RESUMO
The detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) is not only of great significance in the areas of biomedicine and neurochemistry but also helpful in disease diagnosis and pathology research. Due to their diverse structures, designability, and large specific surface areas, metal-organic frameworks (MOFs) have recently caught considerable attention in the electrochemical field. Herein, a family of heterometallic MOFs with amino modification, MIL-125(Ti-Al)-xNH2 (x = 0%, 25%, 50%, 75%, and 100%), were synthesized and employed as electrochemical sensors for the detection of AA, DA, and UA. Among them, MIL-125(Ti-Al)-75%NH2 exhibited the most promising electrochemical behavior with 40% doping of carbon black in 0.1 M PBS (pH = 7.10), which displayed individual detection performance with wide linear detection ranges (1.0-6.5 mM for AA, 5-100 µM for DA and 5-120 µM for UA) and low limits of detection (0.215 mM for AA, 0.086 µM for DA, and 0.876 µM for UA, S/N = 3). Furthermore, the as-prepared MIL-125(Ti-Al)-75%NH2/GCE provided a promising platform for future application in real sample analysis, owing to its excellent anti-interference performance and good stability.
Assuntos
Dopamina , Estruturas Metalorgânicas , Dopamina/análise , Ácido Úrico/análise , Ácido Ascórbico/química , Eletrodos , Titânio , Técnicas EletroquímicasRESUMO
Myometrium plays critical roles in multiple processes such as embryo spacing through peristalsis during mouse implantation, indicating vital roles of smooth muscle in the successful establishment and quality of implantation. Actin, a key element of cytoskeleton structure, plays an important role in the movement and contraction of smooth muscle cells (SMCs). However, the function of peri-implantation uterine smooth muscle and the regulation mechanism of muscle tension are still unclear. This study focused on the molecular mechanism of actin assembly regulation on implantation in smooth muscle. Phalloidin is a highly selective bicyclic peptide used for staining actin filaments (also known as F-actin). Phalloidin staining showed that F-actin gradually weakened in the CD-1 mouse myometrium from day 1 to day 4 of early pregnancy. More than 3 mice were studied for each group. Jasplakinolide (Jasp) used to inhibit F-actin depolymerization promotes F-actin polymerization in SMCs during implantation window and consequently compromises embryo implantation quality. Transcriptome analysis following Jasp treatment in mouse uterine SMCs reveals significant molecular changes associated with actin assembly. Tagln is involved in the regulation of the cell cytoskeleton and promotes the polymerization of G-actin to F-actin. Our results show that Tagln expression is gradually reduced in mouse uterine myometrium from day 1 to 4 of pregnancy. Furthermore, progesterone inhibits the expression of Tagln through the progesterone receptor. Using siRNA to knock down Tagln in day 3 SMCs, we found that phalloidin staining is decreased, which confirms the critical role of Tagln in F-actin polymerization. In conclusion, our data suggested that decreases in actin assembly in uterine smooth muscle during early pregnancy is critical to optimal embryo implantation. Tagln, a key molecule involved in actin assembly, regulates embryo implantation by controlling F-actin aggregation before implantation, suggesting moderate uterine contractility is conducive to embryo implantation. This study provides new insights into how the mouse uterus increases its flexibility to accommodate implanting embryos in the early stage of pregnancy.
Assuntos
Actinas , Receptores de Progesterona , Gravidez , Feminino , Camundongos , Animais , Actinas/metabolismo , Receptores de Progesterona/metabolismo , Progesterona/metabolismo , RNA Interferente Pequeno/metabolismo , Faloidina/metabolismo , Implantação do Embrião , Útero/metabolismo , Músculo Liso/metabolismoRESUMO
Progesterone is required for the establishment and maintenance of mammalian pregnancy and widely used for conservative treatment of luteal phase deficiency in clinics. However, there are limited solid evidences available for the optimal timing and dose of progesterone therapy, especially for the possible adverse effects on implantation and decidualization when progesterone is administrated empirically. In our study, mouse models were used to examine effects of excess progesterone on embryo implantation and decidualization. Our data indicate that excess progesterone is not only harmful for mouse implantation, but also impairs mouse decidualization. In excess progesterone-treated mice, the impaired LIF/STAT3 pathway and dysregulated endoplasmic reticulum stress may lead to the inhibition of embryo implantation and decidualization. It is possible that the decrease in birth weight of excess progesterone-treated mice is due to a compromised embryo implantation and decidualization. Furthermore, excess progesterone compromises in vitro decidualization of human endometrial stromal cells.
Assuntos
Implantação do Embrião/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Endométrio/fisiologia , Progesterona/metabolismo , Progestinas/metabolismo , Animais , Estresse do Retículo Endoplasmático , Feminino , Humanos , Fator Inibidor de Leucemia , Camundongos , Fator de Transcrição STAT3 , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologiaRESUMO
The establishment of decidualization is a prerequisite of successful pregnancy. Lysyl oxidase (Lox) is a copper-containing amine oxidase which catalyzes cross-linking of collagen and elastin in the ECM. Lox is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy. From days 6 to 8, the signals for Lox mRNA and protein are strongly detected in the decidual cells. The expression of Lox is under the control of estrogen via the GSK-3ß/ß-catenin/c-myc pathway. Dtprp is decreased by the inhibition of Lox activity. Furthermore, the inhibition of Lox activity decreases stromal cell migration and embryo adhesion. Our findings highlight the crucial role of Lox in endometrial stromal cells and deepen our understanding of decidualization.
Assuntos
Blastocisto/metabolismo , Decídua/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Prolactina/análogos & derivados , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Animais , Movimento Celular , Implantação do Embrião , Estrogênios/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Gravidez , Prolactina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células Estromais/citologia , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
The aim of this study was to investigate the effects of copper ions on decidualization of human endometrial stromal cells (HESCs) cultured in vitro. Firstly, non-toxic concentrations of copper D-gluconate were screened in HESCs based on cell activity. Then, the effects of non-toxic concentrations of copper ions (0~250 µM) were examined on decidualization of human endometrial stromal cells. Our data demonstrated that the mRNA expressions of insulin-like growth factor binding protein (IGFBP-1), prolactin (PRL), Mn-SOD, and FOXO1were down-regulated during decidualization following the treatments with 100 or 250 µM copper ions. Meanwhile, the amount of malonaldehyde (MDA) in the supernatant of HESCs was increased. These results showed that in vitro decidualization of HESCs was impaired by copper treatment.
Assuntos
Cobre/farmacologia , Células Estromais/efeitos dos fármacos , Cobre/administração & dosagem , Relação Dose-Resposta a Droga , Humanos , Íons/administração & dosagem , Íons/farmacologia , Células Estromais/metabolismoRESUMO
Lipocalin-2 (Lcn2) is a small glycoprotein involved in a number of biological processes such as inflammation and antibacterial response. In our study, Lcn2 is expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy. The expression of Lcn2 in stromal cells is under the control of progesterone through Akt-c-Myc signaling pathway. Data from Lcn2 knockdown and recombinant protein treatments indicate that Lcn2 promotes mPGES-1 expression in stromal cells. The expression of Lcn2 and mPGES-1 is strongly stimulated by lipopolysaccharide (LPS), indicating that Lcn2 mediates LPS-induced inflammation. These findings shed light on the role of Lcn2 during decidualization.
Assuntos
Inflamação/genética , Lipocalina-2/genética , Progesterona/metabolismo , Prostaglandina-E Sintases/biossíntese , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipocalina-2/biossíntese , Lipopolissacarídeos/toxicidade , Camundongos , Gravidez , Prostaglandina-E Sintases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Unfolded or misfolded protein accumulation in the endoplasmic reticulum lumen leads to endoplasmic reticulum stress (ER stress). Although it is known that ER stress is crucial for mammalian reproduction, little is known about its physiological significance and underlying mechanism during decidualization. Here we show that Ire-Xbp1 signal transduction pathway of unfolded protein response (UPR) is activated in decidual cells. The process of decidualization is compromised by ER stress inhibitor tauroursodeoxycholic acid sodium (TUDCA) and Ire specific inhibitor STF-083010 both in vivo and in vitro. A high concentration of ER stress inducer tunicamycin (TM) suppresses stromal cells proliferation and decidualization, while a lower concentration is beneficial. We further show that ER stress induces DNA damage and polyploidization in stromal cells. In conclusion, our data suggest that the GRP78/Ire1/Xbp1 signaling pathway of ER stress-UPR is activated and involved in mouse decidualization.
Assuntos
Decídua/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Decídua/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Camundongos , Gravidez , Transdução de Sinais , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Sulfonamidas/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Tiofenos/farmacologia , Tunicamicina/farmacologia , Resposta a Proteínas não DobradasRESUMO
Decidualization is an essential step in the establishment of pregnancy. However, the functional contributions of long intergenic noncoding RNAs (LincRNAs) to decidualization have not been explored. To explore the regulation and role of LincRNAs during human decidualization, human endometrial stromal cells (HESCs) are induced to undergo in vitro decidualization by treating with estradiol-17ß, db-cAMP and medroxyprogesterone acetate. LINC00473 (LINC473) expression is highly induced in HESCs after decidual stimulus. We found that cAMP-PKA pathway regulates the expression of LINC473 through IL-11-mediated STAT3 phosphorylation. RNA interference-mediated down-regulation of LINC473 inhibits in vitro decidualization. These results suggested that LINC473 might be functionally required for human decidualization. This is the first report demonstrating the presence of LincRNA during human decidualization.
Assuntos
Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Decídua/citologia , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologia , Células Cultivadas , Estradiol/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Acetato de Medroxiprogesterona/metabolismoRESUMO
Receptivity is a limited time in which uterine endometrium can establish a successful dialogue with blastocyst. This study was to investigate the effect of asynchronous embryo transfer on uterine receptivity in mice. Embryos under different stages were transferred into two oviduct sides of a recipient mouse on day 1 of pseudopregnancy. Our results showed the asynchronously transferred embryos can implant in all groups. Compared to zygote-transfer group, the length of implanted embryos is longer in 8-cell embryo- or blastocyst-transfer group. The levels of Snail and COX-2 immunostaining in blastocyst-transfer group are significantly stronger than that in zygote-transfer group. Embryos in blastocyst-transfer group migrate faster than that in zygote-transfer group within uterus. Blastocysts are in a state of developmental delay after they are transferred into oviducts, and they are reactivated and implanted rapidly in uterus. The developmental rate to newborn in zygote-transfer group is obviously higher than that in blastocyst-transfer group, suggesting that a delay in embryo development and implantation will lead to a decrease of litter size. These results indicated that the window of implantation is differentially regulated in two uterine horns of a recipient by embryos at different stages.
Assuntos
Implantação do Embrião/fisiologia , Transferência Embrionária/métodos , Endométrio/fisiologia , Zigoto/transplante , Animais , Blastocisto/citologia , Ciclo-Oxigenase 2/metabolismo , Desenvolvimento Embrionário/fisiologia , Feminino , Masculino , Camundongos , Pseudogravidez/embriologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Zigoto/crescimento & desenvolvimentoRESUMO
Embryo implantation is a highly synchronized process between an activated blastocyst and a receptive uterus. Successful implantation relies on the dynamic interplay of estrogen and progesterone, but the key mediators underlying embryo implantation are not fully understood. Here we show that transcription factor early growth response 1 (Egr1) is regulated by estrogen as a downstream target through leukemia inhibitory factor (LIF) signal transducer and activator of transcription 3 (STAT3) pathway in mouse uterus. Egr1 is localized in the subluminal stromal cells surrounding the implanting embryo on day 5 of pregnancy. Estrogen rapidly, markedly, and transiently enhances Egr1 expression in uterine stromal cells, which fails in estrogen receptor α knock-out mouse uteri. STAT3 is phosphorylated by LIF and subsequently recruited on Egr1 promoter to induce its expression. Our results of Egr1 expression under induced decidualization in vivo and in vitro show that Egr1 is rapidly induced after deciduogenic stimulus. Egr1 knockdown can inhibit in vitro decidualization of cultured uterine stromal cells. Chromatin immunoprecipitation data show that Egr1 is recruited to the promoter of wingless-related murine mammary tumor virus integration site 4 (Wnt4). Collectively, our study presents for the first time that estrogen regulates Egr1 expression through LIF-STAT3 signaling pathway in mouse uterus, and Egr1 functions as a critical mediator of stromal cell decidualization by regulating Wnt4.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Implantação do Embrião , Estrogênios/metabolismo , Fator Inibidor de Leucemia/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína Wnt4/metabolismo , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Primers do DNA , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Imunofluorescência , Técnicas de Silenciamento de Genes , Hibridização In Situ , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Thyroid dysfunction during human pregnancy is closely related to serious pregnancy outcome. However, the regulation and function of thyroid hormones during early pregnancy are largely unknown. We found that type II deiodinase, an enzyme converting T4 to activated T3, is highly expressed in the mouse uterus on days 3 and 4 of pregnancy. Once the embryo implants into the receptive uterus, type III deiodinase (Dio3), a mainly paternally imprinted gene for inactivating T3, is significantly induced in the stromal cells and accompanied by DNA hypermethylation of intergenic differentially CpG methylation regions in the δ-like 1 homolog-Dio3 imprinting cluster. The concentration of uterine free T3 is actually decreased after embryo implantation. T3 induces Dio3 expression both in vivo and in vitro, suggesting a positive feedback loop. T3 addition or Dio3 knockdown compromises decidualization. These results indicate that the Dio3-mediated local T3 decrease is critical for decidualization of stromal cells during early pregnancy. Furthermore, we found that progesterone regulates Dio3 expression through its cognate receptor both in vivo and in vitro. Additionally, cAMP regulates Dio3 transcription through the protein kinase A-cAMP response element-binding protein pathway. The inhibition of the protein kinase A pathway results in decreased Dio3 expression and impaired decidualization. Dio3 opposite strand (Dio3os) expressed in a similar pattern to Dio3, is transcribed from the opposite strand of Dio3 and fine-tunes Dio3 expression during decidualization. Our data indicate that Dio3 is strongly expressed and tightly controlled during decidualization.
Assuntos
Decídua/metabolismo , Iodeto Peroxidase/metabolismo , Tri-Iodotironina/metabolismo , Útero/metabolismo , Animais , Células Cultivadas , Implantação do Embrião , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização In Situ , Iodeto Peroxidase/genética , Masculino , Camundongos , Microscopia de Fluorescência , Ovariectomia , Gravidez , Interferência de RNA , Inibidores da Transcriptase Reversa , Células Estromais/metabolismo , Tri-Iodotironina/farmacologia , Iodotironina Desiodinase Tipo IIRESUMO
Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H2O2). Based on our data, GPX3 may play a major role in reducing H2O2 during decidualization.
Assuntos
Decídua/enzimologia , Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/metabolismo , Animais , Antioxidantes/farmacologia , Hipóxia Celular , Corpo Lúteo/efeitos dos fármacos , Decídua/fisiologia , Implantação do Embrião , Feminino , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Oxirredução , Gravidez , Progesterona/fisiologia , Ácido Selênico/farmacologia , Tiomalatos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Platelet-derived growth factor C (PDGF-C) has been reported to promote angiogenesis independently of vascular endothelial growth factor (VEGF), although its significance in postnatal angiogenesis in vivo remains poorly understood. VEGF has been employed as a major molecular tool to induce therapeutic angiogenesis. However, VEGF therapy is not very effective in models of cardiovascular diseases associated with diabetes, and the mechanisms of this phenomenon still remain to be elucidated. METHODS: We used a murine model of hind limb ischemia and of streptozotocin-induced diabetes. RESULTS: Expression of PDGF-C and its receptor PDGFR-α were markedly upregulated in ischemic limbs. Treatment with a neutralizing antibody against PDGF-C significantly impaired blood flow recovery and neovascularization after ischemia almost to the same extent as a VEGF-neutralizing antibody. Mice deficient in PDGF-C exhibited reduced blood flow recovery after ischemia compared with wild-type mice, confirming a strong proangiogenic activity of PDGF-C. Next, we injected an expression vector encoding PDGF-C into ischemic limbs. Blood flow recovery and neovascularization after ischemia were significantly improved in the groups treated with PDGF-C compared with controls. Attenuation of angiogenic responses to ischemia has been reported in patients with diabetes even after VEGF treatment, although a precise mechanism remains unknown. We hypothesized that PDGF-C might relate to the impaired angiogenesis of diabetes. We tested this hypothesis by inducing diabetes by intraperitoneal injection of streptozotocin. Expression levels of PDGF-C at baseline and after ischemia were significantly lower in limb tissues of diabetic mice than in those of control mice, whereas expression levels of other members of the PDGF family and VEGF were not changed or were even higher in diabetic mice. Introduction of VEGF complementary DNA expression plasmid vector into ischemic limbs did not improve blood flow recovery. However, these changes were effectively reversed by additional introduction of the PDGF-C complementary DNA plasmid vector. CONCLUSIONS: These results indicate that downregulation of PDGF-C expression in limb tissues of diabetic mice contributes to impaired angiogenesis and suggest that introduction of PDGF-C might be a novel strategy for therapeutic angiogenesis, especially in the diabetic state. CLINICAL RELEVANCE: Angiogenesis and arteriogenesis after ischemia are attenuated in most diabetic patients, although the precise mechanisms remain unclear. Platelet-derived growth factors (PDGFs) have a variety of functions on many cell types, and PDGF-C stimulates angiogenesis and revascularizes ischemic tissues. This study indicates the role for PDGF-C as a critical regulator of impaired angiogenesis of diabetes and suggests that PDGF-C might be a novel target for the treatment of ischemic cardiovascular diseases in diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Angiopatias Diabéticas/metabolismo , Isquemia/metabolismo , Linfocinas/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Anticorpos Neutralizantes/administração & dosagem , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Angiopatias Diabéticas/terapia , Técnicas de Transferência de Genes , Membro Posterior , Isquemia/genética , Isquemia/fisiopatologia , Isquemia/terapia , Linfocinas/antagonistas & inibidores , Linfocinas/deficiência , Linfocinas/genética , Linfocinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/deficiência , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Secretin, a classical gastrointestinal and neuroendocrine peptide, plays an important role in maintaining the body fluid balance. However, the expression and regulation of secretin in the reproductive system are still unknown. In our study, secretin is specifically expressed in the decidua on days 5 to 8 of pregnancy. Secretin expression is not detected under delayed implantation but is stimulated after estrogen activation and under artificial decidualization. Progesterone induces secretin expression in ovariectomized mice and cultured stromal cells, which is abrogated by specific LY294002. Because secretin is mainly localized in the decidua and also strongly expressed during in vitro decidualization, secretin may play a role during mouse decidualization through regulating cyclic adenosine monophosphate level.
Assuntos
Implantação do Embrião/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Progesterona/farmacologia , Secretina/biossíntese , Útero/metabolismo , Animais , Células Cultivadas , Implantação do Embrião/efeitos dos fármacos , Feminino , Camundongos , Gravidez , Progesterona/fisiologia , Útero/efeitos dos fármacosRESUMO
Embryo implantation requires a precise synchronism between the receptive uterus and activated blastocyst and is regulated by complicated molecular networks. Although many implantation-related genes have been identified, the crosstalk among them is still unknown. Snail, a transcription repressor, plays a central role during epithelial-mesenchymal transition. Our previous study showed that Snail is highly expressed at implantation site in mouse uterus. This study was to examine how Snail is related with other implantation-related genes in mice. Uterine stromal cells were isolated from mouse uteri on day 4 of pregnancy and treated with HB-EGF. Snail was induced significantly by HB-EGF. By using specific inhibitors and siRNA, we demonstrated that HB-EGF induction on Snail expression is dependent on the EGFR-ERK-Stat3 pathway. Cox-2 was regulated by Snail. The current findings demonstrate that Snail can relate with HB-EGF, Stat3 and Cox-2 and may play a role during mouse embryo implantation and decidualization.
Assuntos
Decídua/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Fatores de Transcrição/genética , Ativação Transcricional , Animais , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Implantação do Embrião , Receptores ErbB/metabolismo , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Fosforilação , Gravidez , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismoRESUMO
Thymosin ß4 (TMSB4X) belongs to a class of highly conserved small proteins that are present in a high abundance in immune tissues, where it participates in various biological activities, including anti-inflammation, wound healing, apoptosis, and cell survival. However, little is known about the expression and regulation of TMSB4X in reproductive tissues. The aim of this study was to examine the expression of rat Tmsb4x and chicken TMSB4X genes during testicular and epididymal development. Rat Tmsb4x was strongly detected in the spermatogonia and spermatocytes of testis at early postnatal development and transmitted to Leydig cells at sexual maturation. Also, rat Tmsb4x was detected at an increased level in the epididymis during postnatal development. When compared to the rat, expression of the chicken TMSB4X gene was low in the testis and epididymal region, and the mRNA localization was also unexpected. Three experiments were performed to examine the regulation of rat Tmsb4x in the epididymis: after elimination of Leydig cells using ethylene dimethane sulfonate (EDS); after destruction of the testis by cryptorchidism; and after castration. EDS-treated and castrated rats were injected with testosterone propionate. The expression of Tmsb4x was significantly reduced in the epididymis of EDS-treated, and castrated rats. In contrast, Tmsb4x was significantly enhanced in the epididymis after testosterone treatment. The expression of rat Tmsb4x was also regulated in the epididymis after cryptorchidism. Collectively, the expression of Tmsb4x was strongly detected in the testis and epididymis of rats, and was highly regulated in the epididymis by testosterone.
Assuntos
Epididimo/metabolismo , Testículo/metabolismo , Testosterona/metabolismo , Timosina/biossíntese , Animais , Galinhas , Regulação da Expressão Gênica no Desenvolvimento , Masculino , RNA Mensageiro/metabolismo , Ratos , Reprodução , Maturidade Sexual/genética , Espermatócitos/metabolismo , Timosina/genéticaRESUMO
Luminal closure and embryo apposition are essential for blastocyst attachment during early pregnancy. In our preliminary microarray results (unpublished data), sodium-potassium adenosine triphosphatase (Na/K-ATPase) ß1 (Atp1b1) was highly expressed in mouse uterus on Days 3 and 4 of pregnancy. However, expression and regulation of Atp1b1 in the mammalian uterus during early pregnancy are unknown. Using in situ hybridization, a strong level of Atp1b1 mRNA was detected in luminal epithelial cells on Days 3 and 4 of pregnancy (Day 1 = day of vaginal plug). The expression pattern of FXYD domain-containing ion transport regulator 4 (Fxyd4) was similar to that of Atp1b1. Real-time reverse transcription polymerase chain reaction confirmed the high expression level of Atp1b1 mRNA. Compared with Day 1, the mRNA level of Atp1b1 on Days 3 and 4 increased by 3.5 ± 0.5 and 4.5 ± 0.5 fold, respectively. When the embryo invaded through epithelial cells into the maternal stromal compartment on day 5, Atp1b1 expression decreased to a basal level. Progesterone stimulated Atp1b1 expression by 2.8 ± 1 fold compared with oil in ovariectomized mice at 24 hours after treatment. Expression of Atp1b1 was further upregulated to 4 ± 0.4 fold by estrogen and progesterone. Based on time-course study, progesterone rapidly induced Atp1b1 expression at 6 and 12 hours (13.7 ± 0.5 and 16.6 ± 1.4, respectively); furthermore, this upregulation was blocked by RU486 (progesterone receptor antagonist). Transcription activity of the Atp1b1 promoter was (Day 1 = day of vaginal plug) stimulated by CCAAT/enhancer binding protein beta (Cebpb). In conclusion, Atp1b1 was highly expressed in luminal epithelium during peri-implantation and upregulated by progesterone.
Assuntos
Implantação do Embrião/genética , Regulação Enzimológica da Expressão Gênica , Progesterona/fisiologia , ATPase Trocadora de Sódio-Potássio/genética , Útero/efeitos dos fármacos , Animais , Implantação do Embrião/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Idade Gestacional , Masculino , Camundongos , Gravidez , Progesterona/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , Distribuição Tecidual , Útero/enzimologia , Útero/metabolismoRESUMO
The epithelial-mesenchymal transition plays a critical role in embryonic development, cancer progression, and metastasis. Decidualization is the process by which the fibroblast-like endometrial stromal cells differentiate into polygonal epithelial-like cells. However, it is still unclear whether mesenchymal-epithelial transition (MET) occurs during decidualization. The aim of this study was to examine whether decidualization causes the downregulation of some mesenchymal markers and upregulation of some epithelial markers in cultured uterine stromal cells. We showed that decidualization causes the downregulation of snail and vimentin expression, and upregulation of E-cadherin and cytokeratin expression. During in vitro decidualization, cultured stromal cells lose elongated shape and show epithelium-like characteristics. Our data suggest that the process of MET may exist during decidualization.
Assuntos
Decídua/citologia , Decídua/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , GravidezRESUMO
The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.
Assuntos
Endométrio/metabolismo , Regulação da Expressão Gênica , Receptores de Progesterona/biossíntese , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Implantação do Embrião , Feminino , Macaca mulatta , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Gravidez , Prenhez , Útero/metabolismoRESUMO
Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.