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1.
Chemosphere ; 286(Pt 2): 131741, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34358888

RESUMO

Airborne particulate matter (PM), polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) are significant contributors leading to many human health issues. Thus, this study was designed to perform chemical analysis and biological impact of airborne particulate matter 10 (PM10) in the World heritage City of Kandy City in Sri Lanka. 12 priority PAHs and 34 metals, including 10 highly toxic HMs were quantified. The biological effects of organic extracts were assayed using an in vitro primary porcine airway epithelial cell culture model. Cytotoxicity, DNA damage, and gene expressions of selected inflammatory and cancer-related genes were also assessed. Results showed that the total PAHs ranged from 3.062 to 36.887 ng/m3. The metals were dominated by Na > Ca > Mg > Al > K > Fe > Ti, while a few toxic HMs were much higher in the air than the existing ambient air quality standards. In the bioassays, a significant cytotoxicity (p < 0.05) was observed at 300 µg/mL treatment, and significant (p < 0.05) DNA damages were noted in all treatment groups. All genes assessed were found to be significantly up-regulated (p < 0.05) after 24 h of exposure and after 48 h, only TGF-ß1 and p53 did not significantly up-regulate (p < 0.05). These findings confirm that the Kandy city air contains potential carcinogenic and mutagenic compounds and thus, exposure to Kandy air may increase the health risks and respiratory tract-related anomalies.

2.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443423

RESUMO

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-ß1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.


Assuntos
Cirrose Hepática Experimental/tratamento farmacológico , Melastomataceae/química , Neovascularização Patológica/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Citocinas/genética , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática Experimental/sangue , Neovascularização Patológica/sangue , Tamanho do Órgão/efeitos dos fármacos , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tioacetamida , Regulação para Cima/efeitos dos fármacos , Água , Perda de Peso/efeitos dos fármacos
3.
Mol Reprod Dev ; 88(3): 201-210, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33559208

RESUMO

Growth hormone (GH) and insulin-like growth factor 1 (IGF1) are crucial for female reproductive functions. The cyclic regulation of the local GH/IGF1 axis in the oviduct and its involvement in oviductal contraction in cattle has not been investigated. Thus, the messenger RNA (mRNA) expression for GH receptor (GHR), IGF1, IGF1 receptor (IGF1R) in the whole oviducts, as well as in cultured bovine oviductal epithelial cells (BOECs) were evaluated. The GHR, IGF1, and IGF1R mRNA expression was significantly higher during postovulatory phase. The luteinizing hormone (LH), estradiol-17ß (E2), and LH + E2 treatments significantly increased GHR and IGF1 mRNA expression in cultured BOECs. Further, GH and combination of GH with LH and E2 upregulated IGF1 mRNA expression in the BOECs. Moreover, IGF1 + LH and combined IGF1 + LH + E2 treatments significantly increased prostaglandin synthesis cascade enzyme mRNA expression in the BOECs. An ex vivo microdialysis assay revealed that GH and IGF1 induced the release of oviductal contraction related prostaglandins, endothelin-1, and angiotensin II in follicular and postovulatory phases. Together, the findings strongly suggest that the presence of the active GH/IGF1 axis during the peri-ovulatory period, regulating the local system for the release of oviductal contraction related substances, which may provide the optimal oviductal environment for gametes and early embryo.

4.
Artigo em Inglês | MEDLINE | ID: mdl-33502284

RESUMO

Fish genetic resources and diversity are very important aspects of environmental management and fisheries and are vital for making decisions on their commercial exploitation as well as conservation. The snakehead fishes in the world have significant economic importance as food and ornamental fish. A clear understanding of species' taxonomic status and genetic diversity is important for the utilization and implementation of conservation and management practices. Channa orientalis is a snakehead endemic to Sri Lanka that is heavily utilized in the ornamental fish export trade. Its genetic diversity has not yet been fully understood and it is difficult to distinguish it from closely resembling species. Therefore, we examined the genetic diversity of C. orientalis and developed a DNA-based marker that permits accurate, low cost, and reliable identification of C. orientalis. Determination of genetic diversity was mainly carried out through genetic analysis of the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene. The development of the DNA-based marker for the identification of C. orientalis was done through Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis. Our analyses confirmed the presence of two distinct genetically divergent and geographically separated lineages of C. orientalis in Sri Lanka. The fast cost-effective gel-based PCR-RFLP marker method developed by us was successful in diagnosing C. orientalis from its closely resembling species. Thus, we believe our findings on the cryptic diversity and diagnostic methods will have important implications for the conservation and management of this endemic species.

5.
Ecotoxicol Environ Saf ; 208: 111606, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396126

RESUMO

Mancozeb is a metal-containing ethylene bis-dithiocarbamate fungicide widely used in agriculture. Ethylene thiourea (ETU) is the primary metabolite of Mancozeb. Mancozeb has been associated with spontaneous abortions and abnormal menstruation in women. However, the effects of Mancozeb and ETU on embryo attachment remain unknown. The human blastocyst surrogate trophoblastic spheroids (JEG-3), endometrial epithelial surrogate adenocarcinoma cells (Ishikawa), or human primary endometrial epithelial cells (EECs) monolayer were used in the spheroid attachment models. Ishikawa and EECs were pretreated with different concentrations of Mancozeb or ETU for 48 h before the attachment assay. Gene expression profiles of Ishikawa cells were examined to understand how Mancozeb modulates endometrial receptivity with Microarray. The genes altered by Mancozeb were confirmed by qPCR and compared with the ETU treated groups. Mancozeb and ETU treatment inhibited cell viability at 10 µg/mL and 5000 µg/mL, respectively. At non-cytotoxic concentrations, Mancozeb at 3 µg/mL and ETU at 300 µg/mL reduced JEG-3 spheroid attachment onto Ishikawa cells. A similar result was observed with human primary endometrial epithelial cells. Mancozeb at 3 µg/mL modified the transcription of 158 genes by at least 1.5-fold in Microarray analysis. The expression of 10 differentially expressed genes were confirmed by qPCR. Furthermore, Mancozeb decreased spheroid attachment possibly through downregulating the expression of endometrial estrogen receptor ß and integrin ß3, but not mucin 1. These results were confirmed in both overexpression and knockdown experiments and co-culture assay. Mancozeb but not its metabolite ETU reduced spheroid attachment through modulating gene expression profile and decreasing estrogen receptor ß and integrin ß3 expression of endometrial epithelial cells.


Assuntos
Adesão Celular/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Fungicidas Industriais/toxicidade , Integrina beta3/metabolismo , Maneb/toxicidade , Esferoides Celulares/efeitos dos fármacos , Zineb/toxicidade , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação para Baixo , Endométrio/citologia , Endométrio/metabolismo , Células Epiteliais/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Integrina beta3/genética , Gravidez , Esferoides Celulares/metabolismo
6.
Biochem Biophys Res Commun ; 527(1): 42-48, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32446389

RESUMO

The fungicide Mancozeb is an endocrine-disrupting chemical and the mode of action of Mancozeb on embryo implantation is largely unknown. Mancozeb (1 and 3 µg/ml) significantly reduced Jeg-3 trophoblastic spheroids attachment to endometrial epithelial Ishikawa cells. Mancozeb treatment from gestation day (GD) 1 to GD8 or from GD4 to GD8 significantly lowered the number of implantation sites with higher incidence of morphological abnormalities in the reproductive tissues. However, these were not seen in the treatment from GD1 to GD4. Mancozeb at 30 mg/kg BW/d did not alter the expression of p53, COX-2, or PGFS transcripts in the uterus, but down-regulated the PGES transcript and protein. Mancozeb treatment in human endometrial stromal cells did not alter the decidualization response, but the morphological transformation was impaired. Taken together, exposure to Mancozeb affected embryo implantation probably through the modulation of decidualization and to delineate the exact mode of action needs further investigations.


Assuntos
Implantação do Embrião/efeitos dos fármacos , Fungicidas Industriais/efeitos adversos , Maneb/efeitos adversos , Zineb/efeitos adversos , Animais , Linhagem Celular , Feminino , Fungicidas Industriais/administração & dosagem , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Masculino , Maneb/administração & dosagem , Camundongos Endogâmicos ICR , Zineb/administração & dosagem
7.
Biol Reprod ; 93(5): 109, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26377223

RESUMO

Successful embryo implantation requires a synchronized dialogue between a competent blastocyst and the receptive endometrium, which occurs in a limited time period known as the "window of implantation." Recent studies suggested that down-regulation of olfactomedin 1 (OLFM1) in the endometrium and fallopian tube is associated with receptive endometrium and tubal ectopic pregnancy in humans. Interestingly, the human chorionic gonadotropin (hCG) induces miR-212 expression, which modulates OLFM1 and C-terminal binding protein 1 (CTBP1) expressions in mouse granulosa cells. Therefore, we hypothesized that embryo-derived hCG would increase miR-212 expression and down-regulate OLFM1 and CTBP1 expressions to favor embryo attachment onto the female reproductive tract. We found that hCG stimulated the expression of miR-212 and down-regulated OLFM1 but not CTBP1 mRNA in both human endometrial (Ishikawa) and fallopian (OE-E6/E7) epithelial cells. However, hCG suppressed the expression of OLFM1 and CTBP1 proteins in both cell lines. The 3'UTR of both OLFM1 and CTBP1 contained binding sites for miR-212. The miR-212 precursor suppressed luciferase expression, whereas the miR-212 inhibitor stimulated luciferase expression of the wild-type (WT)-OLFM1 and WT-CTBP1 reporter constructs. Furthermore, hCG (25 IU/ml) treatments stimulated trophoblastic (Jeg-3) spheroid (blastocyst surrogate) attachment onto Ishikawa and OE-E6/E7 cells. Transfection of miR-212 precursor increased Jeg-3 spheroid attachment onto Ishikawa cells and decreased OLFM1 and CTBP1 protein expressions, whereas the opposite occurred with miR-212 inhibitor. Taken together, hCG stimulated miR-212, which in turn down-regulated OLFM1 and CTBP1 expression in fallopian and endometrial epithelial cells to favor spheroid attachment.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Implantação do Embrião , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , MicroRNAs/metabolismo , Gonadotropina Coriônica , Endométrio/metabolismo , Células Epiteliais/metabolismo , Tubas Uterinas/metabolismo , Feminino , Células HeLa , Humanos , Esferoides Celulares
8.
Fertil Steril ; 104(2): 474-82, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25999259

RESUMO

OBJECTIVE: To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro. DESIGN: Experimental study. SETTING: Reproductive biology laboratory. PATIENT(S): Healthy nonpregnant women. INTERVENTION(S): No patient interventions. MAIN OUTCOME MEASURE(S): Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/ß-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay. RESULT(S): Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/ß-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/ß-catenin signaling pathway but up-regulated Olfm1 expression. CONCLUSION(S): Human chorionic gonadotropin activated both ERK and Wnt/ß-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression.


Assuntos
Gonadotropina Coriônica/farmacologia , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Tubas Uterinas/metabolismo , Glicoproteínas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Trofoblastos/metabolismo , Adulto , Linhagem Celular , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Células Epiteliais/efeitos dos fármacos , Tubas Uterinas/citologia , Tubas Uterinas/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pessoa de Meia-Idade
9.
Reprod Toxicol ; 42: 164-71, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23978332

RESUMO

Exposure of animals to perfluorooctanoic acid (PFOA), a surfactant used in emulsion polymerization processes causes early pregnancy loss, delayed growth and development of fetuses. The mechanisms of action are largely unknown. We studied the effect of PFOA on implantation using an in vitro spheroid-endometrial cell co-culture model. PFOA (10-100µM) significantly reduced Jeg-3 spheroid attachment on RL95-2 endometrial cells. PFOA also suppressed ß-catenin expression in Jeg-3 cells. The Wnt agonist Wnt3a stimulated ß-catenin expression in Jeg-3 cells and reversed the PFOA suppression of the spheroid attachment. The putative PFOA receptors (PPARα, ß, γ) present in both cell lines were not affected by PFOA (0.01-100µM). The PPARα antagonist MK886 restored the ß-catenin and E-cadherin expression levels in Jeg-3 cells and reversed the suppression of the spheroid attachment caused by PFOA. Taken together, PFOA suppresses spheroid attachment through PPARα and Wnt signaling pathways via down-regulation of ß-catenin and E-cadherin expression.


Assuntos
Caprilatos/toxicidade , Disruptores Endócrinos/toxicidade , Endométrio/citologia , Células Epiteliais/efeitos dos fármacos , Fluorcarbonetos/toxicidade , Esferoides Celulares/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Regulação para Baixo , Células Epiteliais/fisiologia , Feminino , Humanos , PPAR alfa/metabolismo , Esferoides Celulares/fisiologia , Células Tumorais Cultivadas , Via de Sinalização Wnt/efeitos dos fármacos
10.
Lab Invest ; 92(2): 256-64, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21968811

RESUMO

Ectopic pregnancy (EP) occurs when the embryo fails to transit to the uterus and attach to the luminal epithelium of the Fallopian tube (FT). Tubal EP is a common gynecological emergency and more than 95% of EP occurs in the ampullary region of the FT. In humans, Wnt activation and downregulation of olfactomedin-1 (Olfm-1) occur in the receptive endometrium and coincided with embryo implantation in vivo. Whether similar molecular changes happen in the FT leading to EP remains unclear. We hypothesized that activation of Wnt signaling downregulates Olfm-1 expression predisposes to EP. We investigated the spatiotemporal expression of Olfm-1 in FT from non-pregnant women and women with EP, and used a novel trophoblastic spheroid (embryo surrogate)-FT epithelial cell co-culture model (JAr and OE-E6/E7 cells) to study the role of Olfm-1 on spheroid attachment. Olfm-1 mRNA expression in the ampullary region of non-pregnant FT was higher (P<0.05) in the follicular phase than in the luteal phase. Ampullary tubal Olfm-1 expression was lower in FT from women with EP compared to normal controls at the luteal phase (histological scoring (H-SCORE)=1.3±0.2 vs 2.4±0.5; P<0.05). Treatment of OE-E6/E7 with recombinant Olfm-1 (0.2-5 µg/ml) suppressed spheroid attachment to OE-E6/E7 cells, while activation of Wnt-signaling pathway by Wnt3a or LiCl reduced endogenous Olfm-1 expression and increased spheroid attachment. Conversely, suppression of Olfm-1 expression by RNAi increased spheroid attachment to OE-E6/E7 cells. Taken together, Wnt activation suppresses Olfm-1 expression, and this may predispose a favorable microenvironment of the retained embryo in the FT, leading to EP in humans.


Assuntos
Regulação para Baixo/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Tubas Uterinas/metabolismo , Glicoproteínas/fisiologia , Gravidez Tubária/fisiopatologia , Proteínas Wnt/fisiologia , Adulto , Western Blotting , Técnicas de Cocultura , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Tubas Uterinas/citologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Gravidez , Transdução de Sinais
11.
Reprod Toxicol ; 33(1): 60-6, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22134133

RESUMO

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects embryo development, implantation and fertility in humans. The underlying molecular mechanism by which TCDD suppresses implantation remains largely unknown. We used the trophoblastic spheroids (embryo surrogate)-endometrial cells co-culture assay to study the attachment of trophoblastic spheroids (BeWo and Jeg-3) onto the endometrial epithelial (RL95-2 and Ishikawa) cells. TCDD dose-dependently induced cytochrome P450 1A1 (Cyp1A1) expression in trophoblastic and endometrial epithelial cells. Moreover, TCDD at 1 and 10 nM suppressed ß-catenin (a Wnt-signaling molecule) and E-cadherin expression, as well as spheroids attachment onto endometrial cells. Interestingly, activation of the canonical Wnt-signaling pathway via Wnt3a or lithium chloride reverted the suppressive effect of TCDD on ß-catenin and E-cadherin expressions in the BeWo and RL95-2 cells, and restored the spheroids attachment rate to be comparable to the untreated controls. Taken together, TCDD induces Cyp1A1 expression, modulates the Wnt-signaling pathway and suppresses spheroids attachment onto endometrial cells.


Assuntos
Adesão Celular/efeitos dos fármacos , Implantação do Embrião/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Células Epiteliais/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Trofoblastos/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocromo P-450 CYP1A1/biossíntese , Relação Dose-Resposta a Droga , Endométrio/metabolismo , Endométrio/patologia , Indução Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Esferoides Celulares , Trofoblastos/metabolismo , Trofoblastos/patologia , beta Catenina/metabolismo
12.
Hum Reprod ; 26(1): 167-75, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21106493

RESUMO

BACKGROUND: Olfactomedin (Olfm) is a member of a diverse group of extracellular matrix proteins important for neuronal growth. Recent microarray studies identified Olfm as one of the down-regulated transcripts in receptive endometrium at the time of embryo attachment and implantation. However, the underlying molecular mechanisms that govern Olfm expression and its effect on embryo attachment and implantation remain unknown. METHODS: The expression of Olfm in the human endometrium was investigated by real-time PCR, western blotting and immunohistochemistry on human endometrial biopsies from natural and ovarian stimulated cycles. To investigate the function of Olfm in trophoblast-endometrial cell attachment, an in vitro spheroid-endometrial cell co-culture study was performed. RESULTS: Human endometrial Olfactomedin-1 and -2(Olfm-1 and -2) transcripts decreased significantly from the proliferative to the secretory phases of the menstrual cycle. Olfm protein was strongly expressed in the luminal and glandular epithelium and moderately in the stromal cells of human endometria. Ovarian stimulation significantly decreased (P < 0.05) the expression of endometrial Olfm-1 and -2 transcripts in patients receiving IVF treatment when compared with those in the natural cycle. Importantly, recombinant Olfm-1 suppressed JAr spheroid attachment onto Ishikawa cells and this was not associated with changes of ß-catenin and E-cadherin expression in trophoblast and endometrial cells. CONCLUSIONS: Decreased expression of Olfm during the receptive phase of the endometrium may allow successful trophoblast attachment for implantation.


Assuntos
Endométrio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Sequência de Aminoácidos , Western Blotting , Caderinas/metabolismo , Linhagem Celular , Implantação do Embrião/fisiologia , Estradiol/sangue , Receptor alfa de Estrogênio/sangue , Receptor beta de Estrogênio/sangue , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Imuno-Histoquímica , Ciclo Menstrual/metabolismo , Dados de Sequência Molecular , Indução da Ovulação , Reação em Cadeia da Polimerase , Progesterona/sangue , RNA Mensageiro/metabolismo , beta Catenina/metabolismo
13.
Hum Reprod ; 25(2): 479-90, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19955106

RESUMO

BACKGROUND: High serum estradiol (E2) levels following ovarian stimulation lead to reduced implantation and pregnancy rates, yet the underlying mechanisms remain unknown. We investigated if aberrant expression of genes in the Wnt-signaling pathway may be involved. METHODS: Microarray and real-time PCR analysis were performed to analyze gene expression profiles of endometrial samples taken at day hCG + 7 in stimulated cycles, and days LH + 7 and LH + 10 in natural cycles. Expression of several Wnt-signaling transcripts, including Dickkopf homolog 1 (DKK1), DKK2 and secreted frizzled-related protein 4 (sFRP4), was analyzed throughout the menstrual cycle. JAr spheroid/Ishikawa endometrial cell co-culture experiments were established to study effects of DKK1 on spheroid attachment in vitro. RESULTS: We identified 351 differentially expressed genes. Endometrial samples taken at hCG + 7 had similar expression profiles to those at LH + 10. DKK1 transcripts were up-regulated and DKK2 and sFRP4 were down-regulated in the stimulated compared with LH + 7 group (all P < 0.05). DKK1 transcripts were low in proliferative phase (PS) and increased in late-secretory phase (LS, P < 0.05), although DKK2 peaked in mid-secretory phase (P < 0.05). sFRP4 transcripts were high in PS. Treatment of spheroid with recombinant human DKK-1 protein dose-dependently suppressed (P < 0.05 versus control) spheroids attachment onto endometrial cells (associated with decreased beta-catenin protein): this suppression was nullified by anti-DKK1 antibody. CONCLUSION: Gene expression patterns in stimulated cycles resembled those of LS in natural cycles, when the implantation window is about to close, suggesting high serum E2 and/or progesterone concentrations may advance endometrial development, altering the implantation window and possibly decreasing pregnancy rate. Aberrant expression of DKK1 might impair embryo attachment and implantation in vivo.


Assuntos
Implantação do Embrião/genética , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Indução da Ovulação/efeitos adversos , Proteínas Wnt/fisiologia , Técnicas de Cocultura , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Proto-Oncogênicas/biossíntese , Regulação para Cima
14.
Reproduction ; 133(6): 1087-94, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17636163

RESUMO

The dynamic action of oviductal secretory compounds on spermatozoa function is well documented. In contrast, the effect of spermatozoa on oviductal function remains poorly characterized. Previously, our lab and others have shown that prostaglandin (PG), together with other vasoactive peptides, plays major roles in oviductal contractions and sperm transport. We therefore examined the effect of spermatozoa on the production of PG by cow oviductal epithelial cells (OEC). A bovine spermatozoa-OEC co-culture system was utilized for this purpose. OECs in the second passage were co-cultured for 0, 1, 3, 6, 12, and 24 h with six doses of motile, killed, or truncated spermatozoa heads (control; without spermatozoa, 10(2)-10(6) spermatozoa/ml medium). The levels of PGE(2) and PGF(2alpha) in the medium were measured using enzyme immunoassays. Messenger RNA expression of cyclooxygenase-2, PGF synthase (PGFS), and PGE synthase (PGES) was investigated using real-time RT-PCR. The results indicated that motile spermatozoa increased the secretion of PG by OEC as well as cellular expression of mRNA for cyclooxygenase, PGES, and PGFS in a dose- and time-dependent manner. A maximum three- to fivefold increased secretion of PG was observed with a dose of 10(5) spermatozoa/ml after a 12-h co-incubation. Neither killed spermatozoa nor truncated spermatozoa heads stimulated oviductal biosynthesis and secretion of PG at any dose or time point observed. The results provide the first evidence that live spermatozoa in the oviduct up-regulate the local PG system, and thereby, enhance oviductal contractions. Thus, spermatozoa may bear a role in accelerating their own transport into the fertilization site.


Assuntos
Tubas Uterinas/metabolismo , Prostaglandinas/biossíntese , Transporte Espermático/fisiologia , Espermatozoides/fisiologia , Regulação para Cima , Animais , Bovinos , Sobrevivência Celular , Técnicas de Cocultura , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/biossíntese , Dinoprosta/metabolismo , Células Epiteliais/metabolismo , Feminino , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Masculino , Gravidez , Prostaglandina-E Sintases , Prostaglandinas/metabolismo , Prostaglandinas E/biossíntese , Prostaglandinas E/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Mol Reprod Dev ; 72(4): 511-20, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16155957

RESUMO

Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability enhancing factor, which shows the highest activity in the oviduct during the periovulatory period of the estrous cycle in cattle. It has also been shown that the contraction activity of oviduct is highest during the periovulatory period. The present study therefore focused on the possible involvement of VEGF in the regulation of biosynthesis and secretion of contraction-relaxation-related substances in the cow oviduct. Possible autonomous VEGF system in the oviduct as well as its endocrine control was also studied. Bovine oviductal epithelial cells (BOEC) in the second passage were cultured with VEGF (1 ng/ml) alone or with luteinizing hormone (LH; 10 ng/ml), estradiol 17-beta (E2; 1 ng/ml), and/or progesterone (P4; 1 ng/ml). The levels of prostaglandins (PGs), endothelin-1 (ET-1), and angiotensin II (Ang II) in the medium were measured using second antibody enzymeimmunoassay (EIA). The mRNA expressions for cycloxygenase-2 (Cox-2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), prepro-ET-1, endothelin converting enzyme-1 (Ece-1), angiotensin converting enzyme-1 (Ace-1), VEGF and its receptors were investigated using real-time RT-PCR. The results indicate that, (1) VEGF dose-dependently stimulated the release of prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha), and ET-1, but not Ang II. VEGF and VEGF with LH, E2, and P4 upregulated mRNA expression for biosynthesis cascade of PG, ET-1 as well as their release. However, only the combination of VEGF with LH, E2, and P4 upregulated mRNA for Ace-1 and Ang II release, but not VEGF alone. (2) Treatments of LH, with E2 and/or P4 increased the mRNA expression for VEGF, Flk-1 and Flt-1, and (3) VEGF itself downregulated the expression of mRNA for VEGF, and LH, E2, and P4 enhanced this downregulatory effect. The results of the present study provide the first evidence that (1) VEGF directly stimulates the biosynthesis and release of PGE2, PGF2alpha, and ET-1 in the bovine oviduct, (2) LH stimulates the oviductal VEGF system, and (3) VEGF downregulates the oviductal VEGF system and this downregulation was further intensified in the presence of LH. The data suggest that the preovulatory LH-surge, together with increasing E2 secretion from the Graffian follicle and basal P4 levels from the regressing corpus luteum (CL), upregulates the oviductal VEGF system, inducing the maximum oviductal production of contraction-relaxation-related substances for active oviduct contraction and rapid transport of gametes to the fertilization site. However, the oviductal VEGF elevation caused by the LH-surge, appears to downregulate the oviductal VEGF system immediately after ovulation thereby may contribute to suppress oviductal contraction to secure slow transport of the embryo to the uterus at the optimal time.


Assuntos
Comunicação Autócrina/fisiologia , Blastocisto/fisiologia , Regulação para Baixo/fisiologia , Células Epiteliais/metabolismo , Tubas Uterinas/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Bovinos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Tubas Uterinas/citologia , Feminino , Relaxamento Muscular/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
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