Harmonized actions of ovarian estrogen (E2) and progesterone (P4) regulate cell proliferation and differentiation in the uterus with a spatiotemporal manner. Imbalance between the actions and levels of two major regulators often lead to infertility and gynecological diseases, such as endometriosis and endometrial cancer. While numerous works have shown that reduced expression and/or deletion of uterine factors associated with P4 signaling could disturb uterine physiology, local factor(s) to mediate E2 actions has not been extensively studied yet. Here we demonstrate that early growth response 1 (Egr1), a transcription factor which is rapidly induced in the uterus by E2, is required to maintain coordinated actions of E2 and P4 for uterine receptivity for embryo implantation. Given exogenous gonadotrophins to overcome LHβ deficiency in the pituitary of Egr1(－/－) mice, ovulation, fertilization and embryo development normally occurred in these mice. However, they showed complete failure of embryo implantation with reduced uterine responses to artificial decidualization stimuli. While serum levels of E2 and P4 in Egr1(－/－) mice were comparable, genes regulated by E2 and/or P4 in uterine epithelial cells (ECs) were aberrantly expressed on day 4 of pregnancy. Impaired P4 signaling along with absence of PR in ECs caused hypersensitive E2 responses shown as enhanced expression of E2-responsive genes such Muc1 and Ltf as well as reduced levels of P4-dependent genes, such as Ihh and Areg, in ECs of Egr1(－/－) mice. This is consistent with persistent proliferation in ECs and severely impaired proliferation in stromal cells (SCs) in Egr1(－/－) mice treated with E2+P4. Furthermore, primary co-culture of Egr1(－/－) ECs with Egr1(+/+) SCs and vice versa supported a notion that Egr1 itself is required for proper responses to two major regulators, E2 and P4, in both uterine cell compartments. Collectively, our results show that E2-induced Egr1 participates in P4-dependent modulation on E2 activities in the uterus by regulating a spectrum of genes essential for uterine receptivity and embryo implantation.

DGCR8 is a RNA-binding protein working with DROSHA to produce pre-microRNA in the nucleus, while DICER does not only mature microRNA but also endogenous siRNAs in the cytoplasm. Here, we have produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8flox/flox; PRcre/+ mice, Dgcr8d/d) and demonstrated that canonical microRNAs dependent of DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8d/d females did not undergo regular reproductive cycle and produce any pups when housed with fertile males, whereas administration of exogenous gonadotropins induced normal ovulation with corpus luteal formation in these mice. Ovulated oocytes from Dgcr8d/d mice had comparable fertilization potentials and were normally developed to the blastocyst after fertilization as compared to those in control Dgcr8f/f mice. Interestingly, PR-Cre-dependent Dgcr8 deletion showed aberrant infiltration of acute inflammatory immune cells to female reproductive organs only when Dgcr8d/d mice were mated with male mice. With respect to uterine development, gross morphology, histology, and weight of Dgcr8d/d uterus were similar to those of control at 3-week-old age. However, multiple uterine abnormalities were noticeable at 4-week-old age when PR expression is significantly increased, and these deformities became severe onwards. Gland formation and myometrial layers were significantly reduced, and stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced cell proliferation in stromal cell compartments of Dgcr8d/d mice. Collectively, our results suggest that DGCR8 dependent-canonical microRNAs are essential for development and physiology of the uterus with respect to morphogenesis, proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.

The transcription factor, early growth response protein 1 (EGR1), act as immediate early response genes to control various cellular and reproductive events. Egr1-deficient female mice show infertility by anovulation resulting from luteinizing hormone-β (LH-β) subunit deficiency. While ovulation, fertilization and embryo development normally occur in Egr1-deficient mice treated with a superovulation regime to rescue LH deficiency, embryo implantation was completely failed. The morphology and ultrastructure of uterine tissues were observed by light and transmission electron microscopy during the peri-implantation period in Egr1-deficient mice. To examine alterations in cellular organelles, the uterine horns were fixed with 2.5% glutaraldehyde and postfixed with 1% osmium tetroxide in PBS. After dehydration and infiltration, the samples were embedded in Epon 812. Semi-thin sections 0.5 μm thick were cut with an ultramicrotome and stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife of the ultramicrotome and placed on copper grids. The sections were double stained and examined under a transmission electron microscope. The height of luminal epithelial cells was decreased and the polarity was poorly differentiated in the Egr1-deficient comparing to the wild mice. The abundant mucinous materials were observed in the surface of luminal epithelial cells of the Egr1-deficient. It was confirmed the microarray and real time qPCR data. The luminal epithelial cells of wild mice had many dense lipophilic granules and healthy mitochondria, but not in the Egr1-deficient. It may related to production and secretion of steroid hormones and prostaglandins in the luminal epithelial cells for successful implantation. These results show that Egr1 is a critical transcription factor to fine-tune subcellular morphological and functional changes for the receptive phase of peri-implantation period of uterine tissue in mice.

Early growth response 1 (Egr1) is a zinc-finger transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. While it is well-known that Egr1 controls transcription of an array of targets in various cell types, downstream target gene(s) whose transcription is regulated by Egr1 in the uterus has not been identified yet. Thus, we have tried to identify a list of potential target genes of Egr1 in the uterus by performing multi-step in silico promoter analyses. Analyses of mRNA microarray data provided a cohort of genes (102 genes) which were differentially expressed (DEGs) in the uterus between Egr1(+/+) and Egr1(–/–) mice. In mice, the frequency of putative EGR1 binding sites (EBS) in the promoter of DEGs is significantly higher than that of randomly selected non-DEGs, although it is not correlated with expression levels of DEGs. Furthermore, EBS are considerably enriched within –500 bp of DEG’s promoters. Comparative analyses for EBS of DEGs with the promoters of other species provided power to distinguish DEGs with higher probability as EGR1 direct target genes. Eleven EBS in the promoters of 9 genes among analyzed DEGs are conserved between various species including human. In conclusion, this study provides evidence that analyses of mRNA expression profiles followed by two-step in silico analyses could provide a list of putative Egr1 direct target genes in the uterus where any known direct target genes are yet reported for further functional studies.

The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulates cell growth, differentiation, and apoptosis. While it is clear that loss of Egr1 leads to anovulatory infertility due to LHβ deficiency in female mice, molecular function of Egr1 in male reproduction has not been clearly investigated. Here, we demonstrate that Egr1 acts as an intrinsic transcription factor in Leydig cells to regulate their proliferation and steroidogenesis in the testis as well as an extrinsic factor for male reproduction via LHβ transcription in the pituitary. Egr1 is predominantly expressed in spermatogonia and Leydig cells in immature testes and later detected in some of these cell types in mature testes. The fertility potential of Egr1(-/-) male mice is relatively deteriorated even at 2 month-old age and aggravated with aging. The incidence of abnormalities of seminiferous tubules such as Sertoli cell only was dramatically increased with aging. The number and mean size of Leydig cells were significantly reduced in Egr1(-/-) testes. The impairment of Leydig cells is consistent with significant reduction in levels of testosterone and expression of factors critical for steroidogenesis such as StAR in Egr1(-/-) testes. Exogenous administration of hCG rapidly and transiently induced Egr1 expression in Leydig cells culture in vitro. hCG could reinstate reduced mean size of Leydig cells but not reduced number of Leydig cells and aberrantly low StAR expression, suggesting that Egr1 has critical functions for Leydig cell proliferation and their steroidgenesis. In addition, daily sperm production and in vitro fertilization (IVF) competence were significantly reduced, and apoptosis was facilitated in these mice. Furthermore, hCG administration to compensate for relatively low LH levels in Egr1(-/-) males could not restore the compromised reproductive phenotypes such as IVF competence and apoptosis in these mice. Interestingly, expression of Egr2, a member of Egr family, is significantly elevated in Egr1(-/-) Leydig cells suggesting that genetic compensation of Egr2 may alleviate phenotypic aberration of Egr1(-/-) male testes. Collectively, these results suggest that Egr1 act as an intrinsic transcription factor required for proliferation and steroidogenesis of Leydig cells to govern spermatogenesis in the testis.

Early growth response 1 (Egr1) belongs to the Egr family of zinc finger transcription factors (Egr1 to Egr4) that regulates cell growth, differentiation, and apoptosis. Egr1(-/-) female mice are infertile due to anovulation resulting from luteinizing hormone β subunit (LHβ) deficiency. While it is clear that Egr1 is critical for LHβ transcription in the pituitary gland, function of Egr1 in uterus still remain unexplored. Uteri on various experimental conditions or days of pregnancy were collected for mRNA microarrays, realtime-RT-PCR, Western blotting, and histological analyses for immunofluorescence and BrdU staining. Egr1 and other Egr family members, Egr2 and Egr3 are highly expressed in the uterus on day 4 of pregnancy (Day 4). While ovulation, fertilization and embryo development normally occur in Egr1(-/-) mice treated with a superovulation regime to rescue LH deficiency, embryo implantation was completely failed. In addition to implantation failure, oviductal transport of embryos is also impaired in these mice. 17/24 Egr1(-/-) mice (71%) retained blastocysts in the oviduct as well as in the uterus of Egr1(-/-) mice on Day 4 whereas all Egr1(+/+) mice have them in the uterus. While serum levels of E2 and P4 in Egr1(-/-) mice on Day 4 were comparable to those of wildtypes, expression of E2 responsive genes which are expressed in luminal epithelium, such as Mucin 1 and lactoferrin, is aberrantly increased in Egr1(-/-) mice with embryos in the oviduct on Day 4. In contrast, P4 responsive genes such as Hoxa10 and amphiregulin are normally expressed in these mice. Collectively, these data suggest that Egr1 deficiency in the oviduct and uterus leads to estrogen hypersensitivity. BrdU incorporation experiments provided evidence that epithelial cells undergo hyperproliferation in Egr1(-/-) mice. This is consistent with microarray data that several key factors for cell cycle progression such as cyclin Ds and E2F1 are overexpressed in these mice. Furthermore, in the uteri of Egr1(-/-) mice treated with E2+P4, stromal cell proliferation is severely impaired and epithelial cells persistently proliferating. With respect to decidualization, Egr1 as well as Egr2 and Egr3 are induced immediately after decidualization stimuli were given. Although the responses were relatively less than those of wildtype mice, decidualization does occur in Egr1(-/-) mice. Relatively compromised decidualization responses seems to result from functional compensation of Egr2 and Egr3 in Egr1(-/-) deficient uteri. Collectively, our results show that Egr1 is a critical transcription factor to fine-tune estrogen responses via regulation of a spectrum of genes for embryo implantation in the uterus.

Estrogen is a primary steroid hormone to govern cell fates in the endometrium. It induces expression of a spectrum of genes such as early growth response 1 (Egr1) critical for dynamic change of uterine environments for embryo implantation. Egr1 belongs to the Egr family of zinc finger transcription factors consisting of 4 members (Egr1 to Egr4) that are co-expressed in many different tissues, suggesting that they may have some redundant functions. Bisphenol A (BPA) is a well-known endocrine disruptor with potent estrogenic activity on reproductive system. Here we have demonstrated molecular pathway(s) by which estrogen (17β estradiol, E2) and BPA regulates Egr1 in uterus. Eight-week-old female mice were ovariectomized (OVX) and rested for a week. Uteri of OVX mice treated with E2, BPA and/or progesterone (P4) were collected 2 h after hormone treatment unless otherwise indicated. ICI 182,780 [estrogen receptor (ER) antagonist] and RU486 [progesterone receptor (PR) antagonist] were pretreated 30 min before hormone treatment. Collected uteri were mainly utilized for RT-PCR, realtime-RT-PCR and Western blotting. Egr1 mRNA was rapidly induced with the highest level at 2h after E2 treatment and gradually decreased to basal levels at 12 h. Pretreatment of ICI 182,780 effectively inhibited E2-induced phosphorylation of ERK1/2 and AKT as well as Egr1 transcription. U0126 (a pharmacological ERK1/2 inhibitor), but not Watmannin (a AKT inhibitor), significantly blocked E2-induced Egr1 expression as well as ERK1/2 phosphorylation in the uterus. P4 effectively dampened E2-dependent Egr1 transcription, and its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, Egr2 and Egr3 showed similar hormone-dependent expression profiles to that of Egr1 in the uterus. BPA (100 mg/kg) was able to induce immediate expression of Egr1 as effective as E2 at 2 h after treatment. ICI 182,780 and P4 considerably reduced BPA-induced expression of Egr1. In addition, RU486 counteracted inhibitory action of P4 on BPA-induced expression of Egr1. While overall patterns of BPA- induced expression of Egr2 and Egr3 were similar to that of Egr1, BPA was not as effective as E2 for induction of Egr2 and Egr3. BPA could induce phosphorylation of ERK1/2 as well as expression of Egr family members, too. Collectively, these results strongly suggest that BPA as well as E2 can activate concurrent expression profiles of Egr family members via ER-ERK1/2 pathways in the uterus.

DGCR8 is a RNA-binding protein working with DROSHA involved in critical processes for microRNA production in the nucleus. To understand function of miRNAs in the uterus, we have produced uterus-specific Dgcr8 conditional knock-out mice using two well-known Cre mouse models, anti-Mullerian hormone receptor 2 (Amhr2)-Cre and progesterone receptor (PR)-Cre. Dgcr8flox/flox;PRcre/+ mice were mainly analyzed and considered as uDgcr8 KO in this study unless otherwise indicated as Dgcr8flox/flox;Amhr2cre/+ mice. Morphological and histological analyses, embryo cultures, genomic DNA PCR, realtime RT-PCR and Western blotting were performed. uDgcr8 KO females bred with fertile males did not produce any offspring, suggesting that these mice are infertile. Vaginal smear analyses showed that these mice do not undergo estrous cycle, whereas Dgcr8flox/flox;Amhr2cre/+ mice exhibited regular estrous cyclicity. In vitro culture of 2-cell stage embryos and histological analyses for CL in uDgcr8 KO demonstrated that they can respond to gonadotrophins to ovulate healthy oocytes with comparable fertilization potentials as compared to those in Dgcr8flox/flox mice (Control). Gross morphology, histology, and weight of uteri of uDgcr8 KO mice were similar to those of control at 3-week-old stage. However, uterus become extremely thinner and shorter from 4-week-old stage onward. Histological examination showed significant reduction in gland numbers and stromal area from 4-week-old stage. Interestingly, this phenotype is reflected by significant increase of PR expression in the uteri of 4-week-old mice. In addition, stromal cell proliferation of uDgcr8 KO is severely impaired. BrdU incorporation experiments showed that while epithelial cells undergo proliferation by E2 treatment, stromal cells do not incorporate BrdU under the uterine conditions provided with E2+P4. Collectively, these results conclude that microRNAs are essential for uterine stromal cell proliferation in mice.

Early growth response 1 (Egr1) is an immediate early response gene which is induced by various external stimuli and acts as transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. It is well known that Egr1 regulates transcription of a cluster of genes in cancers and luteinizing hormone (LH) beta subunit in the pituitary. In addition to function of Egr1 in cancers and pituitary, we recently showed that Egr1 acts as a local master regulator to mediate estrogenic actions in the uterus. However, regulatory mechanism by which Egr1 directs transcription of its downstream target genes in the uterus remains to be yet explored. Thus, we have tried to identify direct target genes of Egr1 in the uterus by analyzing mRNA microarray data sets followed by in silico promoter analyses with chromatin immunoprecipitation (CHIP). mRNA expression profiles of Egr1(－/－) uteri and Egr1(－/－) ovaries were compared to those of wildtype mice to provide a potential list of direct target genes of Egr1 in the uterus. Whereas Egr1 is rapidly and transiently induced in the ovary and the uterus by external stimuli, LH and estrogen, respectively with a similar manner, a list of differentially expressed genes between Egr1(+/+) and Egr1(－/－) mice were barely overlapped between these two datasets. This result suggests that the transcriptional network of Egr1 in the uterus is quite different from that in the ovary. The list of differentially expressed genes in Egr1(－/－) uterus was enriched by RT-PCR. In silico analyses with MatInspector provided evidence that Egr1 binding sites are relatively enriched in －500 bp promoter regions of genes in the list. CHIP assays for Egr1 antibody with uterine tissues 2 h after estrogen treatment reinforced the possibility that genes identified in this study such as Gadd45g and Lbh could be directly regulated by Egr1 in uterine context. Collectively, we show that bioinformatic analyses of expression profiles with in silico analyses could be a useful tool to enrich potential candidates of direct target genes of transcription factors.

Early growth response 1 (Egr1) belongs to the Egr family of zinc finger transcription factors that regulates cell growth, differentiation, and apoptosis. Egr1(－/－) female mice are infertile due to anovulation resulting from luteinizing hormone β subunit (LHβ) deficiency. While it is clear that Egr1 is a critical factor to regulate transcription of LHβ in the pituitary gland, function of Egr1 and mechanisms by which estrogen (E2) and/or progesterone (P4) regulates Egr1 in uterus still remain unexplored. Using multiple approaches, here we have characterized regulatory mechanism of Egr1 induction in the uterus and uterine phenotypes of Egr1(－/－) mice. Eight-week-old female mice were ovariectomized (OVX) and rested for a week. Uteri of OVX mice treated with various concentrations of E2 and/or other hormones were collected at 2h after hormone treatment unless otherwise indicated. Collected uteri were utilized for mRNA microarrays, realtime-RT-PCR, Western blotting, and histological analyses for immunofluorescence and BrdU staining. Egr1 mRNA was rapidly induced with the highest level at 2h after E2 treatment and gradually decreased to basal levels at 12 h. E2-induced phosphorylation of ERK1/2 and AKT, and Egr1 transcription were effectively inhibited by pretreatment of ICI 182,780. Pharmacological inhibition of ERK1/2, but not AKT significantly blocked E2-induced Egr1 expression in the uterus. P4 effectively dampened E2-dependent Egr1 transcription and its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, BrdU incorporation experiments provided evidence that epithelial cells undergo hyperproliferation in Egr1(－/－) mice. This is consistent with microarray data that several key factors for cell cycle progression such as cyclin Ds and E2F1 are overexpressed in these mice. Furthermore, in the uteri of OVX Egr1(－/－) mice treated with E2+P4, stromal cell proliferation is severely impaired and epithelial cells persistently proliferating. While ovulation, fertilization and embryo development normally occur in Egr1(－/－) mice treated with a superovulation regime to rescue LH deficiency, embryo implantation is severely impaired. Blastocysts were not able to implant even on day 6 of pregnancy in Egr1(－/－) mice. In addition to embryo implantation, uterine response to artificial decidualization in hormone-primed Egr1(－/－) OVX mice was relatively less than that of wildtype mice. Collectively, our results show that Egr1, which is rapidly induced by E2-ER-ERK1/2 pathways, is a critical factor to control E2-dependent cell proliferation via regulation of a spectrum of genes for embryo implantation in the uterus.

MicroRNAs (miRNAs) are single-stranded, small non-coding RNAs which are critical for gene regulatory networks by directing the translational repression or degradation of complementary target mRNAs. They can be divided into two subclasses: canonical and non-canonical miRNAs. Canonical miRNAs are produced from long primary transcripts by sequential complex events in which RNA III enzymes such as Drosha and Dicer and accessory factors such as DGCR8 and Argonautes work cooperatively. DGCR8 is a RNA-binding protein that assists Drosha to process canonical miRNAs in the nucleus. To understand function of canonical miRNAs in uterine physiology, we have characterized uterine phenotypes of uterine-specific DGCR8 knock-out mice (uDGCR8 KO, DGCR8flox/flox; PRcre/+), and hormonal regulation of expression profiles of major factors working for miRNA biogenesis in the uterus. Gross morphological and histological analyses, immunohistochemistry, PCR and realtime RT-PCR were performed. While DGCR8 and Drosha do not seem to be regulated by ovarian steroid hormones, expression of Dicer, Exportin 5 and Argonaute 2 was transiently increased by E2 but not by P4. Combination of E2+P4 did not have any additional effects on their expression profiles. Genomic DNA PCR analyses showed that while DGCR8 gene is not completely deleted in the uterus, DGCR8 is specifically deleted in the uterus where PR is strongly expressed. uDGCR8 KO females bred with fertile males did not produce any offspring, suggesting that these mice are infertile. Vaginal smear analyses provided evidence that these mice do not undergo estrous cycle. Whereas gross morphology and histological analyses of uteri of 3-week-old uDGCR8 KO mice is similar to that of DGCR8flox/flox mice (control), uteri of 5- and 8-week-old mice are extremely thinner and shorter than those of control mice. These results were supported by significant decrease in uterine weight/body weight of uDGCR8 KO mice at 5-week-old age onward. Interestingly, this phenotype is reflected by significant increase of PR expression in the uteri of 4-week-old mice. Expression of DGCR8 and Dicer is significantly increased after birth. BrdU incorporation experiments showed that cell proliferation governed by ovarian steroid hormones does not normally occur in these mutant mice. Furthermore, artificial decidualization does not occur in these mice. Collectively, these results conclude that canonical miRNAs plays critical roles in normal uterine development and steroid hormone-dependent uterine function.

The Egr family of zinc finger transcription factors consisting of 4 members (Egr1 to -4) regulates critical genetic programs involved in cellular growth, differentiation, and function. Especially, the critical role for Egr1 in regulating luteinizing hormone responsiveness was demonstrated by using gene-targeted mouse models. Other members of Egr family were shown to be involved in other cellular and developmental processes. To understand if Egr3 is implicated in ovarian functions, we focused on identifying cell type-specific and subcellular localization of Egr3 in cycling mouse ovaries and oocytes. RT-PCR analyses show that Egr3 mRNA is expressed in the mouse ovary and oocytes. By immunofluorescence staining, we observed that Egr3 is weakly expressed in subsets of granulosa cells. Interestingly, Egr3 seems to be co-localized with meiotic spindle in some oocytes in the ovarian section. Therefore, we examined Egr3 localization in MI oocytes cultured in vitro. We confirmed co-localization of Egr3 and microtubule in the mouse oocyte during meiosis I. Egr3 localization is noted around condensing chromosomes during prometaphase I (PMI). At metaphase I (MI) and MII, Egr3 is localized on meiotic spindle and also around each cytosolic microtubule organizing centers (MTOCs) in a punctate pattern. To examine if microtubule is required for correct positioning of Egr3 on this structure, we observed the pattern of Egr3 in oocytes matured under taxol or nocodazole. In taxol-treated oocyte, Egr3 and gamma-tubulin complex are enlarged. In nocodazole-treated oocyte, Egr3 localization on spindle and MTOCs are abolished. Thus, Egr3 localization seems to require the presence of intact microtubule. Collectively, our result shows for the first time that Egr3, a transcription factor, is localized on meiotic spindle of maturing mouse oocytes. The work suggests a novel role for Egr3 as a factor involved in MTOC dynamics during meiosis.

The Egr family of zinc finger transcription factors consisting of 4 members (Egr1 to Egr4) regulates critical genetic programs involved in cellular growth, differentiation, and function. They are co-ex-pressed in many different tissues, suggesting that they may have some redundant functions. While it is clear that estrogen regulates Egr1 in estrogen sensitive breast cancer cells, function of Egr1 and mechanisms by which estrogen (E2) and/or progesterone (P4) regulates Egr1 in uterus still remain unexplored. Thus, we have examined regulatory mechanisms by which Egr1 is regulated in the uterus and abnormal uterine phenotypes of Egr1(－/－) mice. Eight-week-old female mice were ovariectomized (OVX) and rested for a week. Uteri of OVX mice treated with various concentrations of E2 and/or other hormones were collected at 2 h after hormone treatment unless otherwise indicated. ICI 182,780 [estrogen receptor (ER) antagonist] or RU486 [progesterone receptor (PR) antagonist] was injected to OVX mice 30 min prior to hormone treatments. OVX Egr1(+/+) and Egr1(－/－) mice were treated with E2 and/or P4 to examine expression patterns of genes important for estrogen responses, and steroid hormone-induced cell proliferation in the uterus. Collected uteri were utilized for RT-PCR, realtime RT-PCR, Western blotting and histological analyses. Egr1 mRNA was rapidly induced with the highest level at 2h after E2 treatment and gradually deceased to basal levels at 12 h. Pretreatment of ICI 182,780 significantly reduced E2-induced increase of Egr1. However, an agonist for GPR30, a membrane estrogen receptor failed to induce mRNA expression of Egr1, suggesting that E2-dependent Egr1 transcription is mainly regulated via nuclear estrogen receptor, ER. P4 effectively dampened E2-dependent Egr1 transcription and its antagonistic effects were partially interfered with RU486 pretreatment. Histological analyses with BrdU incorporation experiments showed that vascular permeability (an early estrogen response) but not cell proliferation (a late response) was significantly impaired in the uteri of E2 treated OVX Egr1(－/－) mice. Interestingly, some genes involved in early estrogen responses such as Bip and HIF-1a but not those in late responses are dysregulated in uteri of Egr1(－/－) mice. Collectively, our results show that E2 transiently induces Egr1 via activation of nuclear ER. P4 antagonizes E2-dependent Egr1 regulation via PR. Impaired early estrogenic responses in Egr1(－/－) uteri could be due to aberrant gene expression affected by loss of Egr1 which act as a master regulator of estrogen actions in the uterus.-ex

In particular, maternal prostacyclin (PGI2) is critical for embryo implantation and the action of PGI2 is not mediated via its G protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome proliferator-activated receptor δ (PPARδ). Recently, several studies have shown that PGI2 enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI2 improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI2-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, RXRs (heterodimeric partners of PPARδ) and PGI2 synthase are temporally induced after zygotic gene activation and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (cPGI, a stable analogue of PGI2) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI2-induced PPARδ activation accelerates blastocyst hatching in mice.