| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Molecular and Integrative Physiology (M.K.B.), Department of Veterinary Biosciences (Y.-P.C., Q.L., I.C.B.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Department of Molecular and Cellular Biology (X.X., F.J.D.), Baylor College of Medicine, Houston, Texas 77030
Address all corresponence and requests for reprints to: Dr. Milan Bagchi, Department of Molecular and Integrative Physiology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 534 Burrill Hall, MC-114, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: mbagchi{at}life.uiuc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The cellular actions of P are mediated through two PR isoforms, PR-A and PR-B, which are hormone-inducible transcription factors (7, 8). In a P-responsive cell, the hormone binds to its intracellular receptor and triggers its gene regulatory function (5). The hormone receptor complex interacts with specific cellular target genes to modulate their expression (5). It is likely that P triggers the expression of a network of genes in the endometrium during early stages of pregnancy, and these eventually lead to the synthesis of new proteins, which prepare the uterus for establishment and maintenance of gestation. Although P profoundly influences various stages of uterine physiology, such as implantation and decidualization, previous studies in rodents have identified only a few genes that are under P regulation in the uterus. These include the genes encoding the growth factor amphiregulin, the homeobox proteins Hoxa-10 and Hoxa-11, peptide hormones calcitonin and proenkephalin, and the enzyme histidine decarboxylase (4, 9, 10, 11, 12, 13). Although targeted mutation in mice indicated that Hoxa-10 is essential for implantation (14), the roles of the other known P-regulated genes during this process are less clear.
To understand how P regulates implantation, it is essential to identify a broader spectrum of genes that are regulated by PR during this process. To achieve this goal, we employed RU486, a well-characterized antagonist of PR function during pregnancy. RU486 counteracts PR-dependent pathways by binding to the receptor and impairing its gene regulatory function (15, 16). We used oligonucleotide microarrays to identify the genes whose uterine expression is markedly altered at the time of implantation by RU486 complexed PR. In this paper, we report the identification and characterization of an array of novel PR-regulated gene pathways in the preimplantation mouse uterus.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
To examine global changes in uterine mRNA expression profiles in response to RU486, mice on d 3 of pregnancy were treated with either vehicle (sesame oil) or RU486 (8 mg/kg body weight) and uterine tissues were collected on d 4, the day of implantation. The uteri were freed of embryos by repeated flushing. Total RNA was isolated from these uteri and polyadenylated RNA was prepared. The RNA was used to create cDNA, which was then employed as a template to generate a biotinylated RNA probe by in vitro transcription. The transcripts were hybridized to high-density oligonucleotide arrays (Murine GeneChip Expression arrays, Affymetrix, Santa Clara, CA). The microarray analysis used probe arrays that contained oligonucleotides corresponding to approximately 6000 known mouse genes and many unnamed expressed sequence tags (ESTs) (Fig. 1
). The arrays were washed and stained with fluorescent streptavidin conjugates, and the fluorescent signal within each probe was analyzed as described in the Materials and Methods section. To generate reproducible gene expression data, three independent replicates of the control-test pair were performed. We applied a threshold of a 2-fold change in expression level between RU486-treated samples and untreated controls for identifying putative P-regulated mRNAs. Applying this cut off, we identified a total of 148 mRNAs corresponding to known genes whose expression altered significantly in the uterus at the time of implantation in response to RU486. Seventy-eight known genes were down-regulated, and 70 known genes were up-regulated in all three experiments (Table 1). Genes with altered expression were categorized based on their known biological function. Although many EST tags also showed significant alteration in expression in response to RU486, they were not analyzed in this study.
|
|
|
, therefore, represent potential novel targets of P action in the uterus. We found that the putative P-regulated genes included those encoding transcription factors, cell adhesion molecules, peptide hormones, growth factors, proteases, protease inhibitors, metabolic enzymes, signal transduction molecules, cytoskeleton proteins, and molecules involved in immune function and angiogenesis (Table 1). Consistent with its complex physiological effects during pregnancy, P appears to influence a diverse range of cellular pathways and functions.
Confirmation of RU486 Down-Regulation of Microarray-Derived Genes by Northern Blotting
To verify the results of microarray analysis by an independent method, we performed Northern blotting using randomly selected cDNAs representative of different categories of genes (shown in Table 1) as probes. In Fig. 2, we describe the Northern blot analysis using total RNA obtained from uteri of d 4 pregnant mice treated with or without RU486. When the blot was probed with seven different 32P-labeled cDNAs, in each case a signal of marked intensity was observed on d 4 of pregnancy in the absence of RU486 (lane 1), whereas none or much weaker mRNA signal was observed in the presence of RU486 (lane 2). Hybridization of the same blot with a control probe [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] indicated equal loading of mRNAs in these lanes. The RU486 down-regulation was also confirmed for three additional target genes: CTLA2ß, tissue plasminogen activator and follistatin (data not shown). Therefore, in all ten cases tested so far, Northern blots confirmed mRNA alterations predicted by the microarray analysis.
|
, transcripts of p12, L-12/15 lipoxygenase, fisp 12, calcyclin, proenkephalin, osteoblast-specific transcription factor 2 (Osf2), and immune-responsive gene 1 (Irg1) were barely detectable in the uteri of ovariectomized mice upon treatment with vehicle alone. The levels of these transcripts were markedly elevated after treatment with P, confirming that this hormone is a critical regulator of these genes.
|
). These results indicated that P-bound PR indeed positively regulated the selected RU486-down-regulated genes.
|
, these genes displayed two distinct time-courses of induction in response to P. While an early induction pattern was observed for Irg1, L-12/15 lipoxygenase, calcyclin, and Osf2 mRNAs, a late induction pattern was noted for those encoding p12 and fisp 12. A significant increase in the level of Irg1, L-12/15 lipoxygenase, calcyclin, and Osf2 mRNAs was detected within 4 h of P treatment. While the levels of Irg1 and L-12/15 lipoxygenase mRNAs did not alter significantly at 8 and 24 h, those of calcyclin and Osf2 declined at 24 h. The levels of p12 and fisp 12 mRNAs did not significantly increase at 4 or 8 h of P treatment but were markedly elevated at 24 h. Based on these results, we speculate that Irg1, L-12/15 lipoxygenase, calcyclin, and Osf2, which are induced early, are primary targets of regulation by P-bound PR. In contrast, p12 and fisp 12, which are induced late, are likely to be downstream targets of the early hormone-inducible genes.
|
. One pattern, displayed by four genes: L-12/15 lipoxygenase, fisp 12, Osf2, and Irg1, consisted of a transient burst of mRNA expression in the uterus immediately preceding implantation. These transcripts were undetectable on d 1–3 of pregnancy. Their levels rose and attained a peak on d 4, and then started to decline by d 5 of pregnancy. The profile of Osf2, which is a representative of this group, is shown in Fig. 6, (top, left and right panels).
|
, middle, left and right panels). The mRNA signal first appeared on d 4, immediately after embryo attachment. Its intensity progressively increased with days of gestation, reaching a maximal expression on d 5–8. The expression dramatically declined around d 10 of pregnancy. Interestingly, this profile precisely overlapped with the time frame of stromal cell differentiation (decidualization) and trophoblast invasion during pregnancy.
Still another pattern of expression was displayed by the proenkephalin mRNA (Fig. 6, bottom, left and right panels). This mRNA was induced around d 3–4 of gestation, reached peak expression by d 5–6, and remained high beyond d 11 (data not shown). We have not monitored its expression during the second half of the pregnancy.
Spatial Expression of PR and its Target Genes in Mouse Uterus during Early Pregnancy
We next investigated the cell type-specific expression of P-regulated mRNAs in mouse uterus during early pregnancy. Because P mediates its gene regulatory activity via its nuclear receptors, it is of interest to compare the expression of PR in different uterine cell types with that of its target genes.
We first monitored the expression of PR in the pregnant uterus by immunohistochemistry. Sections of pregnant mouse uteri at different days of gestation were incubated with PR antiserum or control serum. As shown in Fig. 7, the uteri of d 2 pregnant mice exhibited low levels of PR-specific staining in the epithelial and stromal cells (panel A). A dramatic increase in the level of PR was noted in the uterine luminal epithelium of d 3 pregnant mice (panel B). On the d 4 of pregnancy, the level of PR in the luminal epithelial cells decreased slightly, but a marked increase in the level of stromal PR was observed (panel C). Whereas a high level of PR continued to express in the stromal cells during the postimplantation period, the level of PR in the luminal epithelial cells declined sharply (panels D–F, respectively). These results, consistent with those of a similar study published previously (19), indicated that PR is expressed in a stage- and cell-specific manner in the uterus during early pregnancy.
|
Our studies revealed distinct patterns of spatial expression of P-regulated genes in mouse uterus during early pregnancy. The mRNAs corresponding to Osf2 and L-12/15-lipoxygenase were transiently expressed in the preimplantation phase uterus. These mRNAs were predominantly localized in the luminal epithelium between d 3 and 5 of pregnancy (Fig. 8, A and B, respectively). Glandular and some stromal staining of L-12/15-lipoxygenase was also observed upon closer examination of the images. Interestingly, the expression of these genes closely followed that of epithelial PR during the preimplantation phase (Fig. 7).
|
). This mRNA was undetectable between d 1 and 3 of pregnancy. Its expression was first detected in the stromal cells surrounding the embryo on d 5 of gestation. The expression of calcyclin mRNA increased dramatically on d 6 (Fig. 8C). The mRNA declined to undetectable levels by d 10 of gestation with the cessation of the decidual phase of pregnancy (data not shown). Taken together, these results indicated that discrete subsets of P-regulated genes identified by the oligonucleotide microarray show distinct cell type-specific expression patterns. Most importantly, the spatio-temporal expression of these genes closely followed that of PR in various uterine compartments during pregnancy.
Genes Up-Regulated by RU486: Evidence for a Complex Interplay of E and P to Control Uterine Gene Expression
We identified by microarray analysis, a surprisingly large number of genes (70 of a total of 148 known genes) whose expression in the pregnant uterus was enhanced by RU486 (Table 2). The RU486 upregulated genes included extracellular matrix/cell adhesion molecules, metabolic enzymes, signal transduction molecules immune modulators, transcription factors, cell surface receptors, and cytoskeletal/structural proteins. To the best of our knowledge, none of these genes except Muc-1 (20) was previously reported to be regulated by RU486 in the pregnant uterus.
|
|
by Northern blotting (Fig. 9). The genes, lactotransferrin, Muc-1, carbonic anhydrase II, and epithelial zinc finger protein, showed minimal expression in the pregnant uterus on d 4. Administration of RU486 led to a marked increase in the expression of each of these genes, confirming the findings of the microarray analysis.
|
). These results indicated that E-complexed ER rather than a partial agonistic activity of RU486-complexed PR is likely responsible for the observed enhancement of uterine expression of these selected genes. Most interestingly, administration of P together with E suppressed their E-induced expression.
|
, a significant induction of all the four genes was observed in the uteri of d 4 pregnant animals upon administration of RU486 (compare lanes 1 and 2). This RU486-induced induction was, however, dramatically reduced in the presence of ICI 182780, presumably due to the inactivation of transcriptional activity of ER (compare lanes 2 and 4). These results are consistent with the view that hormone-occupied PR represses E- and ER-regulated expression of these genes in normal pregnant uterus. Binding of RU486 to PR releases this repression, allowing ER-induced gene expression to take place. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
. It is likely that many of the listed genes are downstream targets of primary PR-responsive genes. Previously known P-regulated genes, such as amphiregulin, Hoxa 11, proenkephalin, and histidine decarboxylase, have appeared in our analysis, supporting the validity of the genomic approach to identify the P-regulated genes during implantation (9, 11, 12, 13). In addition, previous reports indicated that several microarray-derived genes, such as follistatin, c-myc, IGF binding protein-3 (IBFBP-3), and endothelin receptor type B, are expressed in the periimplantation uterus (21, 22, 23, 24). It is pertinent to mention that two previously reported P-regulated genes, Hoxa 10 and calcitonin (CT), are absent from the panel of mouse genes identified by the microarray analysis. CT is induced in response to P in the pregnant rat uterus (11, 25) and also present in the receptive human uterus (26). Its absence from the microarray profile is, however, not surprising in light of our previous finding that only low levels of CT mRNA is detectable in pregnant mouse uterus (27).
The analysis of spatio-temporal expression of several microarray-derived genes revealed multiple distinct patterns of gene expression. Most interesting among these are the expression of clusters of genes overlapping the implantation (d 3–5) and decidual (d 5–10) phases. We are tempted to speculate that the genes that are induced by PR in the epithelium within the preimplantation window of receptivity (d 3–5) are critical regulators of embryo-uterine interactions during implantation. Some of these gene products may also act as signals to induce decidualization of stroma. The PR-regulated genes that are expressed during d 5–10 in the stromal compartment might be involved in various functional aspects of the decidualization process. The abundant expression of PR in the stromal cells may directly control the expression of certain of these genes. It is also likely that PR-regulated gene products synthesized in the stromal cells may act in a paracrine manner within the microenvironment of the uterine tissue to regulate gene expression in other cell-types.
The microarray-based analysis provides, for the first time, a comprehensive description of PR-regulated gene networks that are expressed during implantation. We have identified a variety of novel PR regulated molecules, such as growth factors, protease inhibitors, metabolic enzymes, peptide hormones, transcription factors, immune response molecules, cytoskeletal proteins, and cell adhesion molecules, which may play important roles in the uterus during implantation. The current challenge is to link these molecules and their pathways to previously well-characterized morphological, physiological, and biochemical events that are associated with the process of implantation. We provide below a brief discussion of the plausible roles that some of these molecules might play in mediating P effects in the pregnant uterus.
It is known that during the preimplantation period, a rise in the P level suppresses the E-regulated proliferation of epithelial cells (3). P simultaneously promotes differentiation of epithelial cells and induces proliferation of stromal cells. This sequential triggering of P-controlled differentiation and proliferation of different uterine cell-types is essential for acquisition of receptivity (28). It is, therefore, not surprising that a number of growth factors and cytokines (amphiregulin, fisp 12, Hoxa 11, c-myc, c-kit), growth factor/cytokine receptors (platelet-derived growth factor-
receptor, IL-1 receptor), and an IGF binding protein-3, are under the influence of P in the uterus at the time of implantation (Table 1). Prominent among these novel and potentially important targets of PR is fisp 12, a member of the family of connective tissue growth factors (29). Recent studies have indicated that besides cell proliferation, fisp 12 is involved in a variety of processes such as cell adhesion, extracellular matrix formation, and angiogenesis (29, 30).
Our initial studies also confirmed calcyclin, Osf2, p12, and CTLA2ß as novel targets of P-regulation in the pregnant uterus. Interestingly, calcyclin, which is a small calcium-binding protein, was previously detected in the natural killer cells within the mouse decidua (31). Although its precise function during pregnancy remains unclear, it has been implicated in paracrine regulation of the trophoblast. Addition of calcyclin stimulated placental lactogen secretion from isolated trophoblast cells in vitro (31). Osf2 was originally isolated as a transcriptional activator of osteoblast differentiation (32). During development, its expression is restricted to cells of the mesenchymal condensations and of the osteoblast lineage, and is regulated by calciotropic agents. Our finding that it is expressed in the uterine epithelium of pregnant mouse is intriguing and the physiological significance of this expression is unclear. p12, a serine protease inhibitor, was first discovered as an androgen-regulated 6-kDa protein expressed primarily in ventral prostrate and seminal vesicle (33, 34). It exhibits extensive sequence homology at the amino acid level with members of the Kazal family of secretory serine protease inhibitors, and appears to be the mouse homolog of human pancreatic secretory trypsin inhibitor. CTLA2ß, a cysteine protease inhibitor, is secreted from T lymphocytes and mast cells (35). Initial studies indicate that both protease inhibitors are expressed in the maternal decidua (data not shown). It is possible that they might control invasion by trophoblast-derived proteases.
P-regulated molecules may control cell shape, motility, and adhesion during implantation. During early stages of implantation, the embryonic trophectoderm cells become closely apposed to the luminal epithelium. In mammals, especially the rodent, a generalized stromal edema occurs before the beginning of apposition (36). This event leads to the closure of the uterine lumen, which results in interdigitation of microvilli of the trophectoderm and luminal epithelia (37). A P-induced change in the profiles of various cyoskeletal proteins such as procollagens, tetranectin, chondroitin sulfate proteoglycan 2, and several different types of laminins (Tables 1 and 2) in pregnant uterus, could play an important role at this stage.
Adhesion molecules expressed at the surface of the luminal epithelial cell also could potentially facilitate the implantation process. It is noteworthy that the P markedly induced the expression of Irg1, a gene harboring motifs for glycosaminoglycan attachment site (Ref. 38 , Table 1, and Fig. 3). Preliminary studies indicated that Irg1 mRNAs appear in the luminal epithelial cells precisely on d 4 of pregnancy, coinciding with the adhesive phase of the uterus (Cheon, Y.-P., and I. C. Bagchi, unpublished results). It is also interesting that treatment with RU486 led to a robust induction of the mRNA for the Muc-1 mucin (Table 2 and Fig. 9). This result is consistent with a previous report indicating similar induction of Muc-1 in pregnant mouse uterus upon RU486 treatment (20). It has been proposed that Muc-1 expression impairs access of the blastocyst to the surface of uterine epithelia. In the P-dominated uterus, repression of Muc-1 expression at the time of implantation may increase apical access and promote embryo attachment (4).
Previous studies have shown that, during the preimplantation phase in mice, the oxygen consumption by the uterine tissue increases and glucose incorporation reaches a peak at the time of implantation (39). Consistent with this paradigm, uterine expression of various metabolic enzymes, such as pyruvate carboxylase, PEPCK, peptidylarginine deiminase, and carbonic anhydrase, are altered in response to RU486 at the time of implantation (Tables 1 and 2). We find it interesting that the lipid metabolizing enzymes, leukocyte- and epidermal-12/15 lipoxygenases, which are involved in oxidative metabolism of arachidonic and linoleic acids are induced by P at the time of implantation. These enzymes are known to generate metabolites, such as hydroxy-eicosatetraenoic acids and hydroxy-octadecadienoic acids, which serve as cell differentiation signals (40).
Our study identified two peptide hormones, follistatin and proenkephalin, as P-regulated endocrine signals in the uterus. Both of these hormones were previously reported to be present in the periimplantation uterus (12, 21). It has been speculated that follistatin, which is predominantly expressed in the decidual tissue after embryo attachment, may act by modulating maternal FSH secretion during early pregnancy (21). It is, however, possible that follistatin has an as-yet undiscovered paracrine role in the pregnant uterus. The P-regulated expression of proenkephalin was reported to increase dramatically in the mouse endometrium at the onset of implantation and continued during gestation (Ref. 12 and Fig. 6). Interestingly, enkephalin appears to be involved in regulating peristalsis of the intestines and in inhibiting contractions of the vas deferens (41). A similar role for enkephalins in controlling muscle contractility during implantation is conceivable. This scenario is particularly attractive because P is known for its role in inhibition of uterine contractility and maintenance of tranquil environment during gestation.
We have also identified an array of genes that are up-regulated in the preimplantation uterus in response to RU486 (Table 2). Although RU486, as an antiprogestin, is expected to inhibit the expression of genes that are positively regulated by PR, relatively little is known about the molecules that are up-regulated in the pregnant uterus in response to this drug. These genes may represent molecules that are negatively regulated by P-occupied PR in the pregnant uterus. Alternatively, they may represent genes that are positively regulated by RU486-occupied PR, which manifests its partial agonist activity under certain physiological scenarios (42, 43, 44). Our analysis of four randomly selected genes, lactotransferrin, Muc-1, carbonic anhydrase II, and epithelial zinc finger protein, indicated that RU486-dependent stimulation involves reversal of P-mediated repression of their expression. Interestingly, E stimulated the expression of these RU486-inducible genes in the uteri of ovariectomized mice, whereas simultaneous administration of E and P reversed this effect (Fig. 10A). Moreover, RU486 failed to induce the expression of these genes in the presence of the antiestrogen ICI 182780 (Fig. 10B). Collectively, these findings are consistent with the hypothesis that hormone-occupied PR represses the expression of a set of E-regulated genes during pregnancy. This interpretation is consistent with previous reports that P represses E-induced uterine expressions of Muc1 and lactotransferrin (20, 45, 46). Additionally, studies in PRKO mice indicated that P suppresses E-induced uterine hyperplasia (6). In the presence of RU486, this P-regulated repression is released, leading to the expression of E-inducible genes. Although the existence of an inhibitory cross-talk between the P- and E-dependent pathways in steroid-responsive tissues, such as breast and uterus, is well documented, the mechanism underlying this phenomenon is not entirely clear. A competition between hormone-occupied PR and ER for a limited pool of cellular coactivators has been suggested as a plausible mechanism for this cross-talk (47). We must caution, however, that based on the limited number of RU486-up-regulated genes that we have analyzed so far, it would be premature to conclude that this scenario is valid for the majority of genes in this category. Nevertheless, our study strengthens the view that a complex interaction between E- and P-regulated pathways within the uterus controls the expression of a set of genes, which might be critical for maintenance of pregnancy.
Recently, three other studies also employed DNA microarrays to identify molecules regulating the events during early pregnancy (48, 49, 50). Among these studies, those by Reese et al. (48) and Yashioka et al. (49) sought to identify molecules involved during implantation in mice. The study by Kao et al. (50) compared gene expression profiles of secretory and proliferative human endometrium during the menstrual cycle.
Yoshioka et al. (49) identified genes with differential expression between preimplantation (d 3.5) and postimplantation (d 5.0) stages. They found that the expression of 192 genes increased and that of 207 genes decreased as the pregnant uterus made the transition from the preimplantation to the postimplantation phase. It is reasonable to assume that the genes with increased expression on the postimplantation d 5 represent mostly decidual rather than implantation stage-specific genes. This approach contrasts with our study, which focuses on the P-regulated genes precisely at the time (d 4) of implantation. It is, however, important to note that several genes, whose expression was detected in the preimplantation uterus and declined markedly in the postimplantation phase, also appeared in our screen as RU486-down-regulated genes (Table 1). These genes encoded amphiregulin, fisp 12, Irg1, epidermal-12/15-lipoxygenase, cathepsin D, pyruvate carboxylase, follistatin, Rho B, and laminin-22 chain.
Reese et al. (48) compared gene expression profiles between implantation and interimplantation sites on d 4 of pregnancy. They reported 36 up-regulated and 27 down-regulated genes at the implantation site. The implanting blastocysts were, however, present in the uterine samples analyzed by these workers. Whereas the interimplantation sites were devoid of blastocysts, the implantation sites included them. It is, therefore, likely that a substantial number of genes that are differentially expressed at the implantation sites are of embryonic origin. In contrast, the gene expression profile in our study, which used embryo-free uteri, is likely to be solely of uterine origin. In spite of this obvious difference, a total of eight genes, which showed altered expression at the implantation sites in the study by Reese et al. (48), also showed differential expression in response to RU486 in our analysis. While genes encoding follistatin, spermidine synthase, snail homolog, IGF binding protein-3, cathepsin F, and endothelin receptor type B, were down-regulated by RU486, those encoding CCAAT/enhancer binding protein ß and Lisch7, were up-regulated by this drug (Tables 1 and 2).
Reese et al. (48) also compared the uterine gene expression profile of P-treated, delayed-implanting mice to that of mice in which delayed implantation was terminated by E. They identified 128 up-regulated and 101 down-regulated transcripts upon termination of delayed implantation by E. We noted that seven genes (endothelin receptor type B, Ia-associated invariant chain, pyruvate carboxylase, MHC class II H2-I-A ß, cathepsin F, Aquaporin 1, membrane metallo-endopeptidase, which were down- regulated in response to E during delayed implantation, were also down-regulated in response to RU486 in normal pregnant uterus (Ref. 49 and Table 1). Additionally, two genes (small proline-rich protein 2A and spermidine synthase), which showed increased expression during E-induced implantation, were up-regulated by RU486 in our screen (Ref. 49 and Table 2). Although our strategy to identify implantation-specific genes is quite different from that employed by Reese et al. (48), it is interesting that these studies generated a limited but significant amount of mutually overlapping information.
Kao et al. (50) used high-density oligonucleotide microarrays containing 12,686 gene probes to identify genes that were differentially expressed in mid secretory vs. late proliferative stage human endometrial biopsies. This study reported significant up-regulation of 156 genes and down-regulation of 377 genes within the putative window of implantation. Despite the species difference and the associated difficulties in identifying the homologous genes, we noted that several P-regulated genes (listed in Table 1) such as c-myc, apolipoprotein E, prostaglandin E2 receptor, metallothionein I, and pyruvate carboxylase, were also up-regulated in the human endometrium during the window of implantation.
In conclusion, we have identified a broad diversity of gene networks, which are potentially regulated by PR in the uterus during implantation. Further analysis of hormonal regulation, spatio-temporal expression, and function of these diverse molecules will provide valuable insights into the molecular pathways underlying the complex physiological effects of P during this process.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Animals and Tissue Collection
All experiments involving animals were conducted in accordance with the NIH standards for the use and care of animals. Female mice (CD-1 from Charles River Laboratories, Inc., Wilmington, MA), in proestrus, were mated with adult males. The presence of a vaginal plug after mating was designated as d 1 of pregnancy. To examine changes in uterine mRNA expression profiles in response to RU486, mice on d 3 of pregnancy (1600 h) were injected with either vehicle (sesame oil) (n = 6) or RU486 (n = 6) (8 mg/kg body weight). The injections were repeated after 16 h and the mice were killed (8 h after the second injection) on d 4 (1600 h) to collect the uteri. The uteri were freed of embryos by repeated flushing as described previously (11, 25). The tissues were then flash frozen and stored at -80 C.
The PRKO mice were bred and homozygotes were confirmed by genotyping as described previously (6).
Preparation of RNA for GeneChip Analysis
In RU486-treated or untreated group, embryo-free uterine tissues from six female mice were pooled to isolate RNA. For microarray analysis, RNA samples were processed after the Affymetrix protocol. Poly (A)+ RNA was isolated from the tissue samples using Oligotex mRNA isolation kit (QIAGEN, Valencia, CA), after the manufacturer’s instructions. The purity of isolated mRNAs was evaluated spectrophotometrically, using the A260/A280 ratio. The RNA was then subjected to cDNA synthesis using a T7-(deoxythymidine)24 primer and the Superscript Choice System (Life Technologies, Inc., Gaithersburg, MD). The resulting cDNA was then used to synthesize biotin-labeled cRNA by in vitro transcription employing the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY). The cRNA was then further purified by RNeasy spin columns (QIAGEN) and subjected to chemical fragmentation in a buffer containing 40 mM Tris (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate. The quality of each cRNA preparation was assessed by hybridization with a Test array (Affymetrix) before they were hybridized to murine Affymetrix GeneChips.
GeneChip Hybridization and Data Analysis
The two cRNA samples, control and test, were hybridized to oligonucleotide arrays corresponding to approximately 6000 known mouse genes and many unnamed expressed sequence tags (ESTs) (Fig. 1). To generate reproducible gene expression data, three independent replicates of the control-test pair were performed. The arrays were washed and stained with fluorescent streptavidin conjugates. The fluorescent signal within each probe was captured by a laser confocal scanner and the changes in the gene expression levels were analyzed with GeneChip Analysis Suite 4.0 (Affymetrix) software. Each gene is represented by 16–20 probe pairs of oligonucleotide probes and each probe pair consists of one 25 oligomer that is a perfect complement to the RNA (a perfect match probe) and a companion oligonucleotide that carries a single base difference in a central position (a mismatch probe). The mismatch probes serve as internal controls for hybridization specificity. Differences in levels of fluorescence intensity between the probe pairs (a perfect match and a mismatch) 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. The mean values were then calculated for each probe-set and a difference of 2-fold was applied to select up-regulated and down-regulated genes. For each gene, the fold change in expression is represented by the mean value of the three replicate scores. The expression profile data were then exported to GeneSpring 4.0 for further analysis. The GeneSpring software allows rank-sum normalization and statistical analysis.
Ovariectomy and Hormone Treatments
Female mice were subjected bilateral ovariectomy and, 2 wk later, were injected sc with either E (2 µg/kg body weight), P (40 mg/kg body weight), or a combination of both hormones or vehicle (sesame oil) as described previously (11, 25). The animals were killed 16 h after final injection.
Northern Blot and RT-PCR Analysis
Northern blot analysis was performed as described previously (11, 25). RT-PCRs were performed using a Stratascript RT-PCR kit as described previously (26). The conditions for PCR were 94 C, 30 sec; 1 cycle followed by 94 C, 30 sec; 65 C, 30 sec; and 68 C, 2 min; 25 cycles. PCR products were then subjected to agarose gel electrophoresis.
In Situ Hybridization
Uterine tissues from pregnant animals were collected and frozen. Tissues were fixed in 4% paraformaldehyde at 4 C. Cryostat sections were cut at 8 µm and attached to 3-amino-propyl triethyl silane (Sigma) coated slides. In situ hybridization was performed with digoxygenin (DIG)-labeled sense or antisense RNA probes complementary to Osf2, 12/15 lipoxygenase, and calcyclin cDNAs. DIG-labeled RNA probes were synthesized from the cDNAs using T3 or T7 RNA polymerase and DIG-labeled nucleotides according to manufacturer’s specifications (Roche Molecular Biochemicals, Indianapolis, IN). Prehybridization was carried out in a damp chamber at 55 C for 60 min in hybridization buffer (50% formamide, 5x sodium chloride-sodium citrate (SSC), 2% blocking reagent, 0.02% sodium dodecyl sulfate, 0.1% N-laurylsarcosine). Hybridization was carried out at 55 C overnight in a damp humidified chamber. To develop the substrate, sections were sequentially washed in 2x SSC, 1x SSC, and 0.1x SSC for 15 min in each buffer at 37 C. Sections were then incubated with anti-DIG alkaline phosphatase conjugated antibody. Excess antibody was washed away and the color substrate (nitroblue tetrazolium salt and 5-bromo-4-chloro-3indoylphosphate) was added. Slides were allowed to develop in the dark, and the color was visualized under light microscopy until maximum levels of staining were achieved. The reaction was stopped and the slides counterstained in Nuclear Fast Red for 5 min. The slides were washed in water, dehydrated, and coverslipped. Control incubations used a DIG-labeled RNA sense strand and were performed under identical conditions.
Immunohistochemistry
Polyclonal antibody against human progesterone receptor (DAKO Corp.) was diluted 1:1000 for immunohistochemistry. Paraffin-embedded uterine tissues were sectioned at 4 µm and mounted on slides. Sections were washed in PBS for 20 min and then incubated in a blocking solution containing 10% normal goat serum for 10 min before incubation in primary antibody overnight at 4 C. Immunostaining was performed using Avidin-Biotin kit for rabbit primary antibody (Vector Laboratories, Inc., Burlingame, CA) and the diaminobenzidine chromogen. Sections were counterstained with hematoxylin, mounted, and examined under bright field. Red deposits indicate the sites of immunostaining.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: CT, Calcitonin; DIG, digoxygenin; E, estrogen; EST, expressed sequence tags; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Irg, immune-responsive gene; Osf2, osteoblast-specific transcription factor 2; P, progesterone; PR, progesterone receptor; PRKO, PR knockout; SSC, sodium chloride-sodium citrate.
Received for publication August 2, 2002. Accepted for publication September 13, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
v ß 3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19:2958–2966NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  J. Kim, I. C. Bagchi, and M. K. Bagchi Signaling by Hypoxia-Inducible Factors Is Critical for Ovulation In Mice Endocrinology, July 1, 2009; 150(7): 3392 - 3400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kondoh, T. Okamoto, T. Higuchi, K. Tatsumi, T. Baba, S. K. Murphy, K. Takakura, I. Konishi, and S. Fujii Stress affects uterine receptivity through an ovarian-independent pathway Hum. Reprod., April 1, 2009; 24(4): 945 - 953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Cloke, K. Huhtinen, L. Fusi, T. Kajihara, M. Yliheikkila, K.-K. Ho, G. Teklenburg, S. Lavery, M. C. Jones, G. Trew, et al. The Androgen and Progesterone Receptors Regulate Distinct Gene Networks and Cellular Functions in Decidualizing Endometrium Endocrinology, September 1, 2008; 149(9): 4462 - 4474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Hull, C. R. Escareno, J. M. Godsland, J. R. Doig, C. M. Johnson, S. C. Phillips, S. K. Smith, S. Tavare, C. G. Print, and D. S. Charnock-Jones Endometrial-Peritoneal Interactions during Endometriotic Lesion Establishment Am. J. Pathol., September 1, 2008; 173(3): 700 - 715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-J. Hu, G. Ren, J.-L. Liu, Z.-A. Zhao, Y.-S. Yu, R.-W. Su, X.-H. Ma, H. Ni, W. Lei, and Z.-M. Yang MicroRNA Expression and Regulation in Mouse Uterus during Embryo Implantation J. Biol. Chem., August 22, 2008; 283(34): 23473 - 23484. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. G Lea and O. Sandra Immunoendocrine aspects of endometrial function and implantation Reproduction, September 1, 2007; 134(3): 389 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.D. Catalano, H.O. Critchley, O. Heikinheimo, D.T. Baird, D. Hapangama, J.R.A. Sherwin, D.S. Charnock-Jones, S.K. Smith, and A.M. Sharkey Mifepristone induced progesterone withdrawal reveals novel regulatory pathways in human endometrium Mol. Hum. Reprod., September 1, 2007; 13(9): 641 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ka, S. Al-Ramadan, D. W. Erikson, G. A. Johnson, R. C. Burghardt, T. E. Spencer, L. A. Jaeger, and F. W. Bazer Regulation of Expression of Fibroblast Growth Factor 7 in the Pig Uterus by Progesterone and Estradiol Biol Reprod, July 1, 2007; 77(1): 172 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.A. Horcajadas, A. Pellicer, and C. Simon Wide genomic analysis of human endometrial receptivity: new times, new opportunities Hum. Reprod. Update, January 1, 2007; 13(1): 77 - 86. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Palanisamy, Y.-P. Cheon, J. Kim, A. Kannan, Q. Li, M. Sato, S. R. Mantena, R. L. Sitruk-Ware, M. K. Bagchi, and I. C. Bagchi A Novel Pathway Involving Progesterone Receptor, Endothelin-2, and Endothelin Receptor B Controls Ovulation in Mice Mol. Endocrinol., November 1, 2006; 20(11): 2784 - 2795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Pan, L. Zhu, Y. Deng, and J. W. Pollard Microarray Analysis of Uterine Epithelial Gene Expression during the Implantation Window in the Mouse Endocrinology, October 1, 2006; 147(10): 4904 - 4916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E.A. Campbell, L. O'Hara, R.D. Catalano, A.M. Sharkey, T.C. Freeman, and M. H. Johnson Temporal expression profiling of the uterine luminal epithelium of the pseudo-pregnant mouse suggests receptivity to the fertilized egg is associated with complex transcriptional changes Hum. Reprod., October 1, 2006; 21(10): 2495 - 2513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Pan, Y. Deng, and J. W. Pollard Progesterone blocks estrogen-induced DNA synthesis through the inhibition of replication licensing PNAS, September 19, 2006; 103(38): 14021 - 14026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mukherjee, S. M. Soyal, R. Fernandez-Valdivia, M. Gehin, P. Chambon, F. J. DeMayo, J. P. Lydon, and B. W. O'Malley Steroid Receptor Coactivator 2 Is Critical for Progesterone-Dependent Uterine Function and Mammary Morphogenesis in the Mouse. Mol. Cell. Biol., September 1, 2006; 26(17): 6571 - 6583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J-W Jeong, K Y Lee, J P Lydon, and F J DeMayo Steroid hormone regulation of Clca3 expression in the murine uterus. J. Endocrinol., June 1, 2006; 189(3): 473 - 484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Li, M. K. Bagchi, and I. C. Bagchi Identification of a Signaling Pathway Involving Progesterone Receptor, Calcitonin, and Tissue Tranglutaminase in Ishikawa Endometrial Cells Endocrinology, May 1, 2006; 147(5): 2147 - 2154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Pooley, C. S. Healey, P. L. Smith, P. D.P. Pharoah, D. Thompson, L. Tee, J. West, C. Jordan, D. F. Easton, B. A.J. Ponder, et al. Association of the progesterone receptor gene with breast cancer risk: a single-nucleotide polymorphism tagging approach. Cancer Epidemiol. Biomarkers Prev., April 1, 2006; 15(4): 675 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Talbi, A. E. Hamilton, K. C. Vo, S. Tulac, M. T. Overgaard, C. Dosiou, N. Le Shay, C. N. Nezhat, R. Kempson, B. A. Lessey, et al. Molecular Phenotyping of Human Endometrium Distinguishes Menstrual Cycle Phases and Underlying Biological Processes in Normo-Ovulatory Women Endocrinology, March 1, 2006; 147(3): 1097 - 1121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Mantena, A. Kannan, Y.-P. Cheon, Q. Li, P. F. Johnson, I. C. Bagchi, and M. K. Bagchi C/EBPbeta is a critical mediator of steroid hormone-regulated cell proliferation and differentiation in the uterine epithelium and stroma PNAS, February 7, 2006; 103(6): 1870 - 1875. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Rios, S. V. Koushik, H. Wang, J. Wang, H.-M. Zhou, A. Lindsley, R. Rogers, Z. Chen, M. Maeda, A. Kruzynska-Frejtag, et al. periostin Null Mice Exhibit Dwarfism, Incisor Enamel Defects, and an Early-Onset Periodontal Disease-Like Phenotype Mol. Cell. Biol., December 15, 2005; 25(24): 11131 - 11144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-W. Jeong, K. Y. Lee, I. Kwak, L. D. White, S. G. Hilsenbeck, J. P. Lydon, and F. J. DeMayo Identification of Murine Uterine Genes Regulated in a Ligand-Dependent Manner by the Progesterone Receptor Endocrinology, August 1, 2005; 146(8): 3490 - 3505. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A White and L. A Salamonsen A guide to issues in microarray analysis: application to endometrial biology Reproduction, July 1, 2005; 130(1): 1 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Catalano, M. H. Johnson, E. A. Campbell, D. S. Charnock-Jones, S. K. Smith, and A. M. Sharkey Inhibition of Stat3 activation in the endometrium prevents implantation: A nonsteroidal approach to contraception PNAS, June 14, 2005; 102(24): 8585 - 8590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Bauersachs, S E Ulbrich, K Gross, S E M Schmidt, H H D Meyer, R Einspanier, H Wenigerkind, M Vermehren, H Blum, F Sinowatz, et al. Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes J. Mol. Endocrinol., June 1, 2005; 34(3): 889 - 908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Chabbert-Buffet, G. Meduri, P. Bouchard, and I. M. Spitz Selective progesterone receptor modulators and progesterone antagonists: mechanisms of action and clinical applications Hum. Reprod. Update, May 1, 2005; 11(3): 293 - 307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. A. Sherwin, T. C. Freeman, R. J. Stephens, S. Kimber, A. G. Smith, I. Chambers, S. K. Smith, and A. M. Sharkey Identification of Genes Regulated by Leukemia-Inhibitory Factor in the Mouse Uterus at the Time of Implantation Mol. Endocrinol., September 1, 2004; 18(9): 2185 - 2195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
Q. Li, Y.-P. Cheon, A. Kannan, S. Shanker, I. C. Bagchi, and M. K. Bagchi A Novel Pathway Involving Progesterone Receptor, 12/15-Lipoxygenase-derived Eicosanoids, and Peroxisome Proliferator-activated Receptor {gamma} Regulates Implantation in Mice J. Biol. Chem., March 19, 2004; 279(12): 11570 - 11581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-P. Cheon, F. J. DeMayo, M. K. Bagchi, and I. C. Bagchi Induction of Cytotoxic T-lymphocyte Antigen-2{beta}, A Cysteine Protease Inhibitor in Decidua: A POTENTIAL REGULATOR OF EMBRYO IMPLANTATION J. Biol. Chem., March 12, 2004; 279(11): 10357 - 10363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-P. Cheon, X. Xu, M. K. Bagchi, and I. C. Bagchi Immune-Responsive Gene 1 Is a Novel Target of Progesterone Receptor and Plays a Critical Role during Implantation in the Mouse Endocrinology, December 1, 2003; 144(12): 5623 - 5630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Chen, D. Zhang, and J. W. Pollard Progesterone Regulation of the Mammalian Ortholog of Methylcitrate Dehydratase (Immune Response Gene 1) in the Uterine Epithelium during Implantation through the Protein Kinase C Pathway Mol. Endocrinol., November 1, 2003; 17(11): 2340 - 2354. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
