| In mammals,the development of a new individual arises from the zygote,which derived from the fertilization between a paternal sperm and a maternal oocyte.In the process of evolution,oocytes developed to the larger one,which provided the material basis for later fertilization and early embryonic development.The oocyte meiotic maturation involves two successive divisions,resulting in a smaller polar body and a larger oocyte with more cytoplasm.In this process,the genetic material tranformed from relatively loose chromatin into chromosomes.With the participation of MTOCs,chromosomes assembled into a spindle and then migrated to the cortical area,after which the oocyte extruded the first polar body.However,if the chromosome alignment is abnormal or the separation process is wrong,polyploid embryos will easily form after fertilization or even fail to mature normally,which is obviously harmful to the development of organisms.At the same time,the spatial distribution of organelles and cytoskeletons throughout the cytoplasm also changes to prepare for subsequent fertilization.However,if the poor qualiy of oocytes caused by abnormalities in the maturation process rises,such as failed cortical granule migration or abnormal exocytosis,the chances of polyspermy fertilization are greatly increased.Therefore,egg quality control is crucial for the development and continuation of species.Although some factors involved in the regulation of oocyte maturation have been reported,the specific mechanism and other regulatory factors involved in the continuous process from oocyte maturation to fertilization are still poorly understood.Rab family members can regulate vesicle transport and play important roles in material transport and signal transduction.In somatic cells,Rab23 distributes extensively and is vital for cell differenciation and development.Rab 10 also participates in vesicle polarized transport and energy supply.But the functions of Rab23 and Rab 10 in oocyte maturation are rarely reported and further studies are needed.In our experiments,ICR mice were the research material,and studies the functions of Rab23 and Rab 10 during meiotic maturation and fertilization of mouse oocytes through siRNA,point mutation mRNA microinjection and other means,so as to reveal the signal pathway and mechanism of Rab23 and Rab 10 in this process.This study is mainly divided into two parts.The experimental results are as follows:The role of Rab23 in mouse oocyte meiotic maturationRab23 is widely distributed in somatic cells,located in cytoplasmic membrane,endosome and cytoplasm,and play important roles in cell differentiation and development.However,little is known about its regulation of oocyte meiosis.In this study,we investigated the effect of Rab23 on oocyte meiotic maturation through Rab23 siRNA and point mutant plasmid in vitro transcribed mRNA microinjection.During the maturation process of mouse oocytes,Rab23 was mainly located on the spindle and has a stable expression at all stages of oocyte maturation.Since Rab family members act as small GTPases,and their active form is binding to GTP and inactive form is binding to GDP,we also constructed Rab23 point mutant plasmid to simulate these two states.We find that active form of Rab23 is also located on the spindle,while inactive form of Rab23 is widely distributed in the cytoplasm.Knockdown of Rab23 can significantly reduce the oocyte Pbl extrusion rate,which indicates that Rab23 is involved in the regulation of oocyte maturation.Furthermore,we found that Rab23 can also regulate the expression and localization ofKif17,a member of the kinesin family.At each stage of oocyte maturation,Kif17 distributed on the spindle and in cytoplasm,and continuously expressed at protein level.It should be noted that Kif17 has a significantly polarized localization pattern on the spindle,and this localization pattern could be affected after overexpression of the inactive form of Rab23.After Kif17 RNAi,oocyte maturation can also be inhibited and a certain proportion of big polar body extrusion or even symmetric division occurs.Due to Kif17's positioning pattern on the spindle and its effect on spindle migration,we observed whether it had an effect on cytoskeleton(actin filaments or microtubules).Our results showed that after Kif17 RNAi,spindle morphology and chromosome alignment were affected,and the distribution of microfilaments in cytoplasm was reduced,but the effect on cortical microfilaments was not significantly affected.Therefore,we tested whether factors that can regulate microfilament assembly and spindle assembly are also regulated by Kif17.We found that the RhoA-ROCK-LIMK-cofilin signaling pathway was affected by Kif17 knockdown,but the influence on the expression of formin and spire was not significant.In addition,our results also showed that Kif17 could regulate the acetylation of ?-tubulin through Sirt2,aTAT and NAT 10.The results of co-ip experiments showed that the tail region of Kif17 could combine with RhoA-ROCK-LIMK-cofilin,Sirt2 and ?TAT.In summary,Rab23 can regulate the maturation process of mouse oocytes by regulating Kif17-mediated transport of factors related to microfilament and microtubule.The role of Rab10 in mouse oocyte maturationAs another member of the Rab family,Rab10 was widely distributed in the endosome,around the endoplasmic reticulum-golgi apparatus and other parts of the cytoplasm.Studies have shown that Rab10 participated in the regulation of endoplasmic reticulum,as well as affect the polar transport of cytoplasmic vesicles,thereby regulating the energy supply and signal transduction of cells.However,its role in oocyte meiotic maturation is poorly understood.In this study,we investigated the effect of Rab10 on meiotic maturation through siRNA and point mutant mRNA microinjection.During the maturation of mouse oocytes,Rab expressed stabely during oocyte maturation active form of Rab10 distributed in the cortical area of oocytes,while inactive form of Rab10 localized mainly in the cytoplasmic area.Knockdown of Rab10 can affect the extrusion of the first polar body,but has no significant effect on spindle assembly and the acetylation of spindle microtubules.As an important indicator of cytoplasmic maturation,the migration and distribution of cortical granules have received extensive attention in recent years.Our results showed that after knockdown of Rab10,the migration of cortical granules to cortical areas failed,and cortical granule free area could not be formed normally.It was reported that the migration of cortical granules is dependent on the cytoskeleton of actin filaments.Further studies also showed that after Rab10 RNAi or over-expression of inactive form of Rab10,the distribution of microfilaments in the cytoplasm decreased,and the protein expression of member in RhoA-ROCK-LIMK-cofilin decreased.In addition,our results showed that the microfilament-dependent transport of cortical granules was regulated by Myosin 5a.Besides,active form of Rab10 could promote the expression of Myosin 5a,while the active state of Rab10 could inhibit its expression.The distribution of cortical granule is abnormal,which have important influence on the subsequent fertilization process.In conclusion,Rab10 can regulate the RhoA-ROCK-LIMK-cofilin.signaling pathway and the expression of Myosin 5a to affect the distribution of cortical granules. |
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