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Endocrinology, doi:10.1210/en.2007-0268
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Endocrinology Vol. 148, No. 9 4173-4184
Copyright © 2007 by The Endocrine Society

The Postimplantation Embryo Differentially Regulates Endometrial Gene Expression and Decidualization

Aki Kashiwagi1, Carla M. DiGirolamo1, Yoshiaki Kanda, Yuichi Niikura, Charles T. Esmon, Thomas R. Hansen, Toshi Shioda and James K. Pru

Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service (A.K., C.M.D., Y.K., Y.N., J.K.P.), and the Molecular Profiling Laboratory (T.S.), Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114; Cardiovascular Biology Research Program (C.T.E.), Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104; and Animal Reproduction and Biotechnology Laboratory and the Department of Biomedical Sciences (T.R.H.), Colorado State University, Fort Collins, Colorado 80523

Address all correspondence and requests for reprints to: Dr. James K. Pru, Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital, Harvard Medical School, Thier Research Building, Room 931, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: jpru{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transcriptomal changes in the uterine endometrium induced in response to the implanting embryo remain largely unknown. In this study, using Affymetrix mRNA expression microarray analysis, we identified genes differentially expressed in the murine endometrium in the presence or absence of the embryo. Compared with the pseudopregnant deciduoma induced by a mechanical stimulus in the absence of an embryo, approximately 1500 genes (753 up-regulated, 686 down-regulated; P < 0.05) were differentially expressed by at least 1.2-fold in the uterine decidua of pregnancy. Most of these genes fall into five major biological categories that include binding (45%), catalysis (24%), signal transduction (10%), transcriptional regulators (5%), and transporters (5%). This strong, embryo-induced transcriptomal impact represented approximately 10% of the total number of genes expressed in the decidualizing endometrium. Validation studies with mRNA and protein confirmed existence of the phylogenetically conserved, embryo-regulated genes involved in the following: 1) hemostasis and inflammation; 2) interferon signaling; 3) tissue growth and remodeling; and 4) natural killer cell function. Interestingly, whereas expression of many growth factors and their cognate receptors were not different between the decidual and deciduomal endometria, a number of proteases that degrade growth factors were selectively up-regulated in the decidual tissue. Increased expression of IGF and activin A neutralizing factors (i.e. HtrA1 and Fstl3) correlated with reduced stromal cell mitosis, tissue growth, and mitogenic signaling in the decidual endometrium. These results support the hypothesis that the implanting murine embryo takes a proactive role in modulating endometrial gene expression and development during early gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREGNANCY FAILURE IS thought to be caused by a variety of factors including poor oocyte or embryo quality (1, 2), acquired or congenital anatomical abnormalities (3), and endocrine disruption (4). Epidemiological studies in humans (5) and livestock (6), as well as genetic studies in rodents (7), support the notion that failed pregnancy also occurs due to faulty uterine function or miscommunication between the embryo and mother during implantation. Increased knowledge of the events leading to adequate preparation of the uterus for embryo attachment and implantation, as well as a better understanding of molecular signals regulating trophoblast invasion of the endometrial stromal compartment, is important to prevent pregnancy failure.

In invasively implanting species such as rodents and humans, the endometrial compartment undergoes a postnatal developmental paradigm during early gestation that involves proliferation, growth, and differentiation of resident stromal cells into large polyploidy decidual cells (8, 9, 10). This differentiated stromal tissue, called the deciduum, plays a variety of functions that are critical for pregnancy. For instance, the deciduum produces many endocrine and paracrine signaling molecules (11, 12, 13), displays immunosuppressive actions (14, 15), provides a nutrient source for the expanding trophectoderm as it undergoes progressive regression by apoptosis (8, 9), and controls trophoblast growth and migration (16). It also provides a rudimentary vascular network that becomes modified by the embryo as the placenta develops (17). The deciduum of rodents and humans as well as nondecidualized stromal tissue in noninvasively implanting species harbors immune cells such as natural killer (NK) cells that are recruited to the endometrium during early gestation (18, 19). The NK cells, which constitute approximately 30% of the total cellular volume of the human deciduum (20), modulate maternal spiral arteries to enhance blood flow to the implantation site (21). Additionally, NK cells release growth-promoting and chemoattractant factors that aid in the development of the placenta as it invades the uterus in its quest for nutrients (22).

In mice, decidualization begins shortly after the blastocyst attaches to the uterine luminal epithelium on day of pregnancy (DOP) 3.5 and continues through DOP 9, at which time the embryo begins to develop its own nutrient/gas exchange apparatus (i.e. placenta). The decidualized endometrium therefore is a placental-like structure that serves as the primary exchange apparatus for the developing embryo until the placenta becomes functionally competent to take on this role. A similar but temporally distinct design exists in humans. The observation that the hormonally primed uterus can be stimulated by mechanical means (e.g. sesame oil) to undergo decidualization, even in the absence of an embryo, supports the notion that stromal cell differentiation is initially controlled by intrinsic maternal factors (23). Although the mechanically decidualized endometrium, known as the deciduoma, is similar to the embryo-induced decidua in many respects, several histological differences (e.g. vasculature) are evident. In humans, spontaneously decidualized stromal cells are commonly found during the secretory phase of the menstrual cycle in the absence of pregnancy in regions surrounding uterine spiral arteries (24). It is, however, still unknown as to what extent the embryo contributes to the development of the decidualized endometrium.

To test the hypothesis that the embryo takes a proactive role in helping to establish the uterine environment during implantation, we attempted to determine whether the early postimplanting embryo has the capacity to regulate not only its own development but also certain aspects of maternal endometrial development during decidualization. To this end, genome-wide DNA microarray analysis was completed initially to identify genes and gene networks that became differentially regulated between the deciduum of early pregnancy formed in response to the embryo and the mechanically induced, embryo-free deciduoma. Postmicroarray validation studies at the mRNA and protein levels provide supporting evidence that the embryo modulates endometrial development during early pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mature (6–8 wk old) ICR female mice purchased from Charles River Laboratories (Wilmington, MA) were placed with intact ICR male mice of proven breeding capacity or with vasectomized ICR male mice. Female mice were considered DOP or day of pseudopregnancy (DOPP) 0.5 on observation of a vaginal seminal plug. For collecting decidual tissue on DOP 7.5, 8.5, and 9.5, whole implantation sites were first cut along the axis of the uterus just beneath the myometrium to expose the decidual tissue. Deciduas were bluntly removed and then cut longitudinally to access the embryonic chamber. The embryo and associated membranes were then removed by scraping the inside of the embryonic chamber with a scalpel blade. The remaining decidual tissue was rinsed in ice-cold PBS. Although it is possible that some residual trophoblast giant cells remained attached to the deciduum because of the highly invasive nature of these cells, this method of isolation results in highly enriched decidual tissue. To obtain sufficient RNA to complete the array analysis, decidual tissue from two implantation sites from the same mother were pooled from each of three separate pregnant female mice. Whole implantation sites were collected on DOP 4.5 for Western blotting or on DOP 7.5 and 8.5 and prepared for histology and immunohistochemistry. To induce decidualization in pseudopregnant female mice, sesame oil (10 µl) was infused into the uterine lumen immediately below the uterotubal junction at 1300 h on the third day after observation of a vaginal plug. Whole uterine tissue or decidualized endometrium (referred to as the deciduoma) was collected at times that correspond developmentally to DOP 7.5, 8.5, and 9.5, respectively. Deciduomal tissue was exposed by first making a longitudinal incision just beneath the myometrium. The myometrium and serosa were then peeled away from the decidualized stromal tissue by blunt dissection. Total RNA was immediately isolated from a 50-mg portion from each of three individual female mice. The remaining deciduomal tissue was partitioned into 50- or 100-mg portions and stored at –80 C for subsequent use. Animal protocols were reviewed and approved by the Massachusetts General Hospital Institutional Care and Use Committee.

RNA isolation and semiquantitative RT-PCR
Total RNA was isolated from uterine tissues using TriReagent (Sigma Chemical Co., St. Louis, MO) and subjected to DNase I digestion (RQ1 RNase-free DNase; Promega, Madison, WI) to eliminate genomic DNA contamination. cDNA was synthesized using SuperScript II reverse transcriptase and oligo-dT primer (Invitrogen, Carlsbad, CA). Uterine gene expression was assessed by semiquantitative RT-PCR of the cDNA using primer sets shown in Table 1Go. Each PCR product was sequenced to confirm specific amplification of the target gene. mRNA encoding the ribosomal protein L7 (Rpl7) was used as an internal control. Semiquantitative analysis was completed after densitometry scanning of all genes analyzed by RT-PCR in which the gene expression ratio of each gene vs. Rpl7 was established. A negative control (i.e. mock reverse transcriptase) was also included for each mRNA sample in which reverse transcriptase was omitted to further confirm the absence of genomic DNA contamination.


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  		TABLE 1. PCR primer sets

 
DNA microarray experiment and data analyses
The total RNA samples for microarray analysis were isolated from decidualizing endometrial tissues on d 7.5 of pregnancy and pseudopregnancy using TriReagent and further purified using the RNeasy Mini Elute clean up kit (QIAGEN, Chatsworth, CA). The RNA quantity, integrity, and purity were determined by microscale UV spectrophotometry, electrophoresis in formaldehyde agarose gels with ethidium bromide staining, and the RNA-nano bioanalyzer microcapillary electrophoresis (Agilent, Palo Alto, CA). RNA samples of three independent experiments, in which tissues were obtained from separate female mice, were first evaluated by real-time quantitative RT-PCR using TaqMan quantitative PCR assay for murine ß-actin gene using master mix and AB7500 real-time PCR system (Applied Biosystems, Framingham, MA). Microarray probe synthesis and hybridization of GeneChip Mouse Genome 430–2.0 DNA array (Affymetrix, Santa Clara, CA) were performed in the Molecular Profiling Laboratory at the Massachusetts General Hospital Center for Cancer Research following the manufacturer’s instructions. Hierarchical clustering analysis of the decidual and deciduomal tissue samples was performed using Cluster software and visualized with TreeView software (25). Gene ontology analysis and identification of differentially expressed genes (P < 0.05, Student’s t test) were performed using the GeneSifter.net online service (visXlabs, Seattle, WA). The GeneSifter software served as a linker tool for further analyses using the Kyoto Encyclopedia of Genes and Genomes database (26) and Gene Ontology Consortium Database (27). Based on prior experience (28), genes whose expression intensity value was less than 100 were eliminated from the study for the reduced accuracy in microarray quantitation for such low amounts of mRNA transcripts as well as technical difficulties in validation studies.

Western blot analysis
Protein lysates were collected from decidualized endometrial tissue of pregnancy and pseudopregnancy for Western blot analysis as described in detail (29). After electrophoretic separation using the NuPage system (Invitrogen), proteins were transferred (30 V, 1 h) onto polyvinylidene difluoride membranes. Nonspecific binding was blocked with 5% fat-free milk in PBST buffer (0.1% Tween 20 in PBS) for 1 h at room temperature. Primary antibodies were diluted in PBST with 5% BSA and applied to membranes for over night incubation at 4 C. Membranes were then washed (3 x 10 min each) in PBST buffer and incubated with secondary antibodies for 1 h at room temperature (see Table 2Go for specific details about each of the primary and secondary antibodies). Membranes were washed in PBST as before, and bound antibody was detected using enhanced chemiluminescent reagents based on the manufacturer’s recommendations (Amersham, Piscataway, NJ). Control blots were also completed in which primary antibody was omitted. To verify equal protein loading, membranes were then stripped [1 M glycine (pH 2.5), 1 h, 37 C] and reprobed with pan-AKT antibody.


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  		TABLE 2. Antibodies and lectin used for Western blotting, immunohistochemistry, or histochemistry

 
Immunohistochemical and histological analyses
For immunohistochemical analyses, whole implantation sites (DOP 7.5 and 8.5) or decidualized uterine tissue (DOPP 7.5 and 8.5) were either fixed in 4% buffered paraformaldehyde (24 h at 4 C) followed by paraffin embedding or prepared for frozen sectioning by briefly (2 h) fixing tissues in 4% paraformaldehyde, incubating overnight in PBS-buffered 0.5 M sucrose, and embedded in optimum cutting temperature compound (Sakura Finetek Inc., Torrance, CA). Frozen sections were cut to 6 µm, equilibrated in PBS, air dried, and then postfixed in ice-cold 4% paraformaldehyde for 10 min. Paraffin-embedded uterine sections (6 µm) were deparaffinized with xylenes followed by graded rehydration in ethanol (100, 95, 80, and 70%) and distilled water. After peroxidase quenching (10 min in methanol containing 0.3% hydrogen peroxide, all tissue sections), antigens were unmasked (paraffin embedded sections only) by high-temperature protein denaturation [10 min boiling in 10 mM sodium citrate buffer (pH 6.0)]. After equilibration in PBS and blocking (2% BSA and 1% normal donkey serum), sections were incubated overnight at 4 C with primary antibody diluted in buffer of 0.1 M Tris (pH 7.6), 0.55 M NaCl, and 0.01 M KCl as outlined in Table 2Go. Sections were then washed (PBS, 3 x 10 min each) and incubated for 1 h at room temperature with biotinylated secondary antibody (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA) followed by washing and a final incubation with horseradish peroxidase-conjugated streptavidin for 45 min at room temperature (Vector Laboratories, Burlingame, CA). After washing as before, sections were exposed to 3,3'-diaminobenzidine substrate, counterstained with hematoxylin, dehydrated in ethanol and xylenes, and mounted for light microscopy. Antibody specificity was confirmed in negative control experiments in which primary antibodies were omitted. For detection of endometrial NK cells, paraffin sections were prepared as before with the exception that biotinylated Dolichos biflorous (DBA) lectin (Table 2Go) (30) was used in place of antibodies. For decidual (DOP 7.5 implantation sites) vs. deciduomal (DOPP 7.5) histological comparisons, uterine tissues were fixed in Dietrick’s fixative (24 h at 4 C; 0.34 N acetic acid, 10% formaldehyde, 28% ethanol), paraffin embedded, sectioned (4 µm), and stained with hematoxylin and eosin. Endometrial tissue area (n = 4 experiments) in deciduomal and decidual sections was calculated using the following equation: endometrial area = ({pi}) [(mesometrial vertical radius + antimesometrial vertical radius)/2] (horizontal radius). The mean percentage of mitotic cells in decidual and deciduomal endometrial tissue sections was established by counting the number of phosphohistone H3-positive cells (see Table 2Go for details of antibody usage) vs. the total number of cells (mean of three tissue sections per sample, with counts taken from five fields of view per section) after standard immunohistochemical detection.

Experimental replication and statistical analysis
Each experiment was independently replicated at least three times with different mice being used in each experiment. Data in all graphs represent the mean ± SEM from replicated experiments. Assignment of mice to each experiment was made randomly. Raw data were analyzed with GraphPad PRISM software (version 4.0; San Diego, CA) by Student’s t test for simple comparisons or one-way ANOVA in which three time points were compared. Mean values were considered statistically different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genome-wide mRNA expression profiling identifies differentially regulated transcripts in the naturally vs. mechanically stimulated decidualized endometrium
Potential gene targets of embryonic signaling in the decidualized endometrium of the mouse were identified by comparing mRNA expression profiles between mechanically induced deciduomal and embryo-induced decidual tissues. Because of consistencies in ß-actin gene expression and probe hybridization obtained across all mRNA samples, sample normalization and data transformation were not completed during the analysis. Therefore, the data presented below reflect raw changes in gene expression between decidual and deciduomal tissues without manipulation of the data. Microarray comparison of transcriptomal profiles of three decidual and three deciduomal specimens obtained from independent animals showed that, among approximately 15,000 significantly expressed genes, 2,943 genes were differentially expressed with at least 1.2-fold changes and confidence interval of 95%. Because potentially important genes may be differentially expressed at limited regions in the embryo-induced decidua (e.g. areas immediately adjacent to the embryo), we selected relative low cutoff criterion (1.2-fold) to ensure that our analysis would not lose information on such genes.

Gene ontology annotation was used for categorizing genes on the basis of their involvement in different biological processes. Of the 2943 differentially regulated gene, 2792 genes were partitioned into nine categories (Fig. 1Go). The five largest categories were binding (45%), catalysis (24%), signal transduction (10%), transcriptional regulators (5%), and transporters (5%). These results implied that a major portion of the differentially expressed genes (i.e. potentially embryo-regulated genes) encode binding proteins to certain targets or enzymes. Cluster analysis successfully separated the decidual and deciduomal endometrial tissues, and 1439 class-discriminating marker genes were identified (Fig. 2Go). Among them, expression of 753 genes was significantly up-regulated (P < 0.05, 1.2-fold or greater) in the decidua and 686 genes were down-regulated. In total, the presence of an embryo impacted on the expression of approximately 10% of the entire set of genes expressed in the decidualizing endometrium during pregnancy. Tables 3Go and 4Go show 53 of the 59 total annotated genes that were up- or down-regulated (P < 0.05), respectively, by 2.5-fold or more in decidual tissue. A detailed list of genes differentially regulated 1.2-fold or more can be found elsewhere (www.VCRB.org).


Figure 1
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  		FIG. 1. Pie chart showing distribution of genes differentially regulated (1.2-fold or greater; P < 0.05) in isolated deciduomal and decidual endometrium on d 7.5 postcoitum based according to biological process annotation.

 

Figure 2
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  		FIG. 2. Heatmap representing relative expression levels of genes in the endometrial during uterine decidualization in response to mechanical stimulation (deciduoma) or the embryo (decidua). Hierarchical clustering analysis of the decidual and deciduomal tissue samples was performed using Cluster software and visualized with TreeView software. Each horizontal line represents a single gene with each column representing a single sample. The listed sample genes identified in the blue box are expressed consistently higher in the decidual tissue, whereas genes listed in the red box are expressed higher in deciduomal tissue. The analysis indicates that the samples cluster according to the presence of an embryo.

 

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  		TABLE 3. Endometrial genes up-regulated 2.5-fold or greater in decidual tissue

 

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  		TABLE 4. Endometrial genes down-regulated 2.5-fold or greater in decidual tissue

 
The RT-PCR method was used to validate findings observed by the microarray method. Of the 25 differentially expressed genes presented in Figs. 3Go, 5Go, 6Go, and 7Go, 20 genes were shown by RT-PCR and semiquantitative analysis (n = 3–4 replicates per gene) to be regulated (P < 0.05) in accordance with the microarray data set. That the microarray data could be validated by RT-PCR with 80% efficiency suggests that the fold change observed by microarray analysis reflects the biological condition with reasonable certainty.


Figure 3
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  		FIG. 3. Semiquantitative RT-PCR was used to validate gene profiling data. The gene for ribosomal protein L7 (Rpl7) was used as an internal control. Gene expression in endometrial tissue from DOPP 7.5 (deciduoma) and DOP 7.5 (decidua) female mice are compared. Right panels are negative controls in which reverse transcriptase was omitted from the reverse transcription reaction. Representative of at least n = 3 independent experiments in which individual mice were used in each experiment. Genes found to be differentially regulated (P < 0.05) by RT-PCR are indicated with an asterisk.

 

Figure 5
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  		FIG. 5. Differential endometrial expression of IFN-regulated genes during pregnancy and pseudopregnancy. A, RT-PCR analysis was used to validate gene array data, which showed an increase in the expression of listed IFN-regulated genes. The ribosomal protein L7 (Rpl7) gene was used as an internal control. Genes found to be differentially regulated (P < 0.05) by RT-PCR are indicated with an asterisk. B and C, ISG15 expression on DOP 4.5, 7.5, and 9.5 by Western blotting. D and E, Protein expression of ISG15 (Western blotting) on DOPP 7.5 (–, embryo) or DOP 7.5 (+, embryo). Statistical analyses of ISG15 expression normalized to pan-AKT during early gestation (C, by one-way ANOVA; mean ± SEM) or in response to the presence of an embryo (E, by Student’s t test). Different letters indicate significant difference in expression between days of pregnancy. *, Significant difference vs. deciduoma. Representative of at least n = 3 independent experiments in which individual mice were used in each experiment.

 

Figure 6
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  		FIG. 6. Tissue growth and remodeling in deciduomal and decidual tissues. A, Hematoxylin and eosin-stained sections (x17 magnification) of decidualizing endometrium on DOPP 7.5 (deciduoma) and DOP 7.5 (decidua). B, Graphical results of endometrial area analysis. C, Comparison of mitotic cells (percentage) in deciduomal and decidual tissues. *, Significant difference vs. decidua. D, RT-PCR analysis was used to validate gene array data, which showed a significant change in the expression of the listed growth and tissue remodeling related genes. Genes found to be differentially regulated (P < 0.05) by RT-PCR are indicated with an asterisk. E, Immunohistochemical detection of HtrA1 in deciduomal (DOPP 8.5, x30 magnification) and decidual (DOP 8.5, x40 magnification) uterine tissues. Control sections were stained in the absence of primary antibody. F, Western analysis showing expression of phospho-proteins AKT (pAKT), FOXO1a (pFOXO1a), and ERK 1/2 (pERK 1/2) as well as total AKT (pan-AKT, internal loading control) in endometrial tissues isolated on DOPP 7.5 (–, embryo) or DOP 7.5 (+, embryo). Results of three independent experiments in which individual mice were used.

 

Figure 7
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  		FIG. 7. The embryo facilitates recruitment of NK cells to the uterine deciduum. A, RT-PCR analysis was used to validate gene array data. Genes found to be differentially regulated (P < 0.05) by RT-PCR are indicated with an asterisk. B, DBA lectin staining for NK cells in deciduomal and decidual tissues on d 7.5 of pseudopregnancy and pregnancy, respectively. C, Analysis of NK cell counts in deciduomal and decidual tissues by Student’s t test. *, Significant difference vs. deciduoma. Representative of at least n = 3 independent experiments in which individual mice were used in each experiment.

 
Transcriptional and translational validation of microarray data
Validation studies of microarray data using RT-PCR confirmed differential expression of genes whose biological importance in the endometrial has been previously reported or suggested. These included genes expressed higher in decidual tissue [e.g. peptidoglycan recognition protein 1; protein C receptor (Procr); secreted phosphoprotein 1; angiopoietin 2; and uterine-specific proline-rich acidic protein] and genes expressed higher in deciduoma (e.g. prolactin-like protein b). Expression of some genes such as forkhead box o1a (Foxo1a, Fig. 3Go) was not different between decidua and deciduoma based on both microarray and RT-PCR analyses.

To demonstrate that changes in mRNA expression were translated at the protein level, we next assessed expression of the Procr product, endothelial protein C receptor (EPCR). Based on the microarray experiment, Procr mRNA was approximately 4 times more abundant in decidua than deciduoma. RT-PCR validation confirmed that Procr mRNA was expressed more strongly in decidua than deciduoma (P < 0.05, Fig. 3Go). Western blotting analysis revealed that endometrial expression of the EPCR protein was low on DOP 7.5, increased significantly by DOP 8.5, and remained elevated through DOP 9.5 (Fig. 4Go, A and B). On d 8.5, EPCR protein was expressed 2.4-fold more strongly (P < 0.05) in decidual tissues than deciduoma (Fig. 4Go, C and D). Immunohistochemical analysis of uterine tissues also confirmed elevated endometrial expression of the EPCR protein in the decidual tissues on DOP 8.5, compared with the deciduomal tissues on the corresponding day of pseudopregnancy (Fig. 4EGo).


Figure 4
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  		FIG. 4. Protein analysis of EPCR during uterine decidualization. A, Representative Western blot showing expression of EPCR on DOP 7.5, 8.5, and 9.5. C, EPCR protein expression shown by Western blotting on DOPP 8.5 (–, embryo) or DOP 8.5 (+, embryo). Statistical analyses of EPCR expression normalized to pan-AKT during early gestation (B, by one-way ANOVA) or in response to the presence of an embryo (D, by Student’s t test). Different letters indicate significant difference in expression compared with DOP 4.5. *, Significant difference vs. deciduoma. E, Immunohistochemical detection of EPCR in decidualizing endometrial tissues on DOPP 8.5 and DOP 8.5 (x40). Control sections were stained in the absence of primary antibody. Representative of at least n = 3 independent experiments in which individual mice were used in each experiment.

 
A pregnancy induced type I interferon-like response
Expression of several interferon (IFN)-stimulated genes (ISGs) has previously been shown to be up-regulated in the endometrium in response to embryo implantation (31). Semiquantitative RT-PCR (Fig. 5AGo) was used to show an increase (P < 0.05) in mRNA expression for interferon regulatory factor-8 (Irf-8, 1.6-fold), interferon {alpha}-responsive protein (Iarp, 2.0-fold), interferon activated gene 202B (Ifi202b, 1.9-fold), interferon {gamma}-inducible GTPase (Iigp1, 1.6-fold), as well as transcripts from genes Isg12 (2.5-fold) and Isg15 (1.6-fold). Among these Iarp, Isg12, and Iigp1 were identified from the microarrays as being up-regulated (P < 0.05) in decidual tissue. The Isg15 has been studied in greatest detail for its up-regulation in the bovine endometrium in response to embryo-derived interferon-{tau} (32, 33). Recently, Isg15 was shown to be expressed more abundantly in decidual tissue of pregnant mice in comparison with deciduomal tissue of pseudopregnancy (34). These findings are extended here at the protein level, which show that uterine ISG15 protein increased from DOP 4.5 through DOP 7.5 and then further by DOP 9 (Fig. 5Go, B and C). A 1.7-fold increase (P < 0.05) in ISG15 was found in isolated decidual tissues of pregnancy, compared with deciduomal tissues (Figs. 5DGo and 3EGo).

Regulated tissue growth and remodeling
Throughout the present study, we consistently noted that deciduomal tissue appeared larger in size than decidual tissue. Histological examination and analysis of medial transverse sections from implantation sites and transverse sections of oil-induced deciduomal tissue of pseudopregnancy demonstrated that the circular area of deciduomal tissue was indeed greater than that of decidual tissue (5.83 ± 0.45 vs. 2.98 ± 0.72, respectively, P = 0.02; Fig. 6Go, A and B). Immunohistochemical staining for phosphohistone H3, a marker of mitosis, revealed a significant increase in the number of mitotic cells in deciduomal tissue, compared with decidual tissue (4.16 ± 0.21 vs. 1.32 ± 0.42, respectively, P = 0.01; Fig. 6CGo).

A number of growth factors have been proposed to regulate various aspects of uterine function during implantation. Among these, IGF-I and bone morphogenic proteins (BMPs) are thought to initiate decidualization and coordinate embryo spacing, respectively, during embryo implantation (35). In this next group of studies, we focused on nine genes encoding proteins that are developmental regulators of tissue growth and remodeling. As before, these genes were identified by microarray as being selectively induced or suppressed in decidualized uterine stromal tissue in response to the presence of an embryo. Three genes encoding proteolytic enzymes, protease serine member S (Prss) 11, Prss25, and matrix metalloproteinase (Mmp) 9, were significantly up-regulated (P < 0.05), whereas tissue inhibitor of matrix metalloproteinase (Timp) 3, a classical marker of uterine stromal cell differentiation that blocks MMP catalytic activity, was concomitantly down-regulated (Fig. 6DGo). Although microarray data suggested up-regulation of insulin-like growth factor binding protein (Igfbp) genes 2 and 5, RT-PCR validation did not confirm this observation. Fstl3, whose protein product neutralizes activin A signaling and potentially other members of the BMP family (36), was up-regulated. Bmp2 and Bmp7 were down-regulated (P < 0.05) by 1.7- and 2.3-fold, respectively. Our microarray analysis did not detect differential expression of IGFs, activins, IGF receptors, and BMP receptors between decidua and deciduoma. To confirm differences in expression at the protein level, focus was given to the high temperature requirement factor A1 (HtrA1), the encoded product of Prss11 and a serine protease thought to be involved in decidual cell involution during invasion of the uterine compartment by the trophectoderm (37, 38). As shown in Fig. 6EGo, HtrA1 was strongly expressed in the differentiated stromal cells at the DOP 8.5 implantation site, whereas its expression was only modest in the stromal cells surrounding the uterine lumen on DOPP 8.5.

A common feature of the BMP and IGF systems is that both pathways control organ size by promoting tissue growth through increased cell cycle progression. The MAPK and AKT pathways often mediate mitogenic signals (39). Figure 6FGo demonstrates that the phosphorylated, and thus active form of AKT (i.e. pAKT), was more profuse in the deciduum of pseudopregnancy than in the decidua of pregnancy (1.72 ± 0.08 vs. 0.75 ± 0.14, ratio normalized against pan-AKT, P = 0.01). Likewise, the transcription factor FOXO1A, a downstream target of pAKT signaling that promotes uterine stromal cell decidualization (40, 41), also showed a greater degree of phosphorylation (P < 0.05) in deciduomal tissues. Interestingly, the phosphorylation status (i.e. activity) of the MAPK ERK 1/2 remained unchanged between decidual and deciduomal tissues (Fig. 6FGo).

Immune cell recruitment
Immune cells contribute positively to pregnancy by producing growth factors and cytokines that promote trophoblast invasion of the uterine environment during early pregnancy (22). In accordance with this concept, our microarray data identified numerous NK cell markers as genes preferentially expressed in the decidua over the deciduoma. For example, as shown in Fig. 7AGo, Nkg7, Perforin, Granzyme E, and Granzyme F were all found to be significantly (P < 0.05) up-regulated in the decidua. The elevated expression of these NK cell markers reflected larger numbers of NK cells present in the decidual endometrium (7.3-fold, P < 0.001) than in the deciduomal tissues, as demonstrated by the NK cell-specific DBA lectin staining (30) of paraffin sections (Fig. 7Go, B and C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several laboratories have used high-density microarrays to catalog genes expressed in the uterus during the estrous (42, 43) or menstrual cycles (44, 45, 46), in response to steroid hormones estrogen (47, 48, 49) and progesterone (46, 49, 50, 51) and during early pregnancy (52, 53, 54, 55). However, to our knowledge, endometrial gene expression during early pregnancy in vivo has been limited to a report using a low-density array consisting of 83 genes (56). Other microarray studies have been completed using in vitro models (57, 58), in species other than mice (52, 53), or at different stages of pregnancy without focus on the endometrial compartment (43, 49). In the present study, we used Affymetrix high-density microarray to compare genome-wide mRNA expression profiles of the embryo-induced decidua of murine pregnancy and the oil-induced deciduoma of pseudopregnancy. Results of this study provided new and potentially important insights into the molecular and cellular events initiated by the implanting embryo in the decidualizing mammalian endometrium.

One caveat of this comparative model is that the deciduogenic stimulus used to initiate decidualization was different during pregnancy and pseudopregnancy. It is possible, for example, that some of the genes identified in this study as being differentially regulated are actually induced or suppressed by mechanical stimulation of the endometrium rather than by embryonic factors. Similarly, the increased size of the deciduoma over the decidua may be more reflective of the amount of stimulation rather than the proposed suppressive actions of the embryo on endometrial expansion. Additional experiments employing in vitro coculture methods and in vivo genetic approaches will be necessary to validate embryonic signaling in the endometrium. Another potential limitation of this model is that the decidualized endometrium is comprised of a heterogeneous population of cells. Differences in gene expression between decidual and deciduomal tissues may occur because of differences in the presence of certain populations of cells. Nonetheless, a number of genes (e.g. Isg15, Procr, and Fstl3) identified in this study were previously reported as embryo-induced genes using in vitro or ex vivo approaches (34, 56, 57, 58). In total, 1439 genes were recognized as being differentially regulated in decidual vs. deciduomal tissue.

Among the most highly regulated genes identified in the profiling experiment was the EPCR, which was up-regulated 2.6-fold in decidual tissue (Fig. 4DGo). EPCR is a component of the protein C anticoagulation pathway that activates protein C by the thrombin-thrombomodulin complex (59, 60). This natural antagonist of blood coagulation prevents microvascular thrombosis and is expressed in endometrial tissue of early pregnancy (59). In human pregnancies thrombosis of the maternal-placental vascular circuit is associated with fetal loss, intrauterine growth restriction, and severe early-onset preeclampsia (61). Genetic studies in mutant mice have confirmed a functional role for EPCR during pregnancy in that mutant fetuses generally die from placental thrombosis (60). It is easy to envision that an anticoagulation system such as the protein C pathway must be in place at the implantation site to prevent thrombosis from blocking nutrient/gas exchange, particularly because, at least in humans, pregnancy is associated with increased thrombin generation (62). Whatever the case, EPCR likely plays an important role during early pregnancy in that its presence in decidualized uterine stromal cells and probable regulation by an embryonic factor(s) is evidently conserved phylogenetically in mice and humans. Indeed, Popovici et al. (58) indicated that primary human decidualized stromal cells cultured in the presence of trophectoderm express 2.5 times more Procr than decidual cells cultured alone.

Six IFN-regulated genes were found to be up-regulated in decidual compared with deciduomal tissue. This IFN-like response is conserved evolutionarily and has now been observed in a number of species including cow (32, 33), sheep (53, 63), mouse (current study and Refs. 34 and 56), baboon (64), and human (57, 64). However, only in ruminants has a bona fide IFN been identified as the embryo-derived paracrine factor regulating uterine responses. We demonstrate here that murine ISG15 protein is more abundant in decidual tissue of pregnancy than in deciduomal tissue, supporting previous findings that Isg15 mRNA was transcriptionally up-regulated by the embryo (34, 54, 57). We also add Irf-8, Iarp, Ifi202b, Iigp1, and Isg12 to the list of endometrial IFN-regulated genes, and among these, only Isg12 has been reported as being regulated in the uterus by an embryonic signaling factor (53).

Another interesting observation made was that deciduomal tissue was substantially larger than decidual tissue three days after provision of a deciduogenic stimulus (Fig. 6BGo). A likely explanation for this increased size is elevated cell cycle progression. We noted a 315% increase in the percentage of mitotic cells in deciduomal tissue, compared with decidual tissue. A number of growth factors have been proposed to stimulate endometrial stromal cell proliferation and differentiation at the time of decidualization. These include, among others, IGF-I (35), activin (65), and BMP2 (35). Two mechanisms potentially regulated by the embryo were identified that could account for a reduction in decidual growth during pregnancy. First, Bmp2 was significantly down-regulated in decidual tissue. BMPs are well-established developmental enhancers of tissue growth (66). BMP2, in combination with other factors such as fibroblast growth factor-2, may promote increased proliferation, hypertrophy, alkaline phosphatase activity, and extracellular matrix synthesis associated with decidualization (35).

It is hypothesized here that the embryo retards decidual production of growth factors such as BMP2 as a means to control endometrial growth. Second, whereas differences were not detected in the expression of other growth factors such as Igf-1, Igf-II, or Activin A or their receptors, several proteases that degrade these paracrine factors were more abundant in decidual than deciduomal tissue. For instance, Prss11 mRNA was up-regulated by the presence of the embryo. Likewise, the Prss11 product, HtrA1, a protease with an IGF binding domain (38, 67), was also more abundant in decidualized stromal cells than in corresponding cells of the deciduoma (Fig. 6EGo). In addition to IGF binding proteins, which have been proposed to modulate IGF signaling at the implantation site (68), IGF gradients may be modulated through proteolytic cleavage by proteins such as HtrA1. Like IGFs, activin A is proposed as a promoter of uterine decidualization (65, 69).

The maternal deciduum is the primary source of activin A in which it is though to stimulate endometrial production of proMMPs-2, -3, -7, and -9, as well as MMP-2 and in doing so is a purported important factor in the dialog that exists between the embryo and mother (69). Fstl3, the encoded product of which neutralizes activin A, myostatin, and some BMPs (70), was also up-regulated in decidual tissue. Similar findings were observed for Fstl3 in human decidualized stromal cells exposed to trophoblast conditioned medium (58). These combined results suggest a second mechanism that is used to regulate endometrial development whereby the maternally derived growth-promoting signaling become attenuated by embryo-induced neutralization of growth factors. An interesting clinical observation was made from human pregnancies in which Fstl3 was found to be significantly more abundant at the maternal-fetal interface of intrauterine growth-restricted pregnancies than normal pregnancies (71). It was concluded from this study that FSTL3 might be important for the pathogenesis of intrauterine growth restriction due to enhanced removal of growth promoting factors.

Whereas measurements of decidual growth factors at the protein level were not made, two downstream signal transduction pathways (i.e. AKT and ERK1/2) linked to activated growth factor receptors were studied. Activation of these pathways correlates with cell proliferation and tissue growth. Within uterine stromal cells, pAKT, activated by IGF-I (72) is proposed as an intracellular mediator of decidualization in human endometrium (72, 73). Whereas ERK signaling was not different in deciduomal and decidual tissues, the phosphorylated and thus active form of AKT (i.e. pAKT) was 2.3-fold more prevalent in the highly mitotic deciduoma. A target of pAKT activity and now well-established promoter of uterine stromal cell differentiation (40, 74), pFOXO1A, was also more abundant in deciduomal than decidual tissue. In its phosphorylated form, the FOXO1A transcription factor remains sequestered in the cytosol bound to 14–3-3 proteins and thus incapable of activating genes that promote differentiation and suppressing genes that promote proliferation (75). The increased presence of pFOXO1A correlates well with the increased proliferation observed in deciduomal tissue. Based on these cumulative findings, it is proposed that once initiated, uterine decidualization is a feed-forward process inherently controlled by maternal signals such as ovarian-derived steroid hormones and growth factors and that through paracrine signaling the embryo modulates this process to reduce stromal cell proliferation in favor of differentiation. This may occur due to limited resources at the implantation site in the absence of a fully developed vascular network. In a somewhat parasitic manner then, the embryo may actively curb endometrial growth to conserve nutrients and oxygen that is needed for its own survival.

Uterine NK cells are the most common leukocyte in the uterus, constituting as much as 30–40% of the total endometrial cell population in pregnant women and other species (18, 20). Several common NK cell markers were observed as being up-regulated in the endometrium of pregnancy, compared with deciduomal tissue. Histochemical staining with the NK cell marker DBA lectin revealed a 7.8-fold increase in decidual vs. deciduomal NK cell numbers. Because more NK cells were found in the decidualized endometrium of pregnancy, it is likely that the embryo regulates NK cell numbers. However, it is also recognized that sesame oil used to induce decidualization in pseudopregnant female mice could attenuate recruitment of NK cells to the uterus.

It is quite clear that mechanistic differences exist in embryo implantation between primate and rodent species. However, when compared with other studies, our results show that many similarities also exist, at least in terms of endometrial gene regulation. The malleability of mouse genetics can be used effectively to evaluate functional contributions of genes or processes that are conserved across species with different forms of implantation. For example, it will be important to further explore the functions of ISG15, FSTL3, EPCR, and HtrA1 in the decidualizing endometrium. Our study adds new information to what is currently known about maternal-embryonic interactions in at least two respects. First, the array approach used here is one that provides a genome-wide analysis of gene expression in differentiating uterine stromal tissue in the presence or the absence of an embryo. Second, information beyond comparative gene expression is used to support the hypothesis that the embryo takes a proactive role in certain aspects of maternal endometrial development during implantation. Future experiments should be designed to prioritize an understanding of conserved genes or pathways, four of which are described here. Studies of maternal-embryonic interactions will be beneficial to clinical sciences, in which diagnostic and therapeutic strategies can be developed to assist in reducing first-trimester pregnancy loss as well as basic science in which new gene functions (e.g. IFN-regulated genes or Procr) are likely to be discovered.


   Acknowledgments
 
We thank Petra Sergent for technical assistance.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants P50 HL54502 (to C.T.E.), HD032475 (to T.R.H.), and ES012070 (to J.K.P.) as well as Vincent Memorial Research Funds (to J.K.P.) and the Leducq International Network Against Thrombosis funded by The Leducq Foundation (to C.T.E.). C.T.E. is an investigator at the Howard Hughes Medical Institute. T.S. is supported by the Foundation of Genetic Profiling and Cancer. C.M.D. is recipient of the Dorothy Rackemann Fellowship for Scholarship in Medicine and the American Society for Reproductive Medicine/Organon USA, Inc. Research Grant in Reproductive Medicine.

Disclosure Summary: A.K., C.M.D., Y.K., Y.N., T.R.H., T.S., and J.K.P. have no conflict of interest related to this research. C.T.E. is an inventor on U.S. Patent 5,852,171 and receives royalties from Eli Lilly (U.S. Patent 5,009,889) and Baxter (U.S. Patent 5,202,253).

First Published Online May 17, 2007

1 A.K. and C.M.D. contributed equally to this study. Back

Abbreviations: BMP, Bone morphogenic protein; DBA, Dolichos biflorous; DOP, day of pregnancy; DOPP, day of pseudopregnancy; EPCR, endothelial protein C receptor; Foxo1a, forkhead box o1a; Iarp, IFN{alpha}-responsive protein; Ifi202b, IFN-activated gene 202B; IFN, interferon; Igfbp, insulin-like growth factor binding protein; Iigp1, IFN{gamma}-inducible GTPase; Irf-8, IFN regulatory factor-8; ISG, IFN-stimulated gene; Mmp, matrix metalloproteinase; NK, natural killer; PBST, Tween 20 in PBS; Procr, protein C receptor; Prss, protease serine member S; Timp, tissue inhibitor of matrix metalloproteinase.

Received February 27, 2007.

Accepted for publication May 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sauer M 1998 The impact of age on reproductive potential: lessons learned from oocyte donation. Marturitas 30:221–225[CrossRef]
  2. Sirard MA, Richard F, Blondin P, Robert C 2006 Contribution of the oocyte to embryo quality. Theriogenology 7:126–136
  3. Stephenson MD 1996 Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 66:24–29[Medline]
  4. Arrendondo F, Nobel LS 2006 Endocrinology of recurrent pregnancy loss. Semin Reprod Med 24:33–39[CrossRef][Medline]
  5. Christiansen OB, Andersen AMN, Bosch E, Daya S, Delves PJ, Hviid TV, Kutteh WH, Laird SM, Li TC, van der Ven K 2005 Evidence-based investigations and treatment of recurrent pregnancy loss. Fertil Steril 83:821–839[CrossRef][Medline]
  6. Inskeep EK, Dailey RA 2005 Embryonic death in cattle. Vet Clin North Am Food Anim Pract 21:437–461[CrossRef][Medline]
  7. Wang H, Dey SK 2006 Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 7:185–199[CrossRef][Medline]
  8. Schlafke S, Enders AC 1975 Cellular basis of interactions between trophoblast and uterus at implantation. Biol Reprod 12:41–65[CrossRef][Medline]
  9. Abrahamsohn PA, Zorn TM 1993 Implantation and decidualization in rodents. J Exp Zool 266:603–628[CrossRef][Medline]
  10. Finn CA, McLaren A 1967 A study of the early stages of implantation in mice. J Reprod Fertil 13:259–267[Abstract/Free Full Text]
  11. Soares M 2004 The prolactin and growth hormone families: pregnancy-specific hormones/cytokines at the maternal-fetal interface. Reprod Biol Endocrinol 2:51[CrossRef][Medline]
  12. Dimitriadis E, White CA, Jones RL, Salamonsen LA 2005 Cytokines, chemokines and growth factors in endometrium related to implantation. Hum Reprod Update 11:613–630[Abstract/Free Full Text]
  13. Yee GM, Squires PM, Cejic SS, Kennedy TG 1993 Lipid mediators of implantation and decidualization. J Lipid Mediat 6:525–534[Medline]
  14. Lala PK, Kearns M 1985 Immunobiology of the decidual tissue. Contrib Gynecol Obstet 14:1–15[Medline]
  15. Clark DA, Slapsys R, Croy BA, Krcek J, Rossant J 1984 Local active suppression by suppressor cells in the decidua: a review. Am J Reprod Immunol 5:78–83[Medline]
  16. Kirby DRS, Cowell TP 1968 Trophoblast-host interactions. In: Fleischmajer R, Billingham RE, eds. Epithelial-mesenchymal interactions. Baltimore: Williams and Wilkins; 64–77
  17. Zygmunt M, Herr F, Munstedt K, Lang U, Liang OD 2003 Angiogenesis and vasculogenesis in pregnancy. Eur J Obstet Gynecol Reprod Biol 110:S10–S18
  18. Croy BA, van den Heuvel MJ, Borzychowski AM, Tayade C 2006 Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones. Immunol Rev 214:161–185[CrossRef][Medline]
  19. Croy BA, Chantakru S, Esadeg S, Ashkar AA, Wei Q 2002 Decidual natural killer cells: key regulators of placental development (a review). J Reprod Immunol 57:151–168[CrossRef][Medline]
  20. King A, Jokhi PP, Burrows TD, Gardner L, Sharkey AM, Loke YW 1996 Functions of human decidual NK cells. Am J Reprod Immunol 35:258–260[Medline]
  21. Leonard S, Murrant C, Tayade C, van den Heuvel M, Watering R, Croy BA 2006 Mechanisms regulating immune cell contributions to spiral artery modification—facts and hypotheses—a review. Placenta 27:S40–S46
  22. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O 2006 Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 12:1065–1074[CrossRef][Medline]
  23. Ledford BE, Rankin JC, Markwald RR, Baggett B 1976 Biochemical and morphological changes following artificially stimulated decidualization in the mouse uterus. Biol Reprod 15:529–535[Abstract]
  24. Lockwood CJ, Krikun G, Hausknecht V, Wang EY, Schatz F 1997 Decidual cell regulation of hemostasis during implantation and menstruation. Ann NY Acad Sci 26:188–193
  25. Eisen MB, Spellman PT, Brown PO, Botstein D 1998 Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci 95:14863–14868[Abstract/Free Full Text]
  26. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M 2006 From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357
  27. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Metese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G 2000 Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet 25:25–29[CrossRef][Medline]
  28. Coser KR, Chesnes J, Hur J, Ray S, Isselbacher KJ, Shioda T 2003 Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray. Proc Nat Acad Sci USA 100:13994–13999[Abstract/Free Full Text]
  29. Pru JK, Lynch MP, Davis JS, Rueda BR 2003 Signaling mechanisms in tumor necrosis factor {alpha}-induced death of microvascular endothelial cells of the corpus luteum. Reprod Biol Endocrinol 1:17[CrossRef][Medline]
  30. Paffaro Jr VA, Bizinotto MC, Joazeiro PP, Yamada AT 2003 Subset classification of mouse uterine natural killer cells by DBA lectin reactivity. Placenta 24:479–488[CrossRef][Medline]
  31. Hansen TR, Pru JK, Han Y, Rempel LA, Austin KJ, Failure of uterine-conceptus interactions in cattle. J Reprod Dev, in press
  32. Hansen TR, Austin KJ, Johnson GA 1997 Transient ubiquitin cross-reactive protein gene expression in the bovine endometrium. Endocrinol 138:5079–5082[Abstract/Free Full Text]
  33. Hansen TR, Austin KJ, Perry DJ, Pru JK Teixeira MG, Johnson GA 1999 Mechanism of action of interferon-{tau} in the uterus during early pregnancy. J Reprod Fertil Suppl 54:329–339[Medline]
  34. Austin KJ, Bany BM, Belden EL, Rempel LA, Cross JC, Hansen TR 2003 Interferon-stimulated gene 15 (Isg15) expression is up-regulated in the mouse uterus in response to the implanting conceptus. Endocrinology 144:3107–3113[Abstract/Free Full Text]
  35. Paria BC, Ma W, Tan J, Raja S, Das SK, Dey SK, Hogan BLM 2001 Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors. Proc Nat Acad Sci USA 98:1047–1052[Abstract/Free Full Text]
  36. Schneyer A, Schoen A, Quigg A, Sidis Y 2003 Differential binding and neutralization of activins A and B by follistatin and follistatin like-3 (FSTL-3/FSRP/FLRG). Endocrinology 144:1671–1674[Abstract/Free Full Text]
  37. Nie G, Hale K, Li Y, Manuelpillai U, Wallace EM, Salamonsen LA 2006 Distinct expression and localization of serine protease HtrA1 in human endometrium and first-trimester placenta. Dev Dyn 235:3448–3455[CrossRef][Medline]
  38. Nie G, Li Y, Salamonsen LA 2005 Serine protease HtrA1 is developmentally regulated in trophoblast and uterine decidual cells during placental formation in the mouse. Dev Dyn 233:1102–1109[CrossRef][Medline]
  39. Chambard JC, Lefloch R, Pouyssegur J, Lenormand P, ERK implication in cell cycle regulation. Biochim Biophys Acta, in press
  40. Buzzio OL, Lu Z, Miller CD, Unterman TG, Kim JJ 2006 FOXO1A differentially regulates genes of decidualization. Endocrinol 147:3870–3876[Abstract/Free Full Text]
  41. Grinius L, Kessler C, Schroeder J, Handwerger S 2006 Forkhead transcription factor FOXO1A is critical for induction of human decidualization. J Endocrinol 189:179–187[Abstract/Free Full Text]
  42. Tan YF, Li FX, Piao YS, Sun XY, Wang YL 2003 Global gene profiling analysis of mouse uterus during the oestrous cycle. Reproduction 126:171–182[Abstract]
  43. Niklaus AL, Pollard JW 2006 Mining the mouse transcriptome of receptive endometrium reveals distinct molecular signatures for the luminal and glandular epithelium. Endocrinology 147:3375–3390[Abstract/Free Full Text]
  44. Yanaihara A, Otsuka Y, Iwasaki S, Koide K, Aida T, Okai T 2004 Comparison in gene expression of secretory human endometrium using laser microdissection. Reprod Biol Endocrinol 2:66[CrossRef][Medline]
  45. Carson DD, Lagow E, Thathiah A, Al-Shami R, Farach-Carson MC, Vernon M, Yuan L, Fritz MA, Lessey B 2002 Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod 8:871–879[Abstract/Free Full Text]
  46. Ace CI, Okulicz WC 2004 Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase. Reprod Biol Endocrinol 2:54[CrossRef][Medline]
  47. Hong SH, Nah HY, Lee JY, Gye MC, Kim CH, Kim MK 2004 Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection. J Endocrinol 181:157–167[Abstract]
  48. Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS 2003 Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol Endocrinol 17:2070–2083[Abstract/Free Full Text]
  49. Pan H, Zhu L, Deng Y, Pollard JW 2006 Microarray analysis of uterine epithelial gene expression during the implantation window in the mouse. Endocrinology 147:4904–4916[Abstract/Free Full Text]
  50. Jeong JW, Lee KY, Kwak I, White LD, Hilsenbeck SG, Lydon JP, DeMayo FJ 2005 Identification of murine uterine genes regulated in a ligand-dependent manner by the progesterone receptor. Endocrinology 146:3490–3505[Abstract/Free Full Text]
  51. Cheon YP, Li Q, Xu X, DeMayo FJ, Bagchi IC, Bagchi MK A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation. Mol Endocrinol 16:2853–2871
  52. Chen Y, Green JA, Antoniou E, Ealy AD, Mathialagan N, Walker AM, Avalle MP, Rosenfeld CS, Hearne LB, Roberts RM 2006 Effect of interferon-{tau} administration on endometrium of nonpregnant ewes: a comparison with pregnant ewes. Endocrinology 147:2127–2137[Abstract/Free Full Text]
  53. Kim S, Choi Y, Bazer FW, Spencer TE 2003 Identification of genes in the ovine endometrium regulated by interferon tau independent of signal transducer and activator of transcription 1. Endocrinology 144:5203–5214[Abstract/Free Full Text]
  54. Reese J, Das SK, Paria BC, Lim H, Song H, Matsumoto H, Knudtson KL, DuBois RN, Dey SK 2001 Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation. J Biol Chem 276:44137–44145[Abstract/Free Full Text]
  55. Yoshioka K, Matsuda F, Takakura K, Noda Y, Imakawa K, Sakai S 2000 Determination of genes involved in the process of implantation: application of GeneChip to scan 6500 genes. Biochem Biophys Res Commun 272:531–538[CrossRef][Medline]
  56. Bany BM, Cross JC 2006 Post-implantation mouse conceptuses produce paracrine signals that regulate the uterine endometrium undergoing decidualization. Dev Biol 294:445–456[CrossRef][Medline]
  57. Hess AP, Hamilton AE, Talbi S, Dosiou C, Nyegaard M, Nayak N, Genbecev-Krtolica O, Mavrogianis P, Ferrer K, Kruessel J, Fazleabas AT, Fisher SJ, Giudice LC 2007 Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators. Biol Reprod 76:102–117[Abstract/Free Full Text]
  58. Popovici RM, Betzler NK, Krause MS, Luo M, Jauckus J, Germeryer A, Bloethner S, Schlotterer A, Kumar R, Strowitzki T, von Wolff M 2006 Gene expression profiling of human endometrial-trophoblast interaction in a coculture model. Endocrinology 147:5662–5675[Abstract/Free Full Text]
  59. Crawley JT, Gu JM, Ferrell G, Esmon CT 2002 Distribution of endothelial cell protein C/activated protein C receptor (EPCR) during mouse embryo development. Thromb Haemost 88:259–266[Medline]
  60. Gu JM, Crawley JTB, Ferrell G, Zhang F, Li W, Esmon NL, Esmon CT 2002 Disruption of the endothelial cell protein C receptor gene in mice causes placental thrombosis and early embryonic lethality. J Biol Chem 277:43336–43343
  61. Lockwood CJ, Silver R 2004 Thrombophilias in pregnancy. In: Creasy RK, Resnik R, ed. Maternal-fetal medicine: principles and practices. Philadelphia: Elsevier Inc., USA; 1005–1021
  62. Delorme MA, Burrows RF, Ofosu FA, Andrew M 1992 Thrombin regulation in mother and fetus during pregnancy. Semin Thromb Hemost 18:81–90[Medline]
  63. Joyce MM, White FJ, Burghardt RC, Muniz JJ, Spencer TE, Bazer FW, Johnson GA 2005 Interferon stimulated gene 15 conjugates to endometrial cytosolic proteins and is expressed at the uterine-placental interface throughout pregnancy in sheep. Endocrinology 146:675–684[Abstract/Free Full Text]
  64. Bebington C, Bell SC, Doherty FJ, Fazleabas AT, Fleming SD 1999 Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy. Biol Reprod 60:920–928[Abstract/Free Full Text]
  65. Jones RL, Salamonsen LA, Findlay JK 2002 Activin A promotes human endometrial stromal cell decidualization in vitro. J Clin Endocrinol Metab 87:4001–4004[Abstract/Free Full Text]
  66. Chen D, Zhao M, Mundy GR 2004 Bone morphogenetic proteins. Growth Factors 22:233–241[CrossRef][Medline]
  67. Nie G, Li Y, Minoura H, Batten L, Ooi GT, Findlay JK, Salamonsen LA 2003 A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placenation. Mol Hum Reprod 9:279–290[Abstract/Free Full Text]
  68. Fazleabas AT, Kim JJ, Strakova Z 2004 Implantation: embryonic signals and the modulation of the uterine environment—a review. Placenta 25:S26–S32
  69. Jones RL, Findlay JK, Farnworth PG, Robertson DM, Wallace E, Salamonsen LA 2006 Activin A and inhibin A differentially regulate human uterine matrix metalloproteinases: potential interactions during decidualization and trophoblast invasion. Endocrinology 147:724–732[Abstract/Free Full Text]
  70. Sidis Y, Mukherjee A, Keutmann H, Delbaere A, Sadatsuki M, Schneyer A 2006 Biological activity of follistatin isoforms and follistatin-like-3 is dependent on differential cell surface binding and specificity for activin, myostatin, and bone morphogenetic proteins. Endocrinology 147:3586–3597[Abstract/Free Full Text]
  71. Okamoto A, Endo H, Kalionis B, Shinya M, Saito M, Nikaido T, Tanaka T 2006 IGFBP1 and follistatin-like 3 genes are significantly up-regulated in expression profiles of the IUGR placenta. Placenta 27:317–321[CrossRef][Medline]
  72. Toyofuku A, Hara T, Taguchi T, Katsura Y, Ohama K, Kudo Y 2006 Cyclic and characteristic expression of phosphorylated Akt in human endometrium and decidual cells in vivo and in vitro. Hum Reprod 21:1122–1128[Abstract/Free Full Text]
  73. Yoshino O, Osuga Y, Hirota Y, Koga K, Yano T, Tsutsumi O, Taketani Y 2003 Akt as a possible intracellular mediator for decidualization in human endometrial stromal cells. Mol Hum Reprod 9:265–269[Abstract/Free Full Text]
  74. Labied S, Kajihara T, Madureira PA, Fusi L, Jones MC, Higham JM, Varshochi R, Francis JM, Zoumpoulidou G, Essafi A, Fernandez de Mattos S, Lam EW, Brosens JJ 2006 Progestins regulate the expression and activity of the forkhead transcription factor FOXO1 in differentiating human endometrium. Mol Endocrinol 20:35–44[Abstract/Free Full Text]
  75. Zhao X, Gan L, Pan H, Kan D, Majeski M, Adam SA, Unterman TG 2004 Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14–3-3-dependent and -independent mechanisms. Biochem J 378:839–849[CrossRef][Medline]



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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals