RESUMEN
Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases.
Asunto(s)
Herpesvirus Humano 1 , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunidad Innata , Proteínas de la Membrana , Pez Cebra , Animales , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pez Cebra/inmunología , Herpesvirus Humano 1/inmunología , Herpesvirus Suido 1/inmunología , Inmunidad Celular , Regulación de la Expresión Génica/inmunología , Transducción de Señal/inmunología , Transcripción Genética/inmunología , Regiones Promotoras Genéticas , Células HEK293 , Línea Celular , Herpes Simple/inmunología , Seudorrabia/inmunologíaRESUMEN
Prostaglandin E2 (PGE2) is considered as a luteoprotective factor, influencing the corpus luteum during the early pregnant period in the bovine species. Cyclic AMP (cAMP) is activated in response to PGE2 and plays a role in many physiological processes. The maternal recognition signal, interferon τ (IFNT), induces PGE2 secretion from the endometrial epithelial cells, the function of which in stroma cells has not been completely understood. In this study, PGE2 was found to activate cAMP in the bovine endometrial stromal cells (STRs). STRs were then treated with forskolin to activate the cAMP signaling, from which RNA extracted was subjected to global expression analysis. Transcripts related to transcription regulatory region nucleic acid binding of molecular function, nucleus of cellular component, and mitotic spindle organization of biological processes were up-regulated in cAMP-activated bovine STRs. An increase in the transcription factors, NFIL3, CEBPA, and HIF1A via the cAMP/PKA/CREB signaling pathway in the bovine STRs was also found by qPCR. Knockdown of NFIL3, CEBPA, or HIF1A blocked forskolin-induced PTGS1/2 and IGFBP1/3 expression. Moreover, NFIL3 and CEBPA were localized in endometrial stroma on pregnant day 17 (day 0 = estrous cycle), but not on cyclic day 17. These observations indicated that uterine PGE2 induced by conceptus IFNT is involved in the early pregnancy-related gene expression in endometrial stromal cells, which could facilitate pregnancy establishment in the bovine.
Asunto(s)
Dinoprostona , Células del Estroma , Embarazo , Femenino , Bovinos , Animales , Dinoprostona/metabolismo , Colforsina/farmacología , Colforsina/metabolismo , Células del Estroma/metabolismo , Células Epiteliales/metabolismoRESUMEN
Pregnancy loss predominantly occurs during the first 3-4 weeks due to fertilization failure or early embryonic losses in cattle. Insufficient biochemical communication between conceptus (embryo plus extraembryonic membranes) and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy losses could be better understood. To identify candidate molecules as detection markers of non-pregnant or females undergoing embryonic loss, peripheral blood from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment), which were subjected to metabolome and global proteome iTRAQ analyses. The metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with top 25 proteins was separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 serum had the highest area under the curve (AUC): 0.983. We also detected SNX5 in day 22 serum from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 serum could predict early pregnancy loss in heifers.
RESUMEN
Pigs have been increasingly recognized as a relevant model for studying many human diseases. However, functions and regulations of numerous critical molecules involved in human diseases are not well characterized in pigs, including the prominent tumor suppressor p53, a transcription factor involved in various anti-proliferative processes. In this study, we systematically characterized porcine p53 (p-p53) in its transcriptional activity and regulation by the E3 ligase Mdm2, in comparison with that of human p53 (h-p53). p-p53 is highly homologous to h-p53 with the N-terminal region showing relative divergence. p-p53 exhibits a comparable transcriptional activity to that of h-p53 towards a diverse range of known target genes, and is subject to ubiquitination and degradation by both human and porcine Mdm2 (h-/p-Mdm2). Utilization of the h-Mdm2 targeting compound Nutlin-3 and protein RPL11 inhibits the negative effect of p-Mdm2 on p-p53. These results suggest that the transcription activity and regulation of p-p53 is very similar to that of h-p53, and that the developed agents targeting the h-p53 pathway could be used in the study of p53 related processes and diseases in pigs.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Genes p53 , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Animales , Línea Celular , Humanos , Ratones , PorcinosRESUMEN
Embryo implantation into the uterine endometrium is required for pregnancy establishment in most mammals. By using global expression analysis, we investigated the molecules that are related to epithelial-mesenchymal transition (EMT) in noninvasive bovine trophoblasts and found that the transcription factor, ovo-like zinc finger 2 ( OVOL2), which is essential for mesenchymal-epithelial transition in various cancers, was down-regulated after trophoblast attachment to the endometrial epithelium in utero. In cultured bovine trophoblast cells, OVOL2 down-regulation occurred only when cells were allowed to attach to bovine endometrial epithelial cells via the TEAD3/YAP signaling pathway. This resulted in the up-regulation of the EMT-associated transcription factors, ZEB1 and SNAI2, and the mesenchymal cell markers, N-cadherin ( CDH2) and vimentin ( VIM), whereas epithelial cell marker, E-cadherin ( CDH1), was down-regulated. In contrast, OVOL2 overexpression in bovine trophoblast cells exhibited a decrease in ZEB1 transcripts and an increase in E-cadherin. These observations revealed that ovo-like protein (OVOL)2 down-regulation occurred concurrently with conceptus implantation into the uterine endometrium via the YAP/TEAD3 signaling pathway, and suggest that the down-regulation of OVOL2 expression contributes to the up-regulation of EMT-related transcription factor expression, which enables EMT progression in the noninvasive bovine trophectoderm postimplantation.-Bai, R., Kusama, K., Nakamura, K., Sakurai, T., Kimura, K., Ideta, A., Aoyagi, Y., Imakawa, K. Down-regulation of transcription factor OVOL2 contributes to epithelial-mesenchymal transition in a noninvasive type of trophoblast implantation to the maternal endometrium.
Asunto(s)
Regulación hacia Abajo/fisiología , Implantación del Embrión/fisiología , Endometrio/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Transcripción/biosíntesis , Trofoblastos/metabolismo , Animales , Bovinos , Endometrio/citología , Femenino , Embarazo , Transducción de Señal/fisiología , Trofoblastos/citologíaRESUMEN
In human trophoblast cells, cyclic AMP or its inducer forskolin (FSK) activates two downstream signaling molecules, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), both of which induce syncytialization, cell fusion, and the production of human chorionic gonadotropin (hCG) and progesterone. However, a transcription factor other than GCM1 and molecular mechanisms associated with these events have not been well characterized. To identify novel transcription factors involved in syncytialization of cAMP-stimulated human choriocarcinoma BeWo cells, the microarray analysis was performed with RNAs extracted from PKA- or EPAC-selective cAMP analog-stimulated BeWo cells, from which two up-regulated transcription factors, STAT5 and NR4A3, were found. The knockdown of STAT5B decreased FSK-induced cell fusion and the expression of syncytialization markers, CGB, syncytin1, syncytin2, GCM1, and OVOL1, but NR4A3 knockdown increased FSK-induced cell fusion and the expression of CGB and syncytin2. These findings indicated that cAMP-PKA up-regulated STAT5B, followed by increase in syncytin2 expression through GCM1 and OVOL1, resulting in cell fusion and hCG production, while cAMP-PKA-up-regulated NR4A3 could decrease syncytin2 expression, and suggested that both positive and negative effects of STAT5B and NR4A3, respectively, are required to control the degree of syncytialization in human trophoblast cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Gestacionales/biosíntesis , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Factor de Transcripción STAT5/metabolismo , Trofoblastos/metabolismo , Fusión Celular , Línea Celular Tumoral , Humanos , Trofoblastos/citologíaRESUMEN
Exosomes, extracellular vesicles, are present in uterine flushing fluids (UFs), which are involved in conceptus-endometrial interactions during peri-implantation periods. Despite several studies on intrauterine exosomes conducted, the roles conceptus and endometrial exosomes play during peri-implantation periods have not been well characterized. To investigate the effect of bovine intrauterine exosomes on conceptus implantation, exosomes isolated from bovine UFs during peri-implantation periods were subjected to global protein analysis. The analysis detected 596 exosomal proteins, including ruminants' pregnancy recognition factor IFNT, and 172 differentially expressed proteins with more than 1.5-fold changes in UFs on days 17, 20 and 22 pregnancy (day of conceptus implantation is initiated on days 19-19.5). Treatment of primary bovine endometrial epithelial cells with exosomes from day 17 UFs up-regulated the expression of apoptosis-related genes, and treatment with exosomes from day 20 and 22 UFs up-regulated the expression of adhesion molecule. Based on these findings, intrauterine exosomes should be considered as an essential constituent for successful implantation.
Asunto(s)
Implantación del Embrión/fisiología , Exosomas/metabolismo , Preñez/fisiología , Proteoma/metabolismo , Útero/fisiología , Útero/ultraestructura , Animales , Bovinos , Exosomas/ultraestructura , Femenino , EmbarazoRESUMEN
Seminal plasma (SP) is considered as a vehicle to carry sperm into female reproductive tract, of which functions have not been completely understood. This study aimed to identify the function of seminal exosomes on porcine endometrium. Exosomes were isolated from the sperm-rich fraction of boar semen and were confirmed by the expression of exosome marker HSP70 and size distribution using nano-sight tracking analysis. Porcine endometrial epithelial cells (EECs) were then treated with seminal exosomes, and RNA extracted were subjected to global expression analysis. Transcripts related to "immune response", "inflammatory response" and their associated signaling pathways were up-regulated in EECs treated with seminal exosome, whereas those associated with "steroid biosynthesis", "metabolic pathways" and "T cell differentiation" were down-regulated. The decrease in PMVK, SC5D, INSIG1, HSD17B7, NSDHL, HMGCR, SQLE and FDFT1, and increase in CCL20, TNFSF15, AMCFII, CXCL2 and CXCL8 were also found in the endometrium from the naturally mated pigs. Moreover, changes in exosome-induced CYP24A1, EBP, CCL20, AMCFII and IL1A expression were not regulated by the exosome removed SP. These observations indicated that exosomes present in SP are involved in the immune-related gene regulation in the uterus, which could pave the passage for sperm and possibly fertilized eggs.
Asunto(s)
Endometrio/inmunología , Exosomas/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/inmunología , Factores Inmunológicos/inmunología , Semen/inmunología , Animales , Citocinas/inmunología , Células Epiteliales/inmunología , Femenino , Masculino , Semen/citología , Sus scrofaRESUMEN
Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell-cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.
Asunto(s)
Retrovirus Endógenos/genética , Regulación del Desarrollo de la Expresión Génica , Placenta/fisiología , Transcripción Genética/fisiología , Vía de Señalización Wnt/fisiología , Animales , Bovinos , Femenino , EmbarazoRESUMEN
The year 2017 marks the 30th year since the discovery was made of amino acid and complementary DNA sequences of ovine trophoblast protein-1 (oTP-1), later renamed as interferon-tau (IFNτ). Ovine TP-1 was originally found as a secretory product of sheep conceptuses that rescues maternal corpus luteum (CL) and in fact, the uterine infusion of oTP-1 extended inter-estrous intervals. Finding this signaling molecule as an IFN-like sequence was surprising to the scientific community in reproduction because a homologous molecule in humans possesses anti-viral and anti-prolific activity and is often used in human medicines. However, since its discovery was made, large efforts have been made in the elucidation of transcriptional regulation and functions of bovine and ovine IFNτs, more importantly, the improvement of pregnancy rates in sheep and cattle, most of which resulted in unsuccessful outcomes. In this review, physiological, cellular and molecular events associated with continued secretion of progesterone, maternal recognition of pregnancy, identification, transcriptional regulation and function of IFNτ, and its future perspectives will be discussed.
Asunto(s)
Interferón Tipo I , Proteínas Gestacionales , Investigación/tendencias , Secuencia de Aminoácidos , Animales , Antivirales , Secuencia de Bases , Bovinos , Cuerpo Lúteo , Ciclo Estral/efectos de los fármacos , Femenino , Humanos , Interferón Tipo I/química , Interferón Tipo I/genética , Interferón Tipo I/farmacología , Interferón Tipo I/fisiología , Embarazo , Proteínas Gestacionales/química , Proteínas Gestacionales/genética , Proteínas Gestacionales/farmacología , Proteínas Gestacionales/fisiología , Progesterona/metabolismo , Ovinos , Factores de Tiempo , Transcripción GenéticaRESUMEN
Immunological tolerance between fetal allograft and mother is crucial for pregnancy establishment and maintenance; however, these mechanisms particularly those during the latter part of pregnancy have not been definitively elucidated. The aim of this study was to examine the presence and potential function of innate immunity characteristic to the middle to late pregnancy. We first characterized up-regulated proteins in decidua from day 11 pregnant (P11) mice using 2D-PAGE, followed by MALDI-TOF/MS analysis. These analyses identified increased complement component 3 (C3) and its derivatives in P11 decidua. We then found that in the decidual tissues, C3 mRNA increased on P15 and remained high on P19. C3 is converted to C3b and then iC3b by complement component factor I (Cfi) and complement receptor 1-like protein (Crry), both of which were present in P19 placentas. In addition, iC3b proteins and its receptor CR3 (Cd11b/Cd18) in decidual and placental tissues increased toward the latter phase of pregnancy. Moreover, CR3 subunit CD11b protein was predominantly localized to spongiotrophoblast layer in the P19 placenta. Because iC3b is known to induce anti-inflammatory cytokine production, the analysis was extended to examine changes in pro- and anti-inflammatory cytokines, Il12, Il10, and Tgfb1. Il12 expression decreased in P15 and P19 placenta, while high mRNA expression of Il10 and Tgfb1 was found in P19 placental tissues. Furthermore, placental Il10 and Tgfb1 mRNAs were down-regulated when pregnant mice were treated with an anti-C3 antibody, detecting C3, C3b and iC3b. These results indicated that C3 derivatives, in particular, iC3b and its receptor CR3 were up-regulated at the fetal-maternal interface, and suggest that iC3b may regulate the placental expression of anti-inflammatory cytokines, IL10 and TGFB1, during the latter phase of pregnancy.
Asunto(s)
Antiinflamatorios/metabolismo , Complemento C3b/metabolismo , Citocinas/metabolismo , Inflamación/metabolismo , Animales , Regulación hacia Abajo/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Placenta/metabolismo , Embarazo , ARN Mensajero/metabolismo , Receptores de Complemento 3b/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
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.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Interferón Tipo I/farmacología , Proteínas Gestacionales/farmacología , Animales , Bovinos , Células Cultivadas , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Endometrio/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Factores Reguladores del Interferón/metabolismo , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/genética , Proteínas Gestacionales/antagonistas & inhibidores , Proteínas Gestacionales/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Interferencia de ARN , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
As placental morphology as well as trophoblast characteristics exhibit wide diversity across mammalian species, underling molecules were also thought to vary greatly. In the majority of cases, however, regardless of the mode of implantation, physiological and biochemical processes in conceptus implantation to the maternal endometrium including the kinds of gene expression and their products are now considered to share many similarities. In fact, recent progress has identified that in addition to the hormones, cytokines, proteases and cell adhesion molecules classically characterized, molecules related to lymphocyte homing and epithelial-mesenchymal transition (EMT) are all required for the progression of conceptus implantation to placentation. In this review, therefore, the newest findings are all incorporated into the molecular and cellular events related to conceptus implantation to the maternal endometrium; primarily from non-invasive bovine placentation and also from invasive human implantation.
Asunto(s)
Implantación del Embrión/fisiología , Endometrio/metabolismo , Animales , Bovinos , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
Dynamic changes in bovine conceptus and endometrium occur during early gestation, in which the conceptus undergoes epithelial to mesenchymal transition (EMT) after the conceptus attachment to endometrium. To characterize EMT inducing factors, we initially undertook iTRAQ analysis with bovine uterine flushing (UF) obtained from pregnant animals on days 17 (P17: pre-attachment) and 20 (P20: post-attachment). The iTRAQ analysis demonstrated that follistatin (FST), an inhibitor of activin A, increased in P20 UF. We then found that FST decreased in P22 conceptuses, whereas elevated activin A found in P20 UF and endometria was further increased on P22. In addition, phosphorylated SMAD2 increased in P22 conceptuses. In bovine trophoblast cells, the treatment with P22 UF or activin An up-regulated EMT marker expressions, which were inhibited by FST. These results suggest that the initiation of bovine conceptus EMT could be regulated through the spatiotemporal expression of FST or activin A during the peri-attachment period.
Asunto(s)
Activinas/metabolismo , Endometrio/embriología , Transición Epitelial-Mesenquimal , Folistatina/metabolismo , Animales , Bovinos , Células Cultivadas , Endometrio/metabolismo , Femenino , Fosforilación , Embarazo , Proteína Smad2/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismoRESUMEN
Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to cell interactions for conceptus implantation to the maternal endometrium.
Asunto(s)
Implantación del Embrión , Endometrio/metabolismo , Exosomas/metabolismo , Interferón Tipo I/metabolismo , Proteínas Gestacionales/metabolismo , Animales , Células Cultivadas , Endometrio/fisiología , Femenino , Embarazo , Proteoma/metabolismo , OvinosRESUMEN
Processes of conceptus implantation and placentation, unique to mammalian reproduction, have been extensively studied. It was once thought that processes of these events varied greatly, notably between invasive and noninvasive modes of implantation and/or placentation. Regardless of the mode of implantation, however, physiological and biochemical processes in conceptus implantation to the maternal endometrium including the kinds of gene expression and their products are now considered not to differ so much. Recent progress has identified that in addition to the hormones, cytokines, proteases and cell adhesion molecules classically characterized, epithelial-mesenchymal transition, molecules related to lymphocyte homing, the expression of endogenous retroviruses and possibly exosomes are all required for the progression of conceptus implantation to placentation. In this review, therefore, new findings related to these events are integrated into the context of conceptus implantation to the maternal endometrium.
RESUMEN
A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0 = day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species.
Asunto(s)
Implantación del Embrión/fisiología , Endometrio/fisiología , Epitelio/fisiología , Feto/fisiología , Selectinas/fisiología , Útero/fisiología , Animales , Factor de Transcripción CDX2 , Bovinos , Células Cultivadas , Femenino , Proteínas de Homeodominio/genética , Interferón Tipo I/genética , Ligandos , Glicoproteínas de Membrana/genética , Embarazo , Proteínas Gestacionales/genética , ARN/genética , ARN Interferente Pequeño/genética , Transactivadores/genéticaRESUMEN
GATA transcription factors are emerging as critical regulators in trophoblast development and its gene regulation. The purpose of this study was to examine the expression and cellular localization of GATA2 in ovine conceptuses during the peri-implantation period. In Western blot analyses, GATA2 proteins were found in days 15, 17 and 21 ovine conceptuses (day 0=day of estrus). Using immunohistochemistry and immunofluorescence analyses, we found that GATA2 was localized in days 15, 17 and 21 ovine conceptuses, and more importantly, GATA2 protein was detected in both nuclear and cytoplasmic regions of the trophectoderm. To our knowledge, the present study is the first to demonstrate that GATA2 is localized in two cellular compartments of the trophectoderm in ovine and many other mammalian species, and suggests that the difference in GATA2 location plays a role in the regulation of down-stream genes during the early pregnancy period.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Embrión de Mamíferos , Desarrollo Embrionario/genética , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA4/fisiología , Ovinos/embriología , Ovinos/genética , Trofoblastos/citología , Animales , Implantación del Embrión , Femenino , Factor de Transcripción GATA2 , Factor de Transcripción GATA4/análisis , Regulación del Desarrollo de la Expresión Génica , EmbarazoRESUMEN
Following bidirectional communication, the conceptus and the uterine epithelium must establish a proper cell-cell interaction, resulting in the progression of implantation processes. To clarify the mechanism of conceptus attachment to the uterine endometrium, we studied whether vascular cell adhesion molecule (VCAM1) was expressed in bovine conceptuses or endometrium during the peri-attachment period. Uterine VCAM1 expression was minimal in day 17 (day 0=day of estrus) cyclic and pregnant animals, but increased between days 20 and 22 of pregnancy. In the intercaruncular regions, VCAM1 protein was localized to the luminal and glandular epithelia, whereas in the caruncular regions, VCAM1 protein was detected in the stroma and endothelia of the uterine endometrium. In cultured endometrial epithelial cells (EECs), VCAM1 expression was up-regulated when treated with uterine flushings or growth factor and further increased when EECs were cocultured with bovine trophoblast CT1 cells. VCAM1 expression in CT1 cells was also up-regulated with the use of uterine flushings, and further increased when these cells were cocultured with EECs. Expression of VCAM1 receptor, integrin α 4 (ITGA4) mRNA, increased significantly in day 22 conceptuses. In day 22 pregnant uteri, VCAM1 protein was found in both EECs and conceptuses, but ITGA4 was localized only to trophoblasts. These observations indicate that cell-cell interactions between conceptuses and uterine epithelial cells are required for sufficient VCAM1 and ITGA4 expression in the bovine species and suggest that uterine VCAM1 and conceptus ITGA4 play a role in the establishment of conceptus adhesion to the uterine endometrium.
Asunto(s)
Blastocisto/metabolismo , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Endometrio/metabolismo , Trofoblastos/metabolismo , Útero/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Blastocisto/citología , Western Blotting , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Implantación del Embrión , Endometrio/citología , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas para Inmunoenzimas , Integrina alfa4/genética , Integrina alfa4/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/citología , Útero/citología , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Bovine primary uterine endometrial epithelial cells (EECs) are not ideal for long-term studies, because primary EECs lose hormone responsiveness quickly, and/or they tend to have a short life span. The aims of this study were to establish immortalized bovine EECs and to characterize these cells following long-term cultures. Immortalized bovine EECs were established by transfecting retroviral vectors encoding human papillomavirus (HPV) E6 and E7, and human telomerase reverse transcriptase (hTERT) genes. Established bovine immortalized EECs (imEECs) showed the same morphology as primary EECs, and could be grown without any apparent changes for over 60 passages. In addition, imEECs have maintained the features as EECs, exhibiting oxytocin (OT) and interferon tau (IFNT) responsiveness. Therefore, these imEECs, even after numbers of passages, could be used as an in vitro model to investigate cellular and molecular mechanisms, by which the uterine epithelium responds to IFNT stimulation, the event required for the maternal recognition of pregnancy in the bovine species.