Blastocyst-Derived Lactic Acid May Regulate S100A6 Expression and Function in Mouse Decidualization via Stimulation of Uterine Epithelial Arachidonic Acid Secretion.

(1) Background: Inflammatory responses are implicated in embryo implantation, decidualization, pregnancy maintenance and labor. Both embryo implantation and decidualization are essential to successful pregnancy in rodents and primates. S100A6 is involved in inflammation, tumor development, apoptosis and calcium homeostasis. S100A6 is strongly expressed in mouse decidua, but the underlying mechanisms of how S100A6 regulates implantation and decidualization are poorly defined. (2) Methods: Mouse endometrial stromal and epithelial cells are isolated from day 4 pseudopregnant mouse uteri. Both immunofluorescence and Western blotting are used to analyze the expression and localization of proteins. The molecular mechanism is verified in vitro by Western blotting and the quantitative polymerase chain reaction. (3) Results: From days 4 to 8 of pregnancy, S100A6 is specifically expressed in mouse subluminal stromal cells. Blastocyst-derived lactic acid induces AA secretion by activating the luminal epithelial p-cPLA2. The epithelial AA induces stromal S100A6 expression through the COX2/PGI2/PPAR δ pathway. Progesterone regulates S100A6 expression through the progesterone receptor (PR). S100A6/RAGE signaling can regulate decidualization via EGFR/ERK1/2 in vitro. (4) Conclusions: S100A6, as an inflammatory mediator, is important for mouse implantation and decidualization.

Embryo-derive TNF promotes decidualization via fibroblast activation.

Decidualization is a process in which endometrial stromal fibroblasts differentiate into specialized secretory decidual cells and essential for the successful establishment of pregnancy. The underlying mechanism during decidualization still remains poorly defined. Because decidualization and fibroblast activation share similar characteristics, this study was to examine whether fibroblast activation is involved in decidualization. In our study, fibroblast activation-related markers are obviously detected in pregnant decidua and under in vitro decidualization. ACTIVIN A secreted under fibroblast activation promotes in vitro decidualization. We showed that arachidonic acid released from uterine luminal epithelium can induce fibroblast activation and decidualization through PGI2 and its nuclear receptor PPARδ. Based on the significant difference of fibroblast activation-related markers between pregnant and pseudopregnant mice, we found that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium. Fibroblast activation is also detected under human in vitro decidualization. Similar arachidonic acid-PGI2-PPARδ-ACTIVIN A pathway is conserved in human endometrium. Collectively, our data indicate that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium to induce fibroblast activation and decidualization.

The regulation and function of acetylated high-mobility group box 1 during implantation and decidualization.

Introduction: High-mobility group box 1 (HMGB1) is a non-histone nuclear protein and can be extracellularly secreted to induce sterile inflammation. Although uterine deletion of HMGB1 causes implantation and decidualization defects, how secreted HMGB1 is involved in mouse early pregnancy is still unknown. Methods: Mouse models, mouse primary endometrial cells and human endometrial cell lines were used in this study. Both immunofluorescence and Western blot were performed to show the localization and relative level of HMGB1 and acetylated HMGB1, respectively. Relative mRNA levels were analyzed by real time RT-PCR. Results: The secreted HMGB1 was detected in uterine lumen fluid in mouse periimplantation uterus. There is an obvious difference for secreted HMGB1 levels in uterine fluid between day 4 of pregnancy and day 4 of pseudopregnancy, suggesting the involvement of blastocysts during HMGB1 secretion. Trypsin is clearly detected in mouse blastocyst cavity and in the supernatant of cultured blastocysts. Trypsin significantly stimulates HB-EGF production through activating PAR2 and ADAM17. Uterine injection of PAR2 inhibitor into day 4 pregnant mice significantly reduces the number of implantation sites. HB-EGF released from luminal epithelium can induce mouse in vitro decidualization. The conditioned medium collected from trypsin-treated luminal epithelium is able to induce in vitro decidualization, which is suppressed by EGFR inhibitor. Intrauterine injection of glycyrrhizin (HMGB1 inhibitor) can significantly inhibit mouse embryo implantation. We also showed that exogenous HMGB1 released from human epithelial cells are able to induce human in vitro decidualization. Conclusion: Trypsin can induce decidualization of stromal cells via PAR2-HMGB1-ADAM17-HB-EGF from luminal epithelium.

IHH, SHH, and primary cilia mediate epithelial-stromal cross-talk during decidualization in mice.

The establishment of pregnancy depends on interactions between the epithelial and stromal cells of the endometrium that drive the decidual reaction that remodels the stroma and enables embryo implantation. Decidualization in mice also depends on ovarian hormones and the presence of a blastocyst. Hedgehog signaling is transduced by primary cilia in many tissues and is involved in epithelial-stromal cross-talk during decidualization. We found that primary cilia on mouse uterine stromal cells increased in number and length during early pregnancy and were required for decidualization. In vitro and in vivo, progesterone promoted stromal ciliogenesis and the production of Indian hedgehog (IHH) in the epithelium and Sonic hedgehog (SHH) in the stroma. Blastocyst-derived TNF-α also induced epithelial IHH, which stimulated the production of SHH in the stroma through a mechanism that may involve the release of arachidonic acid from epithelial cells. In the stroma, SHH activated canonical Hedgehog signaling through primary cilia and promoted decidualization through a mechanism that depended on interleukin-11 (IL-11) and primary cilia. Our findings identify a primary cilia-dependent network that controls endometrial decidualization and suggest primary cilia as a candidate therapeutic target for endometrial diseases.

Human Chorionic Gonadotropin-Stimulated Interleukin-4-Induced-1 (IL4I1) Promotes Human Decidualization via Aryl Hydrocarbon Receptor.

Decidualization is necessary for the successful establishment of early pregnancy in rodents and humans. Disturbed decidualization results in recurrent implantation failure, recurrent spontaneous abortion, and preeclampsia. Tryptophan (Trp), one of the essential amino acids in humans, has a positive effect on mammalian pregnancy. Interleukin 4-induced gene 1 (IL4I1) is a recently identified enzyme that can metabolize L-Trp to activate aryl hydrocarbon receptor (AHR). Although IDO1-catalyzed kynurenine (Kyn) from Trp has been shown to enhance human in vitro decidualization via activating AHR, whether IL4I1-catalyzed metabolites of Trp are involved in human decidualization is still unknown. In our study, human chorionic gonadotropin stimulates IL4I1 expression and secretion from human endometrial epithelial cells through ornithine decarboxylase-induced putrescine production. Either IL4I1-catalyzed indole-3-pyruvic acid (I3P) or its metabolite indole-3-aldehyde (I3A) from Trp is able to induce human in vitro decidualization by activating AHR. As a target gene of AHR, Epiregulin induced by I3P and I3A promotes human in vitro decidualization. Our study indicates that IL4I1-catalyzed metabolites from Trp can enhance human in vitro decidualization through AHR-Epiregulin pathway.

Primary Cilia Restrain PI3K-AKT Signaling to Orchestrate Human Decidualization.

Endometrial decidualization plays a pivotal role during early pregnancy. Compromised decidualization has been tightly associated with recurrent implantation failure (RIF). Primary cilium is an antenna-like sensory organelle and acts as a signaling nexus to mediate Hh, Wnt, TGFß, BMP, FGF, and Notch signaling. However, whether primary cilium is involved in human decidualization is still unknown. In this study, we found that primary cilia are present in human endometrial stromal cells. The ciliogenesis and cilia length are increased by progesterone during in vitro and in vivo decidualization. Primary cilia are abnormal in the endometrium of RIF patients. Based on data from both assembly and disassembly of primary cilia, it has been determined that primary cilium is essential to human decidualization. Trichoplein (TCHP)-Aurora A signaling mediates cilia disassembly during human in vitro decidualization. Mechanistically, primary cilium modulates human decidualization through PTEN-PI3K-AKT-FOXO1 signaling. Our study highlights primary cilium as a novel decidualization-related signaling pathway.

BHPF exposure impairs mouse and human decidualization.

Although BHPF has been widely used in plastic manufacturing as a substitute for BPA, current evidence suggests that BHPF also causes harmful effects on reproduction. However, effects of BHPF on mammalian early pregnancy are still poorly defined. This study aimed to explore the effects of BHPF on early pregnancy, especially decidualization and embryonic development in mice and human beings. The results showed that 50 and 100 mg/kg BHPF exposure reduced birth weight, and implantation site weight on the day 8 of pregnancy in mice. Because BHPF inhibits both embryo development and artificial decidualization in mice, suggesting that the detrimental effects of BHPF should be from its effects on embryo development and decidualization. Under in vitro decidualization, 10 µM BHPF inhibits decidualization and leads to disordered expression of Lamin B1 and collagen in mice. In addition, 10 µM BHPF also inhibits decidualization, and causes disordered expression of both collagen III and Lamin B1 under human in vitro decidualization. However, collagen III supplementation can rescue BHPF inhibition on decidualization. Further, our study demonstrates that BHPF impairs human decidualization through the HB-EGF/EGFR/STAT3/Collagen III pathway. Taken together these data suggest that exposure to BHPF impairs mouse and human decidualization during early pregnancy.

Effects of Aurora kinase A on mouse decidualization via Stat3-plk1-cdk1 pathway.

BACKGROUND: Decidualization is essential to the successful pregnancy in mice. The molecular mechanisms and effects of Aurora kinase A (Aurora A) remain poorly understood during pregnancy. This study is the first to investigate the expression and role of Aurora A during mouse decidualization. METHODS: Quantitative real time polymerase chain reaction, western blotting and in situ hybridization were used to determine the expression of Aurora A in mouse uteri. Aurora A activity was inhibited by Aurora A inhibitor to explore the role of Aurora A on decidualization via regulating the Aurora A/Stat3/Plk1/Cdk1 signaling pathway. RESULTS: Aurora A was strongly expressed at implantation sites compared with inter-implantation sites. Furthermore, Aurora A was also significantly increased in oil-induced deciduoma compared with control. Both Aurora A mRNA and protein were significantly increased under in vitro decidualization. Under in vitro decidualization, Prl8a2, a marker of mouse decidualization, was significantly decreased by TC-S 7010, an Aurora A inhibitor. Additionally, Prl8a2 was reduced by Stat3 inhibitor, Plk1 inhibitor and Cdk1 inhibitor, respectively. Moreover, the protein levels of p-Stat3, p-Plk1 and p-Cdk1 were suppressed by TC-S 7010. The protein levels of p-Stat3, p-Plk1 and p-Cdk1 were also suppressed by S3I-201, a Stat3 inhibitor). SBE 13 HCl (Plk1 inhibitor) could reduce the protein levels of p-Plk1 and p-Cdk1. Collectively, Aurora A could regulate Stat3/Plk1/Cdk1 signaling pathway. CONCLUSION: Our study shows that Aurora A is expressed in decidual cells and should be important for mouse decidualization. Aurora A/Stat3/Plk1/Cdk1 signaling pathway may be involved in mouse decidualization.

Female reproductive abnormalities in mouse adolescent pregnancy.

There are around 300 million adolescent pregnancies worldwide, accounting for 11% of all births worldwide. Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies. However, how and why these abnormalities occur remain to be defined. In this study, pregnancy at different stages was compared between 25- and 30- day-old and mature female mice. We found that the litter size of adolescent pregnancy is significantly decreased from F1 to F3 generations compared to mature pregnancy. On days 8 and 12 of pregnancy, multiple abnormalities in decidual and placental development appear in F3 adolescent pregnancy. On days 5 and 8, uterine endoplasmic reticulum stress is dysregulated in F3 adolescent pregnancy. Embryo implantation and decidualization are also compromised in adolescent pregnancy. Many genes are abnormally expressed in adolescent estrous uteri. The abnormal endocrine environment and abnormal implantation from uterine immaturity may result in multiple pregnancy failures in adolescent pregnancy. The aim of this study is to shed light on human adolescent pregnancy.

In situ Synthesized Monosodium Urate Crystal Enhances Endometrium Decidualization via Sterile Inflammation During Pregnancy.

High level of uric acid (UA) is the major origin of gout, and is highly associated with various pregnant complications, such as preeclampsia and gestational diabetes. However, UA's level and role in the very early stage of pregnancy has not been uncovered. This study aims to investigate the relevance of serum UA and decidualization, an essential process for the establishment and maintenance of pregnancy in women and mice during the early stage of pregnancy. In this study, we first proved that expression level of UA synthase xanthine dehydrogenase (XDH) is highly increased along with decidualization of endometrial stromal cells in both in vitro and in vivo models. Furthermore, serum and endometrial levels of UA are higher in mice with decidualized uterin horn and in vitro decidualized stromal cells. The existence of monosodium urate (MSU) crystal was also confirmed by immunostaining. Next, the roles of MSU on decidualization were explored by both in vitro and in vivo models. Our data shows MSU crystal but not UA enhances the decidualization response of endometrial stromal cells, via the upregulation of inflammatory genes such Ptgs2 and Il11. inhibiting of Cox-2 activity abolishes MSU crystal induced higher expression of decidualization marker Prl8a2. At last, in women, we observed enriched expression of XDH in decidua compare to non-decidualized endometrium, the serum level of UA is significantly increased in women in very early stage of pregnancy, and drop down after elective abortion. In summary, we observed an increased serum UA level in the early stage of women's pregnancy, and proved that the increased level of UA results from the expressed XDH in decidualizing endometrium of both human and mouse, leading to the formation of MSU crystal. MSU crystal can enhance the decidualization response via inflammatory pathways. Our study has uncovered the association between UA, MSU, and decidualization during the early stage of pregnancy.

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/EBPß 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/EBPß pathway.

The detrimental effects of stress-induced glucocorticoid exposure on mouse uterine receptivity and decidualization.

Glucocorticoids (GCs), stress-induced steroid hormones, are released by adrenal cortex and essential for stress adaptation. Recently, there has been renewed interest in the relationship between GCs and pregnancy following the discovery that glucocorticoid receptor is necessary for implantation. It has been widely recognized that stress is detrimental to pregnancy. However, effects of stress-induced GC exposure on uterine receptivity and decidualization are still poorly understood. This study aims to explore the effects of GCs exposure on uterine receptivity, decidualization, and their underlying mechanisms in mice. Single prolonged stress (SPS) and corticosterone (Cort) injection models were used to analyze effects of GC exposure on early pregnancy, respectively. SPS or Cort injection inhibits embryo implantation by interfering Lif signaling and stimulating the uterine deposition of collagen types I, III, and IV on day 4 of pregnancy. Uterine decidualization is also attenuated by SPS or Cort injection through suppressing Cox-2 expression. Cort-induced collagen disorder also suppresses decidualization through regulating mesenchymal-epithelial transition. Our data should shed lights for a better understanding for the effects of GCs on embryo implantation for clinical research.

Blastocyst-induced ATP release from luminal epithelial cells initiates decidualization through the P2Y2 receptor in mice.

Embryo implantation involves a sterile inflammatory reaction that is required for the invasion of the blastocyst into the decidua. Adenosine triphosphate (ATP) released from stressed or injured cells acts as an important signaling molecule to regulate many key physiological events, including sterile inflammation. We found that the amount of ATP in the uterine luminal fluid of mice increased during the peri-implantation period, and this depended on the presence of an embryo. We further showed that the release of ATP from receptive epithelial cells was likely stimulated by lactate released from the blastocyst through connexin hemichannels. The ATP receptor P2y2 was present on uterine epithelial cells during the preimplantation period and increased in the stromal cells during the time at which decidualization began. Pharmacological inhibition of P2y2 compromised decidualization and implantation. ATP-P2y2 signaling stimulated the phosphorylation of Stat3 in uterine luminal epithelial cells and the expression of early growth response 1 (Egr1) and prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox-2), all of which are required for decidualization and/or implantation, in stromal cells. Short exposure to high concentrations of ATP promoted decidualization of primary stromal cells, but longer exposures or lower ATP concentrations did not. The expression of genes encoding ATP-degrading ectonucleotidases increased in the decidua during the peri-implantation period, suggesting that they may limit the duration of the ATP signal. Together, our results indicate that the blastocyst-induced release of ATP from uterine epithelial cells during the peri-implantation period may be important for the initiation of stromal cell decidualization.

Tryptophan and kynurenine stimulate human decidualization via activating Aryl hydrocarbon receptor: Short title: Kynurenine action on human decidualization.

Decidualization is essential for successful pregnancy in rodents and primates. Although L-Tryptophan and its metabolites are essential for mammalian pregnancy, the underlying mechanism is poorly defined. We explored effects of tryptophan and kynurenine on human in vitro decidualization in human endometrial stromal cell line and primary endometrial stromal cells. Tryptophan significantly stimulates the expression of prolactin and insulin growth factor binding protein 1, reliable markers for human decidualization. When stromal cells are treated with tryptophan, tryptophan hydroxylase-1 remains unchanged, but indoleamine 2,3-dioxygenase 1 is significantly increased, suggesting tryptophan is mainly metabolized through kynurenine pathway. Kynurenine significantly stimulates insulin growth factor binding protein 1 expression. Aryl hydrocarbon receptor and its target genes (P450 1A1 and P450 1B1) are significantly increased by tryptophan and kynurenine. The induction of tryptophan and kynurenine on insulin growth factor binding protein 1 is abrogated by CH223191, an aryl hydrocarbon receptor inhibitor. Cytochrome P450 1A1 and P450 1B1 catalyze the oxidative metabolism of estradiol to catechol estrogens (2-hydroxy estradiol and 4-hydroxy estradiol), respectively. Insulin growth factor binding protein 1 is up-regulated by 2-hydroxy estradiol and 4-hydroxy estradiol. Interferon-γ significantly induces the expression of indoleamine 2,3-dioxygenase 1, aryl hydrocarbon receptor and insulin growth factor binding protein 1. All the data are also verified in primary human stromal cells. Our data indicate that Interferon-γ-induced kynurenine pathway promotes human decidualization via aryl hydrocarbon receptor signaling.

Nucleolar stress regulates stromal-epithelial transition via NPM1 during decidualization.

Embryo implantation and decidualization are crucial steps during early pregnancy. We recently showed that nucleolar stress is involved in embryo implantation. This study was to explore whether nucleolar stress participates in mouse and human decidualization. Our data demonstrated that a low dose of actinomycin D (ActD) could induce nucleolar stress in stroma cells. Nucleolar stress promotes the stromal-epithelial transition during mouse in vitro decidualization through nucleophosmin1 (NPM1). Under nucleolar stress, Wnt family member 4 (Wnt4), a decidualization marker, is significantly increased, but decidua/trophoblast prolactin-related protein (Dtprp/Prl8a2) expression remains unchanged. For translational significance, we also examined the effects of nucleolar stress on human decidualization. Nucleolar stress stimulated by a low dose of ActD enhances human stromal-epithelial transition during human decidualization, but has no effects on the expression of insulin-like growth factor-binding protein 1 (IGFBP1). Our study indicates that nucleolar stress may promote only the mesenchymal-epithelial transition (MET), but not for all the molecular changes during decidualization.

Aldosterone from endometrial glands is benefit for human decidualization.

Local renin-angiotensin system (RAS) in female reproductive system is involved in many physiological and pathological processes, such as follicular development, ovarian angiogenesis, ovarian, and endometrial cancer progress. However, studies on the functional relevance of RAS in human endometrium are limited, especially for renin-angiotensin-aldosterone system (RAAS). In this study, we defined the location of RAS components in human endometrium. We found that angiotensin II type-1 receptor (AT1R) and aldosterone synthase (CYP11B2), major components of RAAS, are specifically expressed in endometrial gland during mid-secretory phase. Aldosterone receptor, mineralocorticoid receptor (MR), is elevated in stroma in mid-secretory endometrium. In vitro, MR is also activated by aldosterone during decidualization. Activated MR initiates LKB1 expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. The impact of PDK4 on decidualization is independent on PDHE1α inactivation. Based on co-immunoprecipitation, PDK4 interacts with p-CREB to prevent its ubiquitination for facilitating decidualization via FOXO1. Restrain of MR activation interrupts LKB1/p-AMPK/PDK4/p-CREB/FOXO1 pathway induced by aldosterone, indicating that aldosterone action on decidualization is mainly dependent on MR stimulation. Aldosterone biosynthesized in endometrial gland during mid-secretory phase promotes decidualization via activating MR/LKB1/p-AMPK/PDK4/p-CREB/FOXO1 signaling pathway. This study provides the valuable information for understanding the underlying mechanism during decidualization.

Progesterone-regulated Hsd11b2 as a barrier to balance mouse uterine corticosterone.

Glucocorticoids (GCs) are essential for mouse embryo implantation and decidualization. Excess GCs are harmful for mouse embryo implantation and decidualization. 11ß-Hydroxysteroid dehydrogenases type I and II (Hsd11b1/Hsd11b2) are main enzymes for regulating local level of GCs. Hsd11b2 acts as the placental glucocorticoid barrier to protect the fetus from excessive exposure. Although effects of GCs on the fetus and placenta in late pregnancy have been extensively studied, the effects of these adrenal corticosteroids in early pregnancy are far less well defined. Therefore, we examined the expression, regulation and function of Hsd11b1/Hsd11b2 in mouse uterus during early pregnancy. We found that Hsd11b2 is highly expressed in endometrial stromal cells on days 3 and 4 of pregnancy and mainly upregulated by progesterone (P4). In both ovariectomized mice and cultured stromal cells, P4 significantly stimulates Hsd11b2 expression. P4 stimulation of Hsd11b2 is mainly mediated by the Ihh pathway. The uterine level of corticosterone (Cort) is regulated by Hsd11b2 during preimplantation. Embryo development and the number of inner cell mass cells are suppressed by Cort treatment. These results indicate that P4 should provide a low Cort environment for the development of preimplantation mouse embryos by promoting the expression of uterine Hsd11b2.

ATP mediates the interaction between human blastocyst and endometrium.

OBJECTIVE: Embryo implantation needs a reciprocal interaction between competent embryo and receptive endometrium. Adenosine triphosphate (ATP) produced by stressed or injured cells acts as an important signalling molecule. This study aims to investigate whether adenosine triphosphate (ATP) plays an important role in the dialogue of human blastocyst-endometrium. MATERIALS AND METHODS: The concentration of lactate was analysed in culture medium from human embryos collected from in vitro fertilization patients. Extracellular ATP was measured by ATP Bioluminescent Assay Kit. Ishikawa cells and T-HESCs were treated with ATP, ATP receptor antagonist, ATP hydrolysis enzyme or inhibitors of ATP metabolic enzymes. The levels of gene expression were evaluated by real-time PCR and immunoassay. RESULTS: We showed that injured human endometrial epithelial cells could rapidly release ATP into the extracellular environment as an important signalling molecule. In addition, blastocyst-derived lactate induces the release of non-lytic ATP from human endometrial receptive epithelial cells via connexins. Extracellular ATP stimulates the secretion of IL8 from epithelial cells to promote the process of in vitro decidualization. Extracellular ATP could also directly promote the decidualization of human endometrial stromal cells via P2Y-purinoceptors. More importantly, the supernatants of injured epithelial cells clearly induce the decidualization of stromal cells in time-dependent manner. CONCLUSION: Our results suggest that ATP should play an important role in human blastocyst-endometrium dialogue for the initiation of decidualization.

Nucleolar stress regulation of endometrial receptivity in mouse models and human cell lines.

Embryo implantation is essential to the successful establishment of pregnancy. A previous study has demonstrated that actinomycin D (ActD) could initiate the activation of mouse delayed implantation. However, the mechanism underlying this activation remains to be elucidated. A low dose of ActD is an inducer of nucleolar stress. This study was to examine whether nucleolar stress is involved in embryo implantation. We showed that nucleolar stress occurred when delayed implantation was activated by ActD in mice. ActD treatment also stimulated the Lif-STAT3 pathway. During early pregnancy, nucleolar stress was detected in the luminal epithelial cells during the receptive phase. Blastocyst-derived lactate could induce nucleolar stress in cultured luminal epithelial cells. The inhibition of nucleophosmin1 (NPM1), which was a marker of nucleolar stress, compromised uterine receptivity and decreased the implantation rates in pregnant mice. To translate these mouse data into humans, we examined nucleolar stress in human endometrium. Our data demonstrated that ActD-induced nucleolar stress had positive effects on the embryo attachment by upregulating IL32 expression in non-receptive epithelial cells rather than receptive epithelial cells. Our data should be the first to demonstrate that nucleolar stress is present during early pregnancy and is able to induce embryo implantation in both mice and humans.

Oncostatin M expression in the mouse uterus during early pregnancy promotes embryo implantation and decidualization.

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family, which functions in embryo implantation and decidualization. The expression, function and regulation of Osm in mouse uteri during early pregnancy remain unknown. We show that Osm is mainly expressed in the uterine epithelium from days 1 to 4 of pregnancy and in decidual cells on day 5 of pregnancy. Osm promotes the attachment of Osm-soaked blue beads, which mimic blastocysts, to a pseudopregnant mouse uterus. Prostaglandin E2 (PGE2 )-induced Osm in mouse uterine epithelial cells upregulates the levels of Il-33 expression and phosphorylates Stat3. In vitro decidualization is significantly promoted by Osm. Our results indicate that PGE2 -induced Osm may mediate embryo implantation through Il-33 and participate in decidualization via the Stat3-Egr1 pathway.