본 연구는 생쥐 자궁에 있어서 아라키돈산으로부터 prostaglandin의 생성에 관여하는 것으로 추측되는 acyl-CoA sytnhetase 4 유전자의 임신단계별 발현을 확인하고자 실시하였다. Acyl-CoA sytnhetase 4 유전자는 착상 전에는 발현이 증가하는 경향을 나타내었으며 착상 후에는 감소하였다. 이러한 발현의 양상은 세포막의 인질로부터 아라키돈산을 유리시키는 cPLA2의 발현과 유리된 아라키돈산으로부터 prostaglandin의 생성에 관여하는 COX1과 COX2의 발현 양상과 일치하였다. 이러한 결과는 세포막에서 유리된 아라키돈산이 무한적으로 COX1과 COX2에 의하여 prostaglandin의 생성에 이용되는 것이 아니라 acyl-CoA sytnhetase 4에 의하여 세포막의 인지질로 되돌려져 prostaglandin의 생성을 조절하는 기능을 세포가 수행하고 있는 것으로 추정된다.

These studies were undertaken to examine the interaction of tamoxifen with sex steroid hormones in rat uterine activity. The uterine wet weights of the immature Tat uterus were examined after the administration of estradiol-l7(1g), tamoxifen(50g), progesterone(lmg). The uterotropic activity in immature ovariectomized rats was observed under various treatment conditions following pretreatment with above drugs. The results obtained were as follows:1) Tamoxifen produced significant increase (p <0.01) in uterine wet weight compared with control group, although the increase was not as great as that seen with estradiol-17. Administration of estradiol-17 together with tamoxifen inhibited significantly the increase of uterine wet weight by estradiol-17 (p < 0.01). Coadministration of progresterone with tamoxifen partly blocked the increase of tamoxifen-induced uterine wet weights by progesterone. 2) Estradiol-17after the estradiol-17 pretreatment discontinued the declining uterine wet weights due to the absence of estrogen support, but uteri continued to increase in weight if daily estradiol-17 was maintained. Administration of tamoxifen on the fourth day of estradiol-17 treatment reduced uterine wet weights within 24 hours, and the weights continued to decline with additional tamoxifen. 3) The modest growth of the uterus induced by three daily injections of 5Opg tamoxifen remained stable for five days, with or without additional tamoxifen treatment. Coadministration of tamoxifen with estradiol17 increased slightly the increase of uterine wet weight by tamoxifen. Coadministration of tamoxifen with progesterone inhibited the increase of uterine wet weight by tamoxifen. 4) The modest growth of the uterus induced by three daily injections of lmg progesterone reduced uterine wet weight to the control level for five days. Commencement of tamoxifen or estadiol-17 injections on the fourth day of progesterone treatment rapidly elevated uterine wet weight.

A 40-year-old G1 P0 L0 A1 woman was referred to our clinic with 6-year history of infertility. Before visiting the clinic, she had 3 cycles of In-Vitro Fertilization (IVF) procedures (2 cycles of Controlled Ovarian Stimulation-IVF and 1 cycle of frozen-thawed Embryo Transfer (ET)) at other clinic. She had medical history of abortion at early gestation following FET (frozen-thawed-ET). The patient had complete type of septate uterus, double cervix and longitudinal vaginal septum. Vaginal septotomy was done first and 1 month later, hysteroscopic septoplasty was followed using ballooning filled with dye. After septoplasty, we inserted ballooning and left for several days to compress septal endometrium on the septectomy area. All procedures were done in the ambulatory operating room without laparoscopy or admission. 3 months later, she had in vitro fertilization-embryo transfer (IVF-ET) and FET procedures in our clinic. She had successful pregnancy and now is at 22 weeks of gestation. New ambulatory septoplasty using dye-filled ballooning is easy, safe and minimally invasive surgery for treatment of complete septate uterus.

CREBZF (cAMP-response element binding protein zhangfei) is a member of ATF/CREB family, and which regulates various cellular functions by suppressing major factors with direct interaction. In this study, we have examined the expression of CREBZF on mouse endometrium during uterus estrous cycles and estrogen (E2) treatment. In uterus, CREBZF mRNA expression was higher than other organs and mRNA and protein of CREBZF was increased in proestrus phase and decreased in estrus phase. The expression of CREBZF in 3-weeks old mouse uterus was reduced by E2 injection in endometrium. In addition, the expression of progesterone receptor, a marker of E2 in ovariectomized mice was found to be strongly expressed in stroma, while CREBZF was only expressed in epithelium. Also, we conformed that E2-suppressed CREBZF was restored by co-injection of ICI 182,780, an estrogen receptor antagonist. Overall, these results suggest that CREBZF is regulated by estrogen and involved in ER signaling pathway in mouse uterus.

The Hippo signaling pathway is essential for regulating proliferation, differentiation, and apoptosis in mammalian cells. Hippo signaling pathway exists in most body tissues and organs, where it controls the size of organs and tissues by keeping cell growth in check and promoting cell death as needed. It has been reported that the members of Hippo signaling pathway are highly expressed in mammalian ovaries and uteri. However, the regulatory mechanism of this pathway in the uterus during estrous cycle regulation remains unclear. Serine/Threonine Protein Kinase 4 (STK4, also known as MST1, a homolog of Hippo in Drosophila) is a major factor of Hippo signaling pathway. However STK4 in the mouse uterus has not yet been examined. The purpose of our study was to determine the expression of STK4 during the estrous cycle and regulation by estrogen in the mouse uterus. We found that STK4 was dynamically expressed in uterine endometrium during the estrous cycle. STK4 highly expressed at the estrus, diestrus, and were found to dramatically decrease as it progressed to the proestrus, metestrus stage of uterus during the estrous cycle. Expression of STK4 was dominant in glandular epithelial and luminal epithelial of proestrus, estrus, and diestrus stage, whereas in metestrus stage, expression of gene intensity was faint. Estrogen or estrogen receptor antagonist ICI 182,780 treatment, in ovariectomized mouse uterus, Expression of STK4 and its downstream genes were increased by estrogen. Our results show that the Hippo signaling pathway is estrogen-dependent in the mouse uterus. These informations will give us on sights to understand uterine dynamics during the estrous cycle.

For successful embryo implantation, the stromal cells of the endometrium are morphologically and functionally differentiated into decidual cells. In the endometrium, estrogen induces proliferation of epithelial cells, but progesterone regulates the differentiation of epithelial cells, leading to decidualization of stromal cells. Kruppel like factor (KLF) is a zinc finger DNA binding protein that regulates transcription and has a wide range of functions in the cell cycle, cell apoptosis and differentiation control. In the uterus, KLF9, 13 plays an important role in implantation and decidual cell differentiation. KLF4 and KLF15 regulate the proliferation and differentiation of endometrial epithelial cells, but their role in stromal cells is unknown. In this study, we investigated the role of KLF4 and KLF15 in endometrial stromal cells. In mouse uterus, KLF4 was expressed in proliferative phase of glandular and luminal epithelial cells. However in endometrial stromal cells, KLF4 was highly expressed in secretory phase and secondary decidual zone after implantation. The expression of KLF15 was little in cytoplasm of luminal and glandular epithelial cells and proliferated in nucleus of secretory phase stromal cell.herefore, KLF4 and 15 are thought to be important for decidualization. To investigate the effect of estrogen and progesterone on the expression of KLF4 and KLF15, uterus of ovariectomy (OVX) mice which were injcected 17β- estradiol (E2, 0.3 mg) and progesterone (P4, 1 mg) and both ERα-knock out and wild type (diestrus, estrus) mice were used. KLF4 in OVX+E2 group was significantly higher than OVX+E2 / P4 group was lower than OVX+E2 group. There was no significant difference between ERαKO and WT diestrus group and significantly lower than WT estrus group. Expression of KLF15 was higher in the OVX+ P4 group than in the OVX group and lower in the OVX+E2 group. The OVX+E2 / P4 group was higher than the OVX+E2 group. There was no difference between ERαKO and WT diestrus. The differences in expression of KLF4 and KLF15 by P4 in OVX mouse uterine tissues may be due to the tissue specific expression pattern of epithelial (KLF4) and stromal cells (KLF15). The expression of KLF4 and KLF15 was increased by treatment of cyclic adenosine monophostphate (cAMP, 0.5 mM) and medroxyprogesterone acetate (MPA, 1μM) in human endometrial stromal cells. KLF15 siRNA increased the expression of decidualization markers (BMP2, IGFBP-1 and prolactin) with increasing progesterone receptor A/B (PR A/B), while KLF4 siRNA treatment decreased expression of decidualization markers. There was no significant difference in cell proliferation and apoptosis markers in KLF4 / 15 siRNA treatment. Therefore, progesterone induces KLF4 to promote decidualization, while normally induced KLF15 inhibits progesterone receptor expression. Expression of KLF4 in endometrial epithelium is induced by estrogen but induced by progesterone to promote decidualization, and KLF15 is mainly induced by progesterone in stromal cells and inhibit excessive PR A / B activity.

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.

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.

Autophagy is a major cellular catabolic pathway and is tightly associated with survival and death of cells. The involvement of autophagy during prolonged survival of blastocysts in the uterus is established and it was assumed that ovarian steroid hormones – estrogen (E2) and progesterone (P4) – play important roles in its regulation. The uterus is a major target organ of E2 and P4. To examine if E2 or P4 modulate autophagy in the mouse uterus in vivo, the following three systems were used. 1) Normal pregnancy model (days 1 to 8); 2) delayed implantation model; 3) ovariectomized (OVX) mice model treated with single steroid hormone. Six-week-old virgin ICR mice were used for pregnancy and OXV. OVX mice received P4 (1 mg/0.1 ml) or E2 (100 ng/0.1 ml) after 12 days of rest. Collected uteri were subjected to Western blotting and immunofluorescence staining using anti-LC3B antibody to monitor autophagy. In pregnant mouse uterus, the autophagic response was downregulated after implantation. In OVX model, either E2 or P4 injection downregulated the autophagic response in the uterus within several hours. To confirm whether hormone-induced downregulation is mediated by classical estrogen receptor (ER) and progesterone receptor (PR), receptor antagonists (ICI 182,780 and RU-486) were co-treated. Antagonist-treated uteri showed recovery of autophagic response, suggesting that ER or PR mediates hormonal effects on autophagy. In oder to determine which signaling pathway is involved in autophagic regulation by E2, rapamycin (5 mg/kg), a mTOR inhibitor, and LY294002 (5 mg/kg), a PI3 kinase inhibitor, were used. Rapamycin and LY294002 were injected just before E2 injection to OVX mice. Western blotting was performed by using anti-phospho-mTOR and anti-AKT antibodies. We observed that rapamycin treatment partially antagonized downregulation of autophagic activation by E2, whereas LY294002 treatment did not have any effect. Therefore, downregulation of autophagy by E2 seems to be partially mediated by mTOR pathway. Collectively, this study suggests that ovarian steroid hormones are upstream controllers of autophagic response in the mouse uterus.

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.