Search

The aim of this study was to investigate whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway affects the implantation of mouse embryos by regulating the expression of RhoA. The expression of PI3K, Akt, phosphorylated (p-)Akt, phosphatase and tensin homolog (PTEN) and RhoA in the uterus of mice on day 5 of pregnancy (D5) and in pseudopregnant mice was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot analysis. A functional analysis of these genes was also performed by the intrauterine injection with the PI3K inhibitor, LY294002, on day 2 of pregnancy (D2). The expression levels of PI3K, p-Akt, RhoA at the implantation site were higher than those at the inter-implantation site in the endometrium; however, opposite effects were observed for PTEN expression. The expression levels of the above genes in the pseudopregnant group and in the group injected with the PI3K/Akt inhibitor, LY294002, were markedly lower than those in the pregnant group. Functional experiments revealed that the number of implantation sites had been significantly decreased (P

Chemical reactions with products of high reactivity generally suffer from low yields due to the severe side reactions. This is the case for the synthesis of acrolein from glycerol dehydration. To improve the yield of acrolein, fast removal of high reactive acrolein molecules from the reaction system is desirable but remains a major challenge, due to the high viscosity of glycerol. To tackle this problem, the Pickering emulsion was chosen as the reaction system in this work to accelerate the removal of acrolein. A water-in-oil Pickering emulsion was fabricated for the liquid phase dehydration of glycerol to acrolein, which exhibits twice yield of acrolein compared with the homogeneous system. The combination of the transfer experiments and IR analysis confirmed the drastic restrain of the side reactions between acrolein and glycerol, due to the intensified mass transfer of acrolein from the liquid phase by the larger microinterface of emulsion. Furthermore, the transfer rate of acrolein through the water-oil interface can be tuned via a shift in the interfacial properties of the silica emulsifier by silylation treatment. The highest acrolein yield of nearly 50% was obtained with the highest transfer rate of acrolein on the silica silylated by (MeO)3Si(CH2)11CH3.

Freeze-drying of dilute polymer solution is a widely accepted method to yield disentangled polymer. By this method, it has been pointed out that samples recovered from dilute solution possess a better crystallization ability than the bulk polymer. Here, the important role of the critical overlap concentration c* in polymer solution is firstly stressed to give a more quantitative understanding in the relation between entanglement state and melt crystallization behavior. Crystallization kinetics of poly (lactic acid) freeze-extracted from dichloromethane solution with various concentration below and above c*, as well as the crystalline structure after isothermal crystallization at 110 °C, were investigated by means of differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS). Results indicated that crystallization kinetics and lamellar thickness (Lc) show a strong dependence on precursor solution concentration only when the concentration is lower than the critical value (c*), while above c* the crystallization behavior changes a little. Besides, a more rational method to characterize the chain entanglement state in polymer solution and to select concentrations accordingly was suggested, that is, using a dimensionless variable [η]c which is the product of intrinsic viscosity [η] and solution concentration c.

Recognition of the harmful effects of sodium fluoride (NaF) on human reproduction is increasing, especially as it relates to female reproduction. However, the mechanism by which NaF interferes with female reproduction is unclear. The aims of the present study were to investigate the effects of fluoride exposure on female fertility and to elucidate the mechanisms underlying these effects. Female Sprague-Dawley rats were divided into three groups: one control group and two NaF-treated groups (100 and 200 mg/L in the drinking water for 12 weeks). Several parameters were evaluated, including: (i) fluoride concentrations; (ii) estrogen (E2) and progesterone (P) concentrations; (iii) estrogen receptor alpha protein (ERα); (iv) progesterone receptor (PgR) protein; (v) follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) protein. The results indicated that administration of NaF lead to significant decreases in E2 and P levels in the serum and in the expression of FSHR protein. In addition, fluoride exposure significantly increased Erα and PgR protein expression levels and LHR protein expression. These results suggest that the reproductive hormone reduction and the abnormalities of related receptor proteins expression are important factors underlying the decreased fertility observed in female rats that have been exposed to NaF.

A new electrochemical sensor based on a novel organic-inorganic material (PNFCTs) was proposed for detection of paracetamol in this paper. First, PNFCTs were prepared with multi-walled carbon nanotubes (MWNTs) and a derivative of 3,4,9,10-perylenetetracarboxylic dianhydride (PTC-NH2) via cross-linking method. Then, PNFCTs were coated onto the surface of the glassy carbon electrode (GCE) to form porous organic conducting polymer films (PNFCTs/GCE), which could not only increase the loading of paracetamol efficiently but also provide an interface with exceptional electrical conductivity for paracetamol. Finally, gold nanoparticles (GNPs) were attached to the electrode surface through electrodepositing method, which obtained GNPs/PNFCTs/GCE electrode. The electrochemical behavior of paracetamol on GNPs/PNFCTs/GCE was explored by cyclic voltammetrys (CVs) and differential pulse voltammograms (DPVs). The results showed that the GNPs/PNFCTs/GCE exhibited excellent electrocatalytic activity to paracetamol, which should be attributed to remarkable properties of the new composite nanomaterials with porous nanostructure and exceptional electrical conductivity. The wide liner range and detection limit were 0.3-575 and 0.1 μM, respectively. Finally, it was successfully used to detect paracetamol in dilution human serum and commercial tablets. The sensor shows great promise for simple, sensitive, and selective detection paracetamol and provides a promising approach in paracetamol clinical research and overdose diagnostic applications.

Benzo(a)pyrene (B[a]P) is an environmental carcinogen that induces tumors in many animal species, but the neurotoxic effects of B[a]P have not been well studied. In the present study, we investigated the effects of subacute exposure to B[a]P in Sprague-Dawley (SD) rats. Male rats received daily injections of either B[a]P (0, 1, 2.5, or 6.25 mg/kg, i.p.) or vehicle for 45 days. Exposure to B[a]P affected the behavior of rats in the Morris water maze test. Gene microarray and real-time PCR analyses revealed that exposure to B[a]P affected signal transduction in the rat hippocampus. Protein microarray analysis revealed that altered protein expression played a role in cell death in the functional annotation cluster analysis. Finally, major vault protein was found to display low cDNA and protein expression levels. The present study explored some of the possible mechanisms underlying B[a]P neurotoxicity and provided evidence that B[a]P plays a neurotoxic role in rats.

The direct conversion of chitin biomass to 5-hydroxymethylfurfural (5-HMF) in ZnCl2 aqueous solution was studied systemically. d -Glucosamine (GlcNH2) was chosen as the model compound to investigate the reaction, and 5-HMF could be obtained in 21.9% yield with 99% conversion of GlcNH2. Optimization of the reaction parameters including the screening of 8 co-catalysts was carried out. Among them, AlCl3 and B(OH)3 improved 5-HMF yield, whereas CdCl2, CuCl2 and NH4Cl had no effect. CrCl3, SnCl4 and SnCl2 showed negative effects, i.e. lower yields. Consequently, the optimal reaction conditions were found to be 67 wt.% ZnCl2 aqueous solution, at 120 °C without co-catalyst. The reactions were further studied by in situ NMR, and no intermediate or other byproducts, except humins, were observed. Finally, the substrate scope was expanded from GlcNH2 to N-acetyl- d -glucosamine and various chitosan polymers with different molecular weights, 5-HMF yield from polymers were generally lower than that from GlcNH2.

The aim of this study was to investigate the effects of sodium fluoride (NaF) on female reproductive function and examine the morphology of the ovaries and uteri of rats exposed to NaF. Eighty female Sprague-Dawley (SD) rats were divided randomly into four groups of 20: one control group and three NaF treated groups. The three NaF treated groups received 100, 150, and 200 ppm, respectively, of NaF for 6 months via their drinking water, while the control group (GC) received distilled water. The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), progesterone (P) and estradiol (E2) were measured using an enzyme-linked immunosorbent assay. Pathomorphological evaluation of the uteri and ovaries was conducted after staining with hematoxylin-eosin and immunohistochemistry. The rate of successful pregnancy in the NaF-treated groups declined in a dose-dependent manner. The concentration of reproductive hormones was significantly lower in the three NaF-treated groups, and the endometrium was damaged. The maturation of follicles was inhibited. In addition, the total number of follicles of all types was significantly lower in the NaF-treated groups. These results suggest that female reproductive function is inhibited by NaF and that exposure to NaF causes ovarian and uterine structural damage. NaF may thus significantly reduce the fertility of female rats.

DMSO can improve the selectivity of 5-hydroxymethylfurfural (HMF) in the conversion of carbohydrates. However, one of the bottlenecks in its application is product separation. Thus a one-pot synthesis of 5-ethoxymethylfurfural (EMF) rather than HMF from fructose in ethanol–DMSO was investigated. Phosphotungstic acid was used as an effective catalyst. The yield of EMF can be reached as high as 64% in the mixed solvent system of DMSO and ethanol within 130 min at 140 °C. Ethyl levulinate (LAE) was detected as the main by-product, the yield of which increased with the reaction time, temperature and the amount of catalyst. In addition, the existence of water could significantly reduce the yield of EMF and increased the yield of LAE. Most importantly, it was discovered that EMF could be much more efficiently extracted from the reaction solvent system by some organic solvents than HMF.

Salt effects on the aggregation behavior of tripolar zwitterionic surfactants in aqueous solutions have been investigated using surface tension, dynamic light scattering (DLS), freeze-fracture transmission electron microscopy (FF-TEM), and 1H NMR. The tripolar zwitterionic surfactants with different inter-charge spacers are [C14H29(CH3)2N+CsN+(CH3)2CH2CH2CH2SO3 −]Br− (C14CsTri, Cs = –(CH2)2–, –(CH2)6–, –(CH2)10–, and p-xylyl). It is found that the critical micelle concentration (CMC) values of the corresponding traditional zwitterionic surfactant C14H29(CH3)2N+CH2CH2CH2SO3 − (TPS) are almost constant with the increase of the NaBr concentration. However, the CMC values of C14CsTri decrease sharply at a lower NaBr concentration and then level off at a higher NaBr concentration. Moreover, the decreasing extents of the CMC values for C14C2Tri, C14C6Tri, and C14CpxTri are very close, but more significant than that for C14C10Tri, suggesting that the self-assembly ability of the tripolar zwitterionic surfactants with a longer inter-charge spacer is less sensitive to NaBr. The DLS and FF-TEM results reveal that C14C2Tri, C14C6Tri, and C14CpxTri form micelles without NaBr and that the size slightly increases with the increase of NaBr concentration, whereas micelles and vesicles coexist for C14C10Tri and TPS without NaBr and then transfer to micelles upon the addition of NaBr. The salt-induced morphological transition for C14C10Tri is further studied using 1H NMR. The addition of NaBr reduces both the electrostatic repulsion between the same charged ammoniums and the electrostatic attraction between the oppositely charged ammonium and sulfonate. Thus, the longer inter-charge spacer of C14C10Tri tends to be more bended and the sulfonate group becomes available to contact the ammonium, which promotes micellization.

Studies have shown that metabolic syndrome is associated with reproductive problems. Women with metabolic syndrome, characterized by hyperinsulinemia, have common ovarian dysfunction, but the mechanism remains elusive. The aim of this research is to explore the effects of high levels of insulin on ovary function during early pregnancy. Mice in the high insulin-exposed group were given a subcutaneous injection of human recombinant insulin. After insulin treatment, changes in various hormones were tested using ELISA kits which showed hormones secreted by the ovary were significantly altered in the insulin group. TUNEL staining showed less TUNEL-positive cells in the insulin group. A significant decrease in Bax and an increase in Bcl2 in the ovary were found in the insulin group by immunohistochemical studies. Western blotting showed the expressions of apoptosis related proteins in the ovaries from the insulin group were obviously altered. In addition, expression of p-Akt proteins in the ovaries from the insulin group was significantly upregulated. Moreover, the Akt inhibitor LY294002 reversed the anti-apoptotic effects of high insulin in the ovary tissues in early pregnancy mice. All of these results showed that insulin impaired ovarian function during early pregnancy and ovarian apoptosis is imbalanced under the role of insulin. The PI3K/AKT signalling pathway might participate in this process.

Low temperature

// Xi Lan 1 , Li-Juan Fu 1, 2 , Jun Zhang 3 , Xue-Qing Liu 1 , Hui-Jie Zhang 4 , Xue Zhang 1 , Ming-Fu Ma 5 , Xue-Mei Chen 1 , Jun-Lin He 1 , Lian-Bing Li 5 , Ying-Xiong Wang 1 and Yu-Bin Ding 1 1 Department of Reproductive Biology, School of Public Health, Chongqing Medical University, Chongqing, 400016, P.R. China 2 Department of Immunology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA 3 Center of Molecular Diagnostic Medicine, Life Science Institute, Chongqing Medical University, Chongqing, 400016, P.R. China 4 Ministry of Education Key Laboratory of Diagnostic Medicine, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, P.R. China 5 The Key Laboratory of Birth Defects and Reproductive Health of the National Health and Family Planning Commission, Chongqing Population and Family Planning Science and Technology Research Institute, Chongqing, 401147, P.R. China Correspondence to: Yu-Bin Ding, email: 178384198@qq.com Keywords: bisphenol A, extravillous trophoblasts, cell migration, placentation, MAPK/PI3K signaling pathway Received: September 28, 2016 Accepted: April 19, 2017 Published: May 16, 2017 ABSTRACT In this study, we investigated the effect of Bisphenol A (BPA), an endocrine-disrupting chemical, on the migration of human trophoblasts and mouse placentation by using the primary extravillous trophoblast (EVT) and its cell line HTR-8/SVneo, villous explant cultures, and pregnant mice. BPA increased EVT motility and the outgrowth of villous explants in a dose-dependent manner. BPA also increased the protein levels of integrin-β1 and matrix metalloproteinase (MMP)-9 in human EVTs. Low-dose BPA (≤50 mg) increased the protein levels of MMP-9 and MMP-2 as well as integrin-β1 and integrin-α5 in mouse placenta and decreased the proportion of the labyrinth and spongiotrophoblast layers. Inhibitors of mitogen-activated protein kinase (MAPK) U0126 and phosphatidylinositol-3-kinases (PI3K) LY294002 reversed the protein levels of integrin-β1 and MMP-9 as well as the migratory ability induced by BPA. In conclusion, these results indicated that BPA can enhance trophoblast migration and impair placentation in mice by a mechanism involving upregulation of integrin(s) and MMP(s) as well as the stimulation of MAPK and PI3K/Akt (protein kinase B) signaling pathways.

Ubiquitination is a regulatory mechanism that occurs after protein translation. To date, few studies have reported on ubiquitination during embryo implantation. We used real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blotting analyses to analyze the expression of speckle-type pox virus and zinc finger (POZ) protein (SPOP; an adapter of E3 ligases of ubiquitination) in mouse uteri during early pregnancy and pseudopregnancy using an artificially induced decidualization model and a steroid hormone–processing model. At the same time, we established an artificially induced decidualization in vitro model. We observed that SPOP regulates endometrial stromal cell decidualization in mice and that hormones regulate the expression of SPOP. This study suggests that ubiquitination may be involved in embryonic implantation.

STUDY QUESTION Does nm23 have functional significance in decidualization in mice and humans? SUMMARY ANSWER nm23 affects decidualization via the phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathways in mouse endometrial stromal cells (ESCs; mESCs) and human ESCs. WHAT IS KNOWN ALREADY The function of nm23 in suppressing metastasis has been demonstrated in a variety of cancer types. nm23 also participates in the control of DNA replication and cell proliferation and differentiation. STUDY DESIGN, SIZE AND DURATION We first analyzed the expression profile of nm23 in mice during early pregnancy (n = 6/group), pseudopregnancy (n = 6/group) and artificial decidualization (n = 6/group) and in humans during the menstrual cycle phases and the first trimester. We then used primary cultured mESCs and a human ESC line, T-HESC, to explore the hormonal regulation of nm23 and the roles of nm23 in in vitro decidualization, and as a possible mediator of downstream PI3K-Akt-mTOR signaling pathways. PARTICIPANTS/MATERIALS, SETTINGS AND METHODS We evaluated the dynamic expression of nm23 in mice and humans using immunohistochemistry, western blot and real-time quantitative RT-PCR (RT-qPCR). Regulation of nm23 by steroid hormones was investigated in isolated primary mESCs and T-HESCs by western blot. The effect of nm23 knockdown (using siRNA) on ESC proliferation was analyzed by 5-ethynyl-2'-deoxyuridine staining (EdU) and proliferating cell nuclear antigen protein (PCNA) expression. The influence of nm23 expression on the differentiation of ESCs was determined by RT-qPCR using the mouse differentiation markers decidual/trophoblast PRL-related protein (dtprp, also named prl8a2) and prolactin family 3 subfamily c member 1 (prl3c1) and the human differentiation markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The effects of nm23 siRNA (si-nm23) and the PI3K inhibitor LY294002 on the downstream effects of nm23 on the PI3K-Akt-mTOR signaling pathway were estimated by western blot. MAIN RESULTS AND THE ROLE OF CHANCE NM23-M1 was specifically expressed in the decidual zone during early pregnancy and in artificially induced deciduoma, and NM23-H1 was strongly expressed in human first trimester decidua. The expression of nm23 was upregulated by oestradiol and progesterone (P

DNA cytosine-5 methylation plays a vital role in regulating the expression of E-cadherin, which is encoded by the CDH1 gene. In this study, we characterised the DNA methylation and expression pattern of CDH1 in an extravillous trophoblast cell line (HTR-8/SVneo) and two trophoblast cell lines ­– JEG-3 and JAR. Promoter hypermethylation with reduced E-cadherin expression in HTR-8/SVneo cells and promoter hypomethylation with increased E-cadherin expression in JEG-3 and JAR cells were observed. Demethylation treatment significantly restored E-cadherin expression, contributing to decreases in the motility and invasiveness of HTR-8/SVneo cells. Sense-methylated oligonucleotides (MONs) labelled with Cy5 and complementary to a region of the human CDH1 promoter were designed, with the cytosines in 5′-cytosine-phosphate-guanine-3′ (CpG) dinucleotides being replaced by methylated cytosines. Following MON transfection into JEG-3 cells, the level of CDH1 promoter DNA methylation as well as cell motility and invasiveness were increased and gene expression was significantly repressed. Our results indicate that MON-mediated DNA methylation of the CDH1 promoter and subsequent alterations in gene expression may contribute to trophoblast motility and invasion, suggesting a potential method for controlling the biological function of trophoblasts in vitro through epigenetic modification.

Full development of a receptive uterus is necessary for embryo implantation; however, many genes that are required for the endometrial modifications that occur during this process remain unidentified. To identify novel genes that control endometrial modifications during this period, we investigated the differential gene expression profile in the endometrium of mice on days 2 (D2) (pre-implantation) and 4 (D4) of pregnancy (i.e., the implantation window) using 17-bp long serial analysis of gene expression (LongSAGE). One hundred fifty-six tags were annotated as unique transcripts. Of these, 101 tags were significantly upregulated, and 55 tags were downregulated in the D4 library relative to the D2 library. These differentially expressed genes should therefore be of increased importance in the establishment of uterine receptivity. The differential expressions of certain of the identified genes, namely, Hspa8, Tctp, Sparc, Ifitm1, Ik, serbp1 and Dnmt1, were validated by semi-quantitative RT-PCR and/or immunohistochemistry. Functional grouping analysis classified 86 of the mapped tags into 17 categories, which are closely associated with morphological modifications of the endometrium during pregnancy. Ingenuity pathways analysis revealed that the identified differentially expressed genes fell into six primary networks, which themselves contain numerous factors that are related to key modulators of signaling pathways that are vital for endometrial modifications. These findings will aid in the further understanding of the molecular events that underlie the implantation physiology in mice.

With the increase of environment temperature, more and more attentions are payed to the effects of heat stress. Cells under heat shock either are adapted to the condition or are damaged and dead. In this paper, we found that heat shock induced endoplasmic reticulum (ER) stress. ATF4, PERK, and IRE1α were induced by heat shock of 45 °C in the transcriptional level. Under the stress of 45 °C, PERK was phosphorylated and XBP1s was detected. The result indicated that heat shock could induce the ER stress. We found that heat shock of 45 °C induced the dysregulation of HSP70 and DNA-PKcs, and downregulated the expression of PARP1 and XRCC1. Further results showed that after the knockdown of ATF4 or IRE1α, the expression of DNA-PKcs and XRCC1 were increased. It was indicated that ATF4 and IRE1α could inhibit the expression of DNA-PKcs and XRCC1 under the heat stress. Our results suggested that heat shock could activate ER stress. IRE1α and ATF4, as the important ER stress molecules, could inhibit the expression of DNA repair proteins DNA-PKcs, XRCC1, and HSP70 under heat shock. Downregulation of DNA repair proteins could aggravate the cell damage that may cause cell apoptosis. This may explain that heat shock could increase the lethality of chemotherapeutic drugs on tumor cells.

Effects of calcium ions on the solubility and aggregation behavior of an anionic sulfonate gemini surfactant 1,3-bis(N-dodecyl-N-propylsulfonate sodium)-propane (12-3-12(SO(3))(2)) have been studied in aqueous solution. Compared with single-chain surfactant sodium dodecylsulfate, 12-3-12(SO(3))(2) shows much better performance to the hardness tolerance with calcium ions. Moreover aggregates of the Ca(2+)/12-3-12(SO(3))(2) complexes in clear solutions influence the morphologies of the precipitates. At 12-3-12(SO(3))(2) concentrations lower than 1.5 mM, the small spherical micelles of Ca(2+)/12-3-12(SO(3))(2) in clear solutions generate precipitates of solid particles owing to complexation of surfactant monomers with Ca(2+). At 12-3-12(SO(3))(2) concentrations higher than 1.5mM, the Ca(2+)/12-3-12(SO(3))(2) complexes transform into large compact spherical aggregates and then into long wormlike micelles. These large aggregates are well dispersed in aqueous solutions and efficiently complex calcium ions. In particular, long wormlike micelles are entangled with each other at 100.0 mM CaCl(2) and 100.0 mM 12-3-12(SO(3))(2) exhibiting viscoelastic properties. In addition, the stacking of long wormlike micelles produces precipitates with ordered fibrillar structures. This work reveals that such anionic sulfonate gemini surfactants are better candidates than single-chain surfactants in applications with high hardness levels, and the ordered aggregate structures may have potential applications in materials science.

We have characterized the uterine expression of DNA methyltransferases (DNMTs) during early pregnancy in mice and determined whether a folate-deficient diet (FDD) can affect DNMTs in this context. Within endometrial cells, expressions of DNMT (cytosine-5) 1 (Dnmt1),Dnmt3a, andDnmt3bwere significantly elevated during the prereceptive phase of pregnancy but generally returned to baseline levels during receptive and postimplantation periods. As such, the transcription of DNMT genes is temporally regulated during early pregnancy. When comparisons were made between implantation sites (IS) and inter-IS on day 5 of pregnancy, lower levels ofDnmt3awere detected at IS. Comparisons between IS and inter-IS did not reveal significant expression differences for other DNMT genes. When tissue sections were examined, DNMT3A was specifically lower in the stroma of IS. Reduced DNMT1 and DNMT3B levels were also observed in the luminal and glandular epithelia of IS, whereas no obvious differences in the stroma were detected. In pseudo-pregnant mice subjected to a FDD, levels ofDnmt1andDnmt3a(but notDnmt3b) were significantly upregulated in endometrial tissues, as compared with controls. When tissues from these folate-deficient mice were examined, DNMT1 levels were elevated in both the luminal and glandular epithelia, whereas DNMT3A was upregulated in the luminal epithelium and the stroma. A slight increase in DNMT3B levels was detected in the glandular epithelium. These results indicate that DNMTs may regulate the transcription of endometrial genes associated with embryo implantation and that levels of DNMTs are affected by dietary folate in mice.

The harmful effects of the environmental carcinogen, benzo[a]pyrene (B[a]P), on mammalian neurodevelopment and behavior as yet remain unclear. Several studies have suggested that B[a]P impairs learning and memory. In the present investigation, we investigated the effects of subchronic exposure to B[a]P on rats. Male rats received daily injection of B[a]P (0, 1.0, 2.5, and 6.25 mg/kg, i.p.) or vehicle for 13 weeks. Employing the Morris water maze (MWM) test, we observed that rats exposed to either 2.5 mg/kg or 6.25 mg/kg B[a]P had modified behavior compared to controls as indicated by the increased mean latencies, the decreased number of crossing platform and the decreased swimming time in the target area. B[a]P treatment decreased the levels of malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), acetylcholine (ACh), choline acetyltransferase (ChAT), and increased the activity of acetylcholinesterase (AChE). Endogenous monoamine levels, norepinephrine (NE), adrenaline (A), dopamine (DA) and 5-hydroxytryptamine (5-HT) and their selected metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in hippocampus were measured using high performance liquid chromatography (HPLC). B[a]P at both doses, 2.5 and 6.25 mg/kg, increased NE, DA, DOPAC and 5-HT content in the hippocampus. Our results suggested a close link between the modified levels of neurotransmitters in the hippocampus and the impaired behavioral performance, indicating that B[a]P is a potential neurotoxic pollutant.

Mammalian target of rapamycin (MTOR) is a protein kinase that plays a central role in cell growth and proliferation. It is a part of the signaling network transmitting growth factor signaling to translational control. Previous studies have shown that MTOR is involved in embryo implantation, but its expression in the uterus and its role in implantation are unclear. Here, we have investigated the expression and role of MTOR in mouse uterus during early pregnancy. RT-FQ PCR showed that the mRNA levels of Mtor in endometria of pregnant mice were higher than those of nonpregnant mice. The mRNA levels in the pregnant mice gradually increased from D3 of pregnancy, reached maximum on D5, and then declined afterward. In situ hybridization and immunohistochemical analysis showed that the mRNA and protein of MTOR were mainly located in stromal cells. The levels of the expressed MTOR protein correlate with those of mRNA. The number of implantation sites was greatly decreased by the intrauterine injection with rapamycin in the early morning of D4 of the pregnancy. These findings suggest that MTOR may play an important role in embryo implantation in mice.

A series of cationic gemini surfactants, alkanediyl-α,ω-bis[di(2-hydroxylethyl) dodecylammonium] dibromide (abbreviated as 12-s-12 (OH), with s = 4, 6, 8 and 10 methylenes) have been synthesized, and their aggregation properties in aqueous solution have been studied by surface tension, electrical conductivity, isothermal titration calorimetry, 1H NMR, dynamic light scattering, and transmission electron microscopy. Two critical aggregation concentrations are observed for these surfactants. These surfactants start to form dimers at concentrations well below their critical micelle concentrations (CMC). Above the CMC, these surfactants can form both micelles and vesicles spontaneously with a micelle-to-vesicle transition. The hydrogen bond among the OH groups of headgroups and water molecules as well as the strong hydrophobic interaction among the hydrocarbon side chains should be the main origins for these unique aggregation behaviors of these gemini surfactants.

The expression and distribution of the imprinted paternally expressed gene 10 (PEG10) in decidual tissues of 36 patients with inevitable abortion and 36 normal pregnant (NP) women were examined by RT-PCR, immunohistochemistry, in situ hybridization and Western blot. Decidual PEG10 mRNA expression was significantly lower in the patients than that in the NP group by RT-PCR (P

A series of anionic sulfonate gemini surfactants with the general structure of [(CnH2n+1)(C3H6SO−3) NCsN(C3H6SO−3)(CnH2n+1)]⋅2Na+ have been synthesized. While the spacer group Cs represents p-xylyl or (CH2)3, the surfactants are abbreviated as CnCpxCn(SO3)2 ( n = 8 , 10 , 12 ) or C12C3C12(SO3)2 ( n = 12 ) , respectively. A corresponding monomeric surfactant C12H25N(CH3)(C3H6SO−3)⋅Na+(C12NSO3) has also been prepared. The aggregation behavior of these surfactants has been studied at pH 9.2 and ionic strength of 30 mM. The gemini surfactants exhibit stronger aggregation tendencies and much less endothermic enthalpy changes of micellization ( Δ H mic ) compared with the monomeric surfactant. The critical micelle concentrations (CMC) of the gemini surfactants decrease with the increase of the hydrophobic chain length from C8CpxC8(SO3)2 to C10CpxC10(SO3)2, but the CMC values of C10CpxC10(SO3)2 and C12CpxC12(SO3)2 are very close. The Δ H mic values vary from endothermic for C8CpxC8(SO3)2 to almost zero for C12CpxC12(SO3)2. Besides, vesicles are observed above the CMC for all these surfactants. The water-mediated intermolecular hydrogen bonding between the tertiary nitrogen groups may assist C12NSO3 and C12C3C12(SO3)2 in their vesicle formation, while the π–π interaction between aromatic rings should be another additional driving force for the vesicle formation of CnCpxCn(SO3)2. Meanwhile, the hydrogen bonding, π–π interaction, and strong hydrophobic interaction provide the possibility of a multilayer formation for C12CpxC12(SO3)2 and C12C3C12(SO3)2 at the air/water interface, which is a possible reason for the extremely small minimum area occupied per surfactant molecule at the air/water interface for these two gemini surfactants.

The mechanism of birth defects induced by folate deficiency was focused on mainly in fetal development. Little is known about the effect of folate deficiency on the maternal uterus, especially on decidual angiogenesis after implantation which establishes vessel networks to support embryo development. The aim of this study was to investigate the effects of folate deficiency on decidual angiogenesis. Serum folate levels were measured by electrochemiluminescence. The status of decidual angiogenesis was examined by cluster designation 34 (CD34) immunohistochemistry and the expression of angiogenic factors, including vascular endothelial growth factor A (VEGFA), placental growth factor (PLGF), and VEGF receptor 2 (VEGFR2) were also tested. Serum levels of homocysteine (Hcy), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), progesterone (P4), and estradiol (E2) were detected by Enzyme-linked immunosorbent assay. The folate-deficient mice had a lower folate level and a higher Hcy level. Folate deficiency restrained decidual angiogenesis with significant abnormalities in vascular density and the enlargement and elongation of the vascular sinus. It also showed a reduction in the expressions of VEGFA, VEGFR2, and PLGF. In addition, the serum levels of P4, E2, LH, and PRL were reduced in folate-deficient mice, and the expression of progesterone receptor (PR) and estrogen receptor α (ERα) were abnormal. These results indicated that folate deficiency could impaire decidual angiogenesis and it may be related to the vasculotoxic properties of Hcy and the imbalance of the reproductive hormone.

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

Existing evidence suggests that adverse pregnancy outcomes are closely related with dietary factors. Previous studies in mice have focused on the harm of folate deficiency (FD) on development of embryo, while the effect of low maternal folate levels on maternal intrauterine environment during early pregnancy remains unclear. Since our previous study found that FD treatment of mice causes no apparent defects in embryo implantation but is accompanied by female subfertility, we next chose to investigate a potential role of FD on molecular events after implantation. We observed that the decidual bulges began to be stunted on pregnancy day 6. The results of functional experiments in vivo and in vitro showed that FD inhibited the process of endometrial decidualization. It has been confirmed that DNA methylation participates in decidualization, and folate as a methyl donor could change the methylation patterns of genes. Thus, we hypothesized that FD impairs maternal endometrial decidualization by altering the methylation profiles of related genes. Reduced representation bisulphite sequencing was carried out to detect the methylation profiles of endometrium on pregnancy day 6-8, which is equivalent to the decidualization period in mice. The results confirmed that FD changes the methylation patterns of genome, and GO analysis of the differentially methylated regions revealed that the associated genes mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism and signalling. In addition, we found some candidates for regulators of decidual transformation, such as Nr1h3 and Nr5a1. The data indicate that FD inhibits decidualization, possibly by altering methylation patterns of the genome in mice.

Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy.

Our previous research has shown that the spliced isoform of XBP1 (XBP1s) is an important downstream mediator of BMP2 and is involved in BMP2-stimulated chondrocyte differentiation. Herein, we report that ATF6 and its cleaved N-terminal cytoplasmic domain (known as ATF6a) are expressed in growth plate chondrocytes. We find that these proteins are differentially induced during BMP2-triggered chondrocyte differentiation. This differential expression probably results from the activation of the ATF6 gene by Runx2 and its repression by the Sox6 transcription factor. Runx2 and Sox6 act through their respective binding elements on the ATF6 gene. When overexpressed, ATF6 and ATF6a intensify chondrogenesis; our studies demonstrate that under the stimulation of ATF6 and ATF6a, chondrocytes tend to be hypertrophied and mineralized, a process leading to bone formation. By contrast, lowering expression of ATF6a by use of its specific siRNA suppresses chondrocyte differentiation. Moreover, ATF6a interacts with Runx2 and augments the Runx2-mediated hypertrophication of chondrocytes. Importantly, overexpression and knockdown of ATF6a during the chondrocyte hypertrophy process also led to altered expressions of IHH and PTHrP (also known as PTHLH). Taken together, these findings indicate that ATF6a favorably controls chondrogenesis and bone formation (1) by acting as a co-factor of Runx2 and enhancing Runx2-incited hypertrophic chondrocyte differentiation, and (2) by affecting IHH and PTHrP signaling.

STUDY QUESTION Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans. SUMMARY ANSWER The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered β1,6-N-acetylglucosamine (β1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin β1. WHAT IS KNOWN ALREADY GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported. STUDY DESIGN, SIZE, DURATION Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM. PARTICIPANTS, MATERIALS, SETTING, METHODS Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of β1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5β1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the β1,6-GlcNAc and bis-GlcNAc structures in integrin α5β1 during the first trimester in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the normal group expression and activity of GnT-V and the concentration of its product, β1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and β1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. β1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased β1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin β1 (P < 0.05) were observed in the ESM group. WIDER IMPLICATIONS OF THE FINDINGS Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM. LIMITATIONS, REASONS FOR CAUTION The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests.

This study investigated the expression pattern of Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in the endometrium of early-stage pregnant mice and its function during embryo implantation. The expression of KRAS was measured at the mRNA level using real-time polymerase chain reaction (PCR) and at the protein level using immunohistochemistry and western blotting. The expressions of KRAS mRNA and protein were not significantly different in the endometrium of pseudopregnant and early-stage pregnant mice. However, the immunohistochemistry results showed that KRAS was highly expressed in the decidualizing stromal cells on days 5-7 of mouse pregnancy and was enhanced in the epithelial cells as pregnancy progressed. The expression of KRAS protein was higher after the stromal cell was artificially decidualized in vivo and in vitro. Stromal cell proliferation was attenuated after down-regulating KRAS expression. After silencing KRAS in the mouse uterus, the embryo implantation rate was significantly reduced (P < 0.005). We speculate that KRAS may regulate the stromal cell proliferation and differentiation progress and then affect the embryo implantation process. This study reveals that KRAS plays an important role in regulating the embryo implantation process.

We characterised DNA methylation and gene expression of four tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4, DR5, DcR1 and DcR2 in three choriocarcinoma (JAR, JEG-3, BeWo) and two transformed (HTR-8/SVneo and HPT-8) cell lines. DR4 mRNA was detected in JAR, JEG-3, BeWo and HTR-8/SVneo cells, whereas DR5 was present in all detected cells. DcR1 transcripts were expressed only in JAR, JEG-3 and BeWo cells, whereas DcR2 transcripts were detected only in HTR-8/SVneo and HPT-8 cells. Hypermethylated DR4 promoter was observed in JAR, JEG-3, BeWo and HTR-8/SVneo cells, hypermethylated DcR1 promoter in HTR-8/SVneo and HPT-8 cells and hypermethylated DcR2 promoter in JAR, JEG-3 and BeWo cells. Restoration of DR4, DcR1 and DcR2 expression with decreased DNA methylation of these genes was induced by the DNA demethylation agent 5-aza-2′-deoxycytidine (5-aza-CdR) in trophoblast cells, whereas DR5 expression did not exhibit any change. Significant negative correlation between the expression and DNA methylation of these genes was also observed. In all tested cell lines, only HPT-8 demonstrated sensitivity to TRAIL-induced apoptosis. Combined treatment with 5-aza-CdR and TRAIL resulted in apoptosis in JAR, JEG-3, BeWo and HTR-8/SVneo cells but not in HPT-8 cells. The results indicate that DNA methylation is associated with TRAIL receptor expression and might be involved in trophoblast apoptosis.

The dehydration of inulin biomass to the platform chemicals, 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA), in ZnCl2 molten salt hydrate medium was investigated. The influence of the Lewis acid catalyst, SnCl4, on the product distribution was examined. An in situ(1)H NMR technique was employed to follow the reaction at the molecular level. The experimental results revealed that only 5-HMF was obtained from degradation of inulin biomass in ZnCl2 molten salt hydrate medium, while the LA was gradually becoming the main product when the reaction temperature was increased in the presence of the Lewis acid catalyst SnCl4. In situ NMR spectroscopy could monitor the reaction and give valuable insight.

The racial/ethnic disparities in DNA methylation patterns indicate that molecular markers may play a role in determining the individual susceptibility to diseases in different ethnic groups. Racial disparities in DNA methylation patterns have been identified in prostate cancer, breast cancer and colorectal cancer and are related to racial differences in cancer prognosis and survival.

The toxicity of sodium fluoride (NaF) to female fertility is currently recognized; however, the mechanisms are unclear. Previously, we reported a reduction in successful pregnancy rates, ovarian atrophy and dysfunction following exposure to NaF. The purpose of this study was to elucidate the underlying molecular mechanisms. Female Sprague-Dawley rats (10 rats/group) received 100 or 200mg/L NaF in their drinking water for 6 months or were assigned to an untreated control group. Apoptotic indices and oxidative stress indicators in blood and ovarian tissue were analyzed following sacrifice. The results confirmed the NaF-induced ovarian apoptosis, with concomitant activation of oxidative stress. Further investigations in ovarian granular cells showed that exposure to NaF activated extracellular regulated protein kinase (ERK) and c-Jun NH2 kinase (JNK), disrupting the ERK and JNK signaling pathways, while p38 and PI3K remained unchanged. These data demonstrated that oxidative stress may play a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways.

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating literatures have indicated that T cells are involved in this process. The first signal mediated by T-cell receptor/CD3 complex and the second signal delivered by costimulatory molecules are essential for the differentiation of T cell into an effector cell. Expression and function of CD28, an important costimulatory molecule, during early pregnancy in mice is still unclear. In the present study, we investigated the expression pattern of CD28 in mouse uterus during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, Western blotting, in situ hybridization, and immunohistochemistry (IHC). We found that injection of the uterine horn with CD28 antisense oligodeoxynucleotides leads to a decreased number of implantation sites. The expression pattern of CD3 protein examined by IHC is similar to that of CD28. These findings suggest that CD28 participates in the process of embryo implantation in mice, which might play its role through delivering the second costimulatory signal.

Embryo implantation is a complicated process involving a series of endometrial changes that depend on differential gene expression. MicroRNAs (miRNAs) are important for regulation of gene expression. Previous studies have shown that miRNAs may participate in the regulation of gene expression during embryo implantation. To explore the role of endometrial miRNAs in early murine pregnancy, we used microarrays to investigate whether miRNAs were differentially expressed in the mouse endometrium on pregnancy day 4 (D4) and day 6 (D6). The results demonstrated that 17 miRNAs were upregulated and 18 were downregulated (>2-fold) in D6 endometria compared to D4. We identified that mmu-miR-193 exhibited the highest upregulation on D6, and the upregulation of mmu-miR-193 before embryo implantation could reduce the embryo implantation rate. Further, we demonstrated that mmu-miR-193 influenced embryo implantation by regulating growth factor receptor-bound protein 7 expression. In summary, our study suggests that mmu-miR-193 plays an important role in embryo implantation.

Benzo(a)pyrene (BaP) is a ubiquitous environmental pollutant. Studies have demonstrated it to be an endocrine-disrupting chemical that can cause adverse effects on the female reproductive system. However, the effect of BaP on early pregnancy has not been reported. We investigated the effect of BaP on endometrial receptivity and embryo implantation. Pregnant mice were dosed with BaP at 0.2, 2 and 20 mg/kg/day from day 1 (D1) to day 5 (D5) of gestation. Exposure to BaP impaired the morphology of the endometrium and decreased the number of implantation sites (p0.2=0.006, p2=0.167, p20=0.003). Levels of estrodiol (p0.001, for three treatment group compare with control group) and progesterone-4 in plasma were elevated in BaP-treatment groups (p0.20.001, p20.001, p20=0.032). Expression of estrogen receptor-α was up-regulated (p0.2=0.002, p2=0.131, p20=0.024) whereas expression of the progesterone receptor was down-regulated (p0.20.001, p2=0.064, p20=0.021). Levels of receptivity-related genes HoxA10 (p0.20.001, p2=0.135, p200.001) and E-cadherin (p0.2=0.002, p2=0.624, p20=0.137) were changed by BaP. These results revealed that BaP can disrupt the balance of estrogen and progesterone, influence expression of their receptors and downstream related genes, lead to changes in endometrium receptivity, and reduce of the number of implantation sites.

To study the expression of Toll-like receptors (TLRs) mRNA in human trophoblast HTR-8/SVneo cells and the changes in indoleamine 2,3-dioxygenase (IDO) mRNA expression in response to TLR ligand stimulation.The expressions of TLRs and IDO mRNA in human HTR-8/SVneo cells were tested by RT-PCR, and the changes in IDO mRNA levels after exposure to TLR3, TLR4, TLR7/8, and TLR9 ligands were quantitatively analyzed with real-time PCR.IDO and TLR1-10 mRNAs were expressed in HTR-8/SVneo cells. As the cell culture time extended, IDO mRNA expression level tended to increase within 48 h. After stimulation with the TLR ligands, the expression of TLR-3 mRNA was down-regulated while the expression of TLR-4, 7, 8, and 9 mRNA up-regulated. Stimulation of the cells with poly(I:C) lowered the expression of IDO mRNA while IFN-γ increased its expression.The expression of IDO mRNA is associated with the nutrition of the maternal-fetal interface. Stimulation with the TLR ligands affects the expression of IDO and TLR mRNA expressions in the cells, which verifies the functional activity of TLRs and suggests a role of IDO in TLR pathway-dependent antiviral immunity.