RESUMO
Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft(mito)) that is imported into mitochondria. Ft(mito) binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Polaridade Celular , Proteínas de Drosophila/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Olho/crescimento & desenvolvimento , Genes Supressores de Tumor , Humanos , MAP Quinase Quinase 4/metabolismo , Dados de Sequência Molecular , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Asas de Animais/crescimento & desenvolvimentoRESUMO
DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Animais , Linhagem Celular , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Infertilidade/genética , Masculino , Camundongos Endogâmicos C57BL , Deleção de Sequência , Células Sf9RESUMO
Nuclear envelope membrane proteins (NEMPs) are a conserved family of nuclear envelope (NE) proteins that reside within the inner nuclear membrane (INM). Even though Nemp1 knockout (KO) mice are overtly normal, they display a pronounced splenomegaly. This phenotype and recent reports describing a requirement for NE openings during erythroblasts terminal maturation led us to examine a potential role for Nemp1 in erythropoiesis. Here, we report that Nemp1 KO mice show peripheral blood defects, anemia in neonates, ineffective erythropoiesis, splenomegaly, and stress erythropoiesis. The erythroid lineage of Nemp1 KO mice is overrepresented until the pronounced apoptosis of polychromatophilic erythroblasts. We show that NEMP1 localizes to the NE of erythroblasts and their progenitors. Mechanistically, we discovered that NEMP1 accumulates into aggregates that localize near or at the edge of NE openings and Nemp1 deficiency leads to a marked decrease of both NE openings and ensuing enucleation. Together, our results for the first time demonstrate that NEMP1 is essential for NE openings and erythropoietic maturation in vivo and provide the first mouse model of defective erythropoiesis directly linked to the loss of an INM protein.
Assuntos
Membrana Nuclear , Esplenomegalia , Camundongos , Animais , Eritroblastos/metabolismo , Núcleo Celular/metabolismo , Eritropoese/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos KnockoutRESUMO
Betaine has important roles in preimplantation mouse embryos, including as an organic osmolyte that functions in cell volume regulation in the early preimplantation stages and as a donor to the methyl pool in blastocysts. The origin of betaine in oocytes and embryos was largely unknown. Here, we found that betaine was present from the earliest stage of growing oocytes. Neither growing oocytes nor early preantral follicles could take up betaine, but antral follicles were able to transport betaine and supply the enclosed oocyte. Betaine is synthesized by choline dehydrogenase, and female mice lacking Chdh did not have detectable betaine in their oocytes or early embryos. Supplementing betaine in their drinking water restored betaine in the oocyte only when supplied during the final stages of antral follicle development but not earlier in folliculogenesis. Together with the transport results, this implies that betaine can only be exogenously supplied during the final stages of oocyte growth. Previous work showed that the amount of betaine in the oocyte increases sharply during meiotic maturation due to upregulated activity of choline dehydrogenase within the oocyte. This betaine present in mature eggs was retained after fertilization until the morula stage. There was no apparent role for betaine uptake via the SIT1 (SLC6A20) betaine transporter that is active at the 1- and 2-cell stages. Instead, betaine was apparently retained because its major route of efflux, the volume-sensitive organic osmolyte - anion channel, remained inactive, even though it is expressed and capable of being activated by a cell volume increase.
Assuntos
Betaína , Blastocisto , Oócitos , Animais , Betaína/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Feminino , Camundongos , Blastocisto/metabolismo , Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Folículo Ovariano/metabolismo , Folículo Ovariano/efeitos dos fármacos , Colina Desidrogenase/metabolismoRESUMO
OBJECTIVES: To evaluates the effect of different modes of final follicular maturation triggering on the degree of apoptosis of granulosa cells (GCs) and the potential effect on progesterone secretion. METHODS: Thirty patients undergoing controlled ovarian hyperstimulation for IVF who received hCG, GnRH agonist, or dual trigger for final follicular maturation were included in the study. Granulosa cells were obtained at the time of oocyte retrieval. The proportion of apoptotic cells was evaluated via TUNEL and immunohistochemistry. RESULTS: The proportion of apoptotic cells was significantly higher in the GnRH agonist-alone group compared to hCG-alone and the dual trigger groups (13.5 ± 1.5% vs. 7.8% ± 1.8 vs. 10.1% ± 2, respectively, P < 0.01). Moreover, the expression of active-caspase-3 was also significantly increased in the GnRH agonist-alone group compared with the hCG-alone and the dual trigger groups (15.5% ± 2.9 vs. 8.4% ± 1.6 vs. 12.7% ± 2.6, respectively, P < 0.01). The progesterone levels measured in the granulosa-luteal cell culture medium after 24 h of incubation were similar between the three groups. CONCLUSIONS: The levels of apoptosis are increased after GnRH agonist/dual trigger. The increased apoptosis might be one of the culprit of the subsequent premature demise of the corpus luteum post GnRH agonist trigger.
Assuntos
Apoptose , Gonadotropina Coriônica/farmacologia , Hormônio Liberador de Gonadotropina/agonistas , Infertilidade Masculina/fisiopatologia , Células Lúteas/patologia , Luteólise , Indução da Ovulação/métodos , Adulto , Feminino , Fertilização in vitro/métodos , Humanos , Células Lúteas/efeitos dos fármacos , Masculino , Recuperação de Oócitos , Gravidez , Substâncias para o Controle da Reprodução/farmacologiaRESUMO
Cell death is an essential physiological process required for the proper development and function of the human placenta. Although the mouse is a commonly used animal model for development studies, little is known about the extent and distribution of cell death in the mouse placenta throughout development and its physiological relevance. In the present study, we report the results of a systematic and quantitative assessment of cell death patterns in the placentae of two strains of laboratory mice commonly used for developmental studies-ICR and C57Bl/6. TUNEL staining revealed that ICR and C57Bl/6 placentae exhibited similar cell death patterns to those reported in human placentae during pregnancy, with comparatively infrequent death observed during early gestation, which increased and became more organized towards term. Interestingly, when comparing strain differences, increased cell death was observed in almost all regions of the inbred C57Bl/6 placentae compared to the outbred ICR strain. Finally, since Bcl-2 ovarian killer (Bok) has been reported to be a key player in human placental cell death, we examined its expression in murine placentae throughout gestation. Bok protein expression was observed in all placental regions and increased towards term in both strains. The results of this study indicate that although strain-specific differences in placental cell death exist, the overall rates and patterns of cell death during murine placentation parallel those previously described in humans. Thus, the murine placenta is a useful model to investigate molecular pathways involved in cell death signaling during human placentation.
Assuntos
Placenta , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Caspase 3/metabolismo , Morte Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Modelos Animais , Placenta/citologia , Placenta/metabolismo , GravidezRESUMO
Protein-protein interactions (PPIs) are useful for understanding signaling cascades, predicting protein function, associating proteins with disease and fathoming drug mechanism of action. Currently, only â¼ 10% of human PPIs may be known, and about one-third of human proteins have no known interactions. We introduce FpClass, a data mining-based method for proteome-wide PPI prediction. At an estimated false discovery rate of 60%, we predicted 250,498 PPIs among 10,531 human proteins; 10,647 PPIs involved 1,089 proteins without known interactions. We experimentally tested 233 high- and medium-confidence predictions and validated 137 interactions, including seven novel putative interactors of the tumor suppressor p53. Compared to previous PPI prediction methods, FpClass achieved better agreement with experimentally detected PPIs. We provide an online database of annotated PPI predictions (http://ophid.utoronto.ca/fpclass/) and the prediction software (http://www.cs.utoronto.ca/~juris/data/fpclass/).
Assuntos
Biologia Computacional/métodos , Simulação por Computador , Mineração de Dados/métodos , Mapeamento de Interação de Proteínas/métodos , Humanos , Proteoma , Software , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: Previous studies have shown that androgens, in addition to serving as precursors for ovarian estrogen synthesis, also have a fundamental role in primate ovarian follicular development by augmentation of FSH receptor expression on granulosa cells. Recent studies have shown that aromatase inhibitor, letrozole, improves ovarian response to FSH in normal and poor responder patients, possibly by increasing intraovarian androgen levels. Studies in mice also showed an effect of letrozole to increase pigment epithelium-derived factor (PEDF) and to lower vascular epithelial growth factor (VEGF), which might be expected to reduce the risk of ovarian hyperstimulation syndrome (OHSS) with stimulation. The aim of this study was to compare the VEGF and PEDF levels in the follicular fluids of normal responders treated with letrozole and gonadotropins during the ovarian stimulation with patients treated with gonadotropins only. METHODS: A single center, prospective clinical trial. We collected follicular fluid from 26 patients, on a GnRH antagonist protocol, dual triggered with hCG and GnRH agonist. The patients in one group were co-treated with letrozole and gonadotropins during the ovarian stimulation and the patients in the other group were treated with gonadotropins only. VEGF, PEDF, estrogen, progesterone and testosterone levels were measured by ELISA kits. RESULTS: The age of the patients, the total dose of gonadotropins and the number of oocytes were comparable between the two groups. In the follicular fluid, the estrogen levels (2209 nmol/l vs. 3280 nmol/l, p = 0.02) were significantly decreased, and the testosterone levels (246.5 nmol/l vs. 40.7 nmol/l, p < 0.001) were significantly increased in the letrozole group compared to the gonadotropin only group. The progesterone levels (21.4 µmol/l vs. 17.5 p = NS) were comparable between the two groups. The VEGF levels (2992 pg/ml vs. 1812 pg/ml p = 0.02) were significantly increased and the PEDF levels (9.7 ng/ml vs 17.3 ng/ml p < 0.001) were significantly decreased in the letrozole group. CONCLUSIONS: Opposite to observations in the mouse, we found that VEGF levels were increased and PEDF levels were decreased in the follicular fluid in patients treated with letrozole during the stimulation cycles. Further investigation is required to determine if patients treated with letrozole during the IVF stimulation protocol are at increased risk for developing OHSS as a result of these findings.
Assuntos
Proteínas do Olho/metabolismo , Líquido Folicular/metabolismo , Gonadotropinas/metabolismo , Fatores de Crescimento Neural/metabolismo , Nitrilas/administração & dosagem , Indução da Ovulação/métodos , Serpinas/metabolismo , Triazóis/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto , Inibidores da Aromatase/administração & dosagem , Quimioterapia Combinada , Feminino , Humanos , Infertilidade Feminina/tratamento farmacológico , Infertilidade Feminina/metabolismo , Letrozol , Masculino , Gravidez , Estudos ProspectivosRESUMO
UNLABELLED: The wound healing assay (or scratch assay) is a technique frequently used to quantify the dependence of cell motility-a central process in tissue repair and evolution of disease-subject to various treatments conditions. However processing the resulting data is a laborious task due its high throughput and variability across images. This Robust Quantitative Scratch Assay algorithm introduced statistical outputs where migration rates are estimated, cellular behaviour is distinguished and outliers are identified among groups of unique experimental conditions. Furthermore, the RQSA decreased measurement errors and increased accuracy in the wound boundary at comparable processing times compared to previously developed method (TScratch). AVAILABILITY AND IMPLEMENTATION: The RQSA is freely available at: http://ophid.utoronto.ca/RQSA/RQSA_Scripts.zip The image sets used for training and validation and results are available at: (http://ophid.utoronto.ca/RQSA/trainingSet.zip, http://ophid.utoronto.ca/RQSA/validationSet.zip, http://ophid.utoronto.ca/RQSA/ValidationSetResults.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975Results.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip). Supplementary Material is provided for detailed description of the development of the RQSA. CONTACT: juris@ai.utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Ensaios de Migração Celular , Animais , Humanos , CicatrizaçãoRESUMO
The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fertilidade , Proteínas Nucleares/metabolismo , Espermatogênese , Proteínas Supressoras de Tumor/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Envelhecimento/patologia , Animais , Apoptose/genética , Contagem de Células , Proliferação de Células , Dano ao DNA/genética , Proteínas de Ligação a DNA/deficiência , Feminino , Fertilidade/genética , Regulação da Expressão Gênica , Humanos , Infertilidade Masculina/sangue , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Estresse Oxidativo/genética , Progesterona/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Espermatozoides/patologia , Testículo/metabolismo , Testículo/patologia , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/deficiênciaRESUMO
PURPOSE: The purpose of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained. METHODS: We used dialysis membrane with a pore size of <3 KD to characterize the estrogen-binding capacity of the follicular fluid. We performed PCR, western blot, and ELISA on luteinized granulosa cells to determine if sex hormone-binding globulin (SHBG) is produced by granulosa cells, and finally we used affinity columns and mass spectrometry to identify the estrogen-binding protein in the follicular fluid. RESULTS: We found that a significant estrogen concentration difference is maintained in a cell-free system and is lost with proteolysis of the follicular fluid proteins. Luteinized granulosa cells are likely not a source of SHBG, as we were not able to detect expression of SHBG in these cells. Perlecan was the most highly enriched follicular fluid protein in the affinity columns. CONCLUSIONS: We were able to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid.
Assuntos
Estradiol/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Folículo Ovariano/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Estradiol/isolamento & purificação , Feminino , Líquido Folicular/metabolismo , Células da Granulosa/metabolismo , Proteoglicanas de Heparan Sulfato/isolamento & purificação , Humanos , Soro/metabolismoRESUMO
In addition to its proapoptotic function, caspase-8 is also important for several other processes, including suppressing necroptosis, cell migration, and immune cell survival. In the present study, we report that the loss of caspase-8 in B lymphocytes leads to B-cell malignancies and that the risk for these tumors is further enhanced in the absence of p53. We also report that deficiency of caspase-8 results in impaired cytokinesis and that casp8(-/-) lymphomas display remarkably elevated levels of chromosomal aberrations. Our data support an important role for caspase-8 in the maintenance of genomic integrity and highlight its tumor-suppressive function.
Assuntos
Caspase 8/fisiologia , Instabilidade Cromossômica/genética , Linfoma de Células B/genética , Células 3T3 , Animais , Síndrome Linfoproliferativa Autoimune/complicações , Síndrome Linfoproliferativa Autoimune/genética , Síndrome Linfoproliferativa Autoimune/imunologia , Síndrome Linfoproliferativa Autoimune/patologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Caspase 8/genética , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Genes p53/fisiologia , Predisposição Genética para Doença , Linfoma de Células B/etiologia , Linfoma de Células B/mortalidade , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de SobrevidaRESUMO
Inhibitors of the mammalian target of rapamycin (MTOR) belong to a family of drugs with potent immunosuppressive, antiangiogenic, and antiproliferative properties. De novo or worsening proteinuria can occur during treatment with these agents, but the mechanism by which this occurs is unknown. We generated and characterized mice carrying a podocyte-selective knockout of the Mtor gene. Although Mtor was dispensable in developing podocytes, these mice developed proteinuria at 3 weeks and end stage renal failure by 5 weeks after birth. Podocytes from these mice exhibited an accumulation of the autophagosome marker LC3 (rat microtubule-associated protein 1 light chain 3), autophagosomes, autophagolysosomal vesicles, and damaged mitochondria. Similarly, human podocytes treated with the MTOR inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. Taken together, these results suggest that disruption of the autophagic pathway may play a role in the pathogenesis of proteinuria in patients treated with MTOR inhibitors.
Assuntos
Autofagia/fisiologia , Podócitos/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/deficiência , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Lisossomos/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/patologia , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genéticaRESUMO
Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing ß-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of ß-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The combination of TNF-α + IFN-γ induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent ß-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-α + IFN-γ-induced ß-cell apoptosis. DP5 knock-out mice had increased ß-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human ß-cells to a large extent against TNF-α + IFN-γ, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of ß-cell apoptosis during insulitis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Células Secretoras de Insulina/metabolismo , Interferon gama/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Humanos , Interferon gama/genética , Interferon gama/farmacologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição STAT1/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
How the fetoplacental arterial tree grows and expands during late gestational development is largely unknown. In this study, we quantified changes in arterial branching in the fetal exchange region of the mouse placenta during late gestation, when capillarization increases rapidly. We studied two commonly used mouse strains, CD1 and C57Bl/6 (B6), at embryonic days (E)13.5, 15.5, and 17.5. B6 mice differ from CD1 mice by exhibiting a blunted fetal weight gain in late gestation. We found that B6 capillarization and interhemal membrane thinning were reduced and placental hypoxia-inducible factor-1α and VEGF-A expression were higher than CD1 near term. Automated vascular segmentation of microcomputed tomography data sets revealed that the number of arterial vessels ≥50 µm remained constant during late gestation in both strains, despite large increases in downstream capillary volume quantified by stereology (+65% in B6 mice and +200% in CD1 mice). Arterial diameters expanded in both strains from E13.5 to E15.5; however, diameters continued to expand to E17.5 in B6 mice only. The diameter scaling coefficient at branch sites was near optimal (-3.0) and remained constant in CD1 mice, whereas it decreased, becoming abnormal, in B6 mice at term (-3.5 ± 0.2). Based on arterial tree geometry, resistance remained constant throughout late gestation (â¼0.45 mmHg·s·µl(-1)) in CD1 mice, whereas it decreased by 50% in late gestation in B6 mice. Quantification of the fetoplacental vasculature revealed significant strain-dependent differences in arterial and capillary expansion in late gestation. In both strains, enlargement of the fetoplacental arterial tree occurred primarily by increased arterial diameters with no change in segment numbers in late gestation.
Assuntos
Capilares/embriologia , Feto/irrigação sanguínea , Neovascularização Fisiológica , Placenta/irrigação sanguínea , Circulação Placentária , Animais , Artérias/embriologia , Western Blotting , Capilares/diagnóstico por imagem , Capilares/ultraestrutura , Feminino , Peso Fetal , Genótipo , Idade Gestacional , Hemodinâmica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Tamanho da Ninhada de Vivíparos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Neovascularização Fisiológica/genética , Fenótipo , Gravidez , Especificidade da Espécie , Fator A de Crescimento do Endotélio Vascular/metabolismo , Microtomografia por Raio-XRESUMO
Unraveling molecular pathways responsible for regulation of early embryonic development is crucial for our understanding of female infertility. Maternal determinants that control the transition from oocyte to embryo are crucial molecules that govern developmental competence of the newly conceived zygote. We describe a series of defects that are triggered by a disruption of maternal lethal effect gene, Nlrp5. Previous studies have shown that Nlrp5 hypomorph embryos fail to develop beyond the two-cell stage. Despite its importance in preimplantation development, the mechanism by which the embryo arrest occurs remains unclear. We confirmed that Nlrp5 mutant and wild-type females possess comparable ovarian germ pool and follicular recruitment rates. However, ovulated oocytes lacking Nlrp5 have abnormal mitochondrial localization and increased activity in order to sustain physiological ATP content. This results in an accumulation of reactive oxygen species and increased cellular stress causing mitochondrial depletion. Compromised cellular state is also accompanied by increased expression of cell death inducer Bax and depletion of cytochrome c. However, neither genetic deletion (Bax/Nlrp5 double knockout) nor mimetic interference (BH4 domain or Bax inhibitory peptide) were sufficient to alleviate embryo demise caused by depletion of Nlrp5. We therefore conclude that lack of Nlrp5 in oocytes triggers premature activation of the mitochondrial pool, causing mitochondrial damage that cannot be rescued by inactivation of Bax.
Assuntos
Antígenos/metabolismo , Proteínas do Ovo/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos/genética , Citocromos c/metabolismo , Proteínas do Ovo/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Ovário/embriologia , Ovário/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
The role of various p73 isoforms in tumorigenesis has been controversial. However, as we have recently shown, the generation of TAp73-deficient (TAp73(-/-)) mice reveals that TAp73 isoforms exert tumor-suppressive functions, indicating an emerging role for Trp-73 in the maintenance of genomic stability. Unlike mice lacking all p73 isoforms, TAp73(-/-) mice show a high incidence of spontaneous tumors. Moreover, TAp73(-/-) mice are infertile and produce oocytes exhibiting spindle abnormalities. These data suggest a link between TAp73 activities and the common molecular machinery underlying meiosis and mitosis. Previous studies have indicated that the spindle assembly checkpoint (SAC) complex, whose activation leads to mitotic arrest, also regulates meiosis. In this study, we demonstrate in murine and human cells that TAp73 is able to interact directly with several partners of the SAC complex (Bub1, Bub3, and BubR1). We also show that TAp73 is involved in SAC protein localization and activities. Moreover, we show that decreased TAp73 expression correlates with increases of SAC protein expression in patients with lung cancer. Our results establish TAp73 as a regulator of SAC responses and indicate that TAp73 loss can lead to mitotic arrest defects. Our data suggest that SAC impairment in the absence of functional TAp73 could explain the genomic instability and increased aneuploidy observed in TAp73-deficient cells.
Assuntos
Proteínas Nucleares/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fuso Acromático/fisiologia , Animais , Proteínas de Ciclo Celular , Instabilidade Cromossômica , Feminino , Humanos , Camundongos , Proteínas Nucleares/análise , Proteínas Serina-Treonina Quinases/análise , Fuso Acromático/químicaRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and the main toxicants found in cigarettes. Women are often exposed to PAHs before pregnancy, typically via prepregnancy smoking. To determine how prepregnancy exposure affects the fetoplacental vasculature of the placenta, we exposed female mice to PAHs before conception, perfused the fetoplacental arterial trees with X-ray contrast agent, and imaged the vasculature ex vivo by microcomputed tomography (micro-CT) at embryonic day 15.5. Automated vascular segmentation and flow calculations revealed that in control trees, <40 chorionic plate vessels (diameter>180 µm) gave rise to â¼1,300 intraplacental arteries (50-180 µm), predicting an arterial vascular resistance of 0.37±0.04 mmHg·s·µl(-1). PAH exposure increased vessel curvature of chorionic plate vessels and significantly increased the tortuousity ratio of the tree. Intraplacental arteries were reduced by 17%, primarily due to a 27% decrease in the number of arteriole-sized (50-100 µm) vessels. There were no changes in the number of chorionic vessels, the depth or span of the tree, the diameter scaling coefficient, or the segment length-to-diameter ratio. PAH exposure resulted in a tree with a similar size and dichotomous branching structure, but one that was comparatively sparse so that arterial vascular resistance was increased by 30%. Assuming the same pressure gradient, blood flow would be 19% lower. Low flow may contribute to the 23% reduction observed in fetal weight. New insights into the specific effects of PAH exposure on a developing arterial tree were achieved using micro-CT imaging and automated vascular segmentation analysis.
Assuntos
Artérias/efeitos dos fármacos , Circulação Placentária/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Animais , Artérias/patologia , Arteríolas/efeitos dos fármacos , Arteríolas/patologia , Peso Corporal/efeitos dos fármacos , Córion/irrigação sanguínea , Meios de Contraste , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Hemodinâmica/fisiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Modelos Anatômicos , Neovascularização Patológica , Fenótipo , Placenta/efeitos dos fármacos , Placentação , Gravidez , Receptores de Hidrocarboneto Arílico/metabolismo , Fumar/patologia , Tomografia Computadorizada por Raios X , Resistência Vascular/efeitos dos fármacosRESUMO
PURPOSE: The number of women attempting to conceive between the ages of 36 and 44 has increased significantly in the last decade. While it is well established that women's reproductive success dramatically declines with age, the underlying physiological changes responsible for this phenomenon are not well understood. With assisted reproductive technologies, it is clear that oocyte quality is a likely cause since women over 40 undergoing in vitro fertilization (IVF) with oocytes donated by younger women have success rates comparable to young patients. Apart from oocyte donation, there is no known intervention to improve the pregnancy outcome of older patients. The aim of this paper was the review the relevant data on the potential role of mitochondria in reproductive aging. METHOD: Review of current literature on the subject. RESULTS: We present the current evidence that associate mitochondrial dysfunction with age related decrease in female reproductive outcome. CONCLUSIONS: The aging process is complex, driven by a multitude of factors thought to modulate cellular and organism life span. Although the factors responsible for diminished oocyte quality remain to be elucidated, the present review focuses on the potential role of impaired mitochondrial function.
Assuntos
Envelhecimento , Idade Materna , Mitocôndrias/fisiologia , Adulto , Fatores Etários , Senescência Celular , DNA Mitocondrial , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Fertilidade/fisiologia , Humanos , Mitocôndrias/metabolismo , Mutação , Oócitos/fisiologiaRESUMO
Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.