Egr1 protein acts downstream of estrogen-leukemia inhibitory factor (LIF)-STAT3 pathway and plays a role during implantation through targeting Wnt4.

Embryo implantation is a highly synchronized process between an activated blastocyst and a receptive uterus. Successful implantation relies on the dynamic interplay of estrogen and progesterone, but the key mediators underlying embryo implantation are not fully understood. Here we show that transcription factor early growth response 1 (Egr1) is regulated by estrogen as a downstream target through leukemia inhibitory factor (LIF) signal transducer and activator of transcription 3 (STAT3) pathway in mouse uterus. Egr1 is localized in the subluminal stromal cells surrounding the implanting embryo on day 5 of pregnancy. Estrogen rapidly, markedly, and transiently enhances Egr1 expression in uterine stromal cells, which fails in estrogen receptor α knock-out mouse uteri. STAT3 is phosphorylated by LIF and subsequently recruited on Egr1 promoter to induce its expression. Our results of Egr1 expression under induced decidualization in vivo and in vitro show that Egr1 is rapidly induced after deciduogenic stimulus. Egr1 knockdown can inhibit in vitro decidualization of cultured uterine stromal cells. Chromatin immunoprecipitation data show that Egr1 is recruited to the promoter of wingless-related murine mammary tumor virus integration site 4 (Wnt4). Collectively, our study presents for the first time that estrogen regulates Egr1 expression through LIF-STAT3 signaling pathway in mouse uterus, and Egr1 functions as a critical mediator of stromal cell decidualization by regulating Wnt4.

[Effect of Photoaging on Adsorption of Cu(â ¡) by Polystyrene Microplastics with Different Particle Sizes].

In order to evaluate the effect of aging and particle size on the adsorption of heavy metals by microplastics, the adsorption behavior of Cu(Ⅱ) by three different particle sizes of polystyrene (PS; 1, 50, and 100 µm) under UV irradiation was systematically studied. The results demonstrated that UV aging significantly changed the surface morphology and physicochemical properties of PS, and 1 µm PS had the strongest aging degree. The adsorption kinetics of PS on Cu(Ⅱ) conformed to the pseudo-second-order kinetic model, and the Freundlich model was more suitable for the experimental data of isothermal adsorption of Cu(Ⅱ) by PS. These results indicated that the adsorption of Cu(Ⅱ) by PS occurred on the non-uniform surface of PS, and the adsorption behavior was multilayer adsorption. Parameter "n" of the Freundlich model was less than 1, indicating that the adsorption behavior of PS on Cu(Ⅱ) was a higher intensity physical adsorption behavior. The order of theoretical maximum adsorption capacity of different particle sizes PS for Cu(Ⅱ) was as follows:1 µm > 50 µm > 100 µm, indicating that the size of PS was an important influence factor for the adsorption capacity of PS to pollutants. For the same particle size PS, aging enhanced its adsorption capacity for Cu(Ⅱ). The results on the adsorption of Cu(Ⅱ) by PS under different environmental conditions indicated that the adsorption capacity of PS for Cu (II) increased with the increase in pH, whereas an increase in salinity had the opposite effect. Surface complexation and electrical adsorption were the main mechanisms of adsorption of Cu(Ⅱ) by PS. This study provides an important scientific basis for understanding the adsorption behavior of microplastics to heavy metals in the environment.

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice.

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.

[Analysis of Critical Source of Potentially Harmful Elements in Urban Road Dust During Winter in Taiyuan Based on Multiple Attribute Decision Making Method].

In order to analyze the critical sources of potential harmful elements (PHEs) in road dust from Taiyuan during winter, 40 road dust samples were collected. The contents of PHEs, including As, Cd, Pb, Ni, Cr, Cu, and Zn, in the road dust samples were measured using inductively coupled plasma mass spectrometry and atomic fluorescence spectrometry. The ecological risks and human health risks posed by dust PHEs were assessed using NIRI and a health risk evaluation model recommended by USEPA, respectively. The sources of dust PHEs were identified by using the combination of principal component analysis and positive matrix factorization (PMF); the total PHE contents and the ecological risks and human health risks posed by PHEs in dust were apportioned to the PHE sources based on the PMF results; subsequently, the critical source of dust PHEs was determined using the multiple attribute decision making method (MADM). The results demonstrated that: 1 the average concentrations of As, Cd, Pb, Ni, Cr, Cu, and Zn were 17.92, 0.32, 69.10, 30.06, 107.74, 73.37, and 268.49 mg·kg-1, respectively, which were higher than the corresponding background values of soil in Taiyuan, indicating that the PHEs had accumulated in road dust; the mean value of NIRI was 63.86, demonstrating that PHEs in dust posed moderate risks, and the dust PHEs pollution was controllable. 2 Human health risk assessment indicated that exposure to PHEs in dust did not pose serious non-carcinogenic or carcinogenic risks. Ingestion was the most important pathway for exposure to PHEs in road dust that damages human health, and As and Cr have been found to pose the most risks among the seven PHEs. 3 The present study found three main sources of PHEs measured in the dust: natural, traffic, and industrial, which accounted for 35.95%, 40.25%, and 23.82% of the total concentrations of PHEs, respectively. 4 Industrial emissions contributed the least to the total PHEs contents in dust; however, the PHEs released from industrial sources caused relatively high risks, with the results of MADM indicating that industrial sources were the most critical source for dust PHEs. Our results indicated that the critical source identification of PHEs, which was determined to be the most pernicious source, could provide reference for subsequent pollution source control.

Two new genera (Vittiblatta gen. nov. and Planiblatta gen. nov.) of Blattinae (Blattodea, Blattidae) from Southwest China and the discovery of chirally dimorphic male genitalia in Vittiblattapunctata sp. nov.

This study examines Blattinae samples from Southwest China collected in recent years. Based on morphological characters, we establish two genera, Vittiblattagen. nov. and Planiblattagen. nov., and describe four new species, Vittiblattapunctata Luo & Wang, sp. nov., Vittiblattaferruginea Luo & Wang, sp. nov., Vittiblattaundulata Luo & Wang, sp. nov., and Planiblattacrassispina Luo & Wang, sp. nov. These two new genera resemble Periplaneta s.s., but are easily distinguished from it and other genera of Blattinae by morphological characters (genital sclerite L4C). Our results indicate that sclerites L4C and R1G of male genitalia might be important in species delimitation of Blattinae. In addition, chiral dimorphism is found in male genitalia of Vittiblattapunctata sp. nov.

Differential expression and regulation of integral membrane protein 2b in rat male reproductive tissues.

AIM: To examine the expression and regulation of integral membrane protein 2b (Itm2b) in rat male reproductive tissues during sexual maturation and under different treatments by in situ hybridization. METHODS: Testis, epididymis, and vas deferens were collected on days 1-70 to examine Itm2b expression during sexual maturation. To further examine the regulation of Itm2b, adult rats underwent surgical castration and cryptorchidism. Ethylene dimethane sulfonate and busulfan treatments were carried out to test the regulation of Itm2b after destruction of Leydig cells and germ cells. RESULTS: In testis, Itm2b expression was moderately detected in the adluminal area of seminiferous cords on days 1-10, and detected at a low level in the spermatogonia on days 20 and 30. The Itm2b level was markedly increased in Leydig cells from day 20 to day 70. In epididymis and vas deferens, Itm2b was detected from neonate to adults, and the signal gradually increased in accordance with sexual maturation. Itm2b expression was significantly downregulated in epididymis and vas deferens of castrated rats, and strongly stimulated when castrated rats were treated with testosterone. Cryptorchidism led to a significant decline of Itm2b expression in testis and caput epididymis. Itm2b expression in epididymis and vas deferens was significantly decreased after the Leydig cells were destroyed by ethylene dimethane sulfonate. Busulfan treatment produced no obvious change in Itm2b expression in epididymis or vas deferens. CONCLUSION: Our data suggested that Itm2b expression is upregulated by testosterone and might play a role in rat male reproduction.

Differential expression and regulation of prostaglandin E synthases in the mouse ovary during sexual maturation and luteal development.

Prostaglandin (PGE) 2 is the most common prostanoid and plays an important role in female reproduction. The aim of this study was to examine the expression and regulation of microsomal (m) PGE synthase (PGES)-1 and cytosolic (c) PGES in the mouse ovary during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and immunohistochemistry. Both mPGES-1 mRNA signals and immunostaining were localized in the granulosa cells, but not in the thecal cells and oocytes. cPGES mRNA signals were localized in both granulosa cells and oocytes, whereas cPGES immunostaining was exclusively localized in the oocytes. In our superovulated model of immature mice, there was a basal level of mPGES-1 mRNA signals in the granulosa cells at 48 h after equine chorionic gonadotropin (eCG) treatment. mPGES-1 mRNA level was induced by human chorionic gonadotropin (hCG) treatment for 0.5 h, whereas mPGES-1 immunostaining was slightly induced at 0.5 h after hCG treatment and reached a maximal level at 3 h after hCG treatment. eCG treatment had no obvious effects on either cPGES mRNA signals or immunostaining. A strong level of cPGES immunostaining was present in both unstimulated and eCG-treated groups. Both mPGES-1 mRNA signals and immunostaining were highly detected in the corpus luteum 2 days post-hCG injection and declined from days 3 to 7 post-hCG injection. cPGES immunostaining was at a basal level or not detectable from days 1 to 7 after hCG injection and was highly expressed in the corpus luteum from days 9 to 15 post-hCG injection. PGE2 biosynthesized through the mPGES-1 pathway may be important for follicular development, ovulation and luteal formation.

Progesterone induces the expression of lipocalin-2 through Akt-c-Myc pathway during mouse decidualization.

Lipocalin-2 (Lcn2) is a small glycoprotein involved in a number of biological processes such as inflammation and antibacterial response. In our study, Lcn2 is expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy. The expression of Lcn2 in stromal cells is under the control of progesterone through Akt-c-Myc signaling pathway. Data from Lcn2 knockdown and recombinant protein treatments indicate that Lcn2 promotes mPGES-1 expression in stromal cells. The expression of Lcn2 and mPGES-1 is strongly stimulated by lipopolysaccharide (LPS), indicating that Lcn2 mediates LPS-induced inflammation. These findings shed light on the role of Lcn2 during decidualization.

Non-coding RNA LINC00473 mediates decidualization of human endometrial stromal cells in response to cAMP signaling.

Decidualization is an essential step in the establishment of pregnancy. However, the functional contributions of long intergenic noncoding RNAs (LincRNAs) to decidualization have not been explored. To explore the regulation and role of LincRNAs during human decidualization, human endometrial stromal cells (HESCs) are induced to undergo in vitro decidualization by treating with estradiol-17ß, db-cAMP and medroxyprogesterone acetate. LINC00473 (LINC473) expression is highly induced in HESCs after decidual stimulus. We found that cAMP-PKA pathway regulates the expression of LINC473 through IL-11-mediated STAT3 phosphorylation. RNA interference-mediated down-regulation of LINC473 inhibits in vitro decidualization. These results suggested that LINC473 might be functionally required for human decidualization. This is the first report demonstrating the presence of LincRNA during human decidualization.

Regulation and function of deiodinases during decidualization in female mice.

Thyroid dysfunction during human pregnancy is closely related to serious pregnancy outcome. However, the regulation and function of thyroid hormones during early pregnancy are largely unknown. We found that type II deiodinase, an enzyme converting T4 to activated T3, is highly expressed in the mouse uterus on days 3 and 4 of pregnancy. Once the embryo implants into the receptive uterus, type III deiodinase (Dio3), a mainly paternally imprinted gene for inactivating T3, is significantly induced in the stromal cells and accompanied by DNA hypermethylation of intergenic differentially CpG methylation regions in the δ-like 1 homolog-Dio3 imprinting cluster. The concentration of uterine free T3 is actually decreased after embryo implantation. T3 induces Dio3 expression both in vivo and in vitro, suggesting a positive feedback loop. T3 addition or Dio3 knockdown compromises decidualization. These results indicate that the Dio3-mediated local T3 decrease is critical for decidualization of stromal cells during early pregnancy. Furthermore, we found that progesterone regulates Dio3 expression through its cognate receptor both in vivo and in vitro. Additionally, cAMP regulates Dio3 transcription through the protein kinase A-cAMP response element-binding protein pathway. The inhibition of the protein kinase A pathway results in decreased Dio3 expression and impaired decidualization. Dio3 opposite strand (Dio3os) expressed in a similar pattern to Dio3, is transcribed from the opposite strand of Dio3 and fine-tunes Dio3 expression during decidualization. Our data indicate that Dio3 is strongly expressed and tightly controlled during decidualization.

Cyclic adenosine monophosphate-induced argininosuccinate synthase 1 expression is essential during mouse decidualization.

L-Arginine (L-Arg), a conditional essential amino acid in adults, has been shown to enhance pregnancy outcome. Argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl) are the key enzyme for L-Arginine (L-Arg) biosynthesis. Based our microarray analysis, Ass1 expression is upregulated significantly at implantation site on day 5 of pregnancy compared to that at inter-implantation site. However, the expression, regulation and function of Ass1 during early pregnancy remain unknown. Here we found that Ass1 is highly expressed in mouse decidua and uterine stromal cells undergoing decidualization, and Asl is weakly expressed in mouse decidua and uterine stromal cells undergoing decidualization. α-Methyl-DL-aspartic acid (MDLA), a specific inhibitor for Ass1, can significantly increase the rate of embryonic reabsorption. Under in vitro induced decidualization, MDLA clearly inhibits the expression of decidual/trophoblast prolactin-related protein (Dtprp), a marker for decidualization in mice. Only Ass1 expression is induced by cAMP through PKA/p-Creb signaling pathway. Results from our cell culture models further indicates that the high level of L-Arg enhances stromal proliferation, while enzymatic activity or Ass1 expression level is essential to determine the magnitude of both mouse and human decidualization. Interestingly, L-Arg at high concentration down-regulates Ass1 and Asl expression by negative feedback to maintain L-Arg homeostasis. These findings highlight that cAMP-induced Ass1 expression is important in controlling the magnitude of decidualization through regulating L-Arg level.

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization.

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H2O2). Based on our data, GPX3 may play a major role in reducing H2O2 during decidualization.

The mesenchymal-epithelial transition during in vitro decidualization.

The epithelial-mesenchymal transition plays a critical role in embryonic development, cancer progression, and metastasis. Decidualization is the process by which the fibroblast-like endometrial stromal cells differentiate into polygonal epithelial-like cells. However, it is still unclear whether mesenchymal-epithelial transition (MET) occurs during decidualization. The aim of this study was to examine whether decidualization causes the downregulation of some mesenchymal markers and upregulation of some epithelial markers in cultured uterine stromal cells. We showed that decidualization causes the downregulation of snail and vimentin expression, and upregulation of E-cadherin and cytokeratin expression. During in vitro decidualization, cultured stromal cells lose elongated shape and show epithelium-like characteristics. Our data suggest that the process of MET may exist during decidualization.

Comparative expression and regulation of TMSB4X in male reproductive tissues of rats and chickens.

Thymosin ß4 (TMSB4X) belongs to a class of highly conserved small proteins that are present in a high abundance in immune tissues, where it participates in various biological activities, including anti-inflammation, wound healing, apoptosis, and cell survival. However, little is known about the expression and regulation of TMSB4X in reproductive tissues. The aim of this study was to examine the expression of rat Tmsb4x and chicken TMSB4X genes during testicular and epididymal development. Rat Tmsb4x was strongly detected in the spermatogonia and spermatocytes of testis at early postnatal development and transmitted to Leydig cells at sexual maturation. Also, rat Tmsb4x was detected at an increased level in the epididymis during postnatal development. When compared to the rat, expression of the chicken TMSB4X gene was low in the testis and epididymal region, and the mRNA localization was also unexpected. Three experiments were performed to examine the regulation of rat Tmsb4x in the epididymis: after elimination of Leydig cells using ethylene dimethane sulfonate (EDS); after destruction of the testis by cryptorchidism; and after castration. EDS-treated and castrated rats were injected with testosterone propionate. The expression of Tmsb4x was significantly reduced in the epididymis of EDS-treated, and castrated rats. In contrast, Tmsb4x was significantly enhanced in the epididymis after testosterone treatment. The expression of rat Tmsb4x was also regulated in the epididymis after cryptorchidism. Collectively, the expression of Tmsb4x was strongly detected in the testis and epididymis of rats, and was highly regulated in the epididymis by testosterone.

Progesterone regulation of Na/K-ATPase ß1 subunit expression in the mouse uterus during the peri-implantation period.

Luminal closure and embryo apposition are essential for blastocyst attachment during early pregnancy. In our preliminary microarray results (unpublished data), sodium-potassium adenosine triphosphatase (Na/K-ATPase) ß1 (Atp1b1) was highly expressed in mouse uterus on Days 3 and 4 of pregnancy. However, expression and regulation of Atp1b1 in the mammalian uterus during early pregnancy are unknown. Using in situ hybridization, a strong level of Atp1b1 mRNA was detected in luminal epithelial cells on Days 3 and 4 of pregnancy (Day 1 = day of vaginal plug). The expression pattern of FXYD domain-containing ion transport regulator 4 (Fxyd4) was similar to that of Atp1b1. Real-time reverse transcription polymerase chain reaction confirmed the high expression level of Atp1b1 mRNA. Compared with Day 1, the mRNA level of Atp1b1 on Days 3 and 4 increased by 3.5 ± 0.5 and 4.5 ± 0.5 fold, respectively. When the embryo invaded through epithelial cells into the maternal stromal compartment on day 5, Atp1b1 expression decreased to a basal level. Progesterone stimulated Atp1b1 expression by 2.8 ± 1 fold compared with oil in ovariectomized mice at 24 hours after treatment. Expression of Atp1b1 was further upregulated to 4 ± 0.4 fold by estrogen and progesterone. Based on time-course study, progesterone rapidly induced Atp1b1 expression at 6 and 12 hours (13.7 ± 0.5 and 16.6 ± 1.4, respectively); furthermore, this upregulation was blocked by RU486 (progesterone receptor antagonist). Transcription activity of the Atp1b1 promoter was (Day 1 = day of vaginal plug) stimulated by CCAAT/enhancer binding protein beta (Cebpb). In conclusion, Atp1b1 was highly expressed in luminal epithelium during peri-implantation and upregulated by progesterone.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) induction on Snail expression during mouse decidualization.

Embryo implantation requires a precise synchronism between the receptive uterus and activated blastocyst and is regulated by complicated molecular networks. Although many implantation-related genes have been identified, the crosstalk among them is still unknown. Snail, a transcription repressor, plays a central role during epithelial-mesenchymal transition. Our previous study showed that Snail is highly expressed at implantation site in mouse uterus. This study was to examine how Snail is related with other implantation-related genes in mice. Uterine stromal cells were isolated from mouse uteri on day 4 of pregnancy and treated with HB-EGF. Snail was induced significantly by HB-EGF. By using specific inhibitors and siRNA, we demonstrated that HB-EGF induction on Snail expression is dependent on the EGFR-ERK-Stat3 pathway. Cox-2 was regulated by Snail. The current findings demonstrate that Snail can relate with HB-EGF, Stat3 and Cox-2 and may play a role during mouse embryo implantation and decidualization.

Arachidonic acid regulation of the cytosolic phospholipase A 2α/cyclooxygenase-2 pathway in mouse endometrial stromal cells.

OBJECTIVE: To investigate the role of arachidonic acid (AA) in mouse endometrial stromal cells. DESIGN: Experimental animal study. SETTING: University research laboratory. ANIMAL(S): Sexually mature female CD1-strain mice. INTERVENTION(S): Primary culture of endometrial stromal cells. MAIN OUTCOME MEASURE(S): Western blot and real-time polymerase chain reaction for gene expression and/or phosphorylation analysis. Luciferase assay for Cox-2 promoter analysis. RESULT(S): AA-derived prostaglandins play important roles during embryo implantation and decidualization. However, the function of AA itself in reproduction is largely unknown. In this study, exogenous AA stimulated cPLA(2α) phosphorylation and COX-2 expression, mainly through ERK1/2 in mouse endometrial stromal cells, and p38 inhibitor modestly inhibited cPLA(2α) phosphorylation induced by AA. The induction of COX-2 by AA was diminished by short interfering RNA against C/EBPß and inhibitory C/EBPß (LIP). C/EBPß binding site at -872--864 of Cox-2 promoter contributes to Cox-2 promoter activation induced by C/EBPß transfection. The expression of C/EBPß protein induced by AA was inhibited by p38 inhibitor, and the phosphorylation of C/EBPß induced by AA was inhibited by p38 inhibitor and ERK1/2 inhibitor. A nonmetabolized analogue of AA (ETYA) also enhanced cPLA(2α) phosphorylation and COX-2 expression. The activation of cPLA(2α)/COX-2 by AA was not inhibited by COX inhibitor indomethacin. CONCLUSION(S): AA can induce cPLA(2α)/COX-2 pathway activation in mouse endometrial stromal cells.

Estrogen regulates amiloride-binding protein 1 through CCAAT/enhancer-binding protein-beta in mouse uterus during embryo implantation and decidualization.

Embryo implantation is an intricate interaction between receptive uterus and active blastocyst. The mechanism underlying embryo implantation is still unknown. Although histamine and putrescine are important for embryo implantation and decidualization, excess amount of histamine and putrescine is harmful. Amiloride binding protein 1 (Abp1) is a membrane-associated amine oxidase and mainly metabolizes histamine and putrescine. In this study, we first showed that Abp1 is strongly expressed in the decidua on d 5-8 of pregnancy. Abp1 expression is not detected during pseudopregnancy and under delayed implantation but is detected after estrogen activation. Because Abp1 is mainly localized in the decidua and also strongly expressed during in vitro decidualization, Abp1 might play a role during mouse decidualization. The regulation of estrogen on Abp1 is mediated by transcription factor CCAAT/enhancer-binding protein-ß. Abp1 expression is also regulated by cAMP, bone morphogenetic protein 2, and ERK1/2. Abp1 may be essential for mouse embryo implantation and decidualization.