RESUMO
Aging is a degenerative process involving cell function deterioration, leading to altered metabolic pathways, increased metabolite diversity, and dysregulated metabolism. Previously, we reported that human placenta-derived mesenchymal stem cells (hPD-MSCs) have therapeutic effects on ovarian aging. This study aimed to identify hPD-MSC therapy-induced responses at the metabolite and protein levels and serum biomarker(s) of aging and/or rejuvenation. We observed weight loss after hPD-MSC therapy. Importantly, insulin-like growth factor-I (IGF-I), known prolongs healthy life spans, were markedly elevated in serum. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis identified 176 metabolites, among which the levels of 3-hydroxybutyric acid, glycocholic acid, and taurine, which are associated with health and longevity, were enhanced after hPD-MSC stimulation. Furthermore, after hPD-MSC therapy, the levels of vitamin B6 and its metabolite pyridoxal 5'-phosphate were markedly increased in the serum and liver, respectively. Interestingly, hPD-MSC therapy promoted serotonin production due to increased vitamin B6 metabolism rates. Increased liver serotonin levels after multiple-injection therapy altered the expression of mRNAs and proteins associated with hepatocyte proliferation and mitochondrial biogenesis. Changes in metabolites in circulation after hPD-MSC therapy can be used to identify biomarker(s) of aging and/or rejuvenation. In addition, serotonin is a valuable therapeutic target for reversing aging-associated liver degeneration.
Assuntos
Reprogramação Celular , Metabolismo Energético , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Placenta/citologia , Rejuvenescimento , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Biomarcadores , Proliferação de Células , Feminino , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Modelos Animais , Gravidez , Ratos , Serotonina/biossíntese , Vitamina B 6/metabolismoRESUMO
Decrease in quality of postovulatory aged oocytes occurs due to oxidative stress and leads to low fertilization and development competence. It is one of the main causes that exerting detrimental effect on the success rate in assisted reproductive technology (ART). Auraptene (AUR), a citrus coumarin, has been reported to possess an antioxidant effects in other tissues. In this study, we aimed to confirm the potential of AUR to delay the oocyte aging process by alleviating oxidative stress. Superovulated mouse oocytes in metaphase of second meiosis (MII) were exposed to 0, 1 or 10 µM AUR for 12 h of in vitro aging. AUR addition to the culture medium recovered abnormal spindle and chromosome morphology and mitigated mitochondrial distribution and mitochondrial membrane potential (ΔΨ) in aged oocytes. AUR-treated aged oocytes also showed suppressed oxidative stress, with lower reactive oxygen species (ROS) levels, higher glutathione (GSH) levels and increased expression of several genes involved in antioxidation. Furthermore, AUR significantly elevated the fertilization and embryo developmental rates. Oocytes aged with 1 µM AUR exhibited morphokinetics that were very similar to those of the control group. Altogether, these data allowed us to conclude that AUR improved the quality of aged oocytes and suggest AUR as an effective clinical supplement candidate to prevent postovulatory aging.
RESUMO
Previously, we reported that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation by suppressing mitophagy and inducing mitochondrial dysfunction, resulting in fertilization failure. Here, we show that oocyte aging is accompanied by an increase in meiotic defects associated with chromosome misalignment and abnormal spindle organization. Intriguingly, decreased Gas6 mRNA and protein expression were observed in aged oocytes from older females. We further explored the effect of GAS6 on the quality and fertility of aged mouse oocytes using a GAS6 rescue analysis. After treatment with the GAS6 protein, aged oocytes matured normally to the meiosis II (MII) stage. Additionally, maternal age-related meiotic defects were reduced by GAS6 protein microinjection. Restoring GAS6 ameliorated the mitochondrial dysfunction induced by maternal aging. Ultimately, GAS6-rescued MII oocytes exhibited increased ATP levels, reduced ROS levels and elevated glutathione (GSH) levels, collectively indicating improved mitochondrial function in aged oocytes. Thus, the age-associated decrease in oocyte quality was prevented by restoring GAS6. Importantly, GAS6 protein microinjection in aged oocytes also rescued fertility. We conclude that GAS6 improves mitochondrial function to achieve sufficient cytoplasmic maturation and attenuates maternal age-related meiotic errors, thereby efficiently safeguarding oocyte quality and fertility.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mitocôndrias/fisiologia , Mitofagia/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Animais , Cromossomos/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Metáfase/genética , Metáfase/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Mitofagia/genética , Oócitos/crescimento & desenvolvimento , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Aging has detrimental effects on the ovary, such as a progressive reduction in fertility and decreased hormone production, that greatly reduce the quality of life of women. Thus, the current study was undertaken to investigate whether human placenta-derived mesenchymal stem cell (hPD-MSC) treatment can restore the decreases in folliculogenesis and ovarian function that occur with aging. METHODS: Acclimatized 52-week-old female SD rats were randomly divided into four groups: single hPD-MSC (5 × 105) therapy, multiple (three times, 10-day intervals) hPD-MSC therapy, control (PBS), and non-treated groups. hPD-MSC therapy was conducted by tail vein injection into aged rats. The rats were sacrificed 1, 2, 3, and 5 weeks after the last injection. hPD-MSC tracking and follicle numbers were histologically confirmed. The serum levels of sex hormones and circulating miRNAs were detected by ELISA and qRT-PCR, respectively. TGF-ß superfamily proteins and SMAD proteins in the ovary were detected by Western blot analysis. RESULTS: We observed that multiple transplantations of hPD-MSCs more effectively promoted primordial follicle activation and ovarian hormone (E2 and AMH) production than a single injection. After hPD-MSC therapy, the levels of miR-21-5p, miR-132-3p, and miR-212-3p, miRNAs associated with the ovarian reserve, were increased in the serum. Moreover, miRNAs (miR-16-5p, miR-34a-5p, and miR-191-5p) with known adverse effects on folliculogenesis were markedly suppressed. Importantly, the level of miR-145-5p was reduced after single- or multiple-injection hPD-MSC therapy, and we confirmed that miR-145-5p targets Bmpr2 but not Tgfbr2. Interestingly, downregulation of miR-145-5p led to an increase in BMPR2, and activation of SMAD signaling concurrently increased primordial follicle development and the number of primary and antral follicles. CONCLUSIONS: Our study verified that multiple intravenous injections of hPD-MSCs led to improved ovarian function via miR-145-5p and BMP-SMAD signaling and proposed the future therapeutic potential of hPD-MSCs to promote ovarian function in women at advanced age to improve their quality of life during climacterium.
Assuntos
Envelhecimento , Proteínas Morfogenéticas Ósseas , Células-Tronco Mesenquimais , MicroRNAs , Animais , Feminino , Humanos , MicroRNAs/genética , Placenta , Gravidez , Qualidade de Vida , Ratos , Ratos Sprague-DawleyRESUMO
Bcl2l10, also known as Diva, Bclb and Boo, is a member of the Bcl2 family of proteins, which are involved in signaling pathways that regulate cell apoptosis and autophagy. Previously, it was demonstrated that Bcl2l10 plays a crucial role in the completion of oocyte meiosis and is a key regulator of Aurora kinase A (Aurka) expression and activity in oocytes. Aurka is overexpressed in several types of solid tumors and has been considered a target of cancer therapy. Based on these previous results, in the present study, the authors aimed to investigate the regulatory role of Bcl2l10 in A2780 and SKOV3 human ovarian cancer cells. The protein expression of Bcl2l10 was examined in human cancer tissues and cell lines, including the ovaries, using a tissue microarray and various human ovarian cancer cell lines. It was found that Bcl2l10 regulated the protein stability and activities of Aurka in ovarian cancer cells. Although apoptosis was not affected, the cell cycle was arrested at the G0/G1 phase by Bcl2l10 knockdown. Of note, cell viability and motility were markedly increased by Bcl2l10 knockdown. On the whole, the findings of this study suggest that Bcl2l10 functions as tumor suppressor gene in ovarian cancer.
Assuntos
Aurora Quinase A/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/genética , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Ovário/patologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Previously, we found that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation through mitochondrial overactivation with concurrent failure of pronuclear formation after fertilization. In this study, we report that Gas6 regulates mitophagy and safeguards mitochondrial activity by regulating mitophagy-related genes essential to the complete competency of oocytes. Based on RNA-Seq and RT-PCR analysis, in Gas6-silenced MII oocytes, expressions of mitophagy-related genes were decreased in Gas6-silenced MII oocytes, while mitochondrial proteins and Ptpn11, the downstream target of Gas6, was increased. Interestingly, GAS6 depletion induced remarkable MTOR activation. Gas6-depleted MII oocytes exhibited mitochondrial accumulation and aggregation caused by mitophagy inhibition. Gas6-depleted MII oocytes had a markedly lower mtDNA copy number. Rapamycin treatment rescued mitophagy, blocked the increase in MTOR and phosphorylated-MTOR, and increased the mitophagy-related gene expression in Gas6-depleted MII oocytes. After treatment with Mdivi-1, a mitochondrial division/mitophagy inhibitor, all oocytes matured and these MII oocytes showed mitochondrial accumulation but reduced Gas6 expression and failure of fertilization, showing phenomena very similar to the direct targeting of Gas6 by RNAi. Taken together, we conclude that the Gas6 signaling plays a crucial role in control of oocytes cytoplasmic maturation by modulating the dynamics and activity of oocyte mitochondria.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Mitofagia/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Metáfase/genética , Metáfase/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Modelos Biológicos , Oócitos/crescimento & desenvolvimento , Quinazolinonas/farmacologia , Interferência de RNA , RNA-Seq , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , TranscriptomaRESUMO
BACKGROUND/AIMS: Previously, we found that silencing of growth arrest-specific gene 6 (Gas6) in oocytes impaired cytoplasmic maturation, resulting in failure of sperm chromatin decondensation (SCD) and pronuclear (PN) formation after fertilization. Thus, we conducted this study to determine the effect of Gas6 RNAi on downstream genes and to elucidate the working mechanism of Gas6 on oocyte cytoplasmic maturation and SCD. METHODS: Using RT-PCR, Western blot and immunofluorescence, the expression levels of various target genes and the localization of heparan sulfate (HS) were analyzed after Gas6 RNAi. The roles of Gas6 in HS biosynthesis, production of ATP and GSH, ROS generation and ΔΨm were also investigated. SCD and micrococcal nuclease (MNase) analyses were used to examine the effects of HS on the open chromatin state in sperm and somatic cell nuclei, respectively. RESULTS: Disruption of Gas6 expression led to the inhibition of HS biosynthesis through the reduction of several HS biosynthetic enzymes. The rescue experiment, HS treatment in vitro, significantly recovered SCD and PN formation, confirming that HS had the ability to induce sperm head remodeling during fertilization. Interestingly, excessive mitochondrial activation in Gas6-depleted MII oocytes caused ROS generation and glutathione (GSH) degradation via mitochondrial activation, such as elevated ΔΨm and ATP production. Indeed, HS-treated NIH3T3 cell nuclei showed an open chromatin state, as determined by diffuse DAPI staining and increased sensitivity to MNase. CONCLUSION: We propose that the addition of HS to sperm and/or oocyte maturation would improve the efficiency of in vitro fertilization and somatic cell nuclear transfer (SCNT) reprogramming.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Citoplasma/metabolismo , Heparitina Sulfato/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Feminino , Fertilização in vitro , Glutationa/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Células NIH 3T3 , Oócitos/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
This chapter describes methods for preparing oocytes and embryos to analyze their gene expression at specific developmental stages. We illustrate how to collect germinal vesicles (GVs) and mature metaphase II (MII) stage oocytes, as well as how to collect embryos at specific developmental stages from the pronucleus (PN) to the blastocyst stage from female mice. We also describe how to prepare mRNAs from these precious cells to analyze the expression of the target genes. The materials and methods in this chapter are used mainly for mouse oocytes and embryos, but with subtle modifications, they may be applicable for most mammalian species.
Assuntos
Blastocisto/citologia , Perfilação da Expressão Gênica/métodos , Oócitos/citologia , RNA Mensageiro/genética , Animais , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Oócitos/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Ras dexamethasone-induced protein (RASD1) is a member of Ras superfamily of small GTPases. RASD1 regulates various signaling pathways involved in iron homeostasis, growth hormone secretion, and circadian rhythm. However, RASD1 function in oocyte remains unknown. METHODS: Using immunohistochemistry, immunofluorescence, and quantitative real-time RT-PCR, RASD1 expression in mouse ovary and RASD1 role in oocyte maturation-related gene expression, spindle formation, and chromosome alignment were analyzed. RNAi microinjection and time-lapse video microscopy were used to examine the effect of Rasd1 knockdown on oocyte maturation. RESULTS: RASD1 was highly detected in oocytes transitioning from primordial to secondary follicles. Rasd1 was highly expressed in germinal vesicle (GV), during GV breakdown, and in metaphase I (MI) stage as oocytes mature, and its expression was significantly downregulated in MII stage. With knockdown of Rasd1, maturation in GV oocytes was arrested at MI stage, showing disrupted meiotic spindling and chromosomal misalignment. In addition, Obox4 and Arp2/3, engaged in MI-MII transition and cytokinesis, respectively, were misregulated in GV oocytes by Rasd1 knockdown. CONCLUSION: These findings suggest that RASD1 is a novel factor in MI-MII oocyte transition and may be involved in regulating the progression of cytokinesis and spindle formation, controlling related signaling pathways during oocyte maturation.
Assuntos
Diferenciação Celular , Técnicas de Silenciamento de Genes , Oócitos/citologia , Oócitos/metabolismo , Proteínas ras/genética , Animais , Diferenciação Celular/genética , Cromossomos de Mamíferos/metabolismo , Citocinese , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Metáfase/genética , Camundongos Endogâmicos ICR , Especificidade de Órgãos/genética , Interferência de RNA , Fuso Acromático , Proteínas ras/metabolismoRESUMO
Previously, we reported that Sebox is a new maternal effect gene (MEG) that is required for early embryo development beyond the two-cell (2C) stage because this gene orchestrates the expression of important genes for zygotic genome activation (ZGA). However, regulators of Sebox expression remain unknown. Therefore, the objectives of the present study were to use bioinformatics tools to identify such regulatory microRNAs (miRNAs) and to determine the effects of the identified miRNAs on Sebox expression. Using computational algorithms, we identified a motif within the 3'UTR of Sebox mRNA that is specific to the seed region of the miR-125 family, which includes miR-125a-5p, miR-125b-5p and miR-351-5p. During our search for miRNAs, we found that the Lin28a 3'UTR also contains the same binding motif for the seed region of the miR-125 family. In addition, we confirmed that Lin28a also plays a role as a MEG and affects ZGA at the 2C stage, without affecting oocyte maturation or fertilization. Thus, we provide the first report indicating that the miR-125 family plays a crucial role in regulating MEGs related to the 2C block and in regulating ZGA through methods such as affecting Sebox and Lin28a in oocytes and embryos.
Assuntos
Desenvolvimento Embrionário , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas , Animais , Blastocisto , Linhagem Celular , Biologia Computacional/métodos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Herança Materna , CamundongosRESUMO
Previously, we demonstrated that Bcl-2-like 10 (Bcl2l10) is associated with meiotic spindle assembly and that the gene that is most strongly down-regulated by Bcl2l10 RNAi is targeting protein for Xklp2 (Tpx2). Tpx2 is a well-known cofactor that controls the activity and localization of Aurora kinase A (Aurka) during mitotic spindle assembly. Therefore, this study was conducted (1) to identify the associations among Bcl2l10, Tpx2, and Aurka and (2) to understand how Bcl2l10 regulates meiotic spindle assembly in mouse oocytes. Bcl2l10, Tpx2, and Aurka co-localized on the meiotic spindles, and Bcl2l10 was present in the same complex with Tpx2. Tpx2 and Aurka expression decreased whereas phospho-Aurka increased in Bcl2l10 RNAi-treated oocytes. Counterbalancing changes in the levels of these 2 activators, Tpx2 and phospho-Aurka, resulted in decreased Aurka catalytic activity after Bcl2l10 RNAi treatment. Bcl2l10 RNAi decreased the expression of microtubule organizing center (MTOC)-related proteins, disturbed MTOC formation and disrupted meiotic spindle assembly. Our data demonstrate that Bcl2l10 is a binding partner of Tpx2 and a new regulator of the complex controlling the organization of microtubules and MTOC biogenesis in meiotic spindle assembly. The discovery of Bcl2l10 as a new effector of Aurka suggests that Bcl2l10 may have diverse functions in mitotic cells.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Diferenciação Celular , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Centro Organizador dos Microtúbulos/metabolismo , Modelos Biológicos , Oócitos/citologia , Fosforilação , Ligação Proteica , Transporte Proteico , Interferência de RNA , Fuso Acromático/metabolismoRESUMO
Rad51 is a conserved eukaryotic protein that mediates the homologous recombination repair of DNA double-strand breaks that occur during mitosis and meiosis. In addition, Rad51 promotes mitochondrial DNA synthesis when replication stress is increased. Rad51 also regulates cell cycle progression by preserving the G2/M transition in embryonic stem cells. In this study, we report a novel function of Rad51 in regulating mitochondrial activity during in vitro maturation of mouse oocytes. Suppression of Rad51 by injection of Rad51 dsRNA into germinal vesicle-stage oocytes resulted in arrest of meiosis in metaphase I. Rad51-depleted oocytes showed chromosome misalignment and failures in spindle aggregation, affecting the completion of cytokinesis. We found that Rad51 depletion was accompanied by decreased ATP production and mitochondrial membrane potential and increased DNA degradation. We further demonstrated that the mitochondrial defect activated autophagy in Rad51-depleted oocytes. Taken together, we concluded that Rad51 functions to safeguard mitochondrial integrity during the meiotic maturation of oocytes.
RESUMO
Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Inativação Gênica , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Membrana Nuclear/metabolismo , Oócitos/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Feminino , Imunofluorescência , Proteínas Ligadas por GPI/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Técnicas Imunoenzimáticas , Meiose/fisiologia , Mesotelina , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genéticaRESUMO
Previously, using the Illumina HumanHT-12 microarray we found that ß-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line RWPE-1 upon stimulation with lipoteichoic acid (LTA; a gram-positive bacterial component), than that in the untreated RWPE-1 cells. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. We examined the intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-κB directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-κB activation, which was evident in the phosphorylation of both p38MAPK and IκBα. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-κB, respectively, suppressed LTA-induced DEFB131 expression. The chromatin immunoprecipitation assay showed that NF-κB directly binds to the DEFB131 promoter, suggesting that NF-κB is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, with DEFB131 overexpression in RWPE-1 cells, the accumulation of mRNA and protein secretion of cytokines (IL-1α, IL-1ß, IL-6, and IL-12α) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-κB signaling pathway in RWPE-1 cells during bacterial infection and promotes an innate immune response.
Assuntos
Infecções Bacterianas/imunologia , Imunidade Inata , Próstata/metabolismo , beta-Defensinas/fisiologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos , Masculino , NF-kappa B/metabolismo , Nitrilas , Transdução de Sinais , Ácidos Teicoicos , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In a previous report, we identified Sebox as a new candidate maternal effect gene that is essential for embryonic development and primarily impacts the two-cell (2C) stage. The present study was conducted to determine the mechanism of action for Sebox in this capacity, as shown by changes in the expression levels of other known MEG mRNAs after Sebox RNA interference (RNAi) in oocytes. Sebox-knockdown metaphase II (Mll) oocytes displayed normal morphology, but among the 23 MEGs monitored, 8 genes were upregulated, and 15 genes were unchanged. We hypothesized that the perturbed gene expression of these MEGs may cause the arrest of embryo development at the 2C stage and examined the expression of several marker genes for the degradation of maternal factors and zygotic genome activation. We found that some maternal mRNAs, c-mos, Gbx2, and Gdf9, were not fully degraded in Sebox-knockdown 2C embryos, and that several zygotic genome activation markers, Mt1a, Rpl23, Ube2a and Wee1, were not fully expressed in conjunction with diminished embryonic transcriptional activity. In addition, Sebox may be involved in the formation of the subcortical maternal complex through its regulation of the upstream regulator, Figla. Therefore, we concluded that Sebox is important in preparing oocytes for embryonic development by orchestrating the expression of other important MEGs.
Assuntos
Proteínas do Ovo/genética , Desenvolvimento Embrionário , Proteínas de Homeodomínio/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Marcadores Genéticos/genética , Genômica , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Gravidez , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Zigoto/metabolismoRESUMO
Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.
RESUMO
PURPOSE: The present study aimed to determine the role played by ß-defensin 124 (DEFB124) in the innate immunity of prostate epithelial RWPE-1 cells during bacterial infection. MATERIALS AND METHODS: The expression of DEFB124 was examined by quantitative real-time polymerase chain reaction (PCR), Western blotting, and immunocytochemistry. Enzyme-linked immunosorbent assays and quantitative real-time PCR were performed to determine the production of cytokines and chemokines. Western blotting and chromatin immunoprecipitation studies were performed to assess the interaction between DEFB124 and nuclear factor-kappa B (NF-κB) in peptidoglycan (PGN)-stimulated RWPE-1 cells. By chemotaxis assay, we assessed the effect of DEFB124 on the migration of monocytes. RESULTS: Exposure to PGN induced DEFB124 upregulation and NF-κB activation through IκBα phosphorylation and IκBα degradation. Bay11-7082, an NF-κB inhibitor, blocked PGN-induced DEFB124 production. Also, NF-κB was shown to be a direct regulator and to directly bind to the -3.14 kb site of the DEFB124 promoter in PGN-treated human prostate epithelial RWPE-1 cells. When DEFB124 was overexpressed in RWPE-1 cells, interestingly, the production of cytokines (interleukin [IL] 6 and IL-12) and chemokines (CCL5, CCL22, and CXCL8) was significantly increased. These DEFB124-upregulated RWPE-1 cells markedly induced chemotactic activity for THP-1 monocytes. CONCLUSIONS: Taken together, these results provide strong evidence for the first time that increased DEFB124 expression via NF-κB activation in PGN-exposed RWPE-1 cells enhances the production of cytokines and chemokines, which may contribute to an efficient innate immune defense.
RESUMO
OBJECTIVE: To determine whether sequence variants within the FGF23 gene are associated with the risk of developing prostate cancer in a Korean population. PATIENTS AND METHODS: Five common single nucleotide polymorphisms (SNPs) in the FGF23 gene were assessed in 272 patients with prostate cancer and 173 control subjects with benign prostatic hyperplasia. Single-locus analyses were conducted using conditional logistic regression. In addition, we performed a haplotype analysis for the five FGF23 SNPs tested. RESULTS: Three SNPs in the FGF23 gene (rs11063118, rs13312789 and rs7955866) were associated with an increased risk of prostate cancer in our study population. Odds ratios for homozygous variants vs wild-type variants ranged from 1.68 (95% confidence interval [CI]: 1.15-2.46) to 1.79 (95% CI: 1.16-2.75). CONCLUSION: This is the first study showing that genetic variations in FGF23 increase prostate cancer susceptibility.
Assuntos
Povo Asiático/genética , Fatores de Crescimento de Fibroblastos/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Próstata/genética , Idoso , Estudos de Casos e Controles , Fator de Crescimento de Fibroblastos 23 , Predisposição Genética para Doença/genética , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/etnologia , Neoplasias da Próstata/patologia , República da CoreiaRESUMO
Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs (mESCs) and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mESCs did not affect self-renewal under nondifferentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation toward mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and showed that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mESCs as a molecular rheostat of Nodal-Smad2/3 signaling.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/genética , Fatores de Elongação da Transcrição/genética , Animais , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Perfilação da Expressão Gênica , Fatores de Determinação Direita-Esquerda/genética , Fatores de Determinação Direita-Esquerda/metabolismo , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Proteína Nodal/genética , Proteína Nodal/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C.