| Animal husbandry is the pillar industry of agricultural development in our country,and the level of animal reproductive performance plays a pivotal role in the production of animal husbandry.For female livestock,oocyte quality is a key factor in determining reproductive performance.The decrease of oocyte quality will cause reproductive disorder in female animals,and ultimately affect the fecundity of livestock.Therefore,it is very important to study the molecular mechanism of oocyte quality regulation.Oocyte maturation requires two extremely asymmetric divisions,in which half of the homologous chromosomes and half of the sister chromatids are eliminated in small polar bodies during meiosis I and meiosis II,respectively.Because oocyte cytoplasm contains the energy,maternal mRNA,and proteins which is required for embryo development,so these small polar bodies have a few cytoplasm and usually degenerate before fertilization.This pattern of cytokinesis is dependent on actin and spindle.MTOCs promote microtubule nucleation to form meiotic spindles.The kinetochore microtubules of spindle capture homologous chromosomes and align them on the metaphase plate,then chromosomes are partitioned toward the opposite spindle poles during anaphase.Actin cooperates with spindle to complete the extrusion of polar bodies during meiosis.Actin mediates spindle migration to the cortex,which could ensure asymmetric division.Cortical actin and Myosin II form contractile ring to promote polar body abscission.In addition to the role of the cytoskeleton,organelles also provide a guarantee for the high developmental potential of the oocyte and the storage of cytoplasmic materials for embryonic development.Organelles play an important role in energy metabolism and protein synthesis in oocytes.As the different stages of meiosis progress,organelles undergo appropriate distribution reorganization and these changes are regulated by the cytoskeleton.In conclusion,oocyte maturation requires both the cytoskeleton and organelle.In previous reports,most members of Rho GTPases co-localize with actin and regulate actin-related membrane remodeling during cell migration.The members of Rab GTPases are widely distributed in organelles and participate in different vesicle trafficking processes.The members of Roco GTPase regulate mitochondrial dynamics,lysosomal function,Golgi trafficking,and cytoskeleton assembly.These studies suggest that GTPases are indispensable for the regulation of cytoskeleton and organelle function in somatic cells,but the role of GTPases in oocyte maturation is unclear.In this study,sexually mature female ICR mice over six weeks were used as experiment model.We employed inRNA interference,plasmid construction and point mutation,oocyte microinjectiontechnique,combined with experimental methods such as immunofluorescence staining,real-time quantitative PCR,western blotting,mass spectrometry analysis and co-immunoprecipitation etc.to study the function of Rab8 a,Rab7and LRRK2 GTPase in mouse oocyte meiosis and its regulatory mechanism.This study is divided into three parts,the main research contents are as follows:Experiment 1: Rab8 a GTPase regulates spindle migration and Golgi apparatus distribution via ROCK-mediated actin assembly during oocyte maturation.Actin filaments are widely involved in multiple cellular processes in oocyte meiosis,such as spindle migration and polar body extrusion.The actin nucleators like ARP2/3complex and formins are the most recognized molecules for actin assembly in oocytes.We report that the vesicle trafficking factor,Rab8 a GTPase,is a new regulator critical for actin assembly in meiosis.Previous studies have shown that Rab8 a regulates cell migration by promoting the assembly of actin in filopodia and lamellipodia.In mouse oocytes Rab8 a was localized at both the spindle periphery and cortex,which was similar to the localization patterns of actin filaments.Rab8 a depletion caused spindle migration defects and the failure of polar body extrusion,which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes.Based on mass spectrometry analysis,we showed that Rab8 a promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway.The localization of Rab8 a on the Golgi regulates the forward trafficking of Golgi network,and Rab8 a could maintain the normal Golgi morphology in somatic cells.We demonstrated that Rab8 a colocalized and interacted with GM130 at the spindle periphery and that Rab8 a depletion caused the disruption of GM130-docked Golgi distribution in oocyte.Taken together,our data indicated that Rab8 a was required for Golgi distribution,spindle migration,and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.Experiment 2: Rab7 GTPase regulates actin dynamics for spindle migration and DRP1-mediated mitochondria function during oocyte maturation.Rab7 is a small GTPase that belongs to the Rab family,and as a vesicle trafficking factor it is shown to regulate the transport to late endocytic compartments,mitophagy and organelle function.In present study,we showed the critical roles of Rab7 GTPase on actin dynamics and mitochondria function in oocyte meiosis.We found that Rab7 mainly accumulated at cortex and spindle periphery during oocyte maturation by Myc-Rab7 plasmid injection and antibody staining.Rab7 depletion caused the failure of polar body extrusion and asymmetric division,and Rab7 exogenous mRNA supplement could rescue the defects caused by Rab7 siRNA injection.Based on mass spectrometry analysis,we found that Rab7 associated with several actin-related factors and mitochondria-related proteins in oocytes.The depletion of Rab7 caused the decrease of actin dynamics,which further affected meiotic spindle migration to the oocyte cortex.In addition,we found that Rab7 could maintain mitochondrial membrane potential and the mitochondrial distribution in mouse oocytes,and this might be due to its effects on the phosphorylation of DRP1 at Ser616 domain.It has been reported that actin polymerization is an upstream event to initiate mitochondrial dynamics.Taken together,our data indicated that Rab7 transported actin-related factors for actin dynamics,which further affected the phosphorylation of DRP1 for mitochondria dynamics and the meiotic spindle migration in mouse oocytes.Experiment 3: LRRK2 regulates actin assembly for spindle migration and mitochondrial function during oocyte maturation.LRRK2 belongs to the Roco GTPases family and is a large multi-domain protein harboring both GTPase and kinase activities.LRRK2 plays indispensable roles in many biological processes,such as autophagy and vesicle trafficking in somatic cell.In the present study,we showed the critical roles of LRRK2 in mammalian oocyte meiosis.LRRK2 is mainly accumulated at the meiotic spindle periphery during mouse oocyte maturation.Depleting LRRK2 led to the polar body extrusion defects and also induced large polar bodies in mouse oocytes.Mass spectrometry analysis and co-immunoprecipitation results showed that LRRK2 was associated with several actin-regulating factors such as Fascin and ROCK,and depletion of LRRK2 affected the expression of ROCK,phosphorylated cofilin,and Fascin.Further analysis showed that LRRK2 depletion did not affect spindle organization but caused the failure of spindle migration,which was largely due to the decrease of cytoplasmic actin filaments.Moreover,LRRK2 showed similar localization pattern with mitochondria,and LRRK2 was associated with several mitochondria-related proteins.Indeed,mitochondrial distribution and function were all disrupted in LRRK2-depleted oocytes.In summary,our results indicated the critical roles of LRRK2 in actin assembly for spindle migration and mitochondrial function in mouse oocyte meiosis. |
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