Ovariectomized Mouse Uterus Research Articles

Regulation and Function of Laminin A5 during Mouse and Human Decidualization.

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPβ pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPβ pathway.

Genome-wide analysis and functional prediction of the estrogen-regulated transcriptional response in the mouse uterus†.

The ovarian hormones estrogen and progesterone orchestrate the transcriptional programs required to direct functions of the uterus for initiation and maintenance of pregnancy. Estrogen, acting via estrogen receptor alpha, regulates gene expression by activating and repressing distinct genes involved in signaling pathways that regulate cellular and physiological responses including cell division, water influx, and immune cell recruitment. Historically, these transcriptional responses have been postulated to reflect a biphasic physiological response. In this study, we explored the transcriptional responses of the ovariectomized mouse uterus to 17β-estradiol (E2) by RNA-seq to obtain global expression profiles of protein-coding transcripts (mRNAs) and long noncoding RNAs (lncRNAs) following 0.5, 1, 2, and 6hours of treatment. The E2-regulated mRNA and lncRNA expression profiles in the mouse uterus indicate an association between lncRNAs and mRNAs that regulate E2-driven pathways and reproductive phenotypes in the mouse. The transient E2-regulated transcriptome is reflected in the time-dependent shifting of biological processes regulated in the uterus in response to E2. Moreover, high expression of some conserved lncRNAs that are E2 regulated in the mouse uterus are predictive of low overall survival in endometrial carcinoma patients (e.g., H19, KCNQ1OT1, MIR17HG, and FTX). Collectively, this study (1) describes a genomic approach for identifying E2-regulated lncRNAs that may serve critical function in the uterus and (2) provides new insights into our understanding of the regulation of hormone-regulated transcriptional responses with implications in pregnancy and endometrial pathologies.

CGMP-dependent protein kinase II determines β-catenin accumulation that is essential for uterine decidualization in mice.

In the early phase of pregnancy, decidualization is an indispensable event after mammal embryo implantation, accompanied by proliferation and differentiation of uterine stromal cells. Type II cGMP-dependent protein kinase (Prkg2) belongs to the family of serine/threonine kinase, which plays multiple roles in cellular signaling pathways to control proliferation and differentiation. However, the regulatory function and molecular mechanism of Prkg2 in decidualization are still unknown. In this study, we show that Prkg2 has a gradually increased expression pattern during peri-implantation and artificial decidualization, and the expression of Prkg2 is induced by estrogen and progesterone in the ovariectomized mouse uteri and primary cultured uterine stromal cells, the process of which is blocked by treating with estrogen receptor (ER) antagonist (ICI-182,780) and progesterone receptor (PR) antagonist (RU-486). Inhibition of Prkg2 activity by HA-100 promotes uterine stromal cell proliferation but compromises decidualization with decreased expression of prolactin family 8, subfamily a, member 2. In addition, the functional regulation of decidualization by Prkg2 is accomplished by its induced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at serine-9, which results in accumulation of β-catenin in the decidual cells. Taken together, our findings demonstrate that estrogen and progesterone upregulate the expression of Prkg2 in uterine stromal cells depending on ER and PR; Prkg2 promotes phosphorylation of GSK-3β at serine-9 and inactivates it, leading to the accumulation of β-catenin and promoting the process of decidualization. In addition to revealing the regulatory mechanism of Prkg2 that ensures the success of uterine decidualization, our findings will contribute to the understanding in the maintenance of early pregnancy.

Estrogen-dependent expression of sine oculis homeobox 1 in the mouse uterus during the estrous cycle

The sine oculis homeobox 1 (SIX1) is a member of the Six gene family. SIX1 is involved in tissue development by regulating proliferation, apoptosis, and differentiation. However, function of SIX1 in the uterus remains unknown. Here, we found that Six1 expression is regulated along the estrous cycle in mouse uterus. Six1 expression was significantly increased at estrus stage and decreased at the rest of stages. SIX1 is detected in the luminal and glandular epithelium of uterine endometrium at the estrus stage. Estrogen injection increased Six1 expression in the ovariectomized mouse uterus, whereas progesterone had no effect on its expression. Estrogen receptor antagonist inhibited estrogen-induced Six1 expression. Our findings imply that SIX1 may play a role as an important regulator to orchestrate the dynamic of uterine endometrium in response to estrogen level during the estrous cycle. These results will give us a better understanding of uterine biology.

Expression of DROSHA in the Uterus of Mice in Early Pregnancy and Its Potential Significance During Embryo Implantation.

Previous studies have shown that microRNAs are involved in the process of implantation. They play an important role in cell growth and proliferation. DROSHA is the microRNA-processing enzyme and is required for the maturation of microRNAs. However, its expression and function during early pregnancy in mice still remain unclear. In the present study, we analyzed the expression pattern of DROSHA in the mouse uterus during early pregnancy, pseudopregnancy, artificially induced decidualization, and in the ovariectomized mouse uterus using real-time quantitative polymerase chain reaction, Western blotting analyses, and immunohistochemistry. We found that DROSHA was spatiotemporally expressed in decidualizing stromal cells during early pregnancy and in pseudopregnant mice in which decidualization was artificially induced. In the ovariectomized mouse uterus, the expression of DROSHA was upregulated after progesterone treatment. In a stromal cell culture model, the expression of DROSHA gradually increased with the progression of stromal decidualization. Taken together, our findings suggest that DROSHA is involved in stromal decidualization and may play an important role in embryo implantation in mice.

Expression and regulation of androgen receptor in the mouse uterus during early pregnancy and decidualization.

The androgen receptor (AR) is a ligand-activated transcription factor that is important for both the male and female reproductive systems. The expression and regulation of AR in the uterine endometrium during early pregnancy and decidualization remain relatively under-investigated, so we sought to immunohistochemically examine the spatiotemporal expression of AR in mouse uteri during the peri-implantation period as well as in response to specific steroid hormones. AR protein was found in the nuclei of uterine stromal cells starting on pregnancy Days 1 and 2, with its abundance increasing on Days 3 and 4. From pregnancy Days 5 to 9, however, the expression of AR markedly declined in stromal zones of uteri. No signal was detected in the decidualized cells surrounding the site of embryo implantation; moreover, no AR immunostaining was observed in decidualized uterine cells in an artificial oil-induced model of decidualization. Progesterone significantly inhibited AR protein expression, whereas estrogen dramatically elevated AR abundance in the stroma of ovariectomized mouse uteri. Taken together, our results are the first to demonstrate that decidualization and progesterone significantly inhibited the AR protein expression in vivo, whereas estrogen increased AR protein levels in the stromal cells of mouse uteri. These responses might be advantageous for the proliferation and differentiation of uterine stroma and for embryo implantation during early pregnancy.

Expression, regulation and function of Hmgn3 during decidualization in mice

Although Hmgn3 is involved in the regulation of development and cellular differentiation, its physiological roles on decidualization are still unknown. Here we showed that Hmgn3 was highly expressed in the decidua and decidualizing stromal cells. Overexpression of Hmgn3 variants, Hmgn3a or Hmgn3b, enhanced the expression of decidualization markers Prl8a2 and Prl3c1, whereas inhibition of Hmgn3 reduced their expression. Hmgn3 could mediate the effects of Hoxa10 and cAMP on the expression of Prl8a2 and Prl3c1. Further study found that Hmgn3 directed the process of decidualization through influencing the expression of Hand2. Progesterone could induce the expression of Hmgn3 in the ovariectomized mouse uterus, uterine epithelial cells and stromal cells. Knockdown of Hoxa10 with siRNA alleviated the induction of progesterone and cAMP on Hmgn3 expression. Simultaneously, siRNA-mediated down-regulation of Hmgn3 in the uterine stromal cells could attenuate the effects of progesterone, cAMP and Hoxa10 on the expression of Hand2. Collectively, Hmgn3 may play an important role during mouse decidualization.

Differential expression and regulation of Runx1 in mouse uterus during the peri-implantation period.

Runx1 transcription factor is a key developmental regulator. However, little is known about the effects of Runx1 on embryo implantation and decidualization. The aim of this study is to examine the expression and regulation of Runx1 in mouse uterus during the peri-implantation period. There was no evident Runx1 mRNA signal on days 1-4 of pregnancy. On day 5 of pregnancy, Runx1 mRNA was mainly localized in the subluminal stroma surrounding the implanting blastocyst. A similar result was observed in the estrogen-activated implantation uterus. Simultaneously, a high level of Runx1 mRNA expression was detected on days 6-8 of pregnancy and under artificial decidualization. 8-Br-cAMP could induce the expression of Runx1 mRNA in the uterine stromal cells. Moreover, the induction was obviously blocked by PKA inhibitor H89. Inhibition of Runx1 with specific siRNA could decrease the proliferation of stromal cells and expression of decidual markers Prl8a2 and Prl3c1 in the uterine stromal cells. Further study found that inhibition of Runx1 could also suppress the expression of Cox-2, mPGES-1 and Mmp2 genes in uterine stromal cells. Estrogen and progesterone could induce the expression of Runx1 mRNA in ovariectomized mouse uterus and uterine stromal cells. Taken together, these data suggest that Runx1 may play an important role during mouse decidualization.

Very small embryonic-like stem cells are the elusive mouse endometrial stem cells--a pilot study.

BackgroundEndometrium undergoes dramatic growth, breakdown and regeneration throughout reproductive period in mammals. Stem cells have been implicated in the process however their origin, nature, anatomical localization and characterization still remain obscure. Classical concept of presence of stem cells in the basal layer of endometrium was recently challenged when side population and label retaining cells were found to be distributed throughout endometrium. We have earlier reported very small embryonic-like stem cells (VSELs) in adult mammalian ovary and testis as a small population of cells with nuclear OCT-4 along with progenitors (spermatogonial stem cells and ovarian germ stem cells) with cytoplasmic OCT-4. Present study was undertaken to gauge presence of VSELs in bilaterally ovariectomized mouse uterus and their modulation by hormones.MethodsBilaterally ovariectomized mice were subjected to sequential estradiol and progesterone treatment in order to induce proliferation, differentiation and remodeling (regeneration). Stem cells were studied in tissue smears after H & E staining and after sorting using SCA-1 by immuno-localization and qRT-PCR studies (Oct-4A, Nanog and Sca-1). Flow cytometry studies were also undertaken to confirm the presence of VSELs in mouse uterus.ResultsTwo distinct populations of stem cells with dark stained nucleus and high nucleo-cytoplasmic ratio were detected in ovariectomized mouse uterus. These cells were sorted using SCA-1 and comprised smaller VSELs with nuclear expression of OCT-4 and slightly bigger, more abundant progenitors termed as endometrial stem cells (EnSCs) with cytoplasmic OCT-4. RT-PCR studies showed presence of pluripotent transcripts (Oct-4, Sca-1) and flow cytometry confirmed the presence of 0.069% of LIN-/CD45-/SCA-1+ VSELs. These stem cells were distinctly regulated during endometrial growth, differentiation and regeneration as evidenced by qRT-PCR results.ConclusionsVSELs are present in normal uterus and also under conditions of atrophy induced by bilateral ovariectomy. Marked increase in EnSCs is associated with endometrial growth and regeneration. Further studies are warranted to define the niche for these stem cells and whether EnSCs arising from the pluripotent VSELs are common progenitors for epithelial and stromal cells or not remains to be addressed. Results of the present study will help in better understanding of endometrial pathologies and their management in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-015-0138-2) contains supplementary material, which is available to authorized users.

Expression, regulation and function of Egr1 during implantation and decidualization in mice

Early growth response gene 1 (Egr1), a zinc finger transcriptional factor, plays an important role in regulating cell proliferation, differentiation and angiogenesis. Current data have shown that Egr1 is involved in follicular development, ovulation, luteinization and placental angiogenesis. However, the expression, regulation and function of Egr1 in mouse uterus during embryo implantation and decidualization are poorly understood. Here we showed that Egr1 was strongly expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy. Injection of Egr1 siRNA into the mouse uterine horn could obviously reduce the number of implanted embryos and affect the uterine vascular permeability. Further study found that Egr1 played a role through influencing the expression of cyclooxygenase-2 (Cox-2), microsomal prostaglandin E synthase 1 (mPGES-1), vascular endothelial growth factor (Vegf), transformation related protein 53 (Trp53) and matrix metallopeptidase 9 (Mmp9) genes in the process of mouse embryo implantation. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) might direct the expression of Egr1 in the uterine stromal cells. Under in vivo and in vitro artificial decidualization, Egr1 expression was significantly decreased. Overexpression of Egr1 downregulated the expression of decidual marker decidual/trophoblast PRL-related protein (Dtprp) in the uterine stromal cells, while inhibition of Egr1 upregulated the expression of Dtprp under in vitro decidualization. Estrogen and progesterone could regulate the expression of Egr1 in the ovariectomized mouse uterus and uterine stromal cells. These results suggest that Egr1 may be essential for embryo implantation and decidualization.

Expression and function of kisspeptin during mouse decidualization.

BackgroundPlasma kisspeptin levels dramatically increased during the first trimester of human pregnancy, which is similar to pregnancy specific glycoprotein-human chorionic gonadotropin. However, its particular role in the implantation and decidualization has not been fully unraveled. Here, the study was conducted to investigate the expression and function of kisspeptin in mouse uterus during early pregnancy and decidualization.Methodology/Principal FindingsQuantitative PCR results demonstrated that Kiss1 and GPR54 mRNA levels showed dynamic increase in the mouse uterus during early pregnancy and artificially induced decidualization in vivo. KISS-1 and GPR54 proteins were spatiotemporally expressed in decidualizing stromal cells in intact pregnant females, as well as in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Kiss1 mRNA was upregulated after progesterone or/and estradiol treatment. Moreover, in a stromal cell culture model, the expression of Kiss1 and GPR54 mRNA gradually rise with the progression of stromal cell decidualization, whereas the attenuated expression of Kiss1 using small interfering RNA approaches significantly blocked the progression of stromal cell decidualization.Conclusionour results demonstrated that Kiss1/GPR54 system was involved in promoting uterine decidualization during early pregnancy in mice.

Differential expression and regulation of Tdo2 during mouse decidualization

Tryptophan 2,3-dioxygenase (Tdo2) is a rate-limiting enzyme which directs the conversion of tryptophan to kynurenine. The aim of this study was to examine the expression and regulation of Tdo2 in mouse uterus during decidualization. Tdo2 mRNA was mainly expressed in the decidua on days 6-8 of pregnancy. By real-time PCR, a high level of Tdo2 expression was observed in the uteri from days 6 to 8 of pregnancy, although Tdo2 expression was observed on days 1-8. Simultaneously, Tdo2 mRNA was also detected under in vivo and in vitro artificial decidualization. Estrogen, progesterone, and 8-bromoadenosine-cAMP could induce the expression of Tdo2 in the ovariectomized mouse uterus and uterine stromal cells. Tdo2 could regulate cell proliferation and stimulate the expression of decidual marker Dtprp in the uterine stromal cells and decidual cells. Overexpression of Tdo2 could upregulate the expression of Ahr, Cox2, and Vegf genes in uterine stromal cells, while Tdo2 inhibitor 680C91 could downregulate the expression of Cox2 and Vegf genes in uterine decidual cells. These data indicate that Tdo2 may play an important role during mouse decidualization and be regulated by estrogen, progesterone, and cAMP.

Progesterone Receptor-Mediated Regulation of N-Acetylneuraminate Pyruvate Lyase (NPL) in Mouse Uterine Luminal Epithelium and Nonessential Role of NPL in Uterine Function

N-acetylneuraminate pyruvate lyase (NPL) catalyzes N-acetylneuraminic acid, the predominant sialic acid. Microarray analysis of the periimplantation mouse uterine luminal epithelium (LE) revealed Npl being the most downregulated (35×) gene in the LE upon embryo implantation. In natural pregnant mouse uterus, Npl expression increased 56× from gestation day 0.5 (D0.5) to D2.5. In ovariectomized mouse uterus, Npl was significantly upregulated by progesterone (P4) but downregulated by 17β-estradiol (E2). Progesterone receptor (PR) antagonist RU486 blocked the upregulation of Npl in both preimplantation uterus and P4-treated ovariectomized uterus. Npl was specifically localized in the preimplantation D2.5 and D3.5 uterine LE. Since LE is essential for establishing uterine receptivity, it was hypothesized that NPL might play a critical role in uterine function, especially during embryo implantation. This hypothesis was tested in the Npl (−/−) mice. No significant differences were observed in the numbers of implantation sites on D4.5, gestation periods, litter sizes, and postnatal offspring growth between wild type (WT) and Npl (−/−) females from mating with WT males. Npl (−/−)xNpl (−/−) crosses produced comparable little sizes as that from WTxWT crosses. Comparable mRNA expression levels of several genes involved in sialic acid metabolism were observed in D3.5 uterus and uterine LE between WT and Npl (−/−), indicating no compensatory upregulation in the D3.5 Npl (−/−) uterus and LE. This study demonstrates PR-mediated dynamic expression of Npl in the periimplantation uterus and dispensable role of Npl in uterine function and embryo development.

Differential expression and regulation of Cryab in mouse uterus during preimplantation period

The aim of this study was to examine the expression and regulation of the crystallin, alpha B (Cryab) gene in mouse uterus during the peri-implantation period by in situ hybridization and real-time PCR. There was no detectable Cryab mRNA signal on days 1-4 of pregnancy. On day 5 of pregnancy when embryo implanted, a high level of Cryab mRNA signal was found in the subluminal stroma surrounding the implanting blastocyst. On days 6-8, Cryab mRNA was strongly expressed in the primary decidua. By real-time PCR, a high level of Cryab expression was detected on days 7 and 8 of pregnancy, although Cryab expression was seen from days 1 to 8. Under in vivo and in vitro artificial decidualization, Cryab expression was significantly elevated. Compared with the progesterone-primed delayed implantation uterus, a high level of Cryab mRNA expression was observed in estrogen-activated implantation uterus. In the uterine stromal cells, cAMP, estrogen, and progesterone could induce the expression of Cryab gene. In the ovariectomized mouse uterus, estrogen could also induce the expression of Cryab while progesterone inhibited its expression. Our data suggest that Cryab may play an important role during mouse embryo implantation and decidualization and that estrogen and progesterone can regulate the expression of Cryab gene.

Expression and regulation of Runx3 in mouse uterus during the peri-implantation period

The aim of this study was to investigate the differential expression and regulation of Runt-related transcription factor 3 (Runx3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization. There was a low level of the Runx3 mRNA expression in the mouse uterus on days 1-4 of pregnancy. On day 5 when embryo implanted, Runx3 mRNA signal was obviously observed in the stromal cells surrounding the implanting blastocyst. From day 6 to 8 of pregnancy, Runx3 mRNA was highly expressed in the decidual cells and mesometrial decidual beds. Similarly, Runx3 mRNA was strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Runx3 mRNA signal was detected in the uterus with activated implantation. In the ovariectomized mouse uterus, estrogen could induce the expression of Runx3, while progesterone had no effects. These results suggest that Runx3 may play an important role during mouse implantation and decidualization. Estrogen can induce the expression of Runx3 in the ovariectomized mouse uterus.

HIGH CAPACITY COUNTER CURRENT CHROMATOGRAPHY FOR THE ISOLATION OF ACTIVE INGREDIENTS FROM MEDICINAL PLANTS

s. PHYTOPHARM 2012 M 60 Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] ТОМ 10/2012/2 Schisandrols A and b, schisantherin A, schisandrins A and b were detected in Schisandra oil extract by hPTlC (fig. 1A). These lignans are considered to be the main active substances of Schizandra chinensis. lignans antioxidant activity plays a significant role in pharmacological activity of Schizandra drugs (3). We established that these lignans don’t have fRSA by dPPh. They don’t bleach background of the plate after dPPh solution derivatization (fig. 1b). fRSA of Schizandra oil extract depend on other components of the extract, but not lignans. Thus, Schizandra oil extract is a source not only of the well-known lignans, but of other active compounds with the fRSA. The fRSA of these substances causes of their pharmacological importance.

Differential Expression and Regulation of Angiopoietin‐3 in Mouse Uterus during Preimplantation Period

Angiogenesis is necessary for successful implantation and decidualization. This study was to investigate the differential expression of angiopoietin-3 (Ang-3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). There was no detectable Ang-3 mRNA signal on days 1-5 of pregnancy by in situ hybridization. On day 6 of pregnancy, a low level of Ang-3 mRNA signal was seen in the primary decidua. Ang-3 mRNA expression gradually increased on days 7 and 8 of pregnancy along with the development of decidua, and its expression scope was also expanded. The RT-PCR result indicated that Ang-3 mRNA expression was low on days 1-4 of pregnancy. On day 5, as embryo implanted, Ang-3 mRNA was highly expressed in mouse uterus, and the expression gradually increased on days 6-8 of pregnancy, with peak level on day 8 of pregnancy. Similarly, Ang-3 mRNA was also strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Ang-3 mRNA expression was detected in activated implantation uterus by RT-PCR. In the ovariectomized mouse uterus, Ang-3 mRNA expression increased and reached the highest level at 12 hr after injection of estrogen, progesterone, and estrogen plus progesterone, respectively. These results suggest that Ang-3 may play an important role during the process of mouse decidualization. Both estrogen and progesterone can induce the expression of Ang-3 in ovariectomized mouse uterus.

Differential Expression and Regulation of Angiopoietin‐2 in Mouse Uterus During Preimplantation Period

Angiogenesis is crucial to successful implantation and decidualization, however, as an important angiogenic growth factor, the effect of Ang-2 in the process of implantation and decidualization is still unknown. This study is to investigate the differential expression of Ang-2 in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and RT-PCR. There is no detectable Ang-2 mRNA signal on days 1-5 of pregnancy by in situ hybridization. On days 6-8, Ang-2 mRNA is mainly expressed in the primary decidua of mesometrial side, and the expression gradually increases. By RT-PCR, a significantly higher level of Ang-2 expression is observed on day 8 of pregnancy, although Ang-2 expression can be found through days 1-8. Similarly, Ang-2 is highly expressed in decidualized cells under artificial decidualization. In the ovariectomized mouse uterus, Ang-2 expression gradually increases after estrogen injection and with peak levels at 12 hr, while progesterone injection can cause a decline in uterine Ang-2 mRNA level, which reaches a nadir at 12 hr. These results suggest that Ang-2 may play a key role in the process of mouse decidualization. Estrogen can induce the expression of Ang-2 while progesterone can inhibit its expression in the ovariectomized mouse uterus.

Whole-Genome ERalpha Binding in Mouse Uterine Tissue.

The ovariectomized mouse uterus exhibits rapid biochemical and biological responses to estrogens that have been extensively studied and characterized. Using this model we previously evaluated transcript responses by microarray and have identified several thousand transcripts that are increased or decreased following estrogen treatment. To extend these findings we have mapped ERalpha binding sites in chromatin prepared from uterine tissue one hour after injection of saline or estradiol (E2) using chromatin immunoprecipitation (ChIP) with an anti ERalpha antibody followed by sequencing of enriched chromatin fragments using an Illumina system (ChIP-seq). More than 5000 ERalpha binding sites were apparent in the saline treated uterine chromatin, while more than 17000 were seen after the 1 hour E2 treatment. The ERalpha binding sites in E2 treated samples were mapped to almost 10000 Entrez genes while the ERalpha binding sites in the saline sample could be mapped to more than 5000 genes. The average number of sites per gene was greater in the E2 treated chromatin than in the saline treated chromatin, and almost all of the ERalpha bound genes in the saline sample overlap with those in the E2 samples, indicating that some sites are occupied by unliganded ERalpha, and that ERalpha binding is increased by E2. Other studies in MCF7 breast cancer cells have indicated that most ERalpha binding sites are not proximal to promoter regions but are primarily found in distal (>100 kb) or intronic regions. Similarly, in our analysis, fewer than 10% of the uterine ERalpha binding sites in the E2 chromatin were within 500 bp of transcription start sites and more that 80% of sites are within genes or more that 100 kb distal from mapped genes. A de novo motif analysis of sequences in the ERalpha bound chromatin confirmed that EREs were significantly enriched; in addition other transcription factor motifs were enriched as well. Interestingly, in areas of binding without predicted ERE motifs, homeodomain transcription factor binding sites were significantly enriched. To assess the relationship between ERalpha binding and E2 responsive gene regulation, the uterine ChIP-seq data was compared with data from microarrays of uterine RNA after treatment with saline or with E2 for 2 or 24 hours. About 20% of the genes with ERalpha binding sites in the ChIP-seq analysis also had E2 dependent changes in transcription after 2h, and after 24h more than 70% of genes with ERalpha binding encoded regulated transcripts. E2 regulation of uterine transcripts by tethered DNA binding-independent interaction of ERalpha was previously demonstrated using the KIKO mouse model, which has a DNA binding deficient ERalpha. Candidate ERalpha binding sites for several such transcripts were validated by ChIP-PCR of WT and KIKO chromatin. Overall, these findings have the potential to greatly enhance our understanding of mechanisms governing estrogen response in uterine tissue. (platform)

Progesterone receptor-mediated up-regulation of transthyretin in preimplantation mouse uterus

Transthyretin (TTR), a carrier for thyroxine and retinol, has its messenger RNA (mRNA) expressed in the glandular endometrial epithelium and its protein detected in the glandular endometrial epithelium and the uterine lumen. TTR mRNA is dramatically up-regulated in the preimplantation mouse uterus as well as the P-treated ovariectomized mouse uterus, and in both situations the up-regulation of TTR is blocked by treatment with the P receptor antagonist RU486.