| In livestock production,the fertility of livestock directly affects the production level and economic benefits,the quality of female oocytes is one of the key factors to ensure the fertility,so it is very important to study the meiosis of oocytes.Different from mitosis,the meiosis of mammalian oocytes is a unique asymmetric division.After the breakdown of the germinal vesicle in the center of oocytes,the microtubule-organizing center promotes the aggregation of microtubules near chromosomes in oocyte cytoplasm,forming spindle,and homologous chromosomes are orderly arranged in the center of the equatorial plate of the spindle.Afterwards,the spindle migrates to the oocyte cortex under actin,accompanied by separation of homologous chromosomes,cytokinesis by the contraction of the contractile ring,and excretion of the first polar body by oocytes.So far,the first meiosis is finished.After that,the oocytes are blocked in the metaphase of the second meiosis waiting for fertilization,during which actin plays an important role.Current studies have shown that the main roles of actin in the meiosis of oocytes include long-distance vesicle transport,maintaining the central location of the nucleus in the cytoplasm,promoting the migration of spindle,ensuring the normal arrangement of chromosomes,separation and cytokinesis,as well as maintaining the distribution of organelle.Actin nucleation factors are proteins that regulate the nucleation,aggregation and assembly of actin.In recent years,a great number of studies have focused on the influence of actin nucleation factor on the meiosis of oocytes,but the specific molecular regulatory mechanism and related protein pathway remain unclear.Therefore,it is necessary to elucidate the molecular mechanism of actin nucleation factor in the meiosis of mammalian oocytes.This will be of great value for uncovering mammals' reproductive mechanism and improving the reproductive efficiency of domestic animals.In this study,oocytes of 4-6 week female ICR mice were used as experiment model.We employed in vitro culture,immunofluorescence staining,confocal microscopy,RNAi interfering technology,microinjectiontechnique,real-time PCR,Western blotting,mass spectrometry(ms)and Co-IP etc.to study the role of formin family members FHOD1,FMNL2 and FMNL3 during oocyte meiotic maturation.First of all,we employed immunofluorescence staining or fluorescent protein to label FHOD1,FMNL2 and FMNL3 in each stage during oocyte meiotic maturation.Then we knockdown protein level by microinjection of specific siRNA.In order to study their roles in actin and oocyte spindle assembly,meiotic spindle migration,cytokinesis,organelle distribution,and further investigate the molecular regulation of FHOD1,FMNL2,FMNL3 on mammalian oocyte meiosis.In addition,we use mass spectrometry,Co-IP and western blotting to analysis the upstream and downstream proteins,in order to provide basic evidence for further understanding the molecular mechanisms of formin family members during mammalian oocyte maturation.This study is divided into three parts.The main findings are as follows:Experiment 1:FHOD1 regulates cytoplasmic actin-based spindle migration for oocyte asymmetric cell divisionFHOD1 is a member of Diaphanous-related formins(DRFs)which belongs to the Formin family.Previous studies have shown that the DFRs might affect several cellular functions such as morphogenesis,cytokinesis,cell polarity,and embryonic differentiation.However,there is no evidence showing the functions of FHOD1 during oocyte meiosis.This study is aimed at exploring the roles of FHOD1 during the mammalian oocyte maturation.Immunofluorescent staining showed thatFHOD1 was restricted to the nucleus in germinal vesicle(GV)stage of the oocytes,after the GV breakdown FHOD1 was primarily located at two poles of the spindle at both metaphases I and II stages.Knockdown of FHOD1 by siRNA injection did not affect polar body extrusion but generated the large polar bodies.In addition,we observed the spindle migration failure in metaphase I oocytes,with a large number of meiotic spindles anchoring in the center of cytoplasm.The expression level of cytoplasmic actin but not cortex actin was significantly reduced,indicating that FHOD1 regulates cytoplasmic actin distribution for the spindle movement.Furthermore,we found that the disruption of ROCK(the Rho-dependent protein kinase)with inhibitor Y-27632 caused the decreased FHOD1 protein expression.Therefore,our data indicate that FHOD1 is regulated by ROCK for cytoplasm actin assembly and spindle migration during mouse oocyte meiosis.Experiment 2:FMNL2 promotes actin assembly for spindle migration and organelle distribution in mouse oocytesCytoskeleton actin is essential for oocyte maturation.As an acitn nucleating factor,Formin-like 2(FMNL2)plays an important role in the regulation of actin assembly.However.the functions of FMNL2 during oocytes meiosis remain unclear.In this study,we investigated the role of FMNL2 during mouse oocyte maturation.Our results showed that FMNL2 mainly localized in the oocyte cortex and periphery of spindle.Knockdown(KD)of FMNL2 led to the failure of polar body extrusion,and large polar bodies appeared in the FMNL2-KD oocytes.Live-cell imaging revealed that spindle migration defects during metaphase I(MI),and this might be due to the decreased polymerization of cytoplasmic actin.Furthermore,we found several Endoplasmic reticulum(ER)and mitochondria related potential candidates might be related with FMNL2 from mass spectrometry analysis,we examined the ER and mitochondria distribution in FMNL2-KD oocytes found that ER and mitochondrial agglomerated in cytoplasm.Thus,our results indicate that FMNL2 affects the oocyte maturation not only by regulating actin-mediated spindle migration but also by regulating the distribution of ER and mitochondria.Experiment 3:FMNL3 regulates fascin for actin-mediated spindle migration and cytokinesis in mouse oocytesFormin-like 3(FMNL3)is a member of the formin-like(FMNL),as an F-actin nucleator,FMNL3 is essential for several cellular functions,such as polarity control,invasion,and migration.However,the roles of FMNL3 during oocytes meiosis remain unclear.In this study,we investigated the functions of FMNL3 during mouse oocyte maturation.Our results showed that FMNL3 mainly concentrated in the oocyte cortex and spindle periphery.Depleting FMNL3 led to the failure of polar body extrusion,and we also found large polar bodies in the FMNL3-deleted oocytes,indicating the occurrence of symmetric meiotic division.There was no effect of FMNL3 on spindle organization;however,we observed spindle migration defects at late metaphase I,which might be due to the decreased cytoplasmic actin.Microinjecting Fmnl3-EGFP mRNA into FMNL3-depleted oocytes significantly rescued these defects.In addition,the results of co-immunoprecipitation and the perturbation of protein expression experiments suggested that FMNL3 interacted with the actin-binding protein fascin for the regulation of actin filaments in oocytes.Thus,our results provide the evidence that FMNL3 regulates fascin for actin-mediated spindle migration and cytokinesis during mouse oocyte meiosis.In summary,the three proteins FHOD1,FMNL2 and FMNL3 are all involved in actin mediated spindle migration during the first meiosis of oocytes.They also have their own unique functions at the same time.Therefore,the proteins of the Formin family play a significant role in the meiosis of oocytes and are vital regulators to ensure the normal maturation of the latter. |
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