RESUMEN
Arf6 is a member of ADP-ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging-related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac-tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6-deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging-induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Oocitos , Animales , Oocitos/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Porcinos , Femenino , Meiosis/fisiología , Huso Acromático/metabolismo , Envejecimiento/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.
Asunto(s)
Meiosis , Huso Acromático , Femenino , Ratones , Animales , Huso Acromático/metabolismo , Metafase , Oocitos/metabolismo , Puntos de Control del Ciclo Celular , Proteínas Represoras/metabolismo , Cinesinas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon compound that is generated during combustion processes, and is present in various substances such as foods, tobacco smoke, and burning emissions. BaP is extensively acknowledged as a highly carcinogenic substance to induce multiple forms of cancer, such as lung cancer, skin cancer, and stomach cancer. Recently it is shown to adversely affect the reproductive system. Nevertheless, the potential toxicity of BaP on oocyte quality remains unclear. In this study, we established a BaP exposure model via mouse oral gavage and found that BaP exposure resulted in a notable decrease in the ovarian weight, number of GV oocytes in ovarian, and oocyte maturation competence. BaP exposure caused ribosomal dysfunction, characterized by a decrease in the expression of RPS3 and HPG in oocytes. BaP exposure also caused abnormal distribution of the endoplasmic reticulum (ER) and induced ER stress, as indicated by increased expression of GRP78. Besides, the Golgi apparatus exhibited an abnormal localization pattern, which was confirmed by the GM130 localization. Disruption of vesicle transport processes was observed by the abnormal expression and localization of Rab10. Additionally, an enhanced lysosome and LC3 fluorescence intensity indicated the occurrence of protein degradation in oocytes. In summary, our results suggested that BaP exposure disrupted the distribution and functioning of organelles, consequently affecting the developmental competence of mouse oocytes.
Asunto(s)
Benzo(a)pireno , Chaperón BiP del Retículo Endoplásmico , Oocitos , Animales , Benzo(a)pireno/toxicidad , Oocitos/efectos de los fármacos , Femenino , Ratones , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Orgánulos/efectos de los fármacos , Ratones Endogámicos ICRRESUMEN
Microtubule dynamics ensure multiple cellular events during oocyte meiosis, which is critical for the fertilization and early embryo development. KIF15 (also termed Hklp2) is a member of kinesin-12 family motor proteins, which participates in Eg5-related bipolar spindle formation in mitosis. In present study, we explored the roles of KIF15 in mouse oocyte meiosis. KIF15 expressed during oocyte maturation and localized with microtubules. Depletion or inhibition of KIF15 disturbed meiotic cell cycle progression, and the oocytes which extruded the first polar body showed a high aneuploidy rate. Further analysis showed that disruption of KIF15 did not affect spindle morphology but resulted in chromosome misalignment. This might be due to the reduced stability of the K-fibers, which further induced the loss of kinetochore-microtubule attachment and activated spindle assembly checkpoint, showing with the failed release of Bub3 and BubR1. Based on mass spectroscopy analysis and coimmunoprecipitation data we showed that KIF15 was responsible for recruiting HDAC6, NAT10 and SIRT2 to maintain the acetylated tubulin level, which further affected tubulin acetylation for microtubule stability. Taken together, these results suggested that KIF15 was essential for the microtubule acetylation and cell cycle control during mouse oocyte meiosis.
Asunto(s)
Cinesinas , Tubulina (Proteína) , Acetilación , Animales , Cinesinas/genética , Puntos de Control de la Fase M del Ciclo Celular , Meiosis , Ratones , Microtúbulos/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Polo like kinase 1 (PLK1) is a protein kinase involved in regulating the spindle assembly and cell cycle control in mammalian oocytes. SUMOylation, one way of post-translational modification, regulates oocyte meiosis by controlling several substrates. However, the relation between PLK1 and SUMOylation in oocytes is still unknown. In this study, we investigated that whether PLK1 was modified by SUMOylation in oocytes and its potential relationship with age-related meiotic abnormalities. We showed that PLK1 had colocalization and protein interaction with Small Ubiquitin-Like Modifier (SUMO)-1 and SUMO-2/3 in mouse oocytes, indicating that PLK1 could be modified by SUMO-1 and SUMO-2/3. Overexpression of PLK1 SUMOylation site mutants PLK1K178R and PLK1K191R caused the increase of the abnormal spindle rate of oocytes and the decline of the first polar body extrusion rate with the abnormal localization of PLK1, suggesting that the SUMOylation modification of PLK1 is essential for normal meiosis in oocytes. Compared with young mice, the expression of PLK1 protein increased and the expression of SUMO-1 and SUMO-2/3 protein decreased in the oocytes of aged mice, indicating that the SUMOylation of PLK1 might be related to the mouse aging. Therefore, our data suggested that PLK1 could be SUMOylated by SUMO-1 and SUMO-2/3 in mouse oocytes and SUMOylation of PLK1 regulated the meiosis progression of oocytes which was related with aging.
Asunto(s)
Proteínas de Ciclo Celular , Meiosis , Oocitos , Proteínas Serina-Treonina Quinasas , Sumoilación , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Edad , Quinasa Tipo Polo 1RESUMEN
ADP-ribosylation factor 1 (Arf1) is a small GTPase belonging to the Arf family. As a molecular switch, Arf1 is found to regulate retrograde and intra-Golgi transport, plasma membrane signaling, and organelle function during mitosis. This study aimed to explore the noncanonical roles of Arf1 in cell cycle regulation and cytoskeleton dynamics in meiosis with a mouse oocyte model. Arf1 accumulated in microtubules during oocyte meiosis, and the depletion of Arf1 led to the failure of polar body extrusion. Unlike mitosis, it finds that Arf1 affected Myt1 activity for cyclin B1/CDK1-based G2/M transition, which disturbed oocyte meiotic resumption. Besides, Arf1 modulated GM130 for the dynamic changes in the Golgi apparatus and Rab35-based vesicle transport during meiosis. Moreover, Arf1 is associated with Ran GTPase for TPX2 expression, further regulating the Aurora A-polo-like kinase 1 pathway for meiotic spindle assembly and microtubule stability in oocytes. Further, exogenous Arf1 mRNA supplementation can significantly rescue these defects. In conclusion, results reported the noncanonical functions of Arf1 in G2/M transition and meiotic spindle organization in mouse oocytes.
Asunto(s)
Factor 1 de Ribosilacion-ADP , Huso Acromático , Ratones , Animales , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Huso Acromático/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Meiosis , Oocitos/metabolismo , Aparato de Golgi/metabolismoRESUMEN
During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.
Asunto(s)
Actinas , Retículo Endoplásmico , Forminas , Meiosis , Mitocondrias , Oocitos , Animales , Femenino , Ratones , Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Forminas/metabolismo , Forminas/genética , Mitocondrias/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismo , PorcinosRESUMEN
Zearalenone is a mycotoxin produced by fungi of the genus Fusarium, which has severe toxicity on animal and human health including reproduction. Previous study showed that zearalenone exposure inhibited oocyte polar body extrusion, while in present study we found that high dose zearalenone disturbed oocyte meiosis resumption. Our results showed that a high concentration of 100 µM zearalenone reduced the rate of germinal vesicle (GV) breakdown in mouse oocytes. Further analysis indicated that zearalenone caused the decrease of Cyclin B1 and CDK1 expression, indicating MPF activity was affected, which further induced G2/M arrest, and this could be rescued by the inhibition of Wee1 activity. We found that the oocytes under high concentration of zearalenone showed lower γ-H2A.X expression, suggesting that DNA damage repair was disturbed, which further activated of DNA damage checkpoints. This could be confirmed by the altered expression of CHK1 and CHK2 after zearalenone treatment. Moreover, the organelles such as mitochondria, ribosome, endoplasmic reticulum and Golgi apparatus were diffused from germinal vesicle periphery after zearalenone exposure, indicating that zearalenone affected protein synthesis, modification and transport, which further induced the arrest of G2/M transition. Taken together, our results showed that high dose of zearalenone exposure induced G2/M transition defect by affecting organelle function-related CHK1/2-Wee1-MPF pathway.
Asunto(s)
Zearalenona , Animales , Apoptosis , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Meiosis , Ratones , Oocitos/metabolismo , Zearalenona/toxicidadRESUMEN
Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium fungi commonly found in corn, wheat, and other cereals which can infect food and feed commodities, and ZEA mainly has reproductive toxicity which causes widely reproductive disorders in pigs and other animals. However, the toxicity and the functional ways of ZEA on early embryo development is still unclear. In present study we showed that exposure to ZEA (10 µM) significantly decreased the 2-cell and blastocyst developmental rate in porcine early embryos in vitro. ZEA treatment resulted in the occurrence of oxidative stress, showing with increased reactive oxygen species (ROS) level, following with aberrant mitochondrial distribution. Moreover, we found positive signals of γH2A.X in the ZEA-treated embryos, indicating that ZEA induced DNA damage, and the increased autophagy confirmed this. These results suggested that ZEA induced oxidative stress, which further caused mitochondria dysfunction and DNA damage on early embryonic development. We next investigated the effects of melatonin on the ZEA-treated embryo development, and we found that melatonin supplementation could significantly ameliorate ZEA-induced oxidative stress, aberrant mitochondria distribution and DNA damage. In all, our results showed that ZEA was toxic for porcine embryos cultured in vitro and melatonin supplementation could protect their development from the effects of ZEA.