnancy outcome of implantation beyond the
normal ‘window’ of uterine receptivity, day 4 wild-type blastocysts
were transferred into wild-type pseudopregnant recipients on day 4 or
day 5 of pseudopregnancy. Recipients were examined for subsequent
developmental events on day 12 or observed for delivery of pups at
term.
Gross observation and histological examination of
implantation sites and embryos
Day 12 implantation sites and embryos were examined using the
protocol as previously described with some modification (Hogan
et al., 1994). Isolated day 12 implantation sites were weighed
individually, fixed in 10% formalin overnight and dissected to isolate
embryos. Isolated embryos were weighed individually and their
images were captured to examine the size and gross morphology. For
histological examination, paraffin wax-embedded sections of day 12
implantation sites were stained with Eosin and Hematoxylin.
Measurement of PGs
PGs were quantitated using gas chromatography/negative ion
chemical ionization mass spectrometric assays as described
previously (DuBois et al., 1994).
RT-PCR analysis
RT-PCR analyses were performed using gene-specific primers by
following a protocol previously described (Paria et al., 1993a).
Hybridization probes
Sense or antisense 35S-labeled cRNA probes were generated using
appropriate polymerases from cDNAs to Pla2g4a, Pla2g2d, Pla2g2e,
Pla2g10, Lif, Hoxa10, Hegfl, Areg, Ptgs1 and Ptgs2 for in situ
H. Song and others
2881cPLA2αin embryo implantation and development
hybridization as previously described (Das et al., 1994). Mouse
cDNAs to Pla2g4a, Pla2g2d, Pla2g2e and Pla2g10 were generated
by RT-PCR cloning using TOPO cloning kit (Invitrogen, Carlsbad,
CA).
In situ hybridization
In situ hybridization was performed as previously described (Das et
al., 1994; Song et al., 2000). Frozen sections (12 µm) were mounted
onto poly-L-lysine-coated slides, fixed in cold 4% paraformaldehyde
solution in PBS, acetylated and hybridized at 45°C for 4 hours in
hybridization buffer containing the 35S-labeled antisense cRNA
probes. After hybridization, the sections were treated with RNase A
(20 µg/ml) at 37°C for 20 minutes. RNase A-resistant hybrids were
detected by autoradiography. Sections hybridized with the sense
probes served as negative controls.
RESULTS
Ovulation and fertilization are modestly reduced in
Pla2g4a
–/– mice
To determine the cause(s) of reduced fertility in Pla2g4a–/–
female mice, we examined in detail the reproductive
phenotypes of these females during pregnancy. There is
evidence that genetic background of mice contributes to
different phenotypes (Threadgill et al., 1995). Furthermore, a
number of inbred mouse strains including C57BL/6J and
129/Sv have a natural null mutation in Pla2g2a encoding
sPLA2-IIA (Kennedy et al., 1995; MacPhee et al., 1995). Thus,
defective reproduction in Pla2g4a–/– mice on C57BL/6J
background may reflect deficiency of both cPLA2αand sPLA2-
IIA. By contrast, the outbred CD1 mice with larger litter sizes
have variable genotype (+/+, +/–, –/–) of Pla2g2a mutation
(Kennedy et al., 1995). Therefore, to study the reproductive
events in more detail, we introduced cPLA2αdeficiency in
CD1 mice by crossing with C57BL/6J Pla2g4a–/– mice. The
results on these two strains are described.
C57BL/6J
To examine the ovulation and fertilization status in Pla2g4a–/–
mice, we counted the number of ovulated eggs and fertilized
2-cell embryos on day 2 of pregnancy. All of the wild-type
(n=13) and Pla2g4a–/– (n=10) mice ovulated (Fig. 1A). A
slight, but statistically insignificant, reduction in the number of
ovulated eggs was noted in Pla2g4a–/– mice. A modest
reduction in the fertilization rate in mutant mice was also
noted. This modest reduction was not due to defective sperm
functions in Pla2g4a–/– males, as assessed by in vitro
fertilization and cross-breeding experiments (data not shown).
Thus, subsequent experiments used wild-type and Pla2g4a–/–
females mated with males of the same genotypes.
CD1
Our next objective was to determine if these phenotypes were
retained in Pla2g4a–/– mice on the CD1 background. While
ovulation and fertilization rates were increased in both wild-
type and Pla2g4a–/– mice, their profiles were similar to
C57BL/6J mice (Fig. 1B). These results suggested that
cPLA2αhas a modest role in ovulation and fertilization, but
not to the extent that was observed for COX2 deficient mice
showing profound defects in these processes (Lim et al., 1997).
Normal ‘window’ of implantation is altered in
Pla2g4a
–/– mice
Although ovulation and fertilization were somewhat reduced
in Pla2g4a–/– mice on C57BL/6J or CD1 background, these
reduced rates cannot fully account for the reduced litter size
observed in these mice (Bonventre et al., 1997; Uozumi et al.,
1997). Furthermore, frequent pregnancy failure in plug-
positive Pla2g4a–/– mice (Bonventre et al., 1997) also suggests
uterine defects between fertilization and parturition. We thus
investigated whether cPLA2αdeficiency impedes implantation
and decidualization in mice on these backgrounds. Increased
vascular permeability at the site of blastocyst implantation was
recorded on day 5 of pregnancy by the blue dye method (Paria
et al., 1993b).
C57BL/6J
While an average of approx. nine implantation sites
(9.4±0.4/mouse) were observed in all of the eight wild-type
mice, only about three implantation sites (2.6±0.6/mouse) were
detected in nine out of 10 Pla2g4a–/– mice on day 5 (09:00
hours) of pregnancy. The reduced number of implantation sites
was not due to compromised ovulation and fertilization in
Pla2g4a–/– mice for two reasons. First, the few implantation
sites that were detected in Pla2g4a–/– mice on day 5 of
pregnancy showed a very weak blue reaction, suggesting
defective vascular permeability changes during the attachment
reaction. Second, the number of blastocysts recovered from
these Pla2g4a–/– mice was more than the visible implantation
sites, suggesting that these blastocysts failed to initiate the
attachment reaction.
CD-1
Implantation defects were more prominent in Pla2g4a–/– mice
on this background. On day 5, an average of less than two
implantation sites (1.8±0.3/mouse) was detected in four out of
12 Pla2g4a–/– mice examined (Fig. 2A). Not only was the
number of implantation sites remarkably low, but also a large
number of unimplanted blastocysts was recovered from all 12
mice after flushing their uteri (Fig. 2B). Of the 61 blastocysts
recovered, 55 of them were zona free and only six blastocysts
Fig. 1. Ovulation and fertilization rates in Pla2g4a–/– mice. The rate
of ovulation and fertilization in wild-type and Pla2g4a–/– mice was
examined on day 2 of pregnancy on C57BL/6J (A) and CD1 (B)
genetic backgrounds. The numbers within the bars indicate the
number of mice with ovulation/total number of mice. Results of
ovulation are mean±s.e.m. Statistical significance was evaluated
using unpaired t-test and χ2-test, respectively (*P<0.01).
2882
were zona encased. Morphological appearance of these
blastocysts apparently looked normal. Similar to C57BL/6J
Pla2g4a–/– mice, the permeability changes at the implantation
sites were also poor (Fig. 2C, top). By contrast, an average of
11 distinct implantation sites was detected in seven out of seven
wild-type mice examined (Fig. 2A,C).
It has long been held that implantation in rodents occurs only
for a limited period (~24 hours) defined as the ‘window’ of
receptivity for implantation (Paria et al., 1993b; Dey, 1996). In
mice, the uterus becomes receptive on day 4 of pregnancy with
the initiation of the attachment reaction around midnight (Paria
et al., 1993b; Das et al., 1994; Dey, 1996). We surmised that
unimplanted blastocysts that we observed in Pla2g4a–/– uteri
on day 5 could implant beyond the normal ‘window’ of
implantation. As shown in Fig. 2A, all of the Pla2g4a–/– mice
(12/12) showed distinct implantation sites (6.8±0.6) on day 6
of pregnancy, providing evidence that implantation had
occurred beyond the normal ‘window’. Similar results were
obtained using C57BL/6J mice (data not shown). Collectively,
these results establish that implantation occurs beyond the
normal ‘window’ of implantation in Pla2g4a–/– mice,
suggesting that uterine and/or blastocyst cPLA2αis crucial to
the initial attachment reaction.
Experimentally induced decidualization is normal in
Pla2g4a
–/– mice
The blastocyst attachment reaction is followed by extensive
stromal cell proliferation and differentiation into decidual cells.
Decidualization can also be induced experimentally in
pseudopregnant or steroid hormonally prepared uteri by
intraluminal oil infusion (Lim et al., 1997). Although the
attachment reaction between the blastocyst and uterine luminal
epithelium is deferred in the absence of cPLA2α, whether it is
also crucial for decidualization is not known. Thus, we
examined decidualization in Pla2g4a–/– mice by intraluminal
oil infusion on day 4 of pseudopregnancy (Fig. 2D). The results
show that 11 out of 12 Pla2g4a–/– mice had similar decidual
response as the wild-type mice with respect to increased
uterine weight (14.5±1.6-fold versus 15.6±2.7-fold). Taken
together, the results suggest that the initial attachment reaction
is perturbed in Pla2g4a–/– mice, but not the ability of the
uterine stroma for decidualization. This is a novel finding that
has not been observed in many other mutant mice with peri-
implantation defects (Benson et al., 1996; Lim et al., 1997;
Robb et al., 1998).
Pla2g4a
expression follows dynamic changes in
Ptgs1
and
Ptgs2
expression in the uterus during
implantation
As Pla2g4a–/– mice show implantation defects, it is possible
that the Pla2g4a gene has a cell-specific expression pattern
relevant to implantation. We compared the expression of
Pla2g4a with Ptgs1 (COX1) and Ptgs2 (COX2) during
implantation by in situ hybridization. As shown in Fig. 3A,
Pla2g4a is expressed in the uterine epithelia on day 4 of
pregnancy in a pattern similar to that of Ptgs1 (Chakraborty et
al., 1996). With the initiation and progression of implantation,
the pattern of Pla2g4a expression was similar to that of Ptgs2
(COX2) on days 5-8 of pregnancy. Pla2g4a was expressed in
stromal cells surrounding the implanting blastocyst on day 5
H. Song and others
Fig. 2. Implantation and decidualization in
Pla2g4a–/– mice. (A) The number of
implantation sites was examined on days 5 and
6 of pregnancy in wild-type and Pla2g4a–/–
mice by the blue dye method. The numbers
above the bars indicate the number of mice
with implantation sites/total number of mice
(unpaired t-test, *P<0.001). The uteri of mice
with a few or without implantation sites were
flushed to recover unimplanted blastocysts.
The mice without implantation sites or
blastocysts were excluded from the
experiments. (B) A representative
photomicrograph of blastocysts recovered
from Pla2g4a–/– mice on day 5 of pregnancy
(1000 hours) is shown. Note blastocysts with
(arrow) or without zona pellucida.
(C) Representative photographs of uteri with
implantation sites (blue bands) on days 5 and
6. Note very few or no implantation sites on
day 5, but unevenly spaced implantation sites
on day 6 in Pla2g4a–/– mice. Arrowhead and
arrow indicate ovary and implantation site,
respectively. Brackets indicate crowding of
implantation sites. (D) Decidualization. Wild-
type or Pla2g4a–/– mice received intraluminal
oil infusion on day 4 of pseudopregnancy. On
day 8, uterine weights were recorded. Fold
increases denote comparison of weights
between infused and non-infused uterine
horns. The numbers above the bars indicate the number of responding mice/total number of mice. No significant difference in
decidualization was noted between wild-type and Pla2g4a–/– mice (unpaired t-test; P>0.05).
2883cPLA2αin embryo implantation and development
of pregnancy. On day 6-8, the expression was primarily
restricted to the mesometrial pole of the implantation site.
However, the expression of Pla2g4a was more widespread and
apparently at lower levels than that of Ptgs2. These results
suggest that cPLA2αis available as an AA provider for uterine
PG biosynthesis during implantation.
Expression of implantation-specific genes are
dysregulated in
Pla2g4a
–/– mice
Deferred implantation in Pla2g4a–/– mice could be due to
deficiency of uterine cPLA2αand/or secondary to aberrant
expression of genes considered important for implantation.
Normal implantation requires preparation of the uterus to the
receptive stage and embryo-uterine interactions for the
attachment reaction followed by vascular permeability and
stromal decidualization at the sites of blastocysts (Dey, 1996).
Thus, we examined uterine genes involved in these events.
Genes including Areg (amphiregulin), Ptgs1, Hoxa10 and Lif
are expressed on day 4 morning and implicated in uterine
preparation (Benson et al., 1996; Chakraborty et al., 1996; Lim
et al., 1997; Song et al., 2000). These genes were appropriately
expressed in Pla2g4a–/– mice, suggesting that uterine
preparation was not altered (data not shown). However, Hegfl,
the gene encoding HB-EGF, the earliest known molecular
marker of embryo-uterine interaction for implantation (Das
et al., 1994), was not induced in the luminal epithelium
surrounding the blastocyst prior to the attachment reaction on
day 4 night (data not shown) or on day 5 morning in Pla2g4a–/–
mice not showing implantation sites (blue bands) (Fig. 3B).
Furthermore, Ptgs2 and Lif, which are normally induced in the
uterus surrounding the blastocyst during the attachment
reaction (Chakraborty et al., 1996; Song et al., 2000), were
either undetectable or aberrantly expressed at the sites of
blastocysts in Pla2g4a–/– mice on day 5 in the absence of
implantation (Fig. 3B). These results show that the expression
of genes involved during early implantation is altered when on-
time implantation does not occur in the absence of uterine
cPLA2α, further confirming that the implantation process had
been deferred.
Maternal cPLA2αis crucial for implantation
Although Pla2g4a is expressed in the uterus during
implantation and the attachment reaction is temporarily
deferred in the absence of cPLA2α, it is possible that embryonic
cPLA2αis also a contributing factor in directing proper
embryo-uterine interactions during implantation. Thus, we
performed reciprocal embryo transfer experiments. Day 4 wild-
type or Pla2g4a–/– blastocysts were transferred into wild-type
or Pla2g4a–/– recipients on day 4 of pseudopregnancy and
implantation rate was examined 24 hours later. As shown in
Table 1, day 4 wild-type blastocysts transferred into wild-type
recipient uteri showed normal complementation of implantation
(42.4%, n=6). By contrast, wild-type blastocysts transferred
into Pla2g4a–/– recipients showed considerably reduced
number of implantation sites (21.2%, n=8). However, reduced
implantation rate was not observed when Pla2g4a–/– blastocysts
were transferred into wild-type uteri; over 40% of the
blastocysts transferred showed implantation in all seven mice.
Collectively, the results suggest that maternal cPLA2α, but not
embryonic, is the primary contributor to on-time implantation.
Fig. 3. Expression of Pla2g4a in wild-type uteri during implantation and expression of implantation-specific genes in Pla2g4a–/– uteri.
(A) Comparison of expression of Pla2g4a with Ptgs1 and Ptgs2 in the mouse uterus during implantation. In situ hybridization of Pla2g4a,
Ptgs1 and Ptgs2 on days 4-6 (09:00 hours) of pregnancy is shown. (B) In situ hybridization of Hegfl, Ptgs2, and Lif in uteri of wild-type and
Pla2g4a–/– mice on day 5 of pregnancy (10:00 hours). Note aberrant expression of Hegfl, Ptgs2 and Lif in the luminal epithelium and/or
underlying stroma surrounding the blastocyst in Pla2g4a–/– mice. Arrows indicate the location of blastocysts. ge, glandular epithelium; le,
luminal epithelium; s, stroma; myo, myometrium.
2884
Uterine levels of PGs are reduced in
Pla2g4a
–/– mice
The production of eicosanoids is reduced in Pla2g4a–/–
macrophages (Bonventre et al., 1997; Uozumi et al., 1997). It
is not known whether similar situation exists in Pla2g4a–/–
uteri. Thus, we examined whether cPLA2αdeficiency leads to
the reduced PG levels prior to and during implantation. Our
results show that the levels of PGI2were significantly reduced
in Pla2g4a–/– uteri on day 4, and both PGI2and PGE2were
reduced on day 5 of pregnancy (Fig. 4A,B). PGI2is the major
PG that is produced at the implantation sites in mice (Lim et
al., 1999a). The less significant difference in uterine PGE2
levels on day 4 could be due to the expression of specific
sPLA2family members as providers of AA for COX 1. This is
consistent with co-localization of Pla2g4a and Pla2g10
(sPLA2-X) with Ptgs1 on day 4 prior to the attachment
reaction, and primarily of Pla2g4a with Ptgs2 from the time
of attachment reaction and thereafter (see Fig. 3A).
Furthermore, the reduced levels, but not complete abrogation,
of PGs in Pla2g4a–/– mice suggest that other sPLA2family
members contribute partially to maintain basal levels of uterine
PGs. In this respect, our results show that Pla2g1b (sPLA2-IB),
Pla2g2a (sPLA2-IIA), Pla2g2c (sPLA2-IIC), and Pla2g5
(sPLA2-V) are undetectable in day 4 pregnant uteri by RT-
PCR, although they are detected in other tissues used as
controls. However, RT-PCR detected uterine expression of
three other members, Pla2g2d (sPLA2-IID), Pla2g2e (sPLA2-
H. Song and others
Table 1. Implantation of blastocysts transferred into pseudopregnant wild-type or Pla2g4a–/– mice
Genotypes Number of Number of Number of Number of Number of
Blastocysts Recipients blastocysts transferred recipients mice with IS mice without IS IS (%)
+/+ +/+ 85 6 6 0 36 (42.4)*
+/+ –/– 113 8 7 1 24 (21.2)†
–/– +/+ 94 7 7 0 38 (40.5)*
Day 4 wild-type or Pla2g4a–/– blastocysts were transferred into uteri of wild-type or Pla2g4a–/– recipients on day 4 of pseudopregnancy. Recipients were killed
on day 5 to examine implantation sites (IS) by the blue dye method. Uteri without IS were flushed with saline to recover any unimplanted blastocysts.
* are significantly different from †(χ2-test; P<0.01).
Fig. 4. Uterine status of PGs and expression of
genes encoding sPLA2s, and restoration of normal
implantation in Pla2g4a–/– mice by PGs.
(A,B) The levels of PGs in uteri of wild-type and
Pla2g4a–/– mice on days 4 and 5 of pregnancy,
respectively. PGI2was measured as 6-keto-PGF1α.
N.D., not detectable (unpaired t-test, *P<0.05;
**P<0.01; n=4-5). (C) Expression of genes
encoding sPLA2isoforms in various wild-type
mouse tissues by RT-PCR. Heart (H), intestine (I),
kidney (K), liver (Li), lung (Lu), spleen (S) and
testis (T) tissue samples were used as controls
along with uterine (U) samples obtained on day 4
of pregnancy. Actb, mouse β-actin. (D) In situ
hybridization of Pla2g10 (sPLA2-X) in uteri of
wild-type mice on days 4 and 5 of pregnancy. Note
uterine expression of Pla2g10 similar to that of
Pla2g4a and Ptgs1 on day 4 (compare with Fig.
3A). The arrow indicates the location of a
blastocyst. ge, glandular epithelium; le, luminal
epithelium; s, stroma; myo, myometrium.
(E) Restoration of normal implantation in
Pla2g4a–/– mice. Pla2g4a–/– mice were injected
with saline or PGE2plus cPGI twice (10:00 and
18:00 hours) on day 4 and implantation sites were
examined on day 5 (10:00 hours). The numbers
above the bars indicate the number of Pla2g4a–/–
mice with implantation sites/total number of
Pla2g4a–/– mice used (unpaired t-test; *P<0.001).
The mice without implantation sites or blastocysts
were excluded from the experiments.
(F) Representative photographs of day 5 uteri of
Pla2g4a–/– mice given the vehicle or PGs on day 4
of pregnancy. Note increased number of
implantation sites with prominent blue reaction
after PG treatment. Brackets indicate crowding of
implantation sites.
2885cPLA2αin embryo implantation and development
IIE) and Pla2g10 (sPLA2-X), on day 4 (Fig. 4C). Further
examination by in situ hybridization showed that while the
uterine expression of Pla2g2d and Pla2g2e was insignificant
(data not shown), Pla2g10 was expressed in the uterine
epithelium similarly to Pla2g4a on day 4, but the expression
was very low on day 5 (Fig. 4D). These results suggest that
sPLA2-X could serve as an alternative source of AA in
Pla2g4a–/– mice prior to implantation, but its role during
implantation is questionable.
Exogenous administration of PGs restores normal
‘window’ of implantation in
Pla2g4a
–/– mice
The reduced levels of PGs in Pla2g4a–/– mice led us to restore
normal implantation timing in Pla2g4a–/– mice by
supplementing PGs (Fig. 4E,F). Administration of PGE2and
carbaprostacyclin (cPGI, a more stable analogue of PGI2) to
Pla2g4a–/– mice twice on day 4 (10:00 and 18:00 hours, i.p.)
restored implantation when examined on day 5. For example,
seven out of eight mice injected with PGE2and cPGI showed
an average of approx. eight implantation sites similar to normal
day 5 implantation sites in wild-type mice (Fig. 4E,F). By
contrast, parallel experiment without PG supplementation
again showed poor implantation rate as described above; seven
out of 16 Pla2g4a–/– mice injected with the vehicle had an
average of two implantation sites, although blastocysts were
recovered from all 16 mice examined. These results show that
PG supplementation restores the normal ‘window’ of
implantation in Pla2g4a–/– mice, further reinforcing a major
role of cPLA2αin PG biosynthesis. Although thromboxane B2
(TxB2) levels were drastically reduced in Pla2g4a–/– uteri (Fig.
4A,B), it is not likely to play any significant role in
implantation, as mice deficient in thromboxane receptor do not
show reproductive defects (Thomas et al., 1998).
Implantation beyond the normal ‘window’ of
implantation in
Pla2g4a
–/– mice leads to defective
postimplantation development
We demonstrate here that implantation occurs beyond the
normal ‘window’ of uterine receptivity in Pla2g4a–/– mice
(Fig. 2). This new finding of deferred implantation and small
litter size led us to scrutinize postimplantation embryo
development in Pla2g4a–/– mice. We examined the growth and
development of the implantation sites on day 12 to assess the
effects of deferred implantation on subsequent developmental
processes. As shown in Fig. 5A, while most of the implantation
sites in wild-type or Pla2g4a+/– mice were well spaced and
developed normally, many implantation sites in Pla2g4a–/–
mice were smaller and showed signs of resorption. The median
weight of day 12 implantation sites or isolated embryos was
reduced significantly in Pla2g4a–/– mice when compared with
wild-type or Pla2g4a+/– mice (Fig. 5B). Although there was
only a short delay in the timing of implantation, many of the
isolated embryos from Pla2g4a–/– mice exhibited retarded
growth at varying degrees (Fig. 5C). Furthermore, defective
development of feto-placental unit with hemorrhagic placentas
and preponderance of trophoblast giant cells was frequently
noted, although decidual defect was not apparent (Fig. 5D).
Similar dominance of trophoblast giant cells has previously
been reported in mice with mid-gestational placental defects
(Tremblay et al., 2001). Our results show that a transient delay
in the attachment reaction produced heterogeneous feto-
placental developmental phenotypes ranging from less severe
to markedly retarded growth. Our observation of retarded
postimplantation development and demise of embryos was
reflected in high embryonic mortality (38% versus 2%) and the
reduced number (4.8±0.8 versus 12.2±0.8) of pups delivered at
birth by Pla2g4a–/– mice as compared to wild-type mice,
confirming previous reports (Bonventre et al., 1997; Uozumi
et al., 1997).
If deferred implantation is a cause for defective
postimplantation embryonic development, administration of
PGs prior to the attachment reaction in Pla2g4a–/– mice should
improve later stages of embryo development. Thus, we
examined day 12 embryos from Pla2g4a–/– mice injected with
PGE2and cPGI at 10:00 and 18:00 hours on day 4 of pregnancy.
We noted considerable improvement in embryonic development
with concomitant decreases in the number of retarded embryos
compared to vehicle-treated Pla2g4a–/– mice (Fig. 5E).
Embryo spacing is disturbed in
Pla2g4a
–/– mice
Limited information is available regarding cellular and
molecular basis of embryo spacing in the uterus (McLaren and
Michie, 1959). Previous reports using pharmacological
inhibitors suggested that PGs are involved in embryo spacing
in rats (Wellstead et al., 1989). This prompted us to examine
more closely the spacing of embryos in Pla2g4a–/– mice.
Although we observed increased implantation rates in
Pla2g4a–/– mice on day 6, i.e. after a short delay, embryo
spacing was aberrant (see sites within brackets in Fig. 2C). This
abnormal spacing was more prominent when examined on day
12. Implantation sites were closely apposed or even fused
together (see sites within brackets in Fig. 5A). Upon dissection,
we often observed that two or more embryos were residing in
the same decidual envelope or conjoined by a single placenta
(Fig. 5F). This could be one reason for retarded embryonic
development and resorption in Pla2g4a–/– mice, resulting from
crowding of embryos. However, retarded growth of well-
spaced embryos was also noted in Pla2g4a–/– mice.
Although PG treatment on day 4 of pregnancy restored
normal implantation timing in Pla2g4a–/– mice (Fig. 4E), this
treatment did not rescue altered embryo spacing (Fig. 4F),
suggesting that other PGs or mediators, or AA itself are
involved in normal embryo spacing prior to the attachment
reaction. Alternatively, failure of exogenously delivered PGs to
restore normal spacing could be due to inappropriate delivery
of PGs at the right time at the target tissues responsible for
embryo spacing. Our results provide genetic evidence for a role
of cPLA2αin this important event.
Deferred implantation in wild-type mice leads to late
gestational defects
To reinforce that implantation timing affects postimplantation
development, we used embryo transfers in wild-type mice.
Wild-type day 4 blastocysts were transferred into day 4 or day
5 wild-type pseudopregnant mice. We observed that blastocysts
transferred either on day 4 or day 5, when examined 48 hours
later, showed similar implantation rates (Fig. 6A). However,
severe developmental anomalies and resorption of implantation
sites were noted later in pregnancy if blastocysts were
transferred into day 5 recipients when compared with their
transfer into day 4 recipients (Fig. 6B). Furthermore, the
number of pups born at term was much lower for recipients
2886
receiving blastocyst transfers on day 5 (16/130, 12.5%) than
those receiving transfers on day 4 (44/168, 26%) (Fig. 6C). By
contrast, as in Pla2g4a–/– mice, there was no difference in the
decidual response induced by intraluminal oil infusion either
on day 4 or 5 of pseudopregnancy in wild-type mice (Fig. 6D).
These results in wild-type mice, together with our findings
in Pla2g4a–/– mice, clearly demonstrate that timing of
implantation is a crucial determinant for normal feto-placental
development and pregnancy outcome. It is surmised that an
altered uterine environment resulting from the shifting of the
normal ‘window’ of implantation cannot efficiently support
normal pregnancy. This finding has a major clinical
significance, as implantation in humans beyond the normal
‘window’ of uterine receptivity (8-10 days postovulation) is
H. Song and others
Fig. 5. Defective postimplantation developments in Pla2g4a–/– mice. (A) A composite photograph of uteri in wild-type and Pla2g4a–/– mice on
day 12 of pregnancy. Resorption sites were often noted (arrows) and many implantation sites were closely apposed and even conjoined
(brackets) to each other in Pla2g4a–/– mice. (B) Median weights of implantation sites and their embryos on day 12. The implantation sites
without embryos (resorption sites) were excluded from this computation. (C) Photographs of embryos isolated from implantation sites of one
representative wild-type and two Pla2g4a–/– mice on day 12. Note retarded and asynchronous development of embryos in Pla2g4a–/– mice.
(D) Histological examination of day 12 implantation sites in Pla2g4a–/– mice. Feto-placental units from Pla2g4a–/– mice were examined on day
12. Embryos and placentas show defective development with a preponderance of trophoblast giant cells. Arrowheads and an arrow indicate
trophoblast giant cells and degenerating embryo, respectively. (c,d) Higher magnifications of a,b, respectively. la, labyrinthine trophoblast; sp,
spongiotrophoblast; dec, decidua. (E) Distribution of embryonic weights on day 12 (n=60-75). The horizontal orange lines represent median
values of embryonic weights. Pla2g4a–/– mice were given the vehicle or PGs at 10:00 and 18:00 hours on day 4 of pregnancy and killed on day
12. Note a reduction in numbers of retarded embryos in the PG-treated group. (F) Representative photographs of conjoined embryos in a
placenta (a,c) and three embryos in the same decidual envelope (b,d) from Pla2g4a–/– mice on day 12. (c) A histological section of (a) with two
embryos; embryos shown in (d) are from (b). Yellow arrows indicate the source of the embryos from the decidual envelope.
2887cPLA2αin embryo implantation and development
associated with higher risk of early pregnancy losses (Wilcox
et al., 1999). There is evidence that the number of fetuses and
their developmental stages determine the parturition process
(Yoshinaga, 1983). Thus, retarded embryonic development and
resorption observed in Pla2g4a–/– mice are most likely to be
the causes of small litter size and parturition defects previously
observed (Bonventre et al., 1997; Uozumi et al., 1997).
DISCUSSION
cPLA2αis implicated in diverse biological functions (Clark et
al., 1995; Bonventre et al., 1997; Uozumi et al., 1997;
Fujishima et al., 1999; Gijon and Leslie, 1999). While cPLA2-
derived AA is a substrate for COX and lipoxygenase pathways
for eicosanoid production, intracellular AA also has its own
biological effects (Clark et al., 1995; Gijon and Leslie, 1999).
Recent studies showed depressed PG synthesis, small litters
and defective parturition in Pla2g4a–/– mice (Bonventre et al.,
1997; Uozumi et al., 1997; Fujishima et al., 1999). However,
the cause and underlying mechanism of these reproductive
defects were not defined.
We here demonstrate that it is the initiation of implantation
that is affected in Pla2g4a–/– mice, leading to the derangement
of subsequent developmental processes. This is clearly evident
from our results that while normal implantation is noted after
1-day delay i.e., on day 6 in Pla2g4a–/– mice, a gradual
embryonic demise occurs during the remaining period of
pregnancy. The postimplantation developmental defect is
not due to a defect in the decidualization process, as
decidualization occurs when embryos implant 1 day later or in
response to an artificial stimulus in Pla2g4a–/– or wild-type
mice (Fig. 2D, Fig. 6D). This raises a very intriguing
proposition that initial attachment reaction is crucial to the fate
of subsequent developmental processes. Our present work
emphasizes the necessity for careful examination of mid or late
gestational developmental anomalies resulting from specific
gene mutation in mice.
The initiation of implantation and subsequent progression of
pregnancy are the results of coordinated integration of various
signaling pathways between the embryo and the uterus. The
attachment reaction is followed by uterine decidualization,
angiogenesis, embryonic growth and placentation. A defect in
any of these events affects pregnancy outcome. The role of
decidua and placentas in supporting pregnancy is well
documented (Benson et al., 1996; Luo et al., 1997; Robb et al.,
1998; Barak et al., 1999; Tremblay et al., 2001). For example,
defective decidualization in mice deficient in Hoxa10 or IL11-
Rαleads to pregnancy failures (Benson et al., 1996; Robb et
al., 1998). Likewise, defective placentation attributed by
embryos deficient in PPARγor ERRβalso leads to
midgestational embryonic lethality (Luo et al., 1997; Barak et
al., 1999). Our results provide for the first time a novel concept
that a short delay in the initial attachment reaction propagates
detrimental effects during the later course of the pregnancy.
This observation leads to the conclusion that the state of
activity of the blastocyst and uterine environment conducive to
support the initial stages of implantation must be precisely
synchronized for normal pregnancy outcome.
The deferred implantation observed in Pla2g4a–/– mice is
different from traditional lactational or experimentally induced
delayed implantation that occurs in wild-type mice for an
extended period in the absence of ovarian estrogen (Dey,
1996). In the latter, blastocysts undergo dormancy and uteri
become non-responsive to implantation. The removal of the
suckling stimulus or supplementation of estrogen terminates
Fig. 6. Deferred implantation leading to retarded development and
poor pregnancy outcome in wild-type mice. (A) Implantation of day
4 wild-type blastocysts transferred into wild-type recipients on day 4
(n=5) or 5 (n=7) of pseudopregnancy. The numbers above the bars
indicate the number of implantation sites per total number of
blastocysts transferred. Implantation sites were recorded 48 hours
later by the blue dye method. Implantation rate was similar between
the two groups. (B) Postimplantation developments of wild-type
blastocysts transferred into wild-type recipients on day 4 (n=3) or 5
(n=4) of pseudopregnancy. Implantation sites were examined 8 days
later after blastocyst transfer equivalent to day 12 of pregnancy.
While resorption and retarded feto-placental growth were frequent in
recipients that received blastocyst transfers on day 5, vastly normal
development was noted in those receiving blastocyst transfer on day
4. Normal implantation sites (IS) represent sites with normally
developing embryos, while retarded IS represents resorption sites and
IS with retarded embryo development. On day 12, the number of
normal IS was significantly higher in day 4 recipients than in day 5
recipients (18/23 versus 10/42; χ2-test; P<0.001). (C) The pregnancy
outcome of wild-type blastocysts transferred into wild-type
recipients on day 4 (n=13) or 5 (n=11) of pseudopregnancy. The
numbers above the bars indicate the number of pups delivered at
term/total number of blastocysts transferred (χ2-test; *P<0.01). The
number of pups born was significantly lower for mothers receiving
blastocyst transfers on day 5. (D) Decidualization in wild-type mice.
Mice received intraluminal oil infusion on day 4 or 5 of
pseudopregnancy. Uterine weights were recorded 4 days later. Fold
increases denote comparison of weights between infused and non-
infused uterine horns. The numbers above the bars indicate the
number of responding mice/total number of mice. No significant
difference in decidualization was noted between these two groups
(unpaired t-test; P>0.05).
2888
blastocyst dormancy and resumption of implantation with
normal pregnancy outcome.
A two-way interaction between the blastocyst and the uterus
is essential for successful implantation and decidualization.
Although growth factors, cytokines, transcription factors, and
PGs are implicated in successful implantation and
decidualization (Stewart et al., 1992; Das et al., 1994; Benson
et al., 1996; Chakraborty et al., 1996; Lim et al., 1999a; Lim
et al., 1999b; Song et al., 2000), the molecular interactions
between these local mediators are not clearly understood. Our
observation of aberrant expression of the genes encoding HB-
EGF and LIF at the sites of blastocysts at the anticipated time
of implantation without blue bands in Pla2g4a–/– mice
suggests that cPLA2αderived AA and/or eicosanoids
coordinate these signaling pathways for implantation. It is
possible that the absence of HB-EGF in the luminal epithelium
surrounding the blastocyst before the attachment reaction
in Pla2g4a–/– mice makes the blastocysts implantation
incompetent. HB-EGF and LIF are effective in promoting
blastocyst growth, zona-hatching and/or trophoblast outgrowth
in vitro (Das et al., 1994; Dunglison et al., 1996). Deferred
implantation could be due to either incompetence of
blastocysts and/or uterine insufficiency for implantation.
However, reduced implantation of normal wild-type
blastocysts transferred to Pla2g4a–/– uteri suggests that uterine
deficiency is the major cause of this temporary delay.
In rodents, embryos are evenly spaced along the uterus.
However, little is known regarding the underlying mechanism
regulating this process. Our observation of abnormal uterine
spacing of embryos in Pla2g4a–/– mice provides genetic
evidence for a role of PGs in this process and is consistent with
reports of abnormal embryo spacing in rodents exposed to
pharmacological inhibitors of PG synthesis (Wellstead et al.,
1989). It is possible that embryo spacing is regulated by local
factors associated with PG signaling. In other systems, bone
morphogenetic proteins (BMPs) are implicated in the genesis
of evenly spaced tissue structures during development
(reviewed in Hogan, 1996). We have recently shown that local
delivery of BMP-2 in the mouse uterus alters embryo spacing,
suggesting that BMPs are involved in this process (Paria et al.,
2001). As HB-EGF is an inducer of BMP-2 at the implantation
site (Paria et al., 2001), as the EGF-like growth factors
stimulate PG synthesis (DuBois et al., 1994) and as there is a
relationship between BMP and PG signaling in other systems
(Koide et al., 1999), it is conceivable that these regulatory
pathways are operative during embryo spacing.
Genetic mutation of various components of the PG synthetic
and signaling pathways in mice in recent years has provided
important information on distinct and overlapping functions of
various PGs in female reproduction with the conclusion that
these lipid mediators are essential to successful pregnancy
(Langenbach et al., 1995; Challis, 1997; Lim et al., 1997;
Lim et al., 1999a). However, the reproductive deficiency in
Pla2g4a–/– female mice reported here is different from that
observed in Ptgs1–/– or Ptgs2–/– female mice. For example,
while Ptgs1–/– mice are fertile with limited parturition defect
(Langenbach et al., 1995), Ptgs2–/– female mice show profound
defects in ovulation, fertilization, implantation and
decidualization (Lim et al., 1997). By contrast, while
Pla2g4a–/– mice show only modest effects on ovulation and
fertilization, they clearly exhibit temporary postponement of
the blastocyst attachment reaction leading to striking defects
in subsequent postimplantation development. Distinct
phenotypes between Ptgs2–/– and Pla2g4a–/– mice could be
due to functional redundancy among the members of the PLA2
superfamily. It is intriguing to see that although cPLA2αis
crucial to on-time implantation, implantation still can occur
beyond the normal ‘window’ in both the wild-type and mutant
females. Understanding the molecular basis of embryo-uterine
interactions during the deferred ‘window’ of implantation will
provide further insights regarding normal and abnormal
implantation.
In conclusion, using Pla2g4a–/– mice and differential
embryo transfers in wild-type mice, we show that a short
deferral of implantation leads to late developmental defects.
Our results provide a new concept that an early embryo-uterine
interaction during implantation sets up the subsequent
developmental programming.
Our sincere thanks to Brigid Hogan for her crucial reading and
comments on the manuscript. This work was supported by NIH grants
HD12304, HD29968 and HD 33994 awarded to S. K. D.; HD37394
and HD40193 to B. C. P.; GM15431, DK48831 and CA77839
awarded to J. D. M.; and DK39773 and DK38452 to J. V. B. J. D. M.
is a recipient of Burrough Wellcome Fund Clinical Scientist Award.
S. K. D. is a recipient of an NICHD MERIT award. H. S. was
supported by a KUMC Biomedical Research Training fellowship,
whereas H. L. was supported by a Junior Investigator Award from the
Mellon Foundation and by the KUMC Research Institute. A Center
grant in Mental Retardation and Reproductive Sciences provided core
facilities.
REFERENCES
Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien,
K. R., Koder, A. and Evans, R. M. (1999). PPARγis required for placental,
cardiac, and adipose tissue development. Mol. Cell 4, 585-595.
Benson, G. V., Lim, H., Paria, B. C., Satokata, I., Dey, S. K. and Maas, R.
L. (1996). Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine
homeosis and loss of maternal Hoxa-10 expression. Development 122, 2687-
2696.
Bonventre, J. V., Huang, Z., Taheri, M. R., O’Leary, E., Li, E., Moskowitz,
M. A. and Sapirstein, A. (1997). Reduced fertility and postischaemic brain
injury in mice deficient in cytosolic phospholipase A2.Nature 390, 622-625.
Chakraborty, I., Das, S. K., Wang, J. and Dey, S. K. (1996). Developmental
expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the
peri-implantation mouse uterus and their differential regulation by the
blastocyst and ovarian steroids. J. Mol. Endocrinol. 16, 107-122.
Challis, J. R. (1997). Prostaglandins and reproduction–what do knockouts
really tell us? Nat. Med. 3, 1326-1327.
Clark, J. D., Schievella, A. R., Nalefski, E. A. and Lin, L. L. (1995).
Cytosolic phospholipase A2.J. Lipid Mediat. Cell Signal. 12, 83-117.
Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A.,
Klagsbrun, M., Andrews, G. K. and Dey, S. K. (1994). Heparin-binding
EGF-like growth factor gene is induced in the mouse uterus temporally by
the blastocyst solely at the site of its apposition: a possible ligand for
interaction with blastocyst EGF- receptor in implantation. Development 120,
1071-1083.
Dey, S. K. (1996). Implantation. In Reproductive Endocrinology, Surgery, and
Technology (ed. E.Y. Adashi, J.A. Rock and Z. Rosenwaks), pp. 421-434.
Philadelphia: Lippincott-Raven.
DuBois, R. N., Awad, J., Morrow, J., Roberts, L. J. and Bishop, P. R.
(1994). Regulation of eicosanoid production and mitogenesis in rat intestinal
epithelial cells by transforming growth factor-αand phorbol ester. J. Clin.
Invest. 93, 493-498.
Dunglison, G. F., Barlow, D. H. and Sargent, I. L. (1996). Leukaemia
inhibitory factor significantly enhances the blastocyst formation rates of
human embryos cultured in serum-free medium. Hum. Reprod. 11, 191-196.
H. Song and others
2889cPLA2αin embryo implantation and development
Fujishima, H., Sanchez Mejia, R. O., Bingham, C. O., III, Lam, B. K.,
Sapirstein, A., Bonventre, J. V., Austen, K. F. and Arm, J. P. (1999).
Cytosolic phospholipase A2is essential for both the immediate and the
delayed phases of eicosanoid generation in mouse bone marrow-derived
mast cells. Proc. Natl. Acad. Sci. USA 96, 4803-4807.
Gijon, M. A. and Leslie, C. C. (1999). Regulation of arachidonic acid release
and cytosolic phospholipase A2activation. J. Leukoc. Biol. 65, 330-336.
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). In
Manipulating the Mouse Embryo, A Laboratory Manual, pp. 151-162. New
York: Cold Spring Harbor.
Hogan, B. L. (1996). Bone morphogenetic proteins: multifunctional regulators
of vertebrate development. Genes Dev. 10, 1580-1594.
Kennedy, B. P., Payette, P., Mudgett, J., Vadas, P., Pruzanski, W., Kwan,
M., Tang, C., Rancourt, D. E. and Cromlish, W. A. (1995). A natural
disruption of the secretory group II phospholipase A2gene in inbred mouse
strains. J. Biol. Chem. 270, 22378-22385.
Koide, M., Murase, Y., Yamato, K., Noguchi, T., Okahashi, N. and
Nishihara, T. (1999). Bone morphogenetic protein-2 enhances osteoclast
formation mediated by interleukin-1αthrough upregulation of osteoclast
differentiation factor and cyclooxygenase-2. Biochem. Biophys. Res.
Commun. 259, 97-102.
Langenbach, R., Morham, S. G., Tiano, H. F., Loftin, C. D., Ghanayem,
B. I., Chulada, P. C., Mahler, J. F., Lee, C. A., Goulding, E. H.,
Kluckman, K. D. et al. (1995). Prostaglandin synthase 1 gene disruption
in mice reduces arachidonic acid-induced inflammation and indomethacin-
induced gastric ulceration. Cell 83, 483-492.
Lim, H., Paria, B. C., Das, S. K., Dinchuk, J. E., Langenbach, R., Trzaskos,
J. M. and Dey, S. K. (1997). Multiple female reproductive failures in
cyclooxygenase 2-deficient mice. Cell 91, 197-208.
Lim, H., Gupta, R. A., Ma, W. G., Paria, B. C., Moller, D. E., Morrow, J.
D., DuBois, R. N., Trzaskos, J. M. and Dey, S. K. (1999a). Cyclo-
oxygenase-2-derived prostacyclin mediates embryo implantation in the
mouse via PPARδ.Genes Dev. 13, 1561-1574.
Lim, H., Ma, L., Ma, W. G., Maas, R. L. and Dey, S. K. (1999b). Hoxa-10
regulates uterine stromal cell responsiveness to progesterone during
implantation and decidualization in the mouse. Mol. Endocrinol. 13, 1005-
1017.
Luo, J., Sladek, R., Bader, J. A., Matthyssen, A., Rossant, J. and Giguere,
V. (1997). Placental abnormalities in mouse embryos lacking the orphan
nuclear receptor ERR-β.Nature 388, 778-782.
MacPhee, M., Chepenik, K. P., Liddell, R. A., Nelson, K. K., Siracusa, L.
D. and Buchberg, A. M. (1995). The secretory phospholipase A2gene is
a candidate for the Mom1 locus, a major modifier of ApcMin-induced
intestinal neoplasia. Cell 81, 957-966.
McLaren, A. and Michie, D. (1959). The spacing of implantations in the
mouse uterus. Mem. Soc. Endocrinol. 6, 65-74.
Matsumoto, H., Ma, W.-G., Smalley, W., Trzaskos, J., Breyer, R. M. and
Dey, S. K. (2001). Diversification of cyclooxygenase-2-derived
prostaglandins in ovulation and implantation. Biol. Reprod. 64, 1557-1565.
Mechoulam, R., Fride, E. and di Marzo, V. (1998). Endocannabinoids. Eur.
J. Pharmacol. 359, 1-18.
Murakami, M., Nakatani, Y., Kuwata, H. and Kudo, I. (2000). Cellular
components that functionally interact with signaling phospholipase A2s.
Biochim. Biophys. Acta 1488, 159-166.
Paria, B. C., Das, S. K., Andrews, G. K. and Dey, S. K. (1993a). Expression
of the epidermal growth factor receptor gene is regulated in mouse
blastocysts during delayed implantation. Proc. Natl. Acad. Sci. USA 90, 55-
59.
Paria, B. C., Huet-Hudson, Y. M. and Dey, S. K. (1993b). Blastocyst’s state
of activity determines the ‘window’ of implantation in the receptive mouse
uterus. Proc. Natl. Acad. Sci. USA 90, 10159-10162.
Paria, B. C., Ma, W., Tan, J., Raja, S., Das, S. K., Dey, S. K. and Hogan,
B. L. (2001). Cellular and molecular responses of the uterus to embryo
implantation can be elicited by locally applied growth factors. Proc. Natl.
Acad. Sci. USA 98, 1047-1052.
Reddy, S. T. and Herschman, H. R. (1997). Prostaglandin synthase-1 and
prostaglandin synthase-2 are coupled to distinct phospholipases for the
generation of prostaglandin D2in activated mast cells. J. Biol. Chem. 272,
3231-3237.
Robb, L., Li, R., Hartley, L., Nandurkar, H. H., Koentgen, F. and Begley,
C. G. (1998). Infertility in female mice lacking the receptor for interleukin-
11 is due to a defective uterine response to implantation. Nat. Med. 4, 303-
308.
Serhan, C. N. and Oliw, E. (2001). Unorthodox routes to prostanoid
formation: new twists in cyclooxygenase-initiated pathways. J. Clin. Invest.
107, 1481-1489.
Six, D. A. and Dennis, E. A. (2000). The expanding superfamily of
phospholipase A2enzymes: classification and characterization. Biochim.
Biophys. Acta 1488, 1-19.
Smith, W. L. and DeWitt, D. L. (1996). Prostaglandin endoperoxide H
synthases-1 and -2. Adv. Immunol. 62, 167-215.
Song, C., Chang, X. J., Bean, K. M., Proia, M. S., Knopf, J. L. and Kriz,
R. W. (1999). Molecular characterization of cytosolic phospholipase A2-β.
J. Biol. Chem. 274, 17063-17067.
Song, H., Lim, H., Das, S. K., Paria, B. C. and Dey, S. K. (2000).
Dysregulation of EGF family of growth factors and COX-2 in the uterus
during the preattachment and attachment reactions of the blastocyst with the
luminal epithelium correlates with implantation failure in LIF-deficient
mice. Mol. Endocrinol. 14, 1147-1161.
Stewart, C. L., Kaspar, P., Brunet, L. J., Bhatt, H., Gadi, I., Kontgen, F.
and Abbondanzo, S. J. (1992). Blastocyst implantation depends on
maternal expression of leukaemia inhibitory factor. Nature 359, 76-79.
Takano, T., Panesar, M., Papillon, J. and Cybulsky, A. V. (2000).
Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA
phospholipase A2in COS-1 cells. Prostaglandins Lipid Mediat. 60, 15-26.
Thomas, D. W., Mannon, R. B., Mannon, P. J., Latour, A., Oliver, J. A.,
Hoffman, M., Smithies, O., Koller, B. H. and Coffman, T. M. (1998).
Coagulation defects and altered hemodynamic responses in mice lacking
receptors for thromboxanes A2.J. Clin. Invest. 102, 1994-2001.
Threadgill, D. W., Dlugosz, A. A., Hansen, L. A., Tennenbaum, T., Lichti,
U., Yee, D., LaMantia, C., Mourton, T., Herrup, K., Harris, R. C. et al.
(1995). Targeted disruption of mouse EGF receptor: effect of genetic
background on mutant phenotype. Science 269, 230-234.
Tremblay, G. B., Kunath, T., Bergeron, D., Lapointe, L., Champigny, C.,
Bader, J. A., Rossant, J. and Giguere, V. (2001). Diethylstilbestrol
regulates trophoblast stem cell differentiation as a ligand of orphan nuclear
receptor ERRβ.Genes Dev. 15, 833-838.
Underwood, K. W., Song, C., Kriz, R. W., Chang, X. J., Knopf, J. L. and
Lin, L. L. (1998). A novel calcium-independent phospholipase A2, cPLA2-
γ, that is prenylated and contains homology to cPLA2.J. Biol. Chem. 273,
21926-21932.
Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro, F.,
Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y. et al. (1997). Role of
cytosolic phospholipase A2in allergic response and parturition. Nature 390,
618-622.
Valentin, E. and Lambeau, G. (2000). Increasing molecular diversity of
secreted phospholipases A2and their receptors and binding proteins.
Biochim. Biophys. Acta 1488, 59-70.
Wellstead, J. R., Bruce, N. W. and Rahima, A. (1989). Effects of
indomethacin on spacing of conceptuses within the uterine horn and on fetal
and placental growth in the rat. Anat. Rec. 225, 101-105.
Wilcox, A. J., Baird, D. D. and Weinberg, C. R. (1999). Time of implantation
of the conceptus and loss of pregnancy. New Engl. J. Med. 340, 1796-1799.
Yoshinaga, K. (1983). Superimplantation rats as a tool for study of onset of
parturition. In The Anterior Pituitary Gland (ed. A. J. Bhatnagar), pp 367-
375. New York: Raven.
