LIF mRNA Research Articles

Epigenetic alteration of uterine Leukemia Inhibitory Factor gene after glyphosate or a glyphosate-based herbicide exposure in rats

Glyphosate-based herbicides (GBHs) or its active ingredient, glyphosate (Gly), induce implantation failure in rats. We aimed to elucidate a mechanism of action of these compounds assessing the transcriptional and epigenetic status of the receptivity marker, leukemia inhibitory factor (Lif) gene. F0 rats were orally exposed to GBH or Gly at 3.8 or 3.9 mg Gly/kg/day, respectively, from gestational day (GD) 9 until weaning. F1 females were mated and uterine samples collected at GD5. Methylation-sensitive restriction enzymes (MSRE) sites and transcription factors were in silico predicted in regulatory regions of Lif gene. DNA methylation status and histone modifications (histone 3 and 4 acetylation (H3Ac and H4Ac) and H3 lysine-27-trimethylation (H3K27me3)) were assessed. GBH and Gly decreased Lif mRNA levels and caused DNA hypermethylation. GBH increased H3Ac levels, whereas Gly reduced them; both compounds enhanced H3K27me3 levels. Finally, both GBH and Gly induced similar epigenetic alterations in the regulatory regions of Lif.

Endometrial mRNA expression of key inflammatory genes and progesterone receptor in uterine tissue of bitches with endometritis

Our study aimed to evaluate the gene transcription of Cyclooxygenase 1 (COX1), cytokines (Interleukin 6 [IL6], Interleukin 8 [IL8], and Leukemia Inhibitory Factor [LIF]), and Progesterone Receptor (PR) in clinically healthy bitches with a normal endometrium and with endometritis, without the presence of cystic endometrial hyperplasia. Forty-eight mixed breed bitches in dioestrus were used. Uterine biopsies were collected for diagnosis of a normal endometrium (n=15), and endometritis (n=30). Three samples were excluded from the study (two bitches with cystic endometrial hyperplasia and one bitch with atrophy). Samples were collected by cytobrush to quantify mRNA by qPCR. Data were analyzed using a generalized mixed model. COX1, IL6, and IL8 mRNA expression in bitches with endometritis was significantly higher than in a normal endometrium (P=0.0002, P=0.002, and P=0.003, respectively). In contrast, LIF and PR mRNA expression in bitches with endometritis was significantly lower than with a normal endometrium (P=0.006 and P=0.001, respectively). IL6 (AUC 0.67) was the most favorable biomolecular marker for predicting endometritis in bitches. There was an evident change in the mRNA expression of COX1, cytokines, and PR in clinically healthy bitches with a normal endometrium and endometritis without cystic endometrial hyperplasia. These results suggest that biomolecular markers such as COX1, IL6, IL8, LIF, and PR could help diagnose endometritis in a bitch.

Effect of collagen endometrial patch loaded with adipose-derived mesenchymal stem cells on endometrial regeneration in rats with a thin endometrium.

This study aimed to investigate the effects of a collagen endometrial patch (EM patch) loaded with adipose-derived mesenchymal stem cells (ADSCs) on endometrial regeneration in a rat model with thin endometrium. Thin endometrium was induced in female rats and divided into treatment groups as outlined: control, group 1(G1), local injection of ADSCs into the uterus, group 2 (G2), an EM patch without ADSCs, group 3 (G3), and an EM patch loaded with ADSCs, group 4 (G4). The rats were euthanized at either two weeks or four weeks after modeling and treatment followed by histological and biochemical analyses to examine the regenerative effects on the injured endometrium. Transplantation of the ADSC-loaded EM patch significantly promoted endometrial proliferation and increased the luminal epithelial area. Two weeks after treatment, the mean number of von Villebrand factor (vWF)+ or cluster of differentiation (CD) 31+-stained blood vessels was significantly higher in G4 than in G1 and G2. The mRNA and protein expression levels of TGF-β and FGF2 were significantly upregulated in G4 compared to those in the control. G4 exhibited significantly increased LIF mRNA levels and immunoreactivity compared with the other groups at both two weeks and four weeks after treatment. Cell tracking after ADSCs treatment revealed the presence of a substantial number of ADSCs grafted in the uterine tissues of G4, whereas a low number of ADSCs that were focally clustered were present in G2. Transplantation of EM patches loaded with ADSCs resulted in the histological and biochemical restoration of an injured endometrium. The strategic integration of EM patches and ADSCs holds significant promise as an innovative therapeutic approach for effectively treating impaired endometrial conditions.

Mouse skeletal muscle adaptations to different durations of treadmill exercise after the cessation of FOLFOX chemotherapy.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is a treatment for colorectal cancer that can induce persistent fatigue and metabolic dysfunction. Regular exercise after chemotherapy cessation is widely recommended for cancer patients and has been shown to improve fatigue resistance in mice. However, gaps remain in understanding whether the early systemic and skeletal muscle adaptations to regular exercise are altered by prior FOLFOX chemotherapy treatment. Furthermore, the effects of exercise duration on early metabolic and skeletal muscle transcriptional adaptations are not fully established. Purpose: Investigate the effects of prior FOLFOX chemotherapy treatment on the early adaptations to repeated short- or long-duration treadmill exercise, including the fasting regulation of circulating metabolic regulators, skeletal muscle COXIV activity and myokine/exerkine gene expression in male mice. Methods: Male C57BL6/J mice completed 4 cycles of FOLFOX or PBS and were allowed to recover for 4-weeks. Subsets of mice performed 14 sessions (6d/wk, 18m/min, 5% grade) of short- (10min/d) or long-duration (55min/d) treadmill exercise. Blood plasma and muscle tissues were collected 48-72h after the last exercise bout for biochemical analyses. Results: Long-duration exercise increased fasting plasma osteocalcin, LIF, and IL-6 in healthy PBS mice, and these changes were ablated by prior FOLFOX treatment. Slow-oxidative soleus muscle COXIV activity increased in response to long-duration exercise in PBS mice, which was blocked by prior FOLFOX treatment. Fast-glycolytic plantaris muscle COXIV activity increased with short-duration exercise independent of FOLFOX administration. There was a main effect for long-duration exercise to increase fasting muscle IL-6 and COXIV mRNA expression independent of FOLFOX. FOLFOX administration reduced muscle IL-6, LIF, and BDNF mRNA expression irrespective of long-duration exercise. Interestingly, short-duration exercise suppressed the FOLXOX induction of muscle myostatin mRNA expression. Conclusion: FOLFOX attenuated early exercise adaptations related to fasting circulating osteocalcin, LIF, and IL-6. However, prior FOLFOX treatment did not alter the exercise adaptations of plantaris muscle COXIV activity and plasma adiponectin. An improved understanding of mechanisms underlying exercise adaptations after chemotherapy will provide the basis for successfully treating fatigue and metabolic dysfunction in cancer survivors.

Surgical removal of FIGO type 0 and 1 fibroids ameliorates the expression of endometrial proinflammatory transcription factors and receptivity modulators

To reveal whether hysteroscopic removal of the International Federation of Gynecology and Obstetrics (FIGO) types 0 and 1 fibroids makes any changes in the expression of homeobox genes (HOXA10, HOXA11), leukemia inhibitory factor, and nuclear factor-kappa B (NF-kB). A case-control study. University-based invitro fertilisation center. This study consisted of a total of 29 participants, 21 with FIGO types 0 and 1 fibroids and 8 with normal uterine cavity without fibroids. Patients in FIGO types 0 and 1 fibroids group underwent hysteroscopic myomectomy. The patients in the control group underwent laparoscopic tubal ligation. Endometrial cells were collected by flushing method from all participants before and 3 months after myomectomy. Real-time polymerase chain reaction was used to detect HOXA10, HOXA11, and LIF mRNA expressions in endometrial flushing samples. The relative expressions of homeobox and LIF mRNA were calculated with comparative ΔCt method. Endometrial NF-kB concentration was measured quantitatively by enzyme-linked immunosorbent assay. To compare endometrial HOXA10, HOXA11, and LIF mRNA expressions as well as endometrial NF-kB concentration before and after myomectomy. Premyomectomy NF-kB levels of type 0 (4.22 ± 1.02 ng/mL) and type 1 fibroid (6.44 ± 2.30 ng/mL) were significantly higher than the values of control group (0.54 ± 0.10 ng/mL). Surgical removal of type 0 and 1 fibroids resulted in a significant decrease in endometrial NF-kB levels (1.33 ± 0.02 ng/mL vs 1.65 ± 0.27 ng/mL, respectively). In type 0 fibroid group, after myomectomy, there was a 11.1-fold increase in HOXA10 mRNA, 4.23-fold in HOXA11 mRNA, and 7.63-fold in LIF mRNA. In the type 1 fibroid group, after myomectomy, there was a 16.3-fold increase in HOXA10 mRNA, 8.34-fold in HOXA11 mRNA, and 9.38-fold in LIF mRNA. A nonsignificant change was detected in homeobox and LIF mRNA after tubal sterilization. A negative and significant correlation was found between endometrial NF-kB and HOXA10 (r=-0.67), HOXA11 (r=-0.71) and LIF (r=-0.54). High proinflammatory NF-kB concentration and low homeobox and LIF mRNA expressions were detected in the presence of type 0 or 1 fibroids that returned to normal values after hysteroscopic myomectomy.

Mesenchymal Stem Cells-Induced Trophoblast Invasion Is Reduced in Patients with a Previous History of Preeclampsia.

Endometrial stromal cells play an important role in reproductive success, especially in implantation and placentation. Although Mesenchymal stem cells (MSCs) have been studied to assess decidualization disorders in preeclampsia (PE), their role during trophoblast invasion remains unclear. This study aims to determine: (i) whether MSCs isolated from menstrual fluid (MenSCs) from nulliparous, multiparous, and women with a previous history of preeclampsia exhibited different patterns of proliferation and migration and (ii) whether reproductive history (i.e., prior pregnancy or prior history of PE) was able to produce changes in MenSCs, thus altering trophoblast invasion capacity. MenSCs were collected from nulliparous and multiparous women without a history of PE and from non-pregnant women with a history of PE. Proliferation and migration assays were performed on MenSCs with sulforhodamine B and transwell assays, respectively. Trophoblast invasion was analyzed by culturing HTR-8/SVneo trophospheres on a matrigel overlying MenSCs for 72 h at 5% O2, simulating a 3D implantation model. A previous history of pregnancy or PE did not impact the proliferative capacity or migratory behavior of MenSCs. Following exposure to physiological endometrial conditions, MenSCs demonstrated upregulated expression of IGFBP-1 and LIF mRNA, decidualization and window of implantation markers, respectively. The mRNA expression of VIM, NANOG, and SOX2 was upregulated upon trophosphere formation. Relative to co-culture with multiparous MenSCs, co-culture with PE-MenSCs was associated with reduced trophoblast invasion. The findings of this study suggest a potential role for communication between maternal MenSCs and invading trophoblast cells during the implantation process that could be implicated in the etiology of PE.

BCG-trained innate immunity leads to fetal growth restriction by altering immune cell profile in the mouse developing placenta.

Trained immunity is a new concept illustrating that innate immune cells are able to undergo a long-term metabolic and epigenetic reprogramming after infection or vaccination, thus displaying either a pro- or an anti-inflammatory phenotype during a sequential unrelated challenge. Innate immune cells such as natural killer (NK) cells and macrophages constitute a large part of the decidual leukocyte population at the maternal-fetal interface, playing an important role in placental development and as such in fetal growth and development. In this study, we hypothesized that training the innate immune cells before pregnancy could have an impact on pregnancy. To test this hypothesis, we used CBA/J x DBA/2 mouse model to investigate pregnancy outcomes and leukocyte population at the maternal-fetal interface. Although we were not able to show a beneficial effect of LPS-tolerogenic training on fetal resorption, Bacillus Calmette-Guérin (BCG) training, known to prime innate immune cells to be proinflammatory, led to fetal growth restriction, without aggravating the fetal resorption rate. We also found that BCG training led to less NK cells and macrophages at the maternal-fetal interface at the early stage of placentation (E9.5), associated with a down-regulation of Ccr3 and Lif mRNA expression. This induced altered leucocyte population profile can be an explanation for the subsequent fetal growth restriction. These data suggest that preconceptional infections-induced trained immunity could influence pregnancy outcomes.

Investigation into the effects of leukemia inhibitory factor on the bone repair capacity of BMSCs-loaded BCP scaffolds in the mouse calvarial bone defect model.

Leukemia inhibitory factor (LIF) is known to play a major role in bone physiology. In the present study, we examined the in vitro effects of LIF on osteoblast differentiation of bone marrow stem cells (BMSCs) and explored in vivo effects of LIF on the bone repair capacity of BMSCs-loaded biphasic calcium phosphate (BCP) scaffolds in mouse calvarial bone defect model. The mRNA and protein expression levels in the BMSCs were determined by quantitative real-time PCR and western blot, respectively; the in vitro osteoblast differentiation of the BMSCs was evaluated by using Alizarin Red S staining. The bone volume and bone density in the repaired calvarial bone defect were determined by Micro-CT. Bone regeneration was also histologically evaluated by hematoxylin and eosin staining and Masson's trichrome staining. Hypoxia treatment induced the up-regulation of Lif mRNA and LIF protein in the BMSCs. Lif overexpression up-regulated the mRNA expression levels of osteopontin and Runt-related transcription factor 2, and increased intensity of Alizarin Red S staining in the BMSCs; while Lif silence exerted the opposite effects. The in vivo studies showed that implantation of Lif-overexpressing BMSCs-loaded BCP scaffolds significantly increased the bone volume and bone density at 4 and 8weeks after transplantation, and promoted the regeneration of bone tissues in the mouse calvarial bone defect at 8weeks after transplantation when compared to the BMSCs-loaded BCP scaffolds group; while Lif-silencing BMSCs-loaded BCP scaffolds had the opposite effects. The present study for the first time demonstrated that LIF promoted the in vitro osteoblast differentiation of hypoxia-treated BMSCs; and further studies revealed that LIF exerted enhanced effects on the bone repair capacity of BMSCs-load BCP scaffolds in mouse calvarial bone defect model. However, future studies are warranted to determine the detailed mechanisms of LIF in the large-scale bone defect repair.

Generation and initial characterization of novel tumour organoid models to study human pancreatic cancer-induced cachexia.

BackgroundThe majority of patients with pancreatic cancer develops cachexia. The mechanisms underlying cancer cachexia development and progression remain elusive, although tumour‐derived factors are considered to play a major role. Pancreatic tumour organoids are in vitro three‐dimensional organ‐like structures that retain many pathophysiological characteristics of the in vivo tumour. We aimed to establish a pancreatic tumour organoid biobank from well‐phenotyped cachectic and non‐cachectic patients to enable identification of tumour‐derived factors driving cancer cachexia.MethodsOrganoids were generated from tumour tissue of eight pancreatic cancer patients. A comprehensive pre‐operative patient assessment of cachexia‐related parameters including nutritional status, physical performance, body composition, and inflammation was performed. Tumour‐related and cachexia‐related characteristics of the organoids were analysed using histological stainings, targeted sequencing, and real‐time–quantitative PCR. Cachexia‐related factors present in the circulation of the patients and in the tumour organoid secretome were analysed by enzyme‐linked immunosorbent assay.ResultsThe established human pancreatic tumour organoids presented typical features of malignancy corresponding to the primary tumour (i.e. nuclear enlargement, multiple nucleoli, mitosis, apoptosis, and mutated KRAS and/or TP53). These tumour organoids also expressed variable levels of many known cachexia‐related genes including interleukin‐6 (IL‐6), TNF‐α, IL‐8, IL‐1α, IL‐1β, Mcp‐1, GDF15, and LIF. mRNA expression of IL‐1α and IL‐1β was significantly reduced in organoids from cachectic vs. non‐cachectic patients (IL‐1α: −3.8‐fold, P = 0.009, and IL‐1β: −4.7‐fold, P = 0.004). LIF, IL‐8, and GDF15 mRNA expression levels were significantly higher in organoids from cachectic vs. non‐cachectic patients (LIF: 1.6‐fold, P = 0.003; IL‐8: 1.4‐fold, P = 0.01; GDF15: 2.3‐fold, P < 0.001). In line with the GDF15 and IL‐8 mRNA expression levels, tumour organoids from cachectic patients secreted more GDF15 and IL‐8 compared with organoids from non‐cachectic patients (5.4 vs. 1.5 ng/mL, P = 0.01, and 7.4 vs. 1.3 ng/mL, P = 0.07, respectively).ConclusionsThis novel human pancreatic tumour organoid biobank provides a valuable tool to increase our understanding of the mechanisms driving cancer cachexia. Our preliminary characterization of the secretome of these organoids supports their application in functional studies including conditioned medium approaches and in vivo transplantation models.

The effect of polychlorinated biphenyls (PCBs) on bovine oviductal contractions and LIF synthesis during estrous cycle, in vitro studies

Polychlorinated biphenyls (PCBs) are a group of synthetic xenobiotics that have been used in many industrial applications. Currently, PCBs are among the most prominent environmental contaminants. Previously we showed that PCBs impair secretion of prostaglandins (PGs) at the oviduct. PGs are involved in the regulation of oviductal contractions and the synthesis of leukemia inhibitory factors LIF. Since oviductal contractions are crucial for gamete and embryo transport, and LIF is essential for embryo implantation, the direct effect of PCBs on oviductal motor activity and LIF mRNA expression were investigated. Oviductal strips and cells were taken from cows during the estrous cycle and were treated with PCBs at concentrations close to their environmental ranges. All the studied PCBs decreased the force of the contractions of the longitudinal and circular muscles of the isthmus. Additionally, these PCBs decreased the amplitude of the longitudinal muscle of the oviduct. Moreover, PCB-30-OH and PCB-153 increased the mRNA expression of LIF. Since PCBs inhibit the motor function of the oviduct and stimulate the synthesis of LIF, it is possible that PCBs can slow gamete or embryo transport and increase the potential for pathological embryo implantation in the oviduct.

The ameliorating effects of tokishakuyakusan in a rat model of implantation failure involves endometrial gland leukemia inhibitory factor and decidualization

Ethnopharmacological relevanceTokishakuyakusan (TSS) is a Kampo medicine that is prescribed for the treatment of infertility in Japan. However, its precise mechanism of action remains unclear. Aim of the studyLeukemia inhibitory factor (LIF) in the endometrium plays an indispensable role in embryo implantation and is linked to infertility or implantation failure. Previously, we demonstrated that TSS ameliorated implantation failure induced by mifepristone (RU-486), an antagonist of progesterone, in rats. Herein, we aimed to clarify whether the ameliorating effect of TSS on implantation failure in the rat model involves endometrial LIF. Additionally, we determined whether decidualization, the dysfunction of which is linked to infertility or implantation failure similar to LIF, progesterone, and other implantation-related factors, are involved in the effect of TSS. Materials and methodsThe implantation failure rat model was developed via the subcutaneous administration of RU-486 (7 mg/kg) on day 3 post-coitus. Sesame oil was administered as the vehicle control. Rats were fed a diet containing 1% or 3% TSS or a control diet from day 13 pre-coitus. Subsequently, the implantation sites were assessed, and plasma progesterone levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on day 8 post-coitus. The LIF mRNA of the endometrial gland, which was segmented via laser-microdissection from the endometrial tissue, was measured, and endometrial LIF immunostaining was carried out on day 5. The gene expression of different factors related to implantation, including decidualization and progesterone-responsiveness on days 5 and 6, were measured. The human endometrial Ishikawa cell line derived from human adenocarcinoma was treated with TSS (30–300 μg/mL) for 24 h, and the LIF concentrations in the cell culture supernatants were measured. ResultsRU-486 decreased the number of implantation sites in the uterus of rats; however, the decrease was significantly alleviated by TSS (3%-diet), which tended to increase plasma progesterone. In rats with RU-486-induced implantation failure, endometrial gland LIF mRNA and endometrial LIF protein were markedly decreased while the gene expression of both decidualization-related factors such as interleukin-11, insulin-like growth factor binding protein-1, and cyclooxygenase-2, and progesterone responsive-related factors such as FK506 binding protein 5, were significantly decreased. These changes in the uterus of rats with implantation failure were significantly alleviated by TSS (3%-diet). Additionally, TSS significantly enhanced LIF protein production and LIF mRNA in Ishikawa cells. ConclusionsThe mechanism whereby TSS ameliorates RU-486-induced implantation failure in rats may involve the alleviation of decreased LIF production derived from the endometrial gland, and a dysfunction of decidualization, including lower progesterone responsiveness in the model. These findings may partly contribute to the interpretation of the beneficial effects of TSS on infertility.

Sclerostin expression in trabecular bone is downregulated by osteoclasts

Bone tissues have trabecular bone with a high bone turnover and cortical bone with a low turnover. The mechanisms by which the turnover rate of these bone tissues is determined remain unclear. Osteocytes secrete sclerostin, a Wnt/β-catenin signaling antagonist, and inhibit bone formation. We found that sclerostin expression in cortical bone is more marked than in trabecular bone in Sost reporter mice. Leukemia inhibitory factor (LIF) secreted from osteoclasts reportedly suppressed sclerostin expression and promoted bone formation. Here, we report that osteoclasts downregulate sclerostin expression in trabecular bone and promote bone turnover. Treatment of C57BL/6 mice with an anti-RANKL antibody eliminated the number of osteoclasts and LIF-positive cells in trabecular bone. The number of sclerostin-positive cells was increased in trabecular bone, while the number of β-catenin-positive cells and bone formation were decreased in trabecular bone. Besides, Tnfsf11 heterozygous (Rankl+/−) mice exhibited a decreased number of LIF-positive cells and increased number of sclerostin-positive cells in trabecular bone. Rankl+/− mice exhibited a decreased number of β-catenin-positive cells and reduced bone formation in trabecular bone. Furthermore, in cultured osteoclasts, RANKL stimulation increased Lif mRNA expression, suggesting that RANKL signal increased LIF expression. In conclusion, osteoclasts downregulate sclerostin expression and promote trabecular bone turnover.

Effects of LncRNA Lnc-LIF-AS on cell proliferation, migration and invasion in a human cervical cancer cell line

This study explored the effect of LncRNA Lnc-LIF-AS on cell proliferation, migration and invasion in the human cervical cancer (HCC) cell line SiHa. SiHa cells had the lowest expression of Lnc-LIF-AS in the 4 human cervical cancer cell lines (SiHa, ME-180, C-33A and HeLa) and were transfected and divided into the SiHa/con (transfected with pMIGRI) cell group, SiHa/Lnc-LIF-AS (transfected with pMIGRI-Lnc-LIF-AS) cell group, and SiHa/Lnc-LIF-AS-DN (transfected with pMIGRI-Lnc-LIF-AS-DN, in which the sequences overlapping with LIF mRNA was deleted) cell group. Overexpression of Lnc-LIF-AS could promote the proliferation, colony formation, invasion and migration in SiHa and ME-180 cells. And the low expression of Lnc-LIF-AS suppress the proliferation, colony formation invasion and migration in HeLa cells when the Lnc-LIF-AS expression has been suppressed. In the SiHa/Lnc-LIF-AS cells group, the cell cycle was mainly halted in the S phase and overexpression of Lnc-LIF-AS had no effect on the apoptosis of SiHa cells. Overexpression of Lnc-LIF-AS could promote the secretion of LIF in SiHa cells, and the supernatant from SiHa/Lnc-LIF-AS cells could promote cell proliferation in the SiHa/con cells. The STAT3 inhibitor could inhibit cell proliferation in the SiHa/Lnc-LIF-AS cells. The expression level of Lnc-LIF-AS in cervical cancer tissues was higher than that in normal tissues and the expression level of Lnc-LIF-AS was positively correlated with the level of LIF. In the SiHa/con and SiHa/Lnc-LIF-AS-DN cell groups, there were no significant differences in cell proliferation, cell migration and cell invasion. The overexpression of Lnc-LIF-AS can promote cell proliferation, migration and invasion in cervical cancer cells, and the core function domain of this lncRNA was located in the overlapping a 3′-UTR base sequence of LIF mRNA.

Leukemia inhibitory factor signaling in Xenopus embryo: Insights from gain of function analysis and dominant negative mutant of the receptor

Leukemia inhibitory factor (LIF) is a cytokine member of the interleukin 6 family (IL6) of cytokines. It signals through a heterodimer receptor complex that consists of the LIF receptor (or LIFR formerly known as gp190) and the Interleukin 6 signal transducer (or IL6ST formerly known as gp130). LIF signaling is mediated mainly by signal transducer and activator of transcription 3 (STAT3) and has a wide variety of biological activities with pleiotropic effects on many cell types and organs among which are stem cell renewal and implantation process in mammalian embryo. Despite the wealth of data on LIF in mammalian cells, there is a paucity of information on its functions in lower vertebrates. Here, we provide information on the status and the function of LIF signaling in Xenopus amphibian. The IL6 cytokine family is highly conserved in Xenopus genome both at ligands and receptors levels. All cytokines and receptors of the family, except oncostatin M (OSM) and IL27, can be identified in the genome including the orthologs of LIF, cardiotrophin 1 (CTF1), ciliary neurotrophic factor (CNTF), cardiotrophin like cytokine factor 1 (CLCF1), LIFR, IL6ST, IL6R, IL11RA and CNTFR. Lif mRNA is zygotically expressed after midblastula transition while lifr and il6st are maternally expressed. We have investigated the functions of LIF in Xenopus early development with a gain-of-function analysis combined to the use of a dominant negative form of the receptor. The overexpression of Xenopus lif in embryo activates STAT3 phosphorylation and induces a dramatic phenotype where embryos are ventralised and show a reduction of anterior structures with microcephaly. This results mainly from BMP signal stimulation and antagonism towards IGF signals. In addition, most embryos develop tumor-like cell masses according to both autonomous and non-autonomous processes. Through the use of a dominant negative form of the receptor, we demonstrate for the first time that a functional LIF signaling is required for normal vertebrate kidney development. Owing to its experimental advantages, the Xenopus embryo constitutes a useful model to identify the molecular actors that may account for the pleiotropic functions of LIF and their role in vertebrate development.

53 Pre-implantation exposure to bisphenol A and 4-tert-octylphenol result in disruption of calcium channels

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all miscarriage cases in humans. Calcium (Ca2+) has been shown to involve many cellular signal transduction pathways as well as regulation of cell adhesion, which is necessary for the physiology process of endometrial epithelial cell transformation and stromal cell decidualization during embryo implantation. Exposure to endocrine-disrupting chemicals (ED) can regulate the expression of genes associated with calcium transport in during pregnancy such as TRPV5, TRPV6, PMCA, and NCX1. Additionally, exposure to ED during early gestation results in disrupted intrauterine implantation, uterine receptive, leading to implantation failure. In this study, oestrogen (E2), bisphenol A (BPA), octylphenol (OP), and/or ICI 182,780 (oestrogen receptor antagonist, ICI) were injected subcutaneously from gestation Day 1 to gestation Day 3 post coitus. The number of implantation sites was significantly lower in the OP group, and no implantation site was observed in the E2 and ED+ICI groups. There were differences in the expression of calcium transient transport channel between maternal uterine and implantation. The level of TRPV6 and TRPV5 mRNA and protein was significantly increased by ED and/or ICI treatment in the uterus. The levels of TRPV5 and TRPV6 gene expression were significantly increased by ED with/without ICI treatment in the uterus. However, TRPV5 and TRPV6 gene expression was significantly lower in implantation site samples. The NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Both mRNA and protein levels of MUC1 were markedly higher in all groups, except the BPA group when compared with the vehicle group in the uterus. The LIF and HOXA-10 mRNA were significantly low in E2; BPA+ICI; OP; and/or ICI in both the uterus and implantation site. Expression of the oestrogen receptor (ERa) and progesterone receptor (PR) was significantly lower in all groups except the BPA group when compared with the vehicle group. Taken together, E2, BPA, and OP disrupt the success of implantation through altered expression of calcium transport genes.

Decreased expression of LIF mRNA in patients withmyoma uteri.

The aim of this study was to evaluate endometrial receptivity by measuring HOXA-10, HOXA-11, and LIF gene expressions in women with polycystic ovary syndrome. A total of 48 women were included in this clinical study. Thepatients were allocated to two groups: study group consisted of 28 patients with myoma uteri and control group consisted of 20 patients without myoma uteri. Endometrial sampling was performed during the proliferative phase. The biopsies obtained from the patients with myoma uteri were taken from the place where the fibroids were localized. HOXA-10, HOXA-11, and LIF expressions were measured in the endometrial sampling material. Demographic data of the patients such as age, obstetric and gynecologic history, medical conditions, medications, surgical history, last menstrual period were recorded. Also, the number, size, localization, and type of the myoma were registered. The mean age of the patients was 42.07 and 38.17, respectively. HOXA-11 levels in the study and control groups were 0.004 ± 0.001 and 0.010 ± 0.001, respectively ( P < 0.90). Paradoxically, HOXA-10 levels were found to be higher in the study group than the control group, but the difference was not statistically significant ( P < 0.25). LIF levels were significantly lower in the study group ( P < 0.05). Our results showed that myoma uteri might lead to a decrease in implantation rate by diminishing LIF gene expressions. However, there were no differences between the two groups in terms of HOXA-10 and HOXA-11 levels.

GnRH agonist improves pregnancy outcome in mice with induced adenomyosis by restoring endometrial receptivity.

PurposeAdenomyosis has a negative impact on female fertility. GnRH agonist treatment can improve pregnancy outcomes in women with adenomyosis. However, the impact of GnRH agonist upon endometrium receptivity of patients with adenomyosis remains unclear. In this study, endometrial receptivity and pregnancy outcome were investigated using a mouse model of adenomyosis.Materials and methodsAdenomyosis was induced in 12 female ICR mice, neonatally treated with tamoxifen, while another six female mice (control group) received solvent only. At 75 days, the induced adenomyosis group was randomly divided into two groups: an untreated group and a group treated with GnRH agonist (n = 6 each). Sixty days later, the mice were mated and pregnancy outcomes were observed and compared among the three groups (n = 6 each). In a parallel experiment using the same treatment regimes, uterus samples were collected on day 4 of pregnancy for immunohistochemistry, gene (quantitative polymerase chain reaction) and protein expression (Western blot), and scanning electron microscopy analyses.ResultsWe found that the average live litter size was reduced in the adenomyosis compared with control group (8 ± 0.56 versus 13 ± 0.71; P = 0.03). However, the litter size was significantly increased in the treated with GnRH agonist group compared with the untreated group (12 ± 0.35 versus 8 ± 0.56; P = 0.04). The uterine expression levels of Hoxa10, Hoxa11, Lif and integrin b3 mRNA and protein were decreased in the adenomyosis group, and were significantly increased after GnRH agonist treatment. Additionally, pinopodes were reduced in number and poorly developed in mice with induced adenomyosis. However, pinopodes were abundant and well-developed in the GnRH agonist treatment group.ConclusionAdenomyosis may have an adverse impact on endometrial receptivity and reduce pregnancy outcomes in mice. However, GnRH agonist may improve the pregnancy outcome by partially restoring endometrial receptivity.

IL6 and LIF mRNA expression in skeletal muscle is regulated by AMPK and the transcription factors NFYC, ZBTB14, and SP1.

Adenosine monophosphate-activated protein kinase (AMPK) controls glucose and lipid metabolism and modulates inflammatory responses to maintain metabolic and inflammatory homeostasis during low cellular energy levels. The AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) interferes with inflammatory pathways in skeletal muscle, but the mechanisms are undefined. We hypothesized that AMPK activation reduces cytokine mRNA levels by blocking transcription through one or several transcription factors. Three skeletal muscle models were used to study AMPK effects on cytokine mRNA: human skeletal muscle strips obtained from healthy men incubated in vitro, primary human muscle cells, and rat L6 cells. In all three skeletal muscle systems, AICAR acutely reduced cytokine mRNA levels. In L6 myotubes treated with the transcriptional blocker actinomycin D, AICAR addition did not further reduce Il6 or leukemia inhibitory factor ( Lif) mRNA, suggesting that AICAR modulates cytokine expression through regulating transcription rather than mRNA stability. A cross-species bioinformatic approach identified novel transcription factors that may regulate LIF and IL6 mRNA. The involvement of these transcription factors was studied after targeted gene-silencing by siRNA. siRNA silencing of the transcription factors nuclear transcription factor Y subunit c ( Nfyc), specificity protein 1 ( Sp1), and zinc finger and BTB domain containing 14 ( Zbtb14), or AMPK α1/α2 subunits, increased constitutive levels of Il6 and Lif. Our results identify novel candidates in the regulation of skeletal muscle cytokine expression and identify AMPK, Nfyc, Sp1, and Zbtb14 as novel regulators of immunometabolic signals from skeletal muscle.

DHEA supplementation improves endometrial HOXA-10 mRNA expression in poor responders.

Objective:The study was planned to investigate whether DHEA supplementation had an impact on endometrial receptivity in women who were poor responders (POR).Material and Methods:Twenty-eight POR women who were undergoing hysteroscopy and five fertile control subjects were included. The POR women were equally subdivided into two separate groups as patients who were currently using DHEA and those who were not. Endometrial samples of the subjects were obtained during hysteroscopy at the late follicular phase. Expression levels of endometrial HOXA-10, HOXA-11, and LIF mRNA were measured with the using real-time polymerase chain reaction. Spontaneous clinical pregnancy rates were also noted.Results:Compared with POR women who were not given DHEA, upregulated endometrial HOXA-10 (7.33-fold) and HOXA-11 (2.39-fold) mRNA expression were detected in POR women on DHEA. The increase in HOXA-10 mRNA was significant (p<0.03). The fold increase in HOXA-11 mRNA was found as 2.39, which indicated a positive upregulation. However, this fold increment was insignificant (p<0.45). An insignificant increase in spontaneous clinical pregnancy rates in POR women on DHEA (53.3%) was observed compared with POR women who were not given DHEA (43.8%).Conclusion:Oral DHEA supplementation in POR upregulates endometrial HOXA-10 mRNA expression, which is known to positively modulate endometrial receptivity.

Phorbol-12-myristate-13-acetate-mediated stabilization of leukemia inhibitory factor (lif) mRNA: involvement of Nucleolin and PCBP1.

Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis-acting elements. A 216 nucleotide containing proximal cis-element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein trans-factors interacting with lif mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent in vivo co-association of both these proteins with lif mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic lif mRNA and gfp reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both lif mRNA and protein levels. Collectively, this report establishes the stabilization of lif mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal cis-element.