RESUMEN
Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 µM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1α signaling pathway.
Asunto(s)
Flavonas , Mitocondrias , Sirtuina 1 , Animales , Porcinos , Sirtuina 1/metabolismo , Mitocondrias/metabolismo , Transducción de Señal , Oocitos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, G-protein coupled receptor kinase 2 (GRK2) binds to HSP90 in response to hypoxia or other stresses. In the present study, we investigated the effects of GRK2 knockdown and inhibition on porcine embryonic development from the zygote stage. Immunofluorescence and western blotting were used to determine the localization and expression, respectively, of GRK2 and related proteins. First, GRK2 and p-GRK2 were expressed in both the cytoplasm and membrane and co-localized with HSP90 on the membrane. The mRNA level of GRK2 increased until the 8C- morula stage, suggesting that GRK2 may play an essential role during the early development of the porcine embryos. GRK2 knockdown reduced porcine embryo development capacity and led to significantly decreased blastocyst quality. In addition, inhibition of GRK2 also induced poor ability of embryo development at early stage, indicating that GRK2 is critical for embryonic cleavage in pigs. Knockdown and inhibition of GRK2 reduced HSP90 expression and AKT activation and cAMP levels. Additionally, GRK2 deficiency increased LC3 expression, suggesting enhanced autophagy during embryo development. In summary, we showed that GRK2 binds to HSP90 on the membrane to regulate embryonic cleavage and AKT activation during embryonic development in pigs.
RESUMEN
Glucose-regulated protein 78 (GRP78) binds to and stabilizes melanocortin 4 receptor (MC4R), which activates protein kinase A (PKA) by regulating G proteins. GRP78 is primarily used as a marker for endoplasmic reticulum stress; however, its other functions have not been well studied. Therefore, in this study, we aimed to investigate the function of GRP78 during porcine embryonic development. The developmental quality of porcine embryos, expression of cell cycle proteins, and function of mitochondria were evaluated by inhibiting the function of GRP78. Porcine oocytes were activated to undergo parthenogenesis, and blastocysts were obtained after 7 days of in vitro culture. GRP78 function was inhibited by adding 20 µM HA15 to the in vitro culture medium. The inhibition in GRP78 function led to a decrease in G proteins release, which subsequently downregulated the cyclic adenosine monophosphate (cAMP)/PKA pathway. Ultimately, inhibition of GRP78 function induced the inhibition of CDK1 and cyclin B expression and disruption of the cell cycle. In addition, inhibition of GRP78 function regulated DRP1 and SIRT1 expression, resulting in mitochondrial dysfunction. This study provides new insights into the role of GRP78 in porcine embryonic development, particularly its involvement in the regulation of the MC4R pathway and downstream cAMP/PKA signaling. The results suggest that the inhibition of GRP78 function in porcine embryos by HA15 treatment may have negative effects on embryo quality and development. This study also demonstrated that GRP78 plays a crucial role in the functioning of MC4R, which releases the G protein during porcine embryonic development.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Receptor de Melanocortina Tipo 4 , Femenino , Embarazo , Porcinos , Animales , Desarrollo Embrionario , Partenogénesis , AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico , Proteínas de Unión al GTPRESUMEN
Y-box binding protein 1 (YBX1) is a member of the family of DNA- and RNA-binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one-cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four-cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6-methyladenosine (m6A) writer N6-adenosine-methyltransferase 70 kDa subunit (METTL3) and reader insulin-like growth factor 2 mRNA-binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process.
Asunto(s)
Proteínas de Unión al ADN , Desarrollo Embrionario , Cigoto , Animales , Adenosina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Porcinos , Cigoto/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10â h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.
Asunto(s)
Desarrollo Embrionario , Epigénesis Genética , Animales , Porcinos , Temperatura , MamíferosRESUMEN
Zinc finger and SCAN domain-containing 4 (ZSCAN4), a DNA-binding protein, maintains telomere length and plays a key role in critical aspects of mouse embryonic stem cells, including maintaining genomic stability and defying cellular senescence. However, the effect of ZSCAN4 in porcine parthenogenetic embryos remains unclear. To investigate the function of ZSCAN4 and the underlying mechanism in porcine embryo development, ZSCAN4 was knocked down via dsRNA injection in the one-cell stage. ZSCAN4 was highly expressed in the four- and five- to eight-cell stages in porcine embryos. The percentage of four-cell stage embryos, five- to eight-cell stage embryos, and blastocysts was lower in the ZSCAN4 knockdown group than in the control group. Notably, depletion of ZSCAN4 induced the protein expression of DNMT1 and 5-Methylcytosine (5mC, a methylated form of the DNA base cytosine) in the four-cell stage. The H3K27ac level and ZGA genes expression decreased following ZSCAN4 knockdown. Furthermore, ZSCAN4 knockdown led to DNA damage and shortened telomere compared with the control. Additionally, DNMT1-dsRNA was injected to reduce DNA hypermethylation in ZSCAN4 knockdown embryos. DNMT1 knockdown rescued telomere shortening and developmental defects caused by ZSCAN4 knockdown. In conclusion, ZSCAN4 is involved in the regulation of transcriptional activity and is essential for maintaining telomere length by regulating DNMT1 expression in porcine ZGA.
Asunto(s)
Telómero , Factores de Transcripción , Animales , Ratones , Porcinos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Telómero/genética , Telómero/metabolismo , Acortamiento del Telómero , Proteínas de Unión al ADN/metabolismo , Cigoto/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Activator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/genética , Transporte de Proteínas , Red trans-Golgi , Animales , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Transducción de Señal , Red trans-Golgi/metabolismoRESUMEN
ACSS1/2 converts acetate into acetyl-coenzyme A, which contributes to histone acetylation in the mitochondria and cytoplasm. Zygotic genome activation (ZGA) is critical for embryo development involving drastic histone modification. An efficient crRNAs-Cas13a targeting strategy was employed to investigate the ACSS1/2 function during ZGA. The results showed that nuclear accumulation of ACSS1 and ACSS2 occurs during ZGA. Knockdown of ACSS1/2 did not affect blastocyst formation when using a normal medium. On culturing embryos in a medium with acetate and no pyruvate (-P + Ace), knockdown of ACSS1 did not affect histone acetylation levels but significantly reduced ATP levels, whereas knockdown of ACSS2 significantly reduced histone acetylation levels in porcine embryos. Inhibition of fatty acid beta-oxidation by etomoxir significantly reduced ATP levels, which could be restored by acetate. The histone acetylation levels in the ACSS1 and ACSS2 knockdown groups both decreased considerably after etomoxir treatment. Moreover, acetate showed dose-dependent effects on SIRT1 and SIRT3 levels when under metabolic stress. The C-terminus of ACSS1 regulated the nuclear translocation. In conclusion, ACSS1/2 helps to maintain ATP and histone acetylation levels in porcine early embryos under metabolic stress during ZGA.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Metabolismo Energético , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Cigoto/enzimología , Acetato CoA Ligasa/genética , Acetilación , Adenosina Trifosfato/metabolismo , Animales , Técnicas de Cultivo de Embriones , Partenogénesis , Sirtuina 1/metabolismo , Sirtuina 3/metabolismo , Sus scrofaRESUMEN
The cytoskeleton plays an orchestrating role in polarized cell growth. Microtubules (MTs) not only play critical roles in chromosome alignment and segregation but also control cell shape, division, and motility. A member of the plus-end tracking proteins, end-binding protein 1 (EB1), regulates MT dynamics and plays vital roles in maintaining spindle symmetry and chromosome alignment during mitosis. However, the role of EB1 in mouse oocyte meiosis remains unknown. Here, we examined the localization patterns and expression levels of EB1 at different stages. EB1 protein level was found to be stable during meiosis. EB1 mainly localized along the spindle and had a similar localization pattern as that of α-tubulin. The EB1 protein was degraded with a Trim-Away method, and the results were further confirmed with western blotting and immunofluorescence. At 12 h of culture after EB1 knockdown (KD), a reduced number of mature MII oocytes were observed. EB1 KD led to spindle disorganization, chromosome misalignment, and missegregation; ß-catenin protein binds to actin via the adherens junctional complex, which was significantly reduced in the EB1 KD oocytes. Collectively, we propose that the impairment of EB1 function manipulates spindle formation, thereby promoting chromosomal loss, which is expected to fuel aneuploidy and possibly fertilization failure.
Asunto(s)
Meiosis , Huso Acromático , Animales , Cromosomas , Ratones , Microtúbulos , OocitosRESUMEN
Phosphatase and tensin homolog-induced kinase 1 (PINK1) on the outer membranes of impaired mitochondria promotes mitophagy and regulates mitochondrial morphology. Mammalian oocytes and early embryos are mitochondria rich, but mitochondrial dynamics during preimplantation embryo development is not well-studied. To investigate whether PINK1 is required for mitochondrial dynamics in porcine preimplantation embryos, gene knockdown and inhibitors were used, and mitochondrial dynamics were observed by transmission electron microscopy. PINK1 knockdown significantly impaired blastocyst formation and quality, induced mitochondrial elongation and swelling, and reduced mitochondrial DNA copy number. PINK1 knockdown-induced mitochondrial elongation caused mitochondrial dysfunction, oxidative stress, and ATP deficiency, significantly increasing autophagy and apoptosis. Profission dynamin-related protein 1 overexpression prevented PINK1 knockdown-induced impairment of embryo development, mitochondrial elongation, and dysfunction. Thus, PINK1 promotes mitochondrial fission in porcine preimplantation embryos.-Niu, Y.-J., Nie, Z.-W., Shin, K.-T., Zhou, W., Cui, X.-S. PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.
Asunto(s)
Blastocisto/fisiología , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/fisiología , Proteínas Quinasas/fisiología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Autofagia , Blastocisto/metabolismo , Dinaminas/genética , Dinaminas/fisiología , Desarrollo Embrionario , Dosificación de Gen , Técnicas de Silenciamiento del Gen , Genes Mitocondriales , Técnicas de Maduración In Vitro de los Oocitos , Potencial de la Membrana Mitocondrial , Microinyecciones , Partenogénesis , Proteínas Quinasas/genética , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes , Sus scrofaRESUMEN
Melatonin, a major hormone of the pineal gland, exerts many beneficial effects on mitochondria. Several studies have shown that melatonin can protect against toxin-induced oocyte quality impairment during maturation. However, there is little information regarding the beneficial effects of melatonin on toxin-exposed early embryos, and the mechanisms underlying such effects have not been determined. Rotenone, a chemical widely used in agriculture, induces mitochondrial toxicity, therefore, damaging the reproductive system, impairing oocyte maturation, ovulation, and fertilization. We investigated whether melatonin attenuated rotenone exposure-induced impairment of embryo development by its mitochondrial protection effect. Activated oocytes were randomly assigned to four groups: the control, melatonin treatment, rotenone-exposed, and "rotenone + melatonin" groups. Treatment with melatonin abrogated rotenone-induced impairment of embryo development, mitochondrial dysfunction, and ATP deficiency, and significantly decreased oxidative stress and apoptosis. Melatonin also increased SIRT1 and PGC-1α expression, which promoted mitochondrial biogenesis. SIRT1 knockdown or pharmacological inhibition abolished melatonin's ability to revert rotenone-induced impairment. Thus, melatonin rescued rotenone-induced impairment of embryo development by reducing ROS production and promoting mitochondrial biogenesis. This study shows that melatonin rescues toxin-induced impairment of early porcine embryo development by promoting mitochondrial biogenesis.
Asunto(s)
Embrión de Mamíferos , Desarrollo Embrionario/efectos de los fármacos , Melatonina/farmacología , Mitocondrias , Enfermedades Mitocondriales , Rotenona/efectos adversos , Animales , Embrión de Mamíferos/embriología , Embrión de Mamíferos/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/embriología , Enfermedades Mitocondriales/prevención & control , Rotenona/farmacología , PorcinosRESUMEN
Connexin 43 (CX43) is a component of gap junctions. The lack of functional CX43 induces oxidative stress, autophagy, and apoptosis in somatic cells. However, the role of CX43 in the early development of porcine embryos is still unknown. Thus, the aim of this study was to investigate the role of CX43, and its underlying molecular mechanisms, on the developmental competence of early porcine embryos. We performed CX43 knockdown by microinjecting dsRNA into parthenogenetically activated porcine parthenotes. The blastocyst development rate and the total number of cells in the blastocysts were significantly reduced by CX43 knockdown. Results from FITC-dextran assays showed that CX43 knockdown significantly increased membrane permeability. ZO-1 protein was obliterated in CX43 knockdown blastocysts. Mitochondrial membrane potential and ATP production were significantly reduced following CX43 knockdown. Reactive oxygen species (ROS) levels were significantly increased in the CX43 knockdown group compared to those in control embryos. Moreover, CX43 knockdown induced autophagy and apoptosis. Our findings indicate that CX43 is essential for the development and preimplantation of porcine embryos and maintains mitochondrial function, cell junction structure, and cell homeostasis by regulating membrane permeability, ROS generation, autophagy, and apoptosis in early embryos.
Asunto(s)
Conexina 43/genética , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Mitocondrias/metabolismo , Animales , Apoptosis , Autofagia , Blastocisto/metabolismo , Técnicas de Silenciamiento del Gen , Uniones Intercelulares , Potencial de la Membrana Mitocondrial/fisiología , Oocitos , Estrés Oxidativo , Especies Reactivas de Oxígeno , PorcinosRESUMEN
Spindlin 1 (SPIN1), which contains Tudor-like domains, regulates maternal transcripts via interaction with a messenger RNA (mRNA)-binding protein. SPIN1 is involved in tumorigenesis in somatic cells and is highly expressed in cancer cells. Nevertheless, the role of SPIN1 in porcine oocyte maturation remains totally unknown. To explore the function of SPIN1 in porcine oocyte maturation, knockdown, and overexpression techniques were used. SPIN1 mRNA was identified in maternal stages ranging from GV to MII. SPIN1 was localized in the cytoplasm and to chromosomes during meiosis. SPIN1 knockdown accelerated first polar body extrusion. Oocytes with overexpressed SPIN1 were arrested at the MI stage. SPIN1 depletion caused meiotic spindle defects and chromosome instability. The BUB3 signal was investigated, confirming that SPIN1 affects the stability of Bub3 mRNA as well as BUB3 expression. Further, overexpression of SPIN1 inhibited the degradation and regulation of G2/mitotic-specific cyclin-B1. In summation, SPIN1 regulates the meiotic cell cycle by modulating the activation of the spindle assembly checkpoint.
Asunto(s)
Anafase , Proteínas de Ciclo Celular/metabolismo , Metafase , Proteínas Asociadas a Microtúbulos/metabolismo , Oocitos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Huso Acromático/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Segregación Cromosómica , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Maduración In Vitro de los Oocitos , Proteínas Asociadas a Microtúbulos/genética , Fosfoproteínas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Transducción de Señal , Sus scrofa , Factores de TiempoRESUMEN
Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100 µM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100 µM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species (ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.
Asunto(s)
Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Oocitos/efectos de los fármacos , Pirazoles/farmacología , Animales , Proteína Quinasa CDC2/efectos de los fármacos , Proteína Quinasa CDC2/metabolismo , Ciclina B1/efectos de los fármacos , Citocromos c/efectos de los fármacos , Citocromos c/metabolismo , Daño del ADN/efectos de los fármacos , Femenino , Técnicas In Vitro , Oocitos/citología , Oogénesis/efectos de los fármacos , Plaguicidas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Porcinos , Proteína bcl-X/efectos de los fármacos , Proteína bcl-X/metabolismoRESUMEN
If no fertilization occurs for a prolonged time following ovulation, oocytes experience a time-dependent deterioration in quality both in vivo and in vitro due to processes called postovulatory aging. Because the postovulatory aging of oocytes has marked detrimental effects on embryo development and offspring, many efforts have been made to unveil the underlying mechanisms. Here we showed that translationally controlled tumor protein (TCTP) regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality. Spindle dynamics decreased with reduced TCTP level during aging of mouse oocytes. Knockdown of TCTP accelerated the reduction of spindle dynamics, accompanying with aging-related deterioration of oocyte quality. Conversely, overexpression of TCTP prevented aging-associated decline of spindle dynamics. Moreover, the aging-related abnormalities in oocytes were rescued after TCTP overexpression, thereby improving fertilization competency and subsequent embryo development. Therefore, our results demonstrate that TCTP-mediated spindle dynamics play a key role in maintaining oocyte quality during postovulatory aging and overexpression of TCTP is sufficient to prevent aging-associated abnormalities in mouse oocytes.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Senescencia Celular , Fase Luteínica/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Animales , Biomarcadores de Tumor/genética , Blastocisto/metabolismo , Células Cultivadas , Femenino , Fase Luteínica/genética , Masculino , Ratones , Oocitos/citología , Oogénesis , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long-chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR-associated protein 9 (Cas9) system. Cas9 and single-guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS-positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X-box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR-like ER kinase (PERK), and an increase in the mRNA expression of the ER stress-regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction.
Asunto(s)
Apoptosis/genética , Estrés del Retículo Endoplásmico/genética , Ácido Graso Sintasas/genética , Proteína 1 de Unión a la X-Box/genética , Factor de Transcripción Activador 4/genética , Animales , Desarrollo Embrionario/genética , Retículo Endoplásmico/genética , Femenino , Técnicas de Inactivación de Genes , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Porcinos , Factor de Transcripción CHOP/genética , Respuesta de Proteína Desplegada/genéticaRESUMEN
In 2011, DEHP (plasticizer) was reported to illegally be added in food and beverage products in Taiwan, which caused great concerns about food safety worldwide. DHEP has multiple toxic effects to human and animals such as endocrine disruption, cardiotoxicity, reproductive function, and development defects. However, the toxic effects of DEHP on mammalian oocyte quality are still unclear. Since MEHP is the active metabolite of DEHP in vivo, in this study we used porcine oocyte as model to explore the effects of MEHP on oocyte maturation and we also studied the effects of melatonin administration on MEHP exposure-induced meiosis defects. Our results showed that exposure to MEHP significantly decreased the polar body extrusion rate in porcine oocytes. Further study showed that cell cycle progression, meiotic spindle organization, and actin assembly were all disturbed after MEHP exposure. Moreover, the DNA and histone methylation levels were also affected, showing with altered 5mC and H3K4me2 levels. These results indicated that MEHP affected porcine oocyte maturation, while MEHP exposure-induced meiotic defects were all remarkably ameliorated by the administration of melatonin in porcine oocytes. We further tried to explore the causes of MEHP toxicity on oocytes, and we found that MEHP exposure resulted in significant elevations of oxidative stress and induced early apoptosis as well as elevated autophagy, while melatonin administration could reduce these. Taken together, our results indicated that MEHP exposure induced deterioration of oocyte quality, whereas melatonin supplement showed amelioration on oocyte maturation through its rescue effects on oocyte oxidative stress-mediated apoptosis and autophagy.
Asunto(s)
Antioxidantes/farmacología , Dietilhexil Ftalato/análogos & derivados , Meiosis/efectos de los fármacos , Melatonina/farmacología , Oocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Plastificantes/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Dietilhexil Ftalato/farmacología , Femenino , Oocitos/metabolismo , Oogénesis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
Mammalian oocytes undergo several crucial processes during meiosis maturation, including spindle formation and migration and polar body extrusion, which rely on the regulation of actin. As a small actin-binding protein, profilin 1 plays a central role in the regulation of actin assembly. However, the functions of profilin 1 in mammalian oocytes are uncertain. To investigate the function of profilin 1 in oocytes, immunofluorescent staining was first used to examine profilin 1 localisation. The results showed that profilin 1 was localised around the meiotic spindles and was colocalised with cytoplasmic actin. Knockdown (KD) of profilin 1 with specific morpholino microinjection resulted in failure of polar body extrusion. This failure resulted from an increase of actin polymerisation both at membranes and in the cytoplasm. Furthermore, western blot analysis revealed that the expression of Rho-associated kinase (ROCK) and phosphorylation levels of myosin light chain (MLC) were significantly altered after KD of profilin 1. Thus, the results indicate that a feedback mechanism between profilin, actin and ROCK-MLC2 regulates actin assembly during mouse oocyte maturation.
Asunto(s)
Actinas/metabolismo , Oocitos/metabolismo , Cuerpos Polares/metabolismo , Profilinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Citocinesis/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Meiosis/fisiología , Ratones , Fosforilación , Profilinas/genética , Huso Acromático/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
MicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with in vitro fertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.
Asunto(s)
Blastocisto/metabolismo , Núcleo Celular/metabolismo , MicroARNs/metabolismo , Animales , Bovinos , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas de Transferencia Nuclear , Oocitos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.