RESUMO
During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or symmetrical cell fate occurs in female and male germ cells, respectively. Here, we have identified branched cyst structures in mice, and investigated their formation and function in oocyte determination. In fetal female cysts, 16.8% of the germ cells are connected by three or four bridges, namely branching germ cells. These germ cells are preferentially protected from cell death and cyst fragmentation and accumulate cytoplasm and organelles from sister germ cells to become primary oocytes. Changes in cyst structure and differential cell volumes among cyst germ cells suggest that cytoplasmic transport in germline cysts is conducted in a directional manner, in which cellular content is first transported locally between peripheral germ cells and further enriched in branching germ cells, a process causing selective germ cell loss in cysts. Cyst fragmentation occurs extensively in female cysts, but not in male cysts. Male cysts in fetal and adult testes have branched cyst structures, without differential cell fates between germ cells. During fetal cyst formation, E-cadherin (E-cad) junctions between germ cells position intercellular bridges to form branched cysts. Disrupted junction formation in E-cad-depleted cysts led to an altered ratio in branched cysts. Germ cell-specific E-cad knockout resulted in reductions in primary oocyte number and oocyte size. These findings shed light on how oocyte fate is determined within mouse germline cysts.
Assuntos
Cistos , Oócitos , Masculino , Feminino , Animais , Camundongos , Células Germinativas , Citoplasma , Organelas , Gametogênese , OogêneseRESUMO
Stem cells ensure tissue homeostasis through the production of differentiating and self-renewing progeny. In some tissues, this is achieved by the function of a definitive stem cell niche. However, the mechanisms that operate in mouse spermatogenesis are unknown because undifferentiated spermatogonia (Aundiff) are motile and intermingle with differentiating cells in an 'open' niche environment of seminiferous tubules. Aundiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1)(+) and neurogenin 3 (NGN3)(+) subpopulations, both of which retain the ability to self-renew. However, whereas GFRα1(+) cells comprise the homeostatic stem cell pool, NGN3(+) cells show a higher probability to differentiate into KIT(+) spermatogonia by as yet unknown mechanisms. In the present study, by combining fate analysis of pulse-labeled cells and a model of vitamin A deficiency, we demonstrate that retinoic acid (RA), which may periodically increase in concentration in the tubules during the seminiferous epithelial cycle, induced only NGN3(+) cells to differentiate. Comparison of gene expression revealed that retinoic acid receptor γ (Rarg) was predominantly expressed in NGN3(+) cells, but not in GFRα1(+) cells, whereas the expression levels of many other RA response-related genes were similar in the two populations. Ectopic expression of RARγ was sufficient to induce GFRα1(+) cells to directly differentiate to KIT(+) cells without transiting the NGN3(+) state. Therefore, RARγ plays key roles in the differentiation competence of NGN3(+) cells. We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals.
Assuntos
Diferenciação Celular/fisiologia , Espermatogênese/fisiologia , Células-Tronco/fisiologia , Tretinoína/farmacologia , Deficiência de Vitamina A/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Imunofluorescência , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Ácido Retinoico/metabolismo , Receptor gama de Ácido RetinoicoRESUMO
OBJECTIVE: Involvement of reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase has been documented in the development of hypoxia-induced model of pulmonary arterial hypertension (PAH). Because the PAH-like phenotype was demonstrated in mice deficient in Nox1 gene (Nox1(-/Y)) raised under normoxia, the aim of this study was to clarify how the lack of NOX1/NADPH oxidase could lead to pulmonary pathology. APPROACH AND RESULTS: Spontaneous enlargement and hypertrophy of the right ventricle, accompanied by hypertrophy of pulmonary vessels, were demonstrated in Nox1(-/Y) 9 to 18 weeks old. Because an increased number of α-smooth muscle actin-positive vessels were observed in Nox1(-/Y), pulmonary arterial smooth muscle cells (PASMCs) were isolated and characterized by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In Nox1(-/Y) PASMCs, the number of apoptotic cells was significantly reduced without any change in the expression of endothelin-1, and hypoxia-inducible factors HIF-1α and HIF-2α, factors implicated in the pathogenesis of PAH. A significant decrease in a voltage-dependent K(+) channel, Kv1.5 protein, and an increase in intracellular potassium levels were demonstrated in Nox1(-/Y) PASMCs. When a rescue study was performed in Nox1(-/Y) crossed with transgenic mice overexpressing rat Nox1 gene, impaired apoptosis and the level of Kv1.5 protein in PASMCs were almost completely recovered in Nox1(-/Y) harboring the Nox1 transgene. CONCLUSIONS: These findings suggest a critical role for NOX1 in cellular apoptosis by regulating Kv1.5 and intracellular potassium levels. Because dysfunction of Kv1.5 is among the features demonstrated in PAH, inactivation of NOX1/NADPH oxidase may be a causative factor for pulmonary vascular remodeling associated with PAH.
Assuntos
Hipertensão Pulmonar/enzimologia , NADH NADPH Oxirredutases/deficiência , Artéria Pulmonar/enzimologia , Actinas/metabolismo , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Hipertensão Pulmonar Primária Familiar , Predisposição Genética para Doença , Hemodinâmica , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/etiologia , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Fenótipo , Potássio/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Interferência de RNA , Ratos , TransfecçãoRESUMO
During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.
Assuntos
Cistos/embriologia , Cistos/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Mutação , Oogênese/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Homozigoto , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Beneficial effects of statins on cardiovascular diseases have been attributed to decreased generation of reactive oxygen species (ROS). We tested the hypothesis that atorvastatin protects against the development of hypertension by reducing levels of NADPH oxidase-derived ROS in two hypertensive animal models. Atorvastatin was given to mice chronically infused with angiotensin (Ang) II or to apolipoprotein E (ApoE)-deficient mice fed a high-fat diet. Increased mean blood pressure (MBP) demonstrated in both animal models was significantly suppressed by atorvastatin with reduced ROS production in the aorta. Treatment with atorvastatin did not alter the mRNA level of NOX1, a catalytic subunit of NADPH oxidase, but decreased the levels of other NOX isoforms, NOX2 and NOX4, in the ApoE-deficient mice fed a high-fat diet. In the Ang II-infused model treated with statin, only the NOX4 mRNA level was reduced. Membrane translocation of Rac1 was significantly reduced in the Ang II-infused mice treated with atorvastatin. Finally, atorvastatin administered to Ang II-infused mice lacking the Nox1 gene elicited an additional decline in MBP compared to Nox1-deficient mice treated with vehicle. Together, these findings suggest that reduced expression and activity of the isoforms of NADPH oxidase, involving NOX1, NOX2, and possibly NOX4, mediate the anti-hypertensive effect of atorvastatin.
Assuntos
Anti-Hipertensivos , Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , NADPH Oxidases/metabolismo , Pirróis/farmacologia , Angiotensina II/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/patologia , Apolipoproteínas E/genética , Atorvastatina , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Gorduras na Dieta/efeitos adversos , Etídio , Hipertensão/induzido quimicamente , Hipertensão/prevenção & controle , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rac1 de Ligação ao GTP/biossíntese , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
During mammalian oogenesis, germ cells undergo oocyte differentiation and oocyte development to form mature oocytes that contain essential components for supporting early embryogenesis. However, only a small fraction of germ cells become mature oocytes and the mechanism of this massive germ cell loss has been unclear. Our recent studies suggested that the formation of functional oocytes and germ cell loss are interlinked by a 'nursing' process in germline cysts during oocyte differentiation in mouse fetal ovaries. 80% of the fetal germ cells sacrifice themselves by donating their cytoplasmic contents to the remaining sister germ cells that differentiate into primary oocytes with augmented developmental potential. In this review, we will summarize the process of mouse oocyte differentiation with a particular focus on organelle transport in germline cysts.
Assuntos
Oócitos/citologia , Oogênese , Organelas/metabolismo , Animais , Transporte Biológico , Diferenciação Celular , Citoplasma/metabolismo , Feminino , Células Germinativas , CamundongosRESUMO
In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/ß-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/ß-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.
Assuntos
Diferenciação Celular , Proteínas de Membrana/genética , Espermatogônias/citologia , Espermatogônias/metabolismo , Via de Sinalização Wnt , Animais , Ciclo Celular/genética , Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Túbulos Seminíferos/metabolismo , Espermatogênese/genética , Proteínas Wnt/genéticaRESUMO
The functional significance of NOX1/NADPH oxidase in the heart has not been explored due to its low expression relative to other NOX homologs identified so far. We aimed to clarify the role of NOX1/NADPH oxidase in the septic heart by utilizing mice deficient in the Nox1 gene (Nox1(-/Y)). Sepsis was induced by intraperitoneal administration of lipopolysaccharides (LPS: 25mg/kg) or cecal ligation and puncture (CLP) surgery. A marked elevation of NOX1 mRNA was demonstrated in cardiac tissue, which was accompanied by increased production of reactive oxygen species (ROS). In Nox1(-/Y) treated with LPS, cardiac dysfunction and survival were significantly improved compared with wild-type mice (Nox1(+/Y)) treated with LPS. Concomitantly, LPS-induced cardiomyocyte apoptosis and activation of caspase-3 were alleviated in Nox1(-/Y). The level of phosphorylated Akt in cardiac tissue was significantly lowered in Nox1(+/Y) but not in Nox1(-/Y) treated with LPS or that underwent CLP surgery. Increased oxidation of cysteine residues of Akt and enhanced interaction of Akt with protein phosphatase 2A (PP2A), a major phosphatase implicated in the dephosphorylation of Akt, were demonstrated in LPS-treated Nox1(+/Y). These responses to LPS were significantly attenuated in Nox1(-/Y). Taken together, ROS derived from NOX1/NADPH oxidase play a pivotal role in endotoxin-induced cardiomyocyte apoptosis by increasing oxidation of Akt and subsequent dephosphorylation by PP2A. Marked up-regulation of NOX1 may affect the risk of mortality under systemic inflammatory conditions.
Assuntos
Apoptose , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/fisiologia , NADH NADPH Oxirredutases/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Endotoxemia/complicações , Endotoxemia/imunologia , Endotoxemia/patologia , Expressão Gênica , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/microbiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/enzimologia , Miocárdio/imunologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/imunologia , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Especificidade de Órgãos , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Disfunção Ventricular Esquerda/imunologiaRESUMO
Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.