Molecular Regulation Of Actin Nucleation Related Factors WASH Complex,Arp2/3 Complex And FMNL1 In Oocyte Meiotic Maturation

The molecular regulation of mammalian oocyte meiotic maturation is an accurate and ordered multi-stage regulation process.Any defects and anomalies may disrupt oocyte maturation or zygote development.Cytoskeletons,including microfilament and microtubule,are crucial for oocyte maturation.Actin assembly and its dynamic changes cause oocyte cortical reorganization,polarization,and first polar body extrusion.Oocyte meiotic maturation is a unique asymmetric division.This process is depended on an actin flow which performs spindle positioning at the periphery of oocyte.Therefore,actin assembly and dynamics are the key regulators of oocyte asymmetric division.Actin nucleation factors regulate actin nucleation,polymerization and assembly.Recent studies found that actin nucleation factors mainly comprise three categories:The first category is the Arp 2/3 complex and its nucleation-promoting factors(NPFs).Arp 2/3 complex is considered to be the most critical factor which nucleated actin.Recent studies focused on model animals oocytes meiotic maturation,but its expression and function in large mammals oocyte meiotic maturation is unknown.NPFs can activate Arp 2/3 to participate in a variety of cellular processes which mediated by actin.WASH complex is the latest identified nucleation promoting factor,which was found to regulate actin assembly during Drosophila species oogenesis.The second category is Formin family proteins.Formin family proteins contain an important subclass which called diaphanous-related formins(DRFs).Recent years DRFs were found to involve in many cellular functions associated with actin filaments,such as morphogenesis,embryonic differentiation,cell polarity and cytokinesis.FMNL1 has been reported to position in microtubule organizing center(MTOC)during mitosis and regulate spindle formation and cytokinesis.The third category is the actin nucleation factor which contains WH2.Recently,researches on actin nucleation factors in oocyte meiotic maturation have become popular,but the molecular mechanism and signaling pathway of actin nucleation factors in mammalian oocyte meiosis are still unclear.Elucidating the molecular mechanisms of actin nucleation factors in mammalian oocyte meiosis will provide important research value for revealing the mechanism and law of mammalian reproductive,improving the efficiency of livestock breeding and healing human infertility disease.In this study,female ICR mice and pig were experiment animals.We employed in vitro culture,inhibitor treatment,MO(morpholino)microinjection to block protein translation,immunofluorescence staining,confocal microscopy,Western blotting(western blot),etc.to study the role and signaling pathways of actin nucleation factors during oocyte meiotic maturation.First,we employed immunofluorescence staining to label WASH complex,Arp 2/3 complex and FMNL1 in each stage during oocyte meiotic maturation.Then we blocked protein translation,destroy or inhibit the expression of protein function,respectively by microinjection of specific morpholino,specific antibody injection or by specific protein inhibitors treatment,in order to study their roles in actin and oocyte spindle assembly,meiotic spindle migration and positioning,the regional reorganization of cortical granules,chromosome alignment and separation and the cell cycle,and further investigate the molecular regulation of these actin nucleation factors on mammalian oocyte meiosis.In addition,we use western blot to analysis the upstream and downstream proteins,in order to revealing the signaling pathway of actin nucleation factors,which may provide basic evidence for further understanding the molecular mechanisms of actin nucleation factors during mammalian oocyte maturation.This study is divided into three parts.The main findings are as follows:Experiment 1.Actin nucleation promoting factor WASH complex involves in mouse oocytes meiotic maturation through regulating Arp 2/3 complexPrior to their fertilization,oocytes undergo asymmetric division,which is regulated by actin filaments.Recently,WASH complex were identified as actin nucleation promoting factors(NPF)that activated Arp2/3 complex.However,the roles of WASH complex remain uncertain,particularly for oocyte polarization and asymmetric division.Here,we examined the functions of two important subunits of a WASH complex,WASH1 and Strumpellin,during mouse oocyte meiosis.Depleting WASH1 or disrupting Strumpellin activity by WASH1 morpholino(MO)injection or Strumpellin antibody injection decreased polar body extrusion and caused oocyte symmetric division,and this may have been due to spindle formation and migration defects.Time lapse microscopy showed that actin filaments distribution and relative amount at the membrane and in the cytoplasm of oocytes was significantly decreased after disrupting WASH complex.In addition,Arp2/3 complex expression was reduced after WASH1 depletion.Thus,our data indicated that WASH complex regulated Arp2/3 complex and were required for cytokinesis and following polar body extrusion during mouse oocyte meiotic maturation.Experiment 2.The expression and functions of actin nucleation factor Arp 2/3 complex during porcine oocyte meiotic maturationThe Arp2/3 complex regulates actin nucleation,which is critical for a wide range of cellular processes,such as cell polarity,cell locomotion,and endocytosis.In the present study,we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation.Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes,colocalizing with actin.Treatment with an Arp2/3 complex specific inhibitor,CK666,resulted in a decrease in Arp2/3 complex localization at the oocyte cortex.The maturation rate of porcine oocytes decreased significantly after CK666 treatment,concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion.The fluorescence intensity of F-actin decreased in the cytoplasm,and CK666 also disrupted actin cap formation.In summary,our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation.Experiment 3.Actin nucleation factor FMNL1 regulates cytoskeleton dynamic during mouse oocyte meiosisFormin-like 1(FMNL1)is a member of Formin family proteins which are the actin nucleators.Although FMNL1 activities have been shown to be essential for cell adhesion,cytokinesis,cell polarization and migration in mitosis,the functional roles of mammalian FMNL1 during oocyte meiosis remain uncertain.In this study,we investigated the functions of FMNL1 in mouse oocytes using specific morpholino(MO)microinjection and live cell imaging.Immunofluorescent staining showed that in addition to its cytoplasmic distribution,FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown(GVBD).FMNL1 knockdown caused the low rate of polar body extrusion and resulted in large polar bodies.Time-lapse microscopic and immunofluorescence intensity analysis indicated that this might be due to the aberrant actin expression levels.Cortical polarity was disrupted as shown by a loss of actin cap,which was confirmed by a failure of meiotic spindle positioning.Spindle formation was also disrupted,which might be due to the abnormal localization of p-MAPK.Moreover,FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein,GM130.Finally,we found that the small GTPase RhoA might be the upstream regulator of FMNL1.Taken together,our data indicate that FMNL1 is required for spindle organization and actin assembly through a RhoA-FMNL1-GM130/p-MAPK pathway during mouse oocyte meiosis.