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College of Life Sciences, Northeast Agricultural University, Harbin 150030, China
(Requests for offprints should be addressed to Z-M Yang; Email: zmyang{at}neau.edu.cn)
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Abstract |
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-glutamyl hydrolase, Ras homolog gene family, member U, T-cell immunoglobulin, and mucin domain containing 2, proline–serine–threonine phosphatase-interacting protein 2, 3-monooxgenase/tryptophan 5-monooxgenase activation protein, -polypeptide, and cysteine-rich protein 61 (Cyr61) were upregulated in the luminal epithelium at implantation site on day 5 of pregnancy. These genes may be related to embryo apposition and adhesion during embryo implantation. Cyr61, a gene upregulated at the implantation site, was chosen to examine its expression and regulation during the periimplantation period by in situ hybridization. Cyr61 mRNA was specifically localized in the luminal epithelium surrounding the implanting blastocyst at day 4 midnight and on day 5 of pregnancy, and in the activated uterus, but not expressed in inter-implantation sites and under delayed implantation, suggesting a role for Cyr61 in mediating embryonic–uterine dialog during embryo attachment. Our data could be a valuable source for future study on embryo implantation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Depending on the species, trophoblast penetration of the endometrium may remain superficial or continue deep into the endometrium. Nonetheless, all the implantation processes between species involve the interaction between the blastocyst trophectoderm and the apical surface of the uterine luminal epithelium (Carson et al. 2000). Normally, the apical surface of the luminal epithelium is non-adhesive. However, the uterine transition from pre-receptive to receptive requires fundamental structural and functional changes in epithelial cell organization (Kimber & Spanswick 2000), allowing for successful blastocyst attachment.
Implantation is a complex process involving spatio-temporally regulated endocrine, paracrine, autocrine, and juxtacrine modulators that span cell–cell and cell–matrix interactions. The successful implantation of an embryo depends upon cellular and molecular dialog between the uterus and the embryo (Dey et al. 2004). To date, although many specific factors have been identified during the implantation period, the molecular mechanism of embryo implantation is still unknown. Oligonucleotide microarray analysis has shed insight into a wide range of developmental, oncological, and pharmacological processes by allowing the simultaneous and quantitative assessment of expression levels of thousands of genes (Bethin et al. 2003). Recently, microarray analysis has been used to investigate temporal and spatial gene expression profiles in the mouse uterus during the implantation period (Yoshioka et al. 2000, Reese et al. 2001). However, whole uterine fragments were used in these studies. In the uterus, the luminal epithelium represents about 5–10%, the stroma 30–35%, and the myometrium 60–65% of the major uterine cell types (Finn & Porter 1975). It is difficult to characterize the specific gene expression in the luminal epithelium because of the interference from large amounts of uterine stroma and myometrium. Yoon et al.(2004) used laser capture microdissection (LCM) to isolate the luminal epithelium at implantation and inter-implantation sites for microarray analysis. Although luminal epithelium could be collected accurately by LCM, the mRNA count of luminal epithelium collected from frozen sections is very limited and needs to be amplified several times prior to microarray analysis. Because of the sensitivity in mRNA amplification, artificial errors could be a problem during data analysis. In this study, we used enzymatic digestion to isolate luminal epithelium from the mouse uterus at implantation and inter-implantation sites, of over 100 mice. The luminal epithelium obtained by this method was directly used for mRNA extraction and microarray analysis without any amplification. Differential gene expression profile in uterine luminal epithelium at implantation site was performed using a mouse microarray containing 12 345 unigenes. Microarray data were confirmed by both reverse transcription (RT)-PCR and in situ hybridization.
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Materials and methods |
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Mature mice (Kunming White outbred strain) were caged in a controlled environment with a ratio of 14 h light:10 h darkness cycle. All animal procedures were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Adult females were mated with fertile or vasectomized males of the same strain to induce pregnancy or pseudopregnancy respectively (day 1 = day of vaginal plug). Pregnancy on days 1–4 was confirmed by recovering embryos from the reproductive tracts. The implantation sites on the day 4 midnight (2400 h) and on day 5 were identified by i.v. injection of 0.1 ml 1% Chicago blue (Sigma) in 0.85% sodium chloride.
To induce delayed implantation, pregnant mice were ovariectomized under ether anesthesia between 0830 and 0900 h on day 4 of pregnancy. Progesterone (1 mg/mouse) was injected to maintain delayed implantation, from days 5 to 7. Estradiol-17ß (25 ng/mouse) was given to progesterone-primed delayed-implantation mice to terminate delayed implantation. The mice were sacrificed to collect uteri 24 h after estrogen treatment. Implantation sites were identified by i.v. injection of Chicago blue solution. Delayed implantation was confirmed by flushing the blastocysts from one horn of the uterus.
Hormonal treatments were initiated 2 weeks after mature female mice were ovariectomized. Controls received 0.1 ml of sesame oil per mouse. For hormonal treatments, the ovariectomized mice were treated with an injection of estradiol-17ß (100 ng/mouse; Sigma), progesterone (1 mg/mouse; Sigma), or a combination of the same doses of progesterone and estradiol-17ß for 24 h. All steroids were dissolved in sesame oil and injected subcutaneously.
Isolation of uterine luminal epithelium
Pregnant mice on day 5 (0800–0830 h) of pregnancy were used to collect implantation and inter-implantation sites by tail vein injection of Chicago sky blue. For microarray analysis, the uterine fragments with the strongest blue staining on day 5 of pregnancy were collected for implantation sites, and the blue dye-free areas between implantation sites for inter-implantation sites.
Luminal epithelium of mouse uterus was isolated as previously described (Bigsby et al. 1986, Tan et al. 2004) and slightly modified. Uteri at implantation and inter-implantation sites were split longitudinally and incubated in 5 ml 1% (w/v) trypsin and 6 mg/ml dispase in HBSS solution (Gibco) at 4 °C for 1 h, 20 °C for 1 h, and 37 °C for 10 min respectively. After brief shaking, luminal epithelial cells were collected from the supernatants with a mouth-controlled pipette under a stereomicroscope. After enzymatic digestion, implanted blastocysts were found in the digestion solution. To avoid the contamination of glandular epithelium, detached blastocysts, and suspended cells, only the pieces in a single layer were collected. In order to remove any attached uterine stromal cells, the supernatants were centrifuged for 5 min at 200 g. Then the cell pellets were resuspended in HBSS and spun twice. Finally, the pellets containing uterine luminal epithelial cells were pooled for RNA extraction with TRIZOL reagent (Sigma).
To examine the purity of isolated uterine luminal epithelial cells, an aliquot of cell pellets prior to RNA extraction was resuspended in DMEM/F12 medium (1:1; Sigma) with 10% (v/v) fetal bovine serum (Gibco) and plated on a 35 mm culture dish. After the cells reached around 80% confluence, they were washed three times in PBS and fixed in cold methanol for 10 min. The cells were blocked with 1% (w/v) BSA in PBS for 1 h and incubated in goat anti-cytokeratin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4 °C overnight. After washing three times in PBS, the cells were incubated with biotinylated rabbit anti-goat IgG at 37 °C for 30 min and straptavidin conjugated with alkaline phosphatase for 30 min, followed by three washes in PBS. The cells were then incubated in Vector Red substrate (Vector Laboratories, Burlingame, CA, USA). Positive reaction products were indicated by red color. Based on cytokeratin immunostaining, the purity of the uterine luminal epithelial cells was approximately 99%.
Microarray analysis
Uterine luminal epithelium was isolated from implantation and inter-implantation sites as described above respectively. More than 100 mice were used for this study. After uterine luminal epithelium were isolated and extracted for RNA with TRIZOL reagent, reverse transcription, probe labeling, and hybridization were performed according to the procedures from Biostar Biotech, Inc. (Shanghai, China). Briefly, RNA samples extracted with TRIZOL reagent were labeled with Cy3 or Cy5 by direct incorporation of Cy3-dUTP or Cy5-dUTP in the reverse transcription reaction, i.e. Cy3 for inter-implantation sites and Cy5 for implantation sites. Labeled samples from implantation and inter-implantation sites were co-hybridized with Genechip BiostarM-141s containing 12 345 unigenes with defined functions (Biostar Biotech, Inc.). After hybridization at 42 °C for 16 h, the genechip was washed and air-dried. The fluorescent intensities of Cy3 and Cy5 were scanned with ScanArray 4000 (Packard Bioscience, Billerica, MA, USA) and analyzed with GenePix Pro 3.0 software (Axon Instruments, Foster City, CA, USA). The genes having Cy5:Cy3 ratio within 0.1–10 were chosen for further analysis. In order to eliminate the systematic variation, the Cy3 value of each gene was normalized using a normalization factor (0.976), which was the mean value of log2(Cy5 intensity/Cy3 intensity). The ratio of each gene expression was calculated as Cy5 intensity over normalized Cy3 intensity. The criterion of twofold difference was used to choose the up- and downregulated genes for the following analysis as described previously (Yang et al. 2002).
RT-PCR
RNA was extracted from isolated luminal epithelium of both implantation and inter-implantation sites, using TRIZOL reagent. RNA samples were then treated with RQ1 DNase I (Promega) to remove any genomic DNA contamination, extracted with phenol/chloroform, precipitated, and dissolved in formamide at a final concentration of 1 µg/µl and stored at –70 °C.
Total RNAs were reverse-transcribed in a reaction volume of 20 µl containing 22 units of RT and 2.5 µM oligo-dT using a BcaBEST RNA PCR kit (Takara Biotechniques, Dalian, China). The intensity of amplified bands was scanned with UVP Laboratory Imaging and Analysis System (UVP, Inc., Upland, CA, USA). Primers, annealing temperatures, and expected product sizes for these genes are listed in Table 1
. Band densities for each gene were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) expression and the ratios determined. The amplification cycle number of each gene was chosen based on our previous tests to ensure that the amplification was terminated during the exponent phase. RT-PCR for each gene was repeated on, at least, three different samples. The RT-PCR product of each gene was verified by sequencing. Statistical analysis was performed with SPSS software (SPSS Inc., Chicago, IL, USA).
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Total RNAs from pregnant mouse uteri were reverse-transcribed and amplified with the corresponding primers for each gene listed in Table 1
. The amplified fragment of each gene was recovered from agarose gel and cloned into pGEM-T plasmid (pGEM-T Vector System 1l; Promega). The cloned fragments were further verified by sequencing. The recombinant plasmid was amplified individually with the primers for T7 and SP6 to prepare templates for labeling sense or antisense probe respectively. Digoxigenin (DIG)-labeled antisense or sense cRNA probe was transcribed in vitro using a DIG RNA labeling kit (Roche).
Uteri were cut into 4–6 mm pieces and flash frozen in liquid nitrogen. Frozen sections (10 µm) were mounted on 3-aminopropyltriethoxy silane (Sigma)-coated slides and fixed in 4% paraformaldehyde solution in PBS. The sections were washed twice in PBS, treated in 1% Triton-100 for 20 min, and again washed three times in PBS. Following pre-hybridization in the 50% forma-mide/5x SSC solution (1x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) at room temperature for 15 min, the sections were hybridized as previously described (Ni et al. 2002). Endogenous alkaline phosphatase activity was inhibited with 2 mM levami-sole (Sigma). All the sections were counter-stained with 1% methyl green. The positive signal was visualized as a dark brown color. In situ hybridization for each gene was repeated at least three times with the uteri from three different animals at each stage.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In order to examine the differential gene expression profile in uterine luminal epithelium on day 5 of pregnancy in the mouse, luminal epithelium was isolated from uterine stroma, glandular epithelium, myometrium, and serosa by enzymatic digestion on day 5 of pregnancy at implantation or inter-implantation sites respectively. Figure 1 showed the structure of mouse uterus on day 5 of pregnancy at implantation site stained with hematoxylin and eosin, showing that the blastocyst was already attached on the luminal epithelium, which was still intact at the implantation site. During enzymatic digestion, the implanting blastocysts were detached from luminal epithelium, since the blastocysts were already found in the digestion solution. Additionally, a single layer of luminal epithelium was collected with a mouth-controlled pipette under a stereomicroscope to avoid the contamination of blastocysts, stromal cells, and glandular epithelium during the enzymatic digestion.
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and 3 respectively. Among the upregulated genes, 24 were upregulated by at least threefold, with protease, serine, 12 neurotrypsin (Prss12) being the most dramatically upregulated gene (6.75-fold). Prss12, also called motopsin, was highly expressed in the mouse brain (Iijima et al. 1999). Only 27 genes were downregulated by at least threefold. The highest change among downregulated genes with known function was N-acetylneuraminate pyruvate lyase (Npl) by 5.26-fold.
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Confirmation by RT-PCR
To verify the data from microarray analysis, we randomly chose seven upregulated and six down-regulated genes to examine their expression patterns by semi-quantitative RT-PCR. After the expression of each gene was normalized to Gapdh expression, the ratio between implantation and inter-implantation sites showed similar expression trends from both RT-PCR and microarray analysis, but the expression ratio between them did not completely match between RT-PCR and microarray analysis (Fig. 2). The ratio from microarray analysis was slightly higher than that from RT-PCR except for Npl, which was decreased by 5.26-fold, in microarray, but 6.66 in RT-PCR.
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In order to further verify the microarray data, seven upregulated genes were randomly chosen to examine their expression on day 5 of pregnancy and pseudo-pregnancy by in situ hybridization. Compared with the inter-implantation site on the day of pregnancy and the uterus on day 5 of pseudopregnancy, all of these seven genes were upregulated in the luminal epithelium at implantation site on day 5 of pregnancy (Fig. 3). However, their expression patterns were slightly different. Prss12 was strongly expressed in the glandular epithelium and the luminal epithelium near the blastocyst (Fig. 3A). Endothelin-1 (Edn1) is a 21-amino acid vasoconstrictor peptide originally isolated from endothelial cells (Rubanyi & Polakoff 1994). Edn1 was highly detected in the luminal epithelium surrounding the implanting blastocyst (Fig. 3D). -Glutamyl hydrolase (Ggh), a lysosomal endopeptidase, was identified as a biomarker for pulmonary neuroendocrine tumors (He et al. 2004). Ggh was strongly expressed in all the luminal epithelium except for that surrounding the implanting blastocyst (Fig. 3G). A low level of Ggh expression was also seen in the luminal epithelium at inter-implantation site (Fig. 3H), but not in day 5 pseudopregnant uterus (Fig. 3I). Ras homolog gene family member U (Rhou; Santin et al. 2005) was detected in both luminal epithelium and subluminal stromal cells on day 5 of pregnancy at implantation site (Fig. 3J), but not at inter-implantation site on day 5 of pregnancy (Fig. 3K), and on day 5 of pseudopregnancy (Fig. 3L). T-cell immunoglobulin and mucin domain containing 2 (Timd2) is critical for the regulation of Th2 responses during autoimmune inflammation (Chakravarti et al. 2005). Timd2 was detected mainly in the luminal epithelium surrounding the implanting blastocyst (Fig. 3M). Proline–serine–threonine phosphatase-interacting protein 2 (Pstpip2) regulates F-actin bundling, and enhances filopodia formation and motility in macrophages (Chitu et al. 2005). A low level of Pstpip2 expression was observed in all the luminal epithelium at implantation site (Fig. 3P). 3-Monooxgenase/tryptophan 5-monooxgenase-activation protein, -polypeptide (Ywhag), also called 14-3-3 , is a member of 14-3-3 family (Takahashi 2003). Ywhag expression was strongly detected in the luminal epithelium surrounding the implanting blastocyst and weakly in the subluminal stromal cells (Fig. 3S).
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In our microarray analysis, Cyr61 was upregulated by 3.77 at the implantation site. Cyr61 is also known as CCN1 (CCN is the initial of Cyr61, connective tissue growth factor (Ctgf), and nephroblastoma-overexpressed, NOV) and belongs to the CCN family (Perbal 2001). Because Cyr61 was highly expressed in human endometrium and upregulated in the endometria of women with endometriosis (Absenger et al. 2004, Punyadeera et al. 2005), Cyr61 was then chosen to verify the expression pattern and to examine its expression during early pregnancy, pseudopregnancy, delayed implantation, and steroid hormonal treatments by in situ hybridization. There was no detectable Cyr61 expression in the uterus from days 1 to 3 of pregnancy (data not shown). Cyr61 expression was not detected in the mouse uterus on the morning of day 4 of pregnancy (Fig. 4A), whereas it was observed in the luminal epithelium immediately surrounding the blastocyst on day 4 midnight when the attachment reaction was just initiated (Fig. 4B). On day 5 of pregnancy, when embryo implantation occurred, Cyr61 mRNA signals were seen only in the luminal epithelium of mouse uterus at the implantation site (Fig. 4C), but not at the inter-implantation site (Fig. 4D). Furthermore, Cyr61 signals were not detected in the mouse uterus on day 5 of pseudopregnancy (Fig. 4E) or during delayed implantation (Fig. 4G). After delayed implantation was terminated by estrogen treatment and the embryos implanted, Cyr61 mRNA expression was once again observed in the luminal epithelium (Fig. 4H), expression similar to that observed at the implantation sites on day 5 of pregnancy. After Cyr61 anti-sense probe was replaced with Cyr61 sense probe, there was no detectable signal in the uterus on day 5 of pregnancy (Fig. 4F). Additionally, there was no detectable Cyr61 mRNA signal in the uterus of ovariectomized mice. After ovariectomized mice were treated with either estrogen or progesterone for 24 h, Cyr61 mRNA expression was not seen in the uterus (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Because the uterus at implantation sites was used in previous microarray studies (Yoshioka et al. 2000, Reese et al. 2001), it was difficult to compare our data with theirs. In the study by Yoon et al.(2004), the luminal epithelium isolated by LCM was used for microarray analysis, and only three identical upregulated genes were detected both by us and by Yoon et al.(2004). Compared with inter-implantation sites, Ggh, inhibitor of DNA-binding 3, and secreted acidic cysteine-rich glycoprotein (Sparc) were increased by 2.44-, 3.33-, and 3.08-folds in our study, but by 1.63-, 1.89-, and 1.48-folds, by Yoon et al.(2004) respectively. Except for these three genes, other up- or downregulated genes were not shared. The main reason for these differences could be due to the methods for isolating luminal epithelium. We isolated the whole luminal epithelium at implantation or inter-implantation sites by enzymatic digestion, whereas Yoon et al.(2004) collected only the luminal epithelium immediately surrounding the implanting blastocyst. The luminal epithelium immediately surrounding the implanting blastocyst differed greatly from the rest of luminal epithelium in its gene expression. For example, cyclooxygenase-2 and basigin were specifically expressed in the luminal epithelium surrounding the implanting blastocyst (Lim et al. 1997, Xiao et al. 2002).
Upregulated genes
In this study, 136 genes were upregulated by at least twofold, of which 24 were upregulated by at least threefold. Prss12 was the highest one upregulated by 6.75-fold and was strongly expressed in the glandular epithelium and the luminal epithelium near the blastocyst at implantation site on day 5 of pregnancy, but not detected in the inter-implantation site on day 5 of pregnancy, and in the day 5 pseudopregnant uterus. It seems that Prss12 in the luminal epithelium was specifically induced by implanting blastocyst. Prss12, also called motopsin, was highly expressed in mouse brain (Iijima et al. 1999). However, the expression, regulation, and function of this gene in early pregnancy still remain unknown.
Edn1 and Ywhag were strongly expressed in the luminal epithelium surrounding the implanting blastocyst, suggesting a role during embryo apposition and attachment. Edn1 stimulates neutrophil adhesion to endothelial cells by an effect on the expression of adhesive molecules on the neutrophil surface. Edn1 also stimulates neutrophil accumulation in vivo and in vitro in the heart (Lopez Farre et al. 1993). Edn1 induces leukocyte rolling and adherence through a predominantly endothelin receptor A-mediated mechanism in the submucosal venules of the intestinal microcirculation (Boros et al. 1998). Human decidual cells in early pregnancy can synthesize and release EDN1. These cells also possess specific functional receptors for EDN1, which are coupled to phosphoinositide hydrolysis, suggesting a possible role for EDN1 in autocrine and/or paracrine function in human decidual cells (Kubota et al. 1992). Ywhag, also called 14-3-3 , is a member of the 14-3-3 family. The 14-3-3 family is a large group of highly conserved 30 kDa acidic proteins (Takahashi 2003). 14-3-3 proteins participate in integrin-activated signaling pathways through their interaction with docking protein p130 (Cas), which may contribute to important biological responses regulated by cell adhesion to the extracellular matrix (Garcia-Guzman et al. 1999). 14-3-3 
can regulate cell adhesion and spreading through the interaction with ADAM22 (Zhu et al. 2005). Whether Ywhag is involved in cell adhesion still remains unknown.
Timd2 was also highly expressed in the luminal epithelium surrounding the implanting blastocyst. However, there are no papers that mention its role in cell adhesion. The T-cell Ig and mucin domain-containing gene locus has been linked to differences in T(h)2 responsiveness and asthma susceptibility in mice (Encinas et al. 2005). Timd2 is expressed preferentially in differentiated Th2 cells and is critical for the regulation of Th2 responses during auto-immune inflammation (Chakravarti et al. 2005). In normal pregnancy, particularly at the maternal–fetal interface, anti-inflammatory, Th-2, cytokines predominate (Wegmann et al. 1993). An appropriate balance between pro- and anti-inflammatory cytokines is thought to be crucial for determining the success or failure of a pregnancy (Formby 1995).
Rhou belongs to Ras homolog gene family. Ras homolog gene family, member I (ARHI) was down-regulated in uterine serous papillary cancer compared with normal endometrial cells (Santin et al. 2005). Ras homolog gene family plays a central role in a wide range of physiological processes, including cell morphology, cell adhesion, cytokinesis, cell motility, and cell growth (Van Aelst & D’Souza-Schorey 1997, Hall 1998). However, the expression and function of Rhou during early pregnancy still remain to be determined.
Pstpip2, also called macrophage actin-associated tyrosine-phosphorylated protein (MAYP), directly regulates F-actin bundling and enhances filopodia formation and motility in macrophages. Filopodia are postulated to increase directional motility by acting as environmental sensors. The MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation (Chitu et al. 2005). In our study, Pstpip2 was expressed in all luminal epithelium at the implantation site, but not in the inter-implantation site on day 5 of pregnancy, and in the uterus on day 5 of pseudopregnancy, suggesting that Pstpip2 may be involved in embryo apposition and attachment.
Downregulated genes
In our microarray, 223 genes were downregulated by at least twofold in the luminal epithelium at implantation site, of which nine were decreased by over threefold, including N-acetylneuraminate pyruvate lyase (5.26-fold), calbindin-28K (4.57-fold), brain acyl-CoA hydrolase (4.35-fold), glutathione S-transferase µ 1 (4.10-fold), solute carrier family 6 (3.85-fold), myeloid differentiation primary response gene 88 (3.61-fold), interleukin 1 receptor type 1 (3.38-fold), neuronal differentiation related protein (3.14-fold), and peripheral myelin protein 22 (3.08-fold). Although N-acetylneuraminate pyruvate lyase was greatly reduced at implantation sites, the expression and regulation of this gene is still unknown.
Calbindin-28K was decreased by 4.57-fold at implantation sites in our study. A previous study showed that calbindin-28K protein was increased in the endometrial epithelium just before implantation, but disappeared at implantation sites after attachment (Luu et al. 2004). Because calbindin-28K is able to inhibit apoptosis in osteoblastic cells (Bellido et al. 2000), the apoptosis caused by the downregulation of calbindin-28K could destabilize the luminal epithelium at the implantation site and facilitate trophoblast invasion (Luu et al. 2004). However, calbindin-28K-deficient mice do not show implantation failure (Airaksinen et al. 1997). Calbindin-9K mRNA was also specifically downregulated at the implantation site (Nie et al. 2000). Although calbindin-28K-deficient mice are fertile, embryo implantation is blocked if both calbindin-9K and calbindin-28K are inhibited just before implantation (Luu et al. 2004).
A role for Cyr61 in mediating embryo attachment at the implantation site
Cyr61 is also known as CCN1 (CCN is the initial of Cyr61, Ctgf, and NOV) and belongs to the CCN family. Other members of the CCN family include CCN2 (Ctgf), CCN3 (NOV), and the Wnt-inducible secreted proteins CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3, Perbal 2001). Cyr61 overexpression in the undifferentiated AN3CA endometrial cancer cell line led to decreased cell growth and increased apoptosis in liquid culture. Moreover, the increased apoptosis in these endometrial cancer cells with Cyr61 over expression was associated with elevated expression of the pro-apoptotic proteins Bax, Bad, and tumor necrosis factor receptor-associated ligand (Chien et al. 2004). In the mouse, uterine epithelial cells surrounding the implanted embryo will undergo apoptotic cell death on day 5 evening or day 6 of pregnancy (Parr et al. 1987, Joswig et al. 2003). It is possible that the Cyr61 expression might be closely related to subsequent apoptosis in the luminal epithelium.
We found that Cyr61 was upregulated by 3.77-fold in the luminal epithelium at implantation sites. Because Cyr61 was specifically induced in the luminal epithelium at the implantation site by the activated blastocyst, it may play a key role during embryo attachment. Cyr61 and Ctgf supported the attachment of endothelial cells through integrin 
vß3 (Kireeva et al. 1998) and cell surface heparan sulfate proteoglycans (Chen et al. 2000, 2004). Integrin vß3 was expressed in the apical pole of the uterine luminal epithelium and on the epithelial surface of blastocyst (Aplin et al. 1996, Lessey et al. 1996). Nevertheless, the normal Mendelian ratio of Cyr61 mutant embryos at E9.5 suggests that Cyr61 is not critical for implantation (Mo et al. 2002). It is possible that Cyr61 function may be compensated by other CCN members. CTGF shared many common functions with Cyr61, including promoting endothelial cell growth, migration, adhesion, and survival (Brigstock et al. 2002). Moreover, CTGF was also expressed in the luminal epithelium at mouse implantation site (Surveyor et al. 1998). In the Ptgs2 null CD-1 mice, Ptgs1 is upregulated and can maintain mouse implantation although cyclooxygenase (COX)-2 is essential for mouse implantation (Wang et al. 2004). However, whether CTGF is upregulated in Cyr61 null mice remains to be determined.
In human endometrium, Cyr61 was more highly expressed during the proliferative than during the secretory phase in endometrium (Punyadeera et al. 2005). Cyr61 was one of the most upregulated genes in endometria of women with endometriosis and in ectopic endometrium (Absenger et al. 2004). Basic fibroblast growth factor, epidermal growth factor, tumor necrosis factor , and interleukin-1 could stimulate Cyr61 expression (Schutze et al. 1998). Furthermore, Cyr61 could upregulate the synthesis of vascular endothelial growth factor (VEGF)-A and VEGF-C to regulate the processes of angiogenesis, inflammation, and matrix remodeling in the context of cutaneous wound healing (Chen et al. 2001, Brigstock 2002). Interestingly, Cyr61 mutation led to impaired Vegf-C expression in the allantoic mesoderm, suggesting that Cyr61-regulated expression of Vegf-C plays a role in vessel bifurcation (Mo et al. 2002). VEGF was strongly expressed in mouse uterus during implantation period and played a key role in regulating vascular permeability and angiogenesis (Chakraborty et al. 1995, Halder et al. 2000). Increased vascular permeability and angiogenesis were crucial to successful implantation, decidualization, and placentation (Dey et al. 2004).
In conclusion, there are 136 genes upregulated and 223 genes downregulated in the luminal epithelium at the implantation site of mouse uterus by at least twofold in this microarray study compared to inter-implantation site. Among these regulated genes, there were 13 genes confirmed by RT-PCR and eight upregulated genes verified by in situ hybridization. Cyr61 is specifically induced in the luminal epithelium at the implantation site by the activated blastocyst and may play a key role during embryo attachment. Our data could be a valuable source for future study on embryo implantation.
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Acknowledgements |
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References |
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Received in final form 21 May 2006
Accepted 5 June 2006
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