RESUMEN
Estradiol-17ß has been shown to promote primordial follicle formation and to involve bone morphogenetic protein 2 (BMP2) as a downstream effector to promote primordial follicle in hamsters. However, the molecular mechanism whereby these factors regulate ovarian somatic cells to pre-granulosa cells transition leading to primordial follicle formation remains unclear. The objective of this study was to determine whether BMP2 and/or estradiol-17ß would regulate the expression of specific ovarian transcriptome during pre-granulosa cells transition and primordial follicle formation in the mouse ovary. BMP2 mRNA level increased during the period of primordial follicle formation with the concurrent presence of BMP2 protein in ovarian somatic cells. Estradiol-17ß but not BMP2 exposure led to increased expression of ovarian BMP2 messenger RNA (mRNA), and the effect of estradiol-17ß could not be suppressed by 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride (LDN) 193189. BMP2 or estradiol-17ß stimulated primordial follicle formation without inducing apoptosis. Ribonucleic acid-sequence analysis (RNA-seq) of ovaries exposed to exogenous BMP2 or estradiol-17ß revealed differential expression of several thousand genes. Most of the differentially expressed genes, which were common between BMP2 or estradiol-17ß treatment demonstrated concordant changes, suggesting that estradiol-17ß and BMP2 affected the same set of genes during primordial follicle formation. Further, we have identified that estradiol-17ß, in cooperation with BMP2, could affect the expression of three major transcription factors, GATA binding protein 2, GATA binding protein 4 and Early growth response 2, and one serine protease, hepsin, in pre-granulosa cells during primordial follicle formation. Taken together, results of this study suggest that estradiol-17ß and BMP2 may regulate ovarian gene expression that promote somatic cells to pre-granulosa cells transition and primordial follicle formation in the mouse ovary.
Asunto(s)
Estradiol , Ovario , Transcriptoma , Animales , Proteína Morfogenética Ósea 2/farmacología , Cricetinae , Estradiol/farmacología , Femenino , Ratones , Ovario/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Malaria remains a significant health problem in Bangladesh affecting 13 of 64 districts. The risk of malaria is variable across the endemic areas and throughout the year. A better understanding of the spatial and temporal patterns in malaria risk and the determinants driving the variation are crucial for the appropriate targeting of interventions under the National Malaria Control and Prevention Programme. METHODS: Numbers of Plasmodium falciparum and Plasmodium vivax malaria cases reported by month in 2007, across the 70 endemic thanas (sub-districts) in Bangladesh, were assembled from health centre surveillance reports. Bayesian Poisson regression models of incidence were constructed, with fixed effects for monthly rainfall, maximum temperature and elevation, and random effects for thanas, with a conditional autoregressive prior spatial structure. RESULTS: The annual incidence of reported cases was 34.0 and 9.6 cases/10,000 population for P. falciparum and P. vivax respectively and the population of the 70 malaria-endemic thanas was approximately 13.5 million in 2007. Incidence of reported cases for both types of malaria was highest in the mountainous south-east of the country (the Chittagong Hill Tracts). Models revealed statistically significant positive associations between the incidence of reported P. vivax and P. falciparum cases and rainfall and maximum temperature. CONCLUSIONS: The risk of P. falciparum and P. vivax was spatially variable across the endemic thanas of Bangladesh and also highly seasonal, suggesting that interventions should be targeted and timed according to the risk profile of the endemic areas. Rainfall, temperature and elevation are major factors driving the spatiotemporal patterns of malaria in Bangladesh.
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Malaria Falciparum/epidemiología , Malaria Vivax/epidemiología , Topografía Médica , Animales , Bangladesh/epidemiología , Humanos , Incidencia , Medición de Riesgo , Factores de Riesgo , Factores de TiempoRESUMEN
Zinc oxide is one of the most widely studied semiconductor metal oxides, which predominantly crystallizes as hexagonal wurtzite and often cubic zinc-blende phases. Here we report the transformation of the highly stable wurtzite ZnO to a new triclinic phase NZO-2 by using metformin as a template during post-synthesis hydrothermal treatment. This crystalline phase of the material NZO-2 has been identified through the refinement of the powder XRD data. NZO-2 possesses porous rod like particle morphology consisting of the self-assembly of 3-7â nm size spherical nanoparticles and interparticle nanoscopic voids spaces. NZO-2 has been surface phosphorylated and the resulting material displayed good proton conductivity. Further, NZO-2 displayed ultra-low band gap of 1.74â eV, thereby responsible for red emission under high energy laser excitation and this may open new opportunities in optoelectronic application of ZnO.
RESUMEN
The objective of this study was to determine whether surface-modified nanoparticles enhance permeability across nasal mucosa, while retaining the effectiveness of the payload. The uptake and permeability of polystyrene nanoparticles (PS-NPs; FluoSpheres) was evaluated across the various regions of the bovine nasal epithelia following conjugation with deslorelin and transferrin. Uptake and transport of PS-NPs, deslorelin-PS-NPs, and transferrin-PS-NPs exhibited regional differences in the order: inferior turbinate posterior (ITP) > medium turbinate posterior (MTP) > medium turbinate anterior (MTA). Uptake and transport also exhibited directionality and temperature dependence in these tissues. Further, uptake as well as transport of functionalized nanoparticles could be inhibited by excess free functionalizing ligand. Confocal microscopy indicated the presence of functionalized nanoparticles in respiratory epithelial cells, as well as other cell types of the nasal tissue. We chose the ITP region for further studies with deslorelin or transferrin-conjugated poly-l-lactide-co-glycolide nanoparticles (PLGA-NPs) encapsulating an anti-VEGF intraceptor (Flt23k) plasmid. Transport of the nanoparticles, as well as the plasmid from the nanoparticles, exhibited the following order: transferrin-PLGA-NPs > deslorelin-PLGA-NPs > PLGA-NPs >> plasmid. The ability of the nanoparticles transported across the nasal tissue to retain the effectiveness of the Flt23k plasmid was evaluated by measuring transfection efficiency (percentage of cells expressing GFP) and VEGF inhibition in LNCaP and PC-3 prostate cancer cells. Transfection efficiencies and VEGF inhibition in LNCaP and PC-3 cells exhibited the following trend: transferrin-PLGA-NPs >or= deslorelin-PLGA-NPs > PLGA-NPs >> plasmid. Further, functionalized nanoparticles exhibited transfection efficiencies and VEGF inhibition significantly superior compared with the routinely used transfecting agent, lipofectamine. Formulating plasmids into nanoparticulate delivery systems enhances the transnasal delivery and gene therapy at remote target cancer cells, which can be further enhanced by nanoparticle functionalization with deslorelin or transferrin.
Asunto(s)
Mucosa Nasal/metabolismo , Administración Intranasal , Animales , Bovinos , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Ácido Láctico/administración & dosificación , Nanopartículas , Ácido Poliglicólico/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Transferrina/administración & dosificación , Pamoato de Triptorelina/administración & dosificación , Pamoato de Triptorelina/análogos & derivadosRESUMEN
We have previously reported that the expression of Angiotensin II (Ang II) type 1 receptors (AT1R) was increased in the rostral ventrolateral medulla (RVLM) of rabbits with chronic heart failure (CHF) and in the RVLM of normal rabbits infused with intracerebroventricular (ICV) Ang II. The present study investigated whether oxidant stress plays a role in Ang II-induced AT1R upregulation and its relationship to the transcription factor activator protein 1 (AP1) in CHF rabbits and in the CATHa neuronal cell line. In CATHa cells, Ang II significantly increased AT1R mRNA by 123+/-11%, P<0.01; c-Jun mRNA by 90+/-20%, P<0.01; c-fos mRNA by 148+/-49%, P<0.01; NADPH oxidase activity by 126+/-43%, P<0.01 versus untreated cells. Tempol and Apocynin reversed the increased expression of AT1R mRNA, c-Jun mRNA, c-fos mRNA, and superoxide production induced by Ang II. We also examined the effect of ICV Tempol on the RVLM of CHF rabbits. Compared to vehicle treated CHF rabbits, Tempol significantly decreased AT1R protein expression (1.6+/-0.29 versus 0.88+/-0.16, P<0.05), phosphorylated Jnk protein (0.4+/-0.05 versus 0.2+/-0.04, P<0.05), cytosolic phosphorylated c-Jun (0.56+/-0.1 versus 0.36+/-0.05, P<0.05), and nuclear phosphorylated c-Jun (0.67+/-0.1 versus 0.3+/-0.08, P<0.01). Tempol also significantly decreased the AP-1-DNA binding activity in the RVLM of CHF rabbits compared to the vehicle group (9.14 x 10(3) versus 41.95 x 10(3) gray level P<0.01). These data suggest that Ang II induces AT1R upregulation at the transcriptional level by induction of oxidant stress and activation of AP1 in both cultured neuronal cells and in intact brain of rabbits. Antioxidant agents may be beneficial in CHF and other states where brain Ang II is elevated by decreasing AT1R expression through the Jnk and AP1 pathway.
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Insuficiencia Cardíaca/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/fisiología , Receptor de Angiotensina Tipo 1/metabolismo , Angiotensina II/metabolismo , Animales , Antioxidantes/farmacología , Células Cultivadas , Enfermedad Crónica , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/patología , MAP Quinasa Quinasa 4/metabolismo , Masculino , NADPH Oxidasas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Conejos , Especies Reactivas de Oxígeno/metabolismo , Marcadores de Spin , Superóxidos/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.
Asunto(s)
Receptor alfa de Estrógeno/deficiencia , Receptor beta de Estrógeno/deficiencia , Genes APC , Neoplasias Intestinales/etiología , Transducción de Señal/fisiología , Animales , Cadherinas/análisis , Colon/química , Ciclina D1/análisis , Estradiol/sangre , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/fisiología , Femenino , Neoplasias Intestinales/genética , Masculino , Ratones , Ovario/patología , Proteínas Wnt/fisiología , beta Catenina/análisis , beta Catenina/fisiologíaRESUMEN
Deslorelin, a luteinizing hormone releasing hormone (LHRH) agonist, is transported via the LHRH-receptor (LHRH-R) and exhibits regional variation as follows: inferior turbinate posterior (ITP)>medium turbinate posterior (MTP)>medium turbinate anterior (MTA) of the bovine nasal mucosa. Differential LHRH-R expression in various regions of the nose is a potential explanation for regional variation in deslorelin transport. Thus, the objective was to determine whether LHRH-R expression exhibits regional variation in bovine nasal mucosa. LHRH-R density (B(max)) and affinity constant (K(d)) were determined by saturation experiments using 0.5mg tissue in the presence of increasing amounts of I(125)-deslorelin (100-100,000 cpm) at 4 degrees C for 4h. The 50% inhibitory concentration (IC(50)) was determined by competition experiments using various amounts of unlabelled deslorelin (0.01-3000 ng) at 4 degrees C for 4h. LHRH-R mRNA and protein expressions were determined using real-time PCR and Western blot analysis, respectively. LHRH-R B(max) and K(d) varied between the regions of excised bovine nasal mucosa: ITP>MTP>MTA. The inhibition experiments yielded two IC(50) concentrations which exhibited trends similar to B(max) and K(d). Real-time PCR and Western blot analysis indicated that LHRH-R expression exhibits similar trends: ITP>MTP>MTA. We identified two deslorelin binding sites in the nasal tissues, with high affinity sites representing approximately 60-70% of the total sites available. In summary, regional differences in nasal deslorelin transport correlate with regional differences in LHRH-R expression, with LHRH-R expression, peptide binding, and transport being the highest in the inferior turbinate posterior region of the nose.
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Mucosa Nasal/metabolismo , Receptores LHRH/metabolismo , Pamoato de Triptorelina/análogos & derivados , Animales , Transporte Biológico , Bovinos , Expresión Génica , ARN Mensajero/metabolismo , Receptores LHRH/genética , Pamoato de Triptorelina/metabolismo , Cornetes Nasales/metabolismoRESUMEN
In most mammals, prostaglandin F2alpha (PGF2alpha) is believed to be a trigger that induces the regression of the corpus luteum (CL), whereby progesterone synthesis is inhibited, the luteal structure involutes, and the reproductive cycle resumes. Studies have shown that the early growth response 1 (EGR1) protein can induce the expression of proapoptotic proteins, suggesting that EGR1 may play a role in luteal regression. Our hypothesis is that EGR1 mediates the actions of PGF2alpha by inducing the expression of TGF beta1 (TGFB1), a key tissue remodeling protein. The levels of EGR1 mRNA and protein were up-regulated in the bovine CL during PGF2alpha-induced luteolysis in vivo and in PGF2alpha-treated luteal cells in vitro. Using chemical and genetic approaches, the RAF/MAPK kinase (MEK) 1/ERK pathway was identified as a proximal signaling event required for the induction of EGR1 in PGF2alpha-treated cells. Treatment with PGF2alpha increased the expression of TGFB1 mRNA and protein as well as the binding of EGR1 protein to TGFB1 promoter in bovine luteal cells. The effect of PGF2alpha on TGFB1 expression was mimicked by a protein kinase C (PKC)/RAF/MEK1/ERK activator or adenoviral-mediated expression of EGR1. The stimulatory effect of PGF2alpha on TGFB1 mRNA and TGFB1 protein secretion was inhibited by blockade of MEK1/ERK signaling and by adenoviral-mediated expression of NAB2, an EGR1 binding protein that inhibits EGR1 transcriptional activity. Treatment of luteal cells with TGFB1 reduced progesterone secretion, implicating TGFB1 in luteal regression. These studies demonstrate that PGF2alpha stimulates the expression of EGR1 and TGFB1 in the CL. We suggest that EGR1 plays a role in the expression of genes whose cognate proteins coordinate luteal regression.
Asunto(s)
Cuerpo Lúteo/efectos de los fármacos , Dinoprost/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Expresión Génica/efectos de los fármacos , Factor de Crecimiento Transformador beta1/genética , Animales , Northern Blotting , Western Blotting , Bovinos , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Cuerpo Lúteo/metabolismo , AMP Cíclico/farmacología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Inmunohistoquímica , Células Lúteas/efectos de los fármacos , Células Lúteas/metabolismo , Hormona Luteinizante/farmacología , MAP Quinasa Quinasa 1/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Estradiol-17beta (E2) plays an important role in the formation and development of primordial follicles, but the mechanisms remain unclear. G protein-coupled receptor 30 (GPR30) can mediate a rapid and transcription-independent E2 signaling in various cells. The objectives of this study were to examine whether GPR30 was expressed in the neonatal hamster ovary and whether it could mediate estrogen action during the formation of primordial follicles. GPR30 mRNA levels decreased from the 13th day of gestation (E13) through the second day of postnatal (P2) life, followed by steady increases from P3 through P6. Consistent with the changes in mRNA levels, GPR30 protein expression decreased from E13 to P2 followed by a significant increase by P7, the day before the first appearance of primordial follicles in the hamster ovary. GPR30 was expressed both in the oocytes and somatic cells, although the expression in the oocytes was low. GPR30 protein was located primarily in the perinuclear endoplasmic reticulum, which was also the site of E2-BSA-FITC (E2-BSA-fluorescein isothiocyanate) binding. E2 or E2-BSA increased intracellular calcium in neonatal hamster ovary cells in vitro. Exposure to GPR30 small interfering RNA in vitro significantly reduced GPR30 mRNA and protein levels in cultured hamster ovaries, attenuated E-BSA binding to cultured P6 ovarian cells, and markedly suppressed estrogen-stimulated primordial follicle formation. These results suggest that a membrane estrogen receptor, GPR30, is expressed in the ovary during perinatal development and mediates E2 action on primordial follicle formation.
Asunto(s)
Estradiol/farmacología , Folículo Ovárico/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Cricetinae , Embrión de Mamíferos , Estradiol/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/fisiología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Distribución TisularRESUMEN
Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Angiotensin II (Ang II) plays an important role in the development and progression of CHF. Many of the important functions of Ang II are mediated by the Ang II type 1 receptor (AT(1)R), including the increase in sympathetic nerve activity in CHF. However, the central regulation of the AT(1)R in the setting of CHF is not well understood. This study investigated the AT(1)R in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway, and its gene regulation by the transcription factor activator protein 1 (AP-1). Studies were performed in 5 groups of rabbits: sham (n=5), pacing-induced (3 to 4 weeks) CHF (n=5), CHF with intracerebroventricular (ICV) losartan treatment (n=5), normal with ICV Ang II treatment (n=5), and normal with ICV Ang II plus losartan treatment (n=5). AT(1)R mRNA and protein expressions, plasma Ang II, and AP-1-DNA binding activity were significantly higher in RVLM of CHF compared with Sham rabbits (240.4+/-30.2%, P<0.01; 206.6+/-25.8%, P<0.01; 280+/-36.5%, P<0.05; 207+/-16.4%, P<0.01, respectively). Analysis of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway showed that phosphorylated c-Jun proteins, phosphorylated JNK proteins, and JNK activity increased significantly in RVLM of CHF compared with sham (262.9+/-48.1%, 213.8+/-27.7%, 148.2+/-10.1% of control, respectively). Importantly, ICV losartan in CHF rabbits attenuated these increases. ICV Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by concomitant ICV losartan. In addition, Ang II-induced AT(1)R expression was blocked by losartan and a JNK inhibitor, but not by extracellular signal-regulated kinase or p38 MAP kinase inhibitors in a neuronal cell culture. These data suggest that central Ang II activates the AT(1)R, SAPK/JNK pathway. AP-1 may further regulate gene expression in RVLM in the CHF state.
Asunto(s)
Gasto Cardíaco Bajo/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Receptor de Angiotensina Tipo 1/biosíntesis , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba , Angiotensina II/administración & dosificación , Angiotensina II/sangre , Angiotensina II/farmacología , Animales , Peso Corporal , Gasto Cardíaco Bajo/enzimología , Gasto Cardíaco Bajo/genética , Línea Celular , Ecocardiografía , Corazón/anatomía & histología , Corazón/fisiología , Inyecciones Intraventriculares , Sistema de Señalización de MAP Quinasas , Masculino , Tamaño de los Órganos , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/metabolismo , Conejos , Receptor de Angiotensina Tipo 1/genéticaRESUMEN
Skeletal health consequences associated with inflammatory diseases of the airways significantly contribute to morbidity. Sex differences have been described independently for lung and bone diseases. Repetitive inhalant exposure to lipopolysaccharide (LPS) induces bone loss and deterioration in male mice, but comparison effects in females are unknown. Using an intranasal inhalation exposure model, 8-week-old C57BL/6 male and female mice were treated daily with LPS (100 ng) or saline for 3 weeks. Bronchoalveolar lavage fluids, lung tissues, tibias, bone marrow cells, and blood were collected. LPS-induced airway neutrophil influx, interleukin (IL)-6 and neutrophil chemoattractant levels, and bronchiolar inflammation were exaggerated in male animals as compared to female mice. Trabecular bone micro-CT imaging and analysis of the proximal tibia were conducted. Inhalant LPS exposures lead to deterioration of bone quality only in male mice (not females) marked by decreased bone mineral density, bone volume/tissue volume ratio, trabecular thickness and number, and increased bone surface-to-bone volume ratio. Serum pentraxin-2 levels were modulated by sex differences and LPS exposure. In proof-of-concept studies, ovarectomized female mice demonstrated LPS-induced bone deterioration, and estradiol supplementation of ovarectomized female mice and control male mice protected against LPS-induced bone deterioration findings. Collectively, sex-specific differences exist in LPS-induced airway inflammatory consequences with significant differences found in bone quantity and quality parameters. Male mice demonstrated susceptibility to bone loss and female animals were protected, which was modulated by estrogen. Therefore, sex differences influence the biologic response in the lung-bone inflammatory axis in response to inhalant LPS exposures.
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Resorción Ósea/inmunología , Huesos/inmunología , Terapia de Reemplazo de Hormonas , Inflamación/inmunología , Pulmón/inmunología , Animales , Resorción Ósea/tratamiento farmacológico , Estradiol/uso terapéutico , Femenino , Inflamación/tratamiento farmacológico , Exposición por Inhalación , Masculino , Ratones , Ratones Endogámicos C57BL , Ovariectomía , Sexo , Tomografía Computarizada por Rayos XRESUMEN
The role of E2 on primordial follicle formation was examined by treating neonatal hamsters with 1 or 2 microg estradiol cypionate (ECP) at age postnatal d 1 (P1) and P4 or by in vitro culture of embryonic d 15 (E15) ovaries with 1, 5, or 10 ng/ml estradiol-17beta (E2). The specificity of E2 action was examined by ICI 182,780. One microgram of ECP maintained serum levels of E2 within the physiological range, significantly reduced apoptosis, and stimulated the formation and development of primordial follicles. In contrast, 2 microg ECP increased serum E2 levels to 400 pg/ml and had significantly less influence on primordial follicle formation. In vivo, ICI 182,780 significantly increased apoptosis and caused a modest reduction in primordial follicle formation. The formation and development of primordial follicles in vitro increased markedly with 1 ng/ml E2, and the effect was blocked by ICI 182,780. Higher doses of E2 had no effect on primordial follicle formation but significantly up-regulated apoptosis, which was blocked by ICI 182,780. CYP19A1 mRNA expression occurred by E13 and increased with the formation of primordial follicles. P4 ovaries synthesized E2 from testosterone, which increased further by FSH. Both testosterone and FSH maintained ovarian CYP19A1 mRNA, but FSH up-regulated the expression. These results suggest that neonatal hamster ovaries produce E2 under FSH control and that E2 action is essential for the survival and differentiation of somatic cells and the oocytes leading to the formation and development of primordial follicles. This supportive action of E2 is lost when hormone levels increase above a threshold.
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Estradiol/farmacología , Folículo Ovárico/embriología , Animales , Animales Recién Nacidos , Aromatasa/genética , Aromatasa/metabolismo , Células Cultivadas , Cricetinae , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Masculino , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Embarazo , ARN Mensajero/metabolismoRESUMEN
The nongenomic actions of estradiol-17beta are mediated by transmembrane estrogen receptors. Recently, G protein-coupled receptor 30 (GPR30) has been suggested to be a transmembrane estrogen receptor that can mediate rapid and transcription-independent estradiol-17beta signaling in different cell types. However, the expression, regulation, or biological relevance of GPR30 in the ovary remains unknown. We examined the expression and hormonal regulation of GPR30 mRNA and protein in hamster ovarian cells during the estrous cycle and after hypophysectomy and hormone replacement. GPR30 protein expression was high in the theca, appreciable in the granulosa, but low in luteal cells. GPR30 protein levels in granulosa and theca cells increased steadily with the development of preantral and antral follicles, respectively. GPR30 mRNA and protein levels increased significantly on diestrous (d 3 of the estrous cycle), but decreased on d 4 at 1600 h after the LH surge. GPR30 mRNA levels increased significantly after hypophysectomy. Although steroid treatment failed to alter ovarian GPR30 mRNA levels, either FSH or LH effectively reduced the levels. Interestingly, the decrease in GPR30 mRNA corresponded to a marked increase in the receptor protein levels. FSH treatment, either alone or together with LH, resulted in a marked increase in GPR30 immunostaining in granulosa cells. LH alone significantly increased immunostaining in theca cells. These results suggest that GPR30 is expressed in the membrane of hamster granulosa and theca cells, and the expression is regulated by gonadotropins. The unique pattern of GPR30 expression suggests that gonadotropin-regulated follicular cell functions may involve GPR30 activity.
Asunto(s)
Gonadotropinas/fisiología , Hormonas/fisiología , Ovario/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cricetinae , ADN Complementario , Sinergismo Farmacológico , Estro/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Gonadotropinas/farmacología , Hormonas/farmacología , Hipofisectomía , Hormona Luteinizante/metabolismo , Hormona Luteinizante/farmacología , Mesocricetus , Datos de Secuencia Molecular , Ovario/fisiología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Distribución TisularRESUMEN
PURPOSE: Previously, we have synthesized and characterized a novel Cu(II) complex, copper N-(2-hydroxy acetophenone) glycinate (CuNG). Herein, we have determined the efficacy of CuNG in overcoming multidrug-resistant cancer using drug-resistant murine and human cancer cell lines. EXPERIMENTAL DESIGN: Action of CuNG following single i.m. administration (5 mg/kg body weight) was tested in vivo on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing mice and doxorubicin-resistant sarcoma 180-bearing mice. Tumor size, ascitic load, and survival rates were monitored at regular intervals. Apoptosis of cancer cells was determined by cell cycle analysis, confocal microscopy, Annexin V binding, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay ex vivo. IFN-gamma and tumor necrosis factor-alpha were assayed in the culture supernatants of in vivo and in vitro CuNG-treated splenic mononuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Source of IFN-gamma and changes in number of T regulatory marker-bearing cells in the tumor site following CuNG treatment were investigated by flow cytometry. Supernatants of in vitro CuNG-treated cultures of peripheral blood mononuclear cells from different drug-insensitive cancer patients were tested for presence of the apoptogenic cytokine IFN-gamma and its involvement in induction of apoptosis of doxorubicin-resistant CEM/ADR5000 cells. RESULTS: CuNG treatment could resolve drug-resistant cancers through induction of apoptogenic cytokines, such as IFN-gamma and/or tumor necrosis factor-alpha, from splenic mononuclear cells or patient peripheral blood mononuclear cells and reduce the number of T regulatory marker-bearing cells while increase infiltration of IFN-gamma-producing T cells in the ascetic tumor site. CONCLUSION: Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers irrespective of multidrug resistance phenotype.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Quelantes/farmacología , Cobre/química , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Animales , Carcinoma de Ehrlich/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Doxorrubicina/farmacología , Humanos , Leucocitos Mononucleares/metabolismo , Ganglios Linfáticos/patología , Ratones , Trasplante de Neoplasias , Bazo/metabolismoRESUMEN
Primordial follicle (PF) pool determines the availability of follicles for ovulation in all mammals. Premature depletion of the PF reserve leads to subfertility or infertility. Bone morphogenetic protein 2 (BMP2) promotes PF formation by facilitating oocyte and granulosa cell development. Estradiol-17ß (E2) upregulates PF formation in developing hamster ovaries. However, if BMP2 mediates E2 effect is not known. We hypothesize that E2 facilitates the effect of BMP2 on somatic to granulosa cell transition. BMP2 and E2 together significantly upregulated the percentage of PFs in hamster fetal ovaries in vitro compared with either of the treatments alone. E2 also promoted BMP2 expression in vivo. Inhibition of BMP2 receptors suppressed E2-stimulation of PF formation while knockdown of BMP2 in vitro significantly suppressed the E2 effect. In contrast, estrogen receptor blocker did not affect BMP2 action. Inhibition of the activity of E2 or BMP2 receptors, either alone or combined during the last two days of the culture (C6-C8) resulted in a significant decrease in PF formation by C8, suggesting that both BMP2 and E2 action is essential for somatic cell differentiation for PF formation. Together, the results suggest that E2 activates BMP2-BMPR system leading to the formation of primordial follicles.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/genética , Estradiol/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Animales , Proteína Morfogenética Ósea 2/antagonistas & inhibidores , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/efectos de los fármacos , Cricetinae , Estradiol/biosíntesis , Antagonistas de Estrógenos/administración & dosificación , Antagonistas del Receptor de Estrógeno/administración & dosificación , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células de la Granulosa/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Ovulación/genética , Ovulación/metabolismoRESUMEN
PURPOSE: To compare Endothelial cell(EC) loss following Phacoemulsification (PKE) in pupils of different sizes. METHODS: A prospective double masked observational study in which a total of 150 eyes of 150 patients between 50 & 70 years of age with senile cataract of nuclear sclerosis grade II were enrolled. Patients were allocated into three groups of 50 eyes each in Group A (pupil size <5 mm), Group B (pupil size 5-7 mm) and Group C (pupil size >7 mm). Pupillary size was measured by determining the height of slit on slit-lamp biomicroscope examination. PKE was done by the same expert surgeon using vertical chop technique and a foldable intraocular lens was implanted in the capsular bag. Corneal EC count and pachymetry were performed twice and average of 2 readings was taken for the purpose of this study. Measurements were taken preoperatively and postoperatively on day 1, day 7 and day 30. RESULTS: The mean EC count loss on postoperative day 1 in Group A was 19.45%, Group B 14.89%, Group C 10.19% with statistical significant difference between Group A and Group B, as also Group A and Group C. The difference was not significant between Group B and Group C, though there was a fall in EC count in Group C as well. Increase in corneal thickness on postoperative day 1 in group A was 5.43%, Group B 3.55%, Group C 2.14% with statistical significant difference between Group A and Group B, as also Group A and Group C with no difference in Group B and Group C. CONCLUSION: PKE done in eyes with maximal pupillary dilatation of <5 mm causes a greater EC loss and results in thicker corneas postoperatively as compared to eyes with pupillary dilatation of >5 mm at the end of one month.
Asunto(s)
Enfermedades de la Córnea/diagnóstico , Endotelio Corneal/patología , Facoemulsificación/efectos adversos , Complicaciones Posoperatorias/diagnóstico , Pupila , Anciano , Recuento de Células , Enfermedades de la Córnea/etiología , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Estudios Prospectivos , Microscopía con Lámpara de Hendidura/métodos , Factores de Tiempo , Agudeza VisualRESUMEN
Postnatal growth differentiation factor 9 (GDF-9) expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNA interference knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal ovaries were cultured for 9 d with or without 1 ng FSH, 1 microl Metafectane, 100 nM control nontargeting small interfering RNA (siRNA), GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a nontargeting control siRNA labeled with Cy3. More than 90% of cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA-induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of bone morphogenetic protein receptor IA or beta-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Furthermore, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation.
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Péptidos y Proteínas de Señalización Intercelular/fisiología , Oocitos/metabolismo , Folículo Ovárico/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , Femenino , Hormona Folículo Estimulante/farmacología , Factor 9 de Diferenciación de Crecimiento , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Mesocricetus , Datos de Secuencia Molecular , Folículo Ovárico/fisiología , ARN Interferente Pequeño/farmacologíaRESUMEN
Exposure to testosterone (T) during d 30-90 of fetal life results in low-birth-weight offspring, hypergonadotropism, multifollicular ovaries, and early cessation of cyclicity. The multifollicular phenotype may result from failure of follicles to regress and consequent follicular persistence or, alternatively, increased follicular recruitment. We tested the hypothesis that prenatal exposure to excess T causes intrauterine growth retardation and increases ovarian follicular recruitment. Time-mated pregnant ewes were treated with 100 mg T propionate in cottonseed oil or vehicle twice weekly from d 30-90 of gestation. Ewes were euthanized near term, from d 139-141 of gestation (term is 147 d). After determining fetal measures and organ weights, ovaries were removed from fetuses of control and T-treated dams, and follicular distribution in each ovary was determined by morphometric quantification. Total number and percentage distribution of the various classes of follicles (primordial, primary, preantral, and antral follicles) were compared between treatment groups. Prenatally T-treated female fetuses were smaller in size, had an increased head circumference to fetal weight ratio (P < 0.01), increased adrenal to fetal weight ratio (P < 0.05), decreased number of follicles (P < 0.05), a decrease in percentage of primordial follicles (P < 0.001), and a corresponding increase in the remaining classes of follicles (P < 0.05). Ovarian findings support decreased ovarian reserve and enhanced follicular recruitment, potential contributors of early reproductive failure. The extent to which metabolic changes associated with intrauterine growth retardation contribute toward altered trajectory of ovarian folliculogenesis remains to be determined.
Asunto(s)
Andrógenos/farmacología , Retardo del Crecimiento Fetal/inducido químicamente , Folículo Ovárico/efectos de los fármacos , Ovario/efectos de los fármacos , Testosterona/farmacología , Animales , Femenino , Peso Fetal/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Folículo Ovárico/patología , Folículo Ovárico/fisiología , Ovario/patología , Ovario/fisiología , Embarazo , OvinosRESUMEN
The usefulness of azaline B, a GnRH antagonist, in suppressing gonadotropin secretion in the golden hamster was examined by examining follicular development, steroidogenesis and expression of steroidogenic enzymes. Serum levels of P and E declined significantly, while FSH or LH was undetectable in azaline B-treated hamsters. FSH significantly increased serum E levels, whereas LH upregulated serum P levels. The formation of antral follicles ceased in azaline-treated hamsters, but was reversed by FSH with or without LH supplement. FSH also activated the primordial follicle pool resulting in increased formation of primary and preantral follicles. Further, an increasing trend in the formation of preantral follicles in response to E or E + P, and the formation of antral follicles in response to E + P treatment was evident. The level of Cyp11a1 mRNA increased markedly in LH- or LH + FSH-treated hamsters, whereas FSH with or without LH upregulated Cyp17a1, Cyp19a1 and Fshr mRNA expression. E without or with P also upregulated ovarian Cyp19a1 mRNA expression. The expression of enzyme protein corroborated the mRNA data. In summary, azaline B is an efficient GnRH antagonist in the hamster, and will be useful in studying the selective effect of gonadotropins on ovarian functions without disrupting the physiological functions of other hormones in ovarian cells.