Experimental and bioinformatic analysis of cultured Bovine Endometrial Cells (BEND) responding to interferon tau (IFNT).
ABSTRACT: In ruminants, embryo implantation depends on progesterone (P4) and interferon tau (IFNT) controlling endometrial function. IFNT antagonizes bovine endometrial cells (BEND) response to phorbol 12,13-dibutyrate (PDBU) through posttranscriptional regulation of gene expression. We have previously described microRNAs (miRNAs) profiles in bovine endometrium, detecting miR-106a, relevant for embryo maternal communication. In this study, we investigated the expression miR-106a and genes for prostaglandin-endoperoxide synthase 2 (PTGS2), phospholipase A2, group IVA (PLA2G4A), estrogen receptor 1 (ESR1) and progesterone receptor (PR) in response to IFNT in BEND cells and searched for interferon responsive factors (IRFs) binding sites in their promoter genomic regions. The aim of this study was to unravel the molecular mechanisms involved in IFNT signalling and its regulation of miR-106a.PTGS2 showed increased expression under PDBU, which was antagonized by IFNT. IFNT induced expression of PR and miR-106a and downregulation of ESR1 and PR. Bioinformatic analyses detected that PLA2G4A was associated to IRF-1 and IRF-6, while ESR1, PR and PTGS2 were associated to only IRF-6. All genes exhibit one motif per IRF, except miR-106a that had three binding sites for IRF-6.We report the IFNT regulatory effect on miR-106a expression through IRF-6 in bovine endometrial cells. We identified a set of potential binding sites for IRF-1 and IRF-6 within the bovine genome. A set of candidate gene regions could be characterized where IFNT can act via IRFs to regulate the expression of proteins and miRNAs. Future studies will use these data to detect new IFNT regulatory mechanisms in the endometrium.
Project description:In cattle, conceptus-derived interferon tau (IFNT) is the pregnancy recognition (PR) signal. Our previous studies showed that non-cytopathic bovine viral diarrhoea virus (ncpBVDV) infection inhibited IFNT-induced interferon stimulated gene (ISG) expression, potentially causing early embryonic death. This study investigated the effect of bovine viral diarrhoea virus (BVDV) infection on upstream regulatory pathways of ISG production using an established PR model. Uterine endometrial cells from 10 apparently healthy and BVDV free cows were cultured and treated with 0 or 100 ng/mL IFNT for 24 h in the presence or absence of ncpBVDV infection. Microarray and pathway analysis were used to determine the IFNT-induced upstream regulators. Expression of the genes associated with the identified pathways were quantified with qPCR. IFNT challenge activated the signalling pathways associated with IFN receptors, JAK1/TYK2, IRFs and STATs and ncpBVDV infection inhibited the activation of IFNT on this pathway. Inhibition of this upstream signalling pathway may thus reduce ISG production to disrupt maternal PR. In addition, the reduction of uterine immunity by ncpBVDV infection may predispose the animals to uterine infection, which in turn impairs their reproductive performance. This provides a mechanism of how BVDV infection leads to early pregnancy failure in cows.
Project description:Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased ?H2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.
Project description:This study combined in vitro production of bovine blastocysts, multiple embryo transfer techniques, and a conceptus-endometrial explant co-culture system to test the hypothesis that bovine endometrium exposed to long vs. short day 15 conceptuses would exhibit a different transcriptome profile reflective of potential for successful pregnancy establishment. Bovine endometrial explants collected at the late luteal stage of the estrous cycle were cultured in RPMI medium for 6 h with nothing (control), 100 ng/mL recombinant ovine interferon tau (IFNT), a long day 15 conceptus, or a short day 15 conceptus. Transcriptional profiling of the endometrial explants found that exposure of endometrium to IFNT, long conceptuses, or short conceptuses altered (P < 0.05) expression of 491, 498, and 230 transcripts, respectively, compared to the control. Further analysis revealed three categories of differentially expressed genes (DEG): (i) commonly responsive to exposure to IFNT and conceptuses, irrespective of size (n = 223); (ii) commonly responsive to IFNT and long conceptuses only (n = 168); and genes induced by the presence of a conceptus but independent of IFNT (n = 108). Of those 108 genes, 101 were exclusively induced by long conceptuses and functional analysis revealed that regulation of molecular function, magnesium-ion transmembrane transport, and clathrin coat assembly were the principal gene ontologies associated with these DEG. In conclusion, bovine endometrium responds differently to age-matched conceptuses of varying size in both an IFNT-dependent and -independent manner, which may be reflective of the likelihood of successful pregnancy establishment.
Project description:In ruminants, Interferon tau (IFNT) is the pregnancy recognition protein produced by the mononuclear trophectoderm of the conceptus, and is secreted into the uterine lumen during the peri-attachment period. In our previous study, the high-throughput RNA sequencing (RNA-seq) data obtained from bovine conceptuses during the peri-attachment period identified two IFNT mRNAs, IFNT2 and IFNTc1. However, how each of these IFNT variants regulates endometrial gene expression has not been characterized. Using RNA-seq analysis, we evaluated how IFNT2 and IFNTc1 affected transcript expression in primary bovine endometrial epithelial cells (EECs). IFNT treatment induced 348 differentially expressed genes (DEGs); however, there are few DEGs in IFNT2 or IFNTc1 treated EECs, indicating that IFNT2-induced DEGs were similar to those induced by IFNTc1 treatment. In in silico analysis, we identified four IFNT2- and IFNTc1-induced pathways: 1) type II interferon signaling, 2) proteasome degradation, 3) type III interferon signaling, and 4) DNA damage response. We further demonstrated that IFNT2 and IFNTc1 up-regulated several transcription factors, among which forkhead box S1 (FOXS1) was identified as the most highly expressed gene. Furthermore, the knockdown of FOXS1 in IFNT2- or IFNTc1-treated EECs similarly down-regulated 9 genes including IRF3 and IRF9, and up-regulated 9 genes including STAT1, STAT2, and IRF8. These represent the first demonstration that effects of each IFNT on EECs were studied, and suggest that endometrial response as well as signaling mechanisms were similar between two IFNT variants existed in utero.
Project description:Interferon tau (IFNT), the maternal recognition of pregnancy signal in sheep and other ruminants, is secreted by the conceptus and regulates the expression of a number of genes in a cell-specific manner within the uterus. The response of different endometrial cell types to IFNT appears to be specified by IFN regulatory factors (IRFs). IRF2, a potent repressor of gene transcription, is expressed only by luminal (LE) and superficial glandular epithelia (sGE), whereas IRF1 and IRF9, activators of gene transcription, are expressed only in GE and stromal cells of the uterus during early pregnancy. In the present study, IRF6 was found to be expressed in LE/sGE and middle GE of the ovine uterine endometrium as well as conceptus trophectoderm. IRF family members can regulate transcription via IFN-stimulated response elements (ISREs). Transient transfection analyses found that IRF6 enhanced basal activity of ISRE-containing promoters, but did not enhance IFNT stimulation of ISRE-containing promoters in variety of different cell types. Further, IRF6 did not cooperate with IRF1 or reduce IRF2 repression of ISRE-containing promoter activity. These results establish that IRF6 is a transcriptional activator that is preferentially expressed in the endometrial epithelia and conceptus trophectoderm. IRF6 is hypothesized to play critical roles in endometrial gene expression as well as in conceptus trophectoderm growth and differentiation.
Project description:Endometrial cells secrete various cytokines and the dysfunction of endometrial cells may directly lead to infertility. Interferon tau (IFNT) secreted by trophoblast cells, a well-known pregnancy recognition signal in ruminants, acts on the uterus to prepare for pregnancy. Aging causes cellular and organ dysfunction, and advanced maternal age is associated with reduced fertility. However, few studies have investigated age-dependent changes in the uterus.Using next generation sequencing and real-time PCR, we examined mRNA expression in bovine endometrial cells in vitro obtained from young (mean 45.2 months) and aged (mean 173.5 months) animals and the effects of IFNT depending on the age.We showed that inflammation-related (predicted molecules are IL1A, C1Qs, DDX58, NFKB, and CCL5) and interferon-signaling (predicted molecules are IRFs, IFITs, STATs, and IFNs) pathways were activated in endometrial cells obtained from aged compared to young cows. Also, the activation of "DNA damage checkpoint regulation" and the inhibition of "mitotic mechanisms" in endometrial cells obtained from aged cows were evident. Moreover, we showed lower cell viability levels in endometrial cells obtained from aged compared to young cows. Although treatment with IFNT upregulated various types of interferon stimulated genes both in endometrial cells obtained from young and aged cows, the rate of increase by IFNT stimulus was obviously lower in endometrial cells obtained from aged compared to young cows.Endometrial cells obtained from aged cows exhibited higher levels of inflammatory- and IFN-signaling, and dysfunction of cell division compared with young cows. In addition, a high basal level of IFN-related genes in endometrial cells of aged cows is suggested a concept of "inflammaging".
Project description:Growing evidence indicates that miR-106a is involved in tumor growth and metastasis of cancers, but the participation of miR-106a in endometrial adenocarcinoma (EC) is not clear. BCL2L11 is a member of the BCL-2 family and is located in the outer membrane of mitochondria, where this protein acts as a key regulator of excitotoxic apoptosis, apoptosis-inducing factor translocation, and mitochondrial depolarization. To identify a novel therapeutic target in EC, we studied the roles of miR-106a in the proliferation, apoptosis, and metastasis of EC. The expression levels of miR-106a were measured in tumor tissues of EC by quantitative real-time PCR, and lentiviral transduction was used to verify the function of miR-106a by silencing. Subcutaneous injection of EC cell lines into athymic mice was used to research EC tumor formation. Bioinformatics tools and a luciferase assay were applied to assess the relation between miR-106a and its target. The protein level of the miR-106a target was measured by western blotting. MiR-106a expression was higher in EC tissues compared with their healthy counterparts. Inhibition of expression of miR-106a reduced EC cell migration and invasion <i>in vitro</i> as well as <i>in vivo</i> tumor growth. BCL2L11 mRNA contains a binding site for miR-106a in the 3'untranslated region. <i>BCL2L11</i> was found to be one of miR-106a targets. Altogether, our data suggest that miR-106a inhibits proliferation and invasiveness and induces cell cycle arrest and apoptosis in EC cells by targeting <i>BCL2L11</i>, and therefore miR-106a may serve as a prognostic marker of EC.
Project description:This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P ? 0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P < 0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P < 0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.
Project description:To examine the difference of the bovine cells between PBS and IFNT treatment, gene expression profiles were compared. The expressions of 902 genes were significantly higher in granulocytes with IFNT treament than those PBS treatment, whereas 1810 genes were lower (Fold change<1, P=0.05 or lower). The expressions of 555 genes were significantly higher in Bovine Endometrial Stromal cells (BES) with IFNT treatment than those PBS treatment, whereas 874 genes were lower (Fold change<1, P=0.05 or Low) Bovine granulocytes and BES were treated with PBS or IFNT
Project description:The aim of the present study was to determine the possible roles of chemokines in regulating bovine endometrial function during early pregnancy. The expression of six chemokines, including CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10, was higher in the endometrium at 15 and 18 days of pregnancy than at the same days in non-pregnant animals. Immunohistochemical staining showed that chemokine receptors (CCR1, CCR2, CCR3, and CXCR3) were expressed in the epithelial cells and glandular epithelial cells of the bovine endometrium as well as in the fetal trophoblast obtained from a cow on day 18 of pregnancy. The addition of interferon-? (IFNT) to an endometrial tissue culture system increased CCL8 and CXCL10 expression in the tissues, but did not affect CCL2, CCL11, and CCL16 expression. CCL14 expression by these tissues was inhibited by IFNT. CCL16, but not other chemokines, clearly stimulated interferon-stimulated gene 15 (ISG15) and myxovirus-resistance gene 1 (MX1) expression in these tissues. Cyclooxygenase 2 (COX2) expression decreased after stimulation with CCL8 and CCL14, and oxytocin receptor (OTR) expression was decreased by CCL2, CCL8, CCL14, and CXCL10. Collectively, the expression of chemokine genes is increased in the endometrium during early pregnancy. These genes may contribute to the regulation of endometrial function by inhibiting COX2 and OTR expression, subsequently decreasing prostaglandin production and preventing luteolysis in cows.