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J. Biol. Chem., Vol. 276, Issue 47, 44137-44145, November 23, 2001
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From the Departments of
Received for publication, August 8, 2001, and in revised form, September 6, 2001
Infertility and spontaneous pregnancy
losses are an enduring problem to women's health. The establishment of
pregnancy depends on successful implantation, where a complex series of
interactions occurs between the heterogeneous cell types of the uterus
and blastocyst. Although a number of genes are implicated in
embryo-uterine interactions during implantation, genetic evidence
suggests that only a small number of them are critical to this process.
To obtain a global view and identify novel pathways of implantation, we used a dual screening strategy to analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of implantation and interimplantation sites by a conservative statistical approach revealed 36 up-regulated genes and 27 down-regulated genes at the
implantation site. We also compared the uterine gene expression profile
of progesterone-treated, delayed implanting mice to that of mice in
which delayed implantation was terminated by estrogen. The results show
up-regulation of 128 genes and down-regulation of 101 genes after
termination of the delayed implantation. A combined analysis of these
experiments showed specific up-regulation of 27 genes both at the
implantation site and during uterine activation, representing a broad
diversity of molecular functions. In contrast, the majority of genes
that were decreased in the combined analysis were related to host
immunity or the immune response, suggesting the importance of these
genes in regulating the uterine environment for the implanting
blastocyst. Collectively, we identified genes with recognized roles in
implantation, genes with potential roles in this process, and genes
whose functions have yet to be defined in this event. The
identification of unique genetic markers for the onset of implantation
signifies that genome-wide analysis coupled with functional assays is a
promising approach to resolve the molecular pathways required for
successful implantation.
Early loss of pregnancy is a significant clinical problem for
women and their health care providers. Successful embryo implantation depends upon bi-directional communication between the blastocyst and
the uterus. Recent advances in our ability to define complex biochemical and genetic pathways have begun to unfold the molecular mechanisms underlying the regulation of implantation (1, 2). Although
numerous factors involved in implantation have been identified (3-5),
targeted mutations in mice have revealed only a few genes that are
essential to this process (6-11).
Implantation is defined as a process by which the blastocyst makes the
first physical and physiological contact with the maternal uterine
luminal epithelium. Under the influence of ovarian steroid hormones, an
optimal "window" for implantation is created when the activated
state of the developing blastocyst overlaps with a brief period of
uterine receptivity (12, 13). In pregnant mice, removal of
preimplantation estrogen secretion by ovariectomy postpones the onset
of implantation and induces blastocyst dormancy (14). A single
injection of estrogen can reactivate the signaling network resulting in
implantation in the progesterone (P4)-primed uterus. This
model, termed "delayed implantation," has provided insights into
the cellular communication pathways between the uterine and embryonic
cell types and led to the identification of embryo-induced uterine
genes that correspond to early steps in the establishment of pregnancy
(2).
The blastocyst and uterus generate various factors during implantation,
but it is likely that the molecular "cross-talk" between them
involves many more yet unknown factors. Indeed, it is more realistic to
view the process of implantation as a condition of equilibrium in the
up-regulation and down-regulation of a diverse set of genes.
Identification of other essential regulatory steps is necessary to
further understand the biologic basis for the establishment of
pregnancy or the underlying causes of pregnancy failures. In this
respect, two recent reports highlight gene expression profiling in the
post-implantation period (15, 16). However, to our knowledge, no such
analysis at the onset of implantation has been reported. To address
this issue, we employed two complementary strategies using murine
GeneChip Expression Arrays (Affymetrix, Santa Clara, CA) to determine
global gene expression profiles during implantation in mice. The first
approach compared RNAs from implantation and interimplantation sites to
identify genes that are specifically up- or down-regulated at the
implantation site. A second analysis compared RNA from
P4-primed pregnant uteri with delayed implantation with
that of P4-primed uteri after estrogen activation. Several
genes with known expression status at the implantation site were
detected. In addition, cell-specific expression patterns in the
implantation and interimplantation sites were observed for four
candidate genes, confirming the validity of this approach. Mice with
delayed implantation expressed a large number of genes associated with
immunity or immune responses. The suppression of these genes at the
implantation site also suggests that this site is immunologically
privileged during early pregnancy and that modulation of the immune
response is an active process during implantation. There were 81 genes
whose expression was affected in both analyses. These results suggest
that pan-genomic gene expression profiles are a promising approach for
the identification of markers of uterine receptivity during
implantation and that multiple screening strategies yield a distinct
set of candidate genes that appear to be critical in early pregnancy.
Animals--
All experiments were conducted in accordance with
National Institutes of Health standards for the care and use of
animals. Mice were killed by cervical dislocation or were anesthetized for survival surgery with avertin. Adult virgin CD-1 female mice were
mated with fertile males of the same strain to induce pregnancy (day
1 = vaginal plug).
Increased stromal vascular permeability at the site of initial contact
of the blastocyst with the uterine luminal epithelium is the first
visible sign of the implantation process (11:00-12:00 p.m. on day 4)
and can be monitored by an intravenous injection of a blue dye (12, 13,
17). For the first analysis, implantation and interimplantation sites
were divided by sharp dissection at 11:00-12:00 p.m. on day 4 (n = 12 mice). Uterine segments included uterine
myometrium, stroma, and epithelium. Implantation sites also included
blastocysts. Because there are normal variations in the timing of
implantation, only those uteri with uniformly distinct blue bands were
included, whereas regions with embryo crowding were discarded (Fig.
1).
In the second analysis, P4-primed uteri of mice with
delayed implantation were compared with P4-treated uteri
after estrogen activation. To induce delayed implantation, pregnant
females were ovariectomized on the morning of day 4 of pregnancy (9:00
a.m.) and given daily subcutaneous injections of P4 (2 mg/mouse in 0.1 ml of sesame oil) from days 5 through 7 (13, 14). To
terminate delayed implantation and induce blastocyst activation, a
single subcutaneous injection of estradiol-17 Sample Preparation--
Uterine tissues were flash frozen at the
time of dissection and stored at GeneChip Hybridization and Statistical Analysis--
Each cRNA
(15 µg) preparation was used to inoculate murine U74A GeneChip
expression arrays (Affymetrix, Inc.), and the hybridization, staining,
scan, and analysis were conducted per recommended protocols. An
Affymetrix software filter was applied to mask transcripts with
incorrect orientation in the public data bases. Although numerous
expressed sequence tags were differentially expressed, only those
transcripts with known identities are reported herein. Three replicate
hybridizations were performed using each of the four pooled RNA samples
(implantation, interimplantation, delayed, activated) to establish the
reproducibility of our results. Alterations in RNA transcript levels
were analyzed using the Affymetrix Analysis Suite 4.0 software.
Differences in levels of fluorescence intensity, which represents
levels of hybridization, between the 25 base pair oligonucleotides and
their mismatches, were analyzed by multiple decision matrices to
determine the presence or absence of gene expression and to
derive an average difference score representing the relative level of
gene expression. Background and noise corrections account for
nonspecific binding and minor variations in hybridization conditions.
Values for the mean and standard deviation of the three replicate
average difference scores were calculated for each gene on the
GeneChip. Comparison between groups was performed by Student's
t test (p < 0.05 considered significant).
The -fold change in expression between groups was calculated from the
mean average difference scores.
A second approach was used to verify the identification of
differentially expressed genes. Affymetrix algorithms produced statistical decisions for an increase or decrease in expression when
comparing the hybridization results of any two samples. Thus, paired
comparisons of three replicate hybridizations resulted in nine possible
outcomes for a single analysis (e.g. implantation versus interimplantation). Transcripts with statistically
significant expression (by t test) that were also
differentially expressed in four or more of the nine pair-wise
comparisons (increase or decrease) were considered candidates for
further evaluation. Others have used this counting approach using
somewhat higher cut-off points (18). However, at higher threshold
levels, we noted that a number of genes, known to be expressed at the
implantation site, were excluded.
In Situ Hybridization--
In situ hybridization was
performed as previously described (17). 35S-Labeled cRNA
probes were generated for Sik-SP, Bip,
Cyp1b1, and ptgerep4 (EP4) with the
appropriate polymerases (19-21).
Gene Expression Is Altered at the Onset of Natural
Implantation (Implantation versus Interimplantation
Sites)--
Hybridization intensity to the arrays was uniform for
housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase and
cyclophilin and a large number of ribosomal proteins, indicating that
the expression data in the array hybridization experiments are
consistent with other standards for studying gene expression.
The relative levels of gene expression at the implantation and
interimplantation sites were first compared by plotting the average
difference values for one individual array hybridization experiment
against another and determining the presence or absence of gene
expression for the entire array (Fig. 2).
Genes that were considered absent or marginally expressed were widely
dispersed (black points), whereas genes that were declared
present in both hybridizations were tightly grouped (red
points). An increase of more than 2-fold in the average difference
score indicated genes with significantly higher expression at the
implantation site (above the curve) or at the
interimplantation region (below the curve) (Fig. 2). These
results show that a vast majority of the genes on the chip have similar
expression patterns (present, absent, or marginal) and their relevance
to implantation is questionable. A small number of genes were present
in both (red points) or present in one but marginal or
absent in the other (blue points) that also had greater than
a 2-fold difference in the level of expression. These genes were
considered as potential candidates for further evaluation.
Surprisingly, a symmetrical distribution of these candidate genes was
observed, suggesting that an equal number of genes are up-regulated in
the implantation and interimplantation regions.
Genes with statistically significant differences in expression at
implantation versus interimplantation sites were identified by comparison of replicate hybridizations and by a statistical decision
for an increase or a decrease in gene expression. By t test
alone, there were 293 up-regulated and 370 down-regulated genes at the
implantation site. A second statistical approach was performed to
provide an additional objective analysis. We used a threshold value of
four of the nine possible outcomes, which resulted in 49 up-regulated
and 60 down-regulated genes and included several known genes that are
expressed during implantation or are considered biologically relevant
to the implantation process. A combination of t test and
counting approaches identified 36 genes that were up-regulated
at the implantation site and 27 genes that were down-regulated (Tables
I and
II).
Differentially expressed genes were
categorized based on the best available information regarding their
biologic functions. Genes with multiple functions were assigned to a
single category. Many genes that are known to be associated with the
implantation process fall into categories similar to the genes we
detected with increased expression at the implantation site, including growth factors/cytokines and their receptors, transcription factors, genes encoding structural proteins, or genes associated with cell proliferation. We also observed up-regulation of a group of
calcium-related genes, including Bip, Sik similar protein
(Sik-SP1), and
calcineurin- and calcyclin-related proteins. Genes encoding Bip and
Sik-SP showed highly localized expression at the implantation site,
confirming our array results (Fig. 3).
These genes are of interest, because calcium is an essential modulator
of enzyme functions and signal transduction, and there is evidence that genes involved in calcium regulation play an important role during implantation (21-25).
Previous efforts to identify novel genes during the peri-implantation
period have primarily focused on the implantation site, with less
attention paid to genes that are expressed at the interimplantation region. Genes with increased expression at the interimplantation site
may act to guide the blastocyst to specific sites for implantation or
be important for embryo spacing. Aberrant expression of these genes may
be as detrimental to implantation as the loss of genes that are
expressed at the implantation site. In our gene array experiments, we
observed a 5-fold decrease in levels of Cyp1b1 expression at
the implantation site. Cyp1b1 is a member of the cytochrome P450 system
that converts primary estrogens to their active metabolites,
catecholestrogens, which are important for blastocyst activation (20).
In situ hybridization showed that Cyp1b1 is
restricted to the subepithelial stroma of the interimplantation region
(Fig. 3), in agreement with the decreased expression levels seen in the
array experiments. A similar expression pattern was noted for the
prostaglandin E2 receptor subtype, EP4, which
showed a nearly 2-fold decrease by gene array. The overall diversity of
genes with this pattern of expression suggests that further investigation of interimplantation-specific genes is warranted.
Gene Expression Is Altered in an Experimental Model of Implantation
(Delayed versus Induced Implantation)--
To identify genes that are
up-regulated at the time of blastocyst activation for implantation,
RNAs from P4-primed delayed implanting uteri and
P4-primed uteri after estrogen treatment were obtained for
comparative analysis. Scatterplot analysis of genes considered absent
or present showed that overall gene expression patterns in delayed and
activated uteri were closely correlated (Fig. 2). The tight
distribution of overall expression is similar to that seen in the
implantation versus interimplantation analysis. The overall
similarity of the two different scatterplots is not surprising, because
the ultimate outcome, implantation, is similar in both models and the
experiments were designed to be complementary and provide a dual
approach to identify novel implantation-specific genes.
Statistical analysis by t test alone identified 409 up-regulated and 550 down-regulated genes in comparisons of delayed
versus activated uteri. However, our combined statistical
approach reduced the number of candidate genes to 128 up-regulated and
101 down-regulated transcripts after termination of the delayed
implantation by estrogen (Supplemental Tables IS and IIS). Mice with
delayed implantation expressed a large number of genes associated with
host immunity or the immune response (n = 48) compared
with the uteri of mice after estrogen activation (n = 3) (Supplemental Table IS). Furthermore, nearly 50% of the genes with
significant expression during delayed implantation have some
immune-related function (48/101). Specific roles for these genes during
implantation have not been described.
There were striking differences in the functional categories of delayed
versus activated genes. In general, more DNA processing, cell cycle-associated genes and a larger number of enzymes
were observed after initiating the process of implantation
(Supplemental Table IIS). This shift in gene diversity suggests that
embryonic activation and uterine preparation for the onset of
implantation is mediated by a subset of genes that requires estrogen.
In this respect, the gap junction proteins, connexins 26 and 43, are
implicated in implantation, and their regulation is influenced by
steroid hormones (26). In our array experiments, the expression of the gene encoding connexin 26 increased over 8-fold, whereas the expression of connexin 43 showed a greater than 2.5-fold increase after estrogen activation. The prostaglandin E2 receptor subtype
EP2 (ptgerep2) is the only other gene that was
detected in our delayed versus activated gene array whose
expression is also known to be induced at the site of the implanting
blastocyst after termination of delayed implantation (27). Overall, our
results show that delayed implantation is a valuable model to dissect
the molecular aspects of implantation, and compare the distribution of
genes that are expressed during dormancy or active implantation.
Comparison of Gene Expression in Natural and Delayed Implanting
Models of Pregnancy--
Although implantation is the eventual outcome
of both models, it is unclear whether molecular mechanisms underlying
natural implantation and induced implantation are similar. To determine whether genes up-regulated in the delayed uterus after estrogen activation are similar to the group of genes with increased expression at the implantation site, the results of both hybridization analyses were combined to highlight genes that were differentially expressed in
both comparisons. The intersection of these sets revealed 244 genes
with significantly altered expression (t test only) in both models (Fig. 4). Considering only those
transcripts with a greater than 2-fold difference in average difference
scores, there were 54 genes that had significant expression at the
interimplantation site and during progesterone-primed implantation
delay (Table III). By similar criteria,
we also observed 27 genes that had increased expression at the
implantation site and after estrogen activation (Table
IV). Among these, only connexins 26 and
43, amphiregulin, and nexin-1 are associated with implantation (26, 28,
29). The importance of the remaining genes in implantation awaits
further investigation. Nonetheless, these results show that a
dual screening strategy identifies a small number of candidate genes
that are likely to have significant roles in implantation.
The survival of any species depends on stable mechanisms for
reproduction. Thus, it is assumed that essential mechanisms for embryo
implantation must be supported by redundant pathways to ensure the
conception of new offspring. This predicts that a large number of genes
that are important for implantation remain to be identified. Previous
approaches to investigate implantation have generally relied on the
analysis of individual candidate genes or gene families. We used DNA
microarray technology to screen a large cross-section of the murine
genome to identify novel implantation-specific genes. Our present
investigation has identified genes with recognized roles in
implantation, genes with potential roles in this process, and genes
whose functions have yet to be defined. In addition, a small number of
genes showed significantly altered expression during both natural and
induced implantation.
The process of implantation involves cell-cell interactions between the
blastocyst and uterus, cell-type specific proliferation and
differentiation of the uterus, and immunological responses of the
mother to the "semi-allogenic" embryo. Our data show a broad
diversity of genes that are modulated during implantation. A recent
report described a microarray-based approach to identify genes in the
uterus during the post-implantation period (15). The authors of that
report observed 192 genes with increased expression and 207 genes with
decreased expression levels. Similar to our results, genes typically
showed 1.5- to 3-fold induction at the implantation site. Surprisingly,
there are very few genes mutually identified in both studies. However,
Yoshioka et al. (15) compared uterine gene expression
profiles on the evening of day 4 to those on day 6 of pregnancy. With
this approach, both implantation and interimplantation regions would be
included in a single sample so that no distinction could be made for
gene localization around the implanting blastocyst. Our in
situ hybridization results show the importance of differentiating
these two sites. In addition, uterine horns were flushed in this study
(15), and the uteri were split and the luminal surface was scraped to
remove concepti. Physical disruption of the uterine epithelium
would likely result in different gene expression profiles. We designed
our experiments to include the implanting blastocyst in both analyses.
The presence of blastocysts in our first (implantation
versus interimplantation) and second (delayed
versus activated) analyses serves to strengthen our approach
by including the embryonic genome and profiling the expression of
embryonic factors that may be significant to implantation. Moreover, we
analyzed intact uterine horns to preserve an undisturbed relationship
between the uterine myometria, stroma, and epithelium and its intimate
contact with the blastocyst. In contrast, Yoshioka et al.
(15), focused on two distinct time points in pregnancy, resulting in
the identification of genes with differential expression between
implantation and decidualization rather than implantation site-specific genes.
We also identified genes that are differentially expressed in delayed
versus estrogen-activated uteri. There are previous reports
that genes, which encode the EGF-like growth factors, cytokines and
other inflammatory mediators, extracellular matrix proteins, cell cycle
molecules, and immunoregulatory proteins, are expressed at the site of
estrogen-induced implantation in a pattern similar to their expression
in natural implantation (17, 27, 30, 31-38). In this respect, our
microarray results are consistent with the previously described
expression of several steroid hormone-sensitive and
implantation-specific genes, including the genes encoding Cyp1b1,
connexin 26 and connexin 43, Sik-SP, the prostaglandin receptor
EP2, and histidine decarboxylase (20, 21, 26, 27, 39).
However, we failed to detect the anticipated changes in the expression
of the genes encoding cyclooxygenase-2 (Cox-2), perlecan,
trophinin, HB-EGF, LIF, and a number of other hormone-responsive,
implantation-associated genes at the implantation site. This is perhaps
due to their highly restricted expression around the implanting
blastocyst, resulting in the dilution of implantation-specific RNAs in
a large pool of RNAs derived from other uterine cell types. Indeed, we
have previously observed that changes in the expression of
Cox-2 and HB-EGF and several other implantation-specific
genes could not be detected by Northern hybridization, but showed
discrete up-regulation at the implantation site as observed by in
situ hybridization (17, 30-32).
A significant shift was noted in the diversity of genes expressed in
delayed implantation uteri compared with estrogen-activated uteri. In
particular, mice with delayed implantation expressed a large number of
genes associated with immunity and/or the immune response. The
suppression of these genes at normal implantation sites and after
estrogen-activation (Table III) suggests that the implantation site is
immunologically protected. Although large numbers of maternal natural
killer cells are recruited to the uterine deciduum 48 h after
embryo attachment (40), leukocytes and other bone marrow-derived cells
migrate away from the site of blastocyst attachment at an earlier time
during the onset of implantation (41, 42). Reduced expression of
numerous immune-related genes at the implantation site suggests that
immunomodulatory cells, even if present, remain quiescent with the
onset of implantation. Thus, reduced expression of these genes is an
important finding, because it is still unclear how the embryo escapes
maternal immunological responses during pregnancy (43). The mechanism
of down-regulation of these genes at the implantation site is unknown,
but elaboration of immunosuppressive signals from active blastocysts
cannot be ruled out (44, 45). Our data suggest that modulation of the immune response is an active process prior to or during blastocyst implantation.
There were 81 genes with differential expression at the implantation
site during both natural and induced implantation, suggesting their
importance for implantation. Connexins 26 and 43 are gap junction
proteins that are influenced by ovarian steroids, accumulate in the
stroma around the implantation site, and are up-regulated during
experimentally induced decidualization (26). Their expression was
significantly increased at the implantation site and in the uterus
after estrogen activation. These observations coincide with a growing
body of evidence for the role of structural genes in the establishment
of pregnancy (46). Amphiregulin (Areg) and nexin-1
(serpine2) were the only other implantation-associated genes
that had increased expression during both natural and induced implantation (Table IV). Amphiregulin is a member of the EGF family of
growth factors that becomes intensely localized to the uterine luminal
epithelium surrounding the blastocyst at the onset of implantation
(28). Nexin-1 is a serine protease inhibitor that regulates processing
of plasmin, thrombin, urokinase, plasminogen activators, and other
proteases and is up-regulated during implantation (29). Tight
regulation of proteases and their inhibitors is considered an important
aspect of embryo-uterine interaction at the site of implantation (47).
Collectively, our results suggest that other genes identified in the
composite analysis are physiologically relevant to the implantation process.
The remaining genes identified by the combined screening approach do
not yet have any recognized roles in implantation. However, many of the
genes identified with increased expression in both analyses (Table IV)
are associated with cell proliferation (spermidine synthase,
PCNA); cell cycle regulation (cyclin E2); inflammation or tumor
biology (ribonucleotide reductase M2, NM23); and DNA replication,
synthesis, and/or repair (ribonucleotide reductase M2, CDC46 and
Mcm homologs, topoisomerase, PCNA, FEN-1). Because the process
of implantation is considered a pro-inflammatory response and involves
uterine proliferation, differentiation, and apoptosis, these genes
could well be very important for various aspects of this process. It is
interesting to note that PCNA can directly bind to FEN-1 to stimulate
its nuclease activity during base excision repair of damaged DNA (48)
and that ribonucleotide reductase is the rate-limiting enzyme that
provides the essential deoxynucleotides for new DNA synthesis and DNA
repair. All three of these genes showed up-regulated expression during
implantation and may act in a concerted fashion for remodeling of the
uterine epithelium and stroma during blastocyst attachment and
invasion. However, the diverse functions for each of these genes
preclude any speculation as to their specific significance to implantation.
In summary, we employed a global gene expression strategy to identify
novel genes in the implantation process. Our results show an equal
number of genes that are up-regulated or down-regulated at the
implantation site and a small number of candidate genes that have
significant changes in their expression during natural and
estrogen-induced implantation. A better understanding of the molecular
mechanisms of embryo-uterine interactions during implantation will
provide insight into the high rate of spontaneous pregnancy losses.
Attempts to resolve these complex signaling networks will likely
benefit from a genome-wide approach coupled with functional assays and
facilitate new methods to address infertility and contraceptive challenges in women's health.
We are grateful to Jian Tan and Xuemei Zhao
for their assistance with in situ hybridization and to
Richard Melvin for his assistance with microarray analysis.
*
This work was supported in part by The Mellon Foundation; by
National Institutes of Health (NIH) Grants HD37677 (to J. R.), ES07814
(to S. K. Das), HD37394 (to B. C. P.), DK47297 (to R. N. D.),
HD12304, HD29968, HD33994 (to S. K. Dey); and by NICHD, NIH
Mental Retardation and Developmental Disabilities Center Grant HD02528.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 10, 2001, DOI 10.1074/jbc.M107563200
The abbreviations used are:
Sik-SP, Sik similar protein;
EGF, epidermal growth factor;
PCNA, proliferating
cell nuclear antigen;
MHC, major histocompatibility complex.
Global Gene Expression Analysis to Identify Molecular Markers of
Uterine Receptivity and Embryo Implantation*,
§,§,, and
Pediatrics, ¶ Obstetrics
and Gynecology, and § Molecular and Integrative
Physiology, University of Kansas Medical Center, Kansas City,
Kansas 66160, the ** Department of Internal Medicine,
Diabetes and Endocrinology Research Center, University of Iowa,
Iowa City, Iowa 52242, and the Departments
of Medicine and Gastroenterology, Vanderbilt University Medical
Center, Nashville, Tennessee 37232
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Microarray analysis to identify novel
implantation-specific genes. Increased vascular permeability at
the site of blastocyst attachment demarcates implantation sites after
injection of a macromolecular blue dye. Top panel (natural
pregnancy), comparison of RNAs from implantation and interimplantation
sites at 11:00-12:00 p.m. on day 4 of pregnancy. Bottom
panel (induced pregnancy), comparison of RNAs from mice with
delayed implantation (progesterone only) and estrogen-induced
termination of delayed implantation (progesterone + estrogen).
(25 ng/mouse in 0.1 ml of oil) was given to one group of animals at the same time as P4 injection on the third day of delay (day 7). Whole uteri
(n = 6) were collected from each of these two groups
12 h after the last injection of steroids.
80 °C. Specimens of implantation
and interimplantation regions and of delayed and activated uteri were
separately pooled, and total RNA was extracted in TRIzol reagent (Life
Technologies, Gaithersburg, MD) according to the manufacturer's
recommendations. An additional RNA cleanup step was performed using the
Qiagen (Chatsworth, CA) RNeasy total RNA isolation kit. Total RNA (10 µg) from each group was used to generate cDNA using the
Superscript Choice system (Life Technologies). First-strand synthesis
was performed using a T7-(dT)24 primer (Sigma-Genosys,
Woodlands, TX). The resulting cDNA was used to synthesize
biotin-labeled cRNA via in vitro transcription using the
ENZO BioArray HighYield RNA transcript labeling kit (Affymetrix, Inc.).
The cRNA was fragmented in fragmentation buffer (40 mM Tris
(pH 8.1), 100 mM potassium acetate, and 30 mM
magnesium acetate, final concentration) by heating to 94 °C
for 35 min. The quality of each cRNA preparation was assessed by
analysis with a Test2 array (Affymetrix, Inc.), and all preparations
met Affymetrix's recommended criteria for use on their expression arrays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
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Fig. 2.
Levels of gene expression during
implantation. Fluorescence-labeled cRNAs were hybridized to the
Affymetrix murine U74A GeneChip. x and y axes
indicate values for the average difference score (arbitrary units) of a
single GeneChip hybridization. The average difference score reflects
the relative level of gene expression for a given transcript. The
results of a single representative experiment are shown as a
scatterplot. Algorithms for data analysis indicate genes whose
expression is present, absent, or marginal in either experiment.
Lines indicate 2- and 10-fold differences in the level of
gene expression from the mean. Top panel, comparison of
implantation and interimplantation RNAs. Transcripts that were called
marginal or absent (black, n = 6600),
present in one but absent or marginal in the other (blue,
n = 1317), or present in both analyses (red,
n = 4668) are shown at the time of initial blastocyst
attachment (day 4, 11:00 to 12:00 p.m.). Bottom panel,
comparison of uterine RNAs from delayed implantation and
estrogen-activated mice. Transcripts that were called marginal or
absent (black, n = 7186), present in one but
absent or marginal in the other (blue, n = 2358), or present in both analyses (red, n = 3044) are shown.
Genes with significantly increased expression at the implantation site
Genes with significantly decreased expression at the implantation site
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Fig. 3.
Expression of implantation and
interimplantation-specific genes. In situ hybridization
with 35S-labeled Sik-SP and Bip shows
concentration of autoradiographic signals in the stroma surrounding the
implanting blastocyst (arrows) (top panel, 40×).
Bip signals are also noted in the glandular and luminal
epithelium, whereas Sik-SP signals are absent in the luminal
epithelium and immediate subepithelial stroma in the implantation bed.
In contrast, 35S-labeled Cyp1b1 and
EP4 signals accumulated in the interimplantation
regions, with marked decreases in signal intensity at the implantation
site (bottom panel, 20×). s, stroma;
le, luminal epithelium; ge, glandular epithelium;
myo, myometrium.
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Fig. 4.
Expression levels of candidate
implantation-specific genes. x and y axes
indicate values for the average difference score (arbitrary units).
Combined analysis of average difference scores from both implantation
models (implantation versus interimplantation, delayed
versus activated) shows only those transcripts that have
statistically significant differential expression in both models
(t test only). Lines indicate 2- and 10-fold
differences in the level of gene expression from the mean.
-fold Change of genes that showed decreased expression at the
implantation site and after initiation of estrogen-induced implantation
-fold Change of genes that showed increased expression at the
implantation site and after initiation of estrogen-induced implantation
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Tables IS and IIS.
A recipient of an NICHD (NIH) MERIT award. To whom
correspondence should be addressed: Dept. of Molecular and Integrative Physiology, University of Kansas Medical Center, MRRC 3017, 3901 Rainbow Blvd., Kansas City, KS 66160. Tel.: 913-588-6213; Fax: 913-588-5677; E-mail: sdey@kumc.edu.
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G. X. Rosario, D. N. Modi, G. Sachdeva, D. D. Manjramkar, and C. P. Puri Morphological events in the primate endometrium in the presence of a preimplantation embryo, detected by the serum preimplantation factor bioassay Hum. Reprod., January 1, 2005; 20(1): 61 - 71. [Abstract] [Full Text] [PDF]
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F. L Lopes, J. A Desmarais, and B. D Murphy Embryonic diapause and its regulation Reproduction, December 1, 2004; 128(6): 669 - 678. [Abstract] [Full Text] [PDF]
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N. Brown, K. Deb, B. C. Paria, S. K. Das, and J. Reese Embryo-Uterine Interactions via the Neuregulin Family of Growth Factors During Implantation in the Mouse Biol Reprod, December 1, 2004; 71(6): 2003 - 2011. [Abstract] [Full Text] [PDF]
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 S. Mirkin, G. Nikas, J.-G. Hsiu, J. Diaz, and S. Oehninger Gene Expression Profiles and Structural/Functional Features of the Peri-Implantation Endometrium in Natural and Gonadotropin-Stimulated Cycles J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5742 - 5752. [Abstract] [Full Text] [PDF]
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 K. J. D. A. Excoffon, A. Hruska-Hageman, M. Klotz, G. L. Traver, and J. Zabner A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth J. Cell Sci., September 1, 2004; 117(19): 4401 - 4409. [Abstract] [Full Text] [PDF]
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 D. Pati, B. R. Haddad, A. Haegele, H. Thompson, F. S. Kittrell, A. Shepard, C. Montagna, N. Zhang, G. Ge, S. K. Otta, et al. Hormone-Induced Chromosomal Instability in p53-Null Mammary Epithelium Cancer Res., August 15, 2004; 64(16): 5608 - 5616. [Abstract] [Full Text] [PDF]
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O. A. Mohamed, D. Dufort, and H. J. Clarke Expression and Estradiol Regulation of Wnt Genes in the Mouse Blastocyst Identify a Candidate Pathway for Embryo-Maternal Signaling at Implantation Biol Reprod, August 1, 2004; 71(2): 417 - 424. [Abstract] [Full Text] [PDF]
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S. K. Dey, H. Lim, S. K. Das, J. Reese, B. C. Paria, T. Daikoku, and H. Wang Molecular Cues to Implantation Endocr. Rev., June 1, 2004; 25(3): 341 - 373. [Abstract] [Full Text] [PDF]
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X.-Y. Sun, F.-X. Li, J. Li, Y.-F. Tan, Y.-S. Piao, S. Tang, and Y.-L. Wang Determination of Genes Involved in the Early Process of Embryonic Implantation in Rhesus Monkey (Macaca mulatta) by Suppression Subtractive Hybridization Biol Reprod, May 1, 2004; 70(5): 1365 - 1373. [Abstract] [Full Text] [PDF]
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M. H. Melner, N. A. Ducharme, A. R. Brash, V. P. Winfrey, and G. E. Olson Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes Biol Reprod, February 1, 2004; 70(2): 406 - 414. [Abstract] [Full Text] [PDF]
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S. Tang, H. Han, and V. B. Bajic ERGDB: Estrogen Responsive Genes Database Nucleic Acids Res., January 1, 2004; 32(90001): D533 - 536. [Abstract] [Full Text] [PDF]
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V. C. L. Lin, C. T. Woon, S. E. Aw, and C. Guo Distinct Molecular Pathways Mediate Progesterone-Induced Growth Inhibition And Focal Adhesion Endocrinology, December 1, 2003; 144(12): 5650 - 5657. [Abstract] [Full Text] [PDF]
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S. C. Hewitt, B. J. Deroo, K. Hansen, J. Collins, S. Grissom, C. A. Afshari, and K. S. Korach Estrogen Receptor-Dependent Genomic Responses in the Uterus Mirror the Biphasic Physiological Response to Estrogen Mol. Endocrinol., October 1, 2003; 17(10): 2070 - 2083. [Abstract] [Full Text] [PDF]
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E. C. Wasco, E. Martinez, K. S. Grant, E. A. St. Germain, D. L. St. Germain, and V. A. Galton Determinants of Iodothyronine Deiodinase Activities in Rodent Uterus Endocrinology, October 1, 2003; 144(10): 4253 - 4261. [Abstract] [Full Text] [PDF]
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J. W. Ross, J. R. Malayer, J. W. Ritchey, and R. D. Geisert Characterization of the Interleukin-1{beta} System During Porcine Trophoblastic Elongation and Early Placental Attachment Biol Reprod, October 1, 2003; 69(4): 1251 - 1259. [Abstract] [Full Text] [PDF]
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X. Wu, S.-T. Pang, L. Sahlin, A. Blanck, G. Norstedt, and A. Flores-Morales Gene Expression Profiling of the Effects of Castration and Estrogen Treatment in the Rat Uterus Biol Reprod, October 1, 2003; 69(4): 1308 - 1317. [Abstract] [Full Text] [PDF]
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J. Shao, S. B. Lee, H. Guo, B. M. Evers, and H. Sheng Prostaglandin E2 Stimulates the Growth of Colon Cancer Cells via Induction of Amphiregulin Cancer Res., September 1, 2003; 63(17): 5218 - 5223. [Abstract] [Full Text] [PDF]
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K. J. Austin, B. M. Bany, E. L. Belden, L. A. Rempel, J. C. Cross, and T. R. Hansen Interferon-Stimulated Gene-15 (Isg15) Expression Is Up-Regulated in the Mouse Uterus in Response to the Implanting Conceptus Endocrinology, July 1, 2003; 144(7): 3107 - 3113. [Abstract] [Full Text] [PDF]
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 A. Riesewijk, J. Martin, R. van Os, J. A. Horcajadas, J. Polman, A. Pellicer, S. Mosselman, and C. Simon Gene expression profiling of human endometrial receptivity on days LH+2 versus LH+7 by microarray technology Mol. Hum. Reprod., May 1, 2003; 9(5): 253 - 264. [Abstract] [Full Text] [PDF]
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N. Baran, P. A. Kelly, and N. Binart Decysin, a New Member of the Metalloproteinase Family, Is Regulated by Prolactin and Steroids During Mouse Pregnancy Biol Reprod, May 1, 2003; 68(5): 1787 - 1792. [Abstract] [Full Text] [PDF]
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K. Tamura, T. Hara, M. Yoshie, S. Irie, A. Sobel, and H. Kogo Enhanced Expression of Uterine Stathmin during the Process of Implantation and Decidualization in Rats Endocrinology, April 1, 2003; 144(4): 1464 - 1473. [Abstract] [Full Text] [PDF]
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C. Richard, J. Gao, N. Brown, and J. Reese Aquaporin Water Channel Genes Are Differentially Expressed and Regulated by Ovarian Steroids during the Periimplantation Period in the Mouse Endocrinology, April 1, 2003; 144(4): 1533 - 1541. [Abstract] [Full Text] [PDF]
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M. W. M. Yao, H. Lim, D. J. Schust, S. E. Choe, A. Farago, Y. Ding, S. Michaud, G. M. Church, and R. L. Maas Gene Expression Profiling Reveals Progesterone-Mediated Cell Cycle and Immunoregulatory Roles of Hoxa-10 in the Preimplantation Uterus Mol. Endocrinol., April 1, 2003; 17(4): 610 - 627. [Abstract] [Full Text] [PDF]
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Y.-P. Cheon, Q. Li, X. Xu, F. J. DeMayo, I. C. Bagchi, and M. K. Bagchi A Genomic Approach to Identify Novel Progesterone Receptor Regulated Pathways in the Uterus during Implantation Mol. Endocrinol., December 1, 2002; 16(12): 2853 - 2871. [Abstract] [Full Text] [PDF]
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G.C. Weston, I. Haviv, and P.A.W. Rogers Microarray analysis of VEGF-responsive genes in myometrial endothelial cells Mol. Hum. Reprod., September 1, 2002; 8(9): 855 - 863. [Abstract] [Full Text] [PDF]
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 B. C. Paria, J. Reese, S. K. Das, and S. K. Dey Deciphering the Cross-Talk of Implantation: Advances and Challenges Science, June 21, 2002; 296(5576): 2185 - 2188. [Abstract] [Full Text] [PDF]
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L. C. Kao, S. Tulac, S. Lobo, B. Imani, J. P. Yang, A. Germeyer, K. Osteen, R. N. Taylor, B. A. Lessey, and L. C. Giudice Global Gene Profiling in Human Endometrium during the Window of Implantation Endocrinology, June 1, 2002; 143(6): 2119 - 2138. [Abstract] [Full Text] [PDF]
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