Cdc42 Involved In Cytokinesis During Xenopus Oocyte Maturation

INTRODUCTIONThe oocyte maturation process is controlled by many signal pathways, which play critical roles for the ongoing of GV and GVBD as well as polar body formation.Along side with meiosis of Xenopus oocyte,one pole of metaphaseâ… spindle attaching the cortex spatial and temporally,trigger onset of downstream events,such as furrow locolization,contractile ring formation,cytokinesis and polar body emission. Furthermore,actin filament and other cytoskeleton material also participate in this process,but what signal cause attachment of spindle to the cortex and later cases puzzled biochemist many years.In this study,Cdc42—the regulator of cytoskeleton and cell polarity,contributes to meiosis and oocyte maturation.Cdc42 was first found in Saccharomyces cerevisiae,as a member of Rho family GTPases,which play critical roles in cell signal transduction pathways. In yeast Cdc42 functions in cytoskeleton regulation, especially in the control of the polarity and bud site establishment. In the mammalian cells microinjected active Cdc42 induced the pseudopodia formation, also contributes to cell migration, trafficking and development. Through the regulation of filament actin and other cytoskeleton materials coordinately.Cdc42 controls the cell morphology. In addition, molecular showing physical interactions with Cdc42 include both forming and members of the Par3/Par6/aPKC polarity complex.As forming is required for asymmetrical positioning of the spindle and as both Par3 and Par61ocalize asymmetrically in the oocyte following spindle migration.We considered if Cdc42 and formin,Par3/Par6/aPKC could have an involvement in cell polarity processes?Evidence showed that in Saccharomyces cerevisiae, Cdc42 and Bni1p together involved in spindle localization. We approached this question by using asymmetrical Xenopus oocyte as a model,and study polar body formation during oocyte meiosis.Cdc42 signaling may thus be an evolutionarily conserved mechanism that function on polar body formation and cytokinesis during oocyte meiosis.MATERIALS1. Plasmids for gene cloning and reagents for western blot.2. Related fluorescently-labeled biology reagents.METHODS1. Preparation of xenopus oocytes and extractionPMSG(50IU per frog) was injected to xenopus 3 days before experiment. Frog was killed by cutting the head under animal care rules, both ovaries were removed and isolated into pieces each containing about 10-15 oocytes, then digested in collagenase for about 3 hours, collected stageâ…¥oocytes and transferred to calcium free OR2 media shaking for 4 hours under room temperature. Ice-cold extraction buffer (20mM HEPES [pH 7.3], 80mM glycerophosphate, 20mM EGTA, 15mM MgCl₂, 1mM dithiothreitol, 10μM ATP, 150μM NaF, 10μg of leupeptin per ml, 200μM phenylmethylsulfonyl fluoride, 25μg of benzamidine per ml, 10μl lysis buffer per oocyte). Following centrifugation (13000g for 5 min, 4℃), the clarified extract was removed, mixed with 2X SDS sample buffer plus beta-mercaptoethanol (β-Me), and stored in-20℃for further western blotting. Or else, clarified extract was directly used or immunoprecipitated for kinase assay.)2. MPF Kinase assayMPF assay was performed by following the protocol.Briefly, after progesterone stimulation, at different time point, 8μl of the clarified extract was added to 4μl of the same EB buffer containing 2μg histone H1, 5μCi of [³²P]γ-ATP (Amersham) and 100μM ATP. Kinase reactions were carried out at room temperature for 20 min and stopped by adding 12μl of 2X SDS sample buffer. Proteins were separated on a 15% SDS-PAGE, dried and visualized by autoradiography.3. In vitro synthesis mRNA of Cdc42T17N and GFP-wGBDCdc42T17N and GFP-wGBD plasmids digested with BamH1 restriction engyme to make linerized template, as promoter to synthesis Cdc42T17N and GFP-wGBD mRNA,stored under-70℃4. Observation of cytokinesis process from the control oocytes14nl Alexa-phalloidin (4U/ml) and 10nL Rhodamine-tubulin (3mg/ml) were microinjected into the frog oocytes in the calcium free OR2 media. 8 hours later, 1μM progesterone was used to stimulate oocytes, the appearance of a white spot was considered as GVBD occurance. 90 minutes after GVBD cytokinesis monitored by confocal microscopy under 40×object, 72 seconds and 2μm scanning interval with Time-lapes method.5. Chromosome changes of both the Cdc42T17N mRNA injected and control oocytes observationCollected oocyte at different time point according to GVBD beginning time and transferred into the fresh calcium containing OR2 media for later using. 60 minutes after GVBD oocytes were dyed with Hoechst dying (1:5000 dilution) for 20 minutes, fluorescent microscopy with time-lapes program was employed to monitor the chromosome change with the oocyte maturation process. Cdc42T17N mRNA injected oocytes were observed with the same procedure as control oocytes except the Cdc42T17N mRNA injected oocytes needed overnight expression before experiment.6. Cdc42T17N mRNA injected and control oocytes morphological changes observation14 nl Alexa-phalloidin (4U/ml) and 10nL Rhodamine-tubulin (3mg/ml) were microinjected into oocyte each, same as above mentioned stimulated oocytes with 1uM progesterone and collected different time point oocytes according the GVBD beginning time, adopted the same procedure as mentioned in step 4 to compare the two group oocytes morphological changes with confocal microscopy.7. Cdc42T17N mRNA injected and control oocytes Cdc42 activity monitoring10nL 0.25mg/ml GFP-wGBD mRNAå’Œ10nL Rhodamine-tubulin (3mg/ml) microinjected into oocytes each incubated at RT for about 8 hours for enough expression, then transfer to 16℃incubator overnight to maintain the MI status. With same parameter setting as described in step 4, used of confocal microscopy to observe the signals.8. Colocalization of Cdc42 and g-actin observation and analysisThe contractile ring forming process monitoring operation was the same as above described. The co localization experiment needed to co-injected into oocytes,the parameter setting was same as above.9. Statistic analysisCompare cytokinesis and polar body formation percentage of normal oocytes,oocytes injected with Cdc42T17N mRNA.SPSS 11.5 software was emplyed, and P＜0.05 was considered different significantly.RESULTS1. MPF kinase activityMPF showed no activity 2 hours after progesterone stimulation, then began to increase at 3 hours after stimulation and decreased to lower activity about 1 hour following GVBD.2. Cytokinesis process observation of control oocytes14nl Alexa-phalloidin (4U/ml) and 10nL Rhodamine-tubulin (3mg/ml) co-injected oocytes showed that actin filament signals began to aggregate to around over spindle after one pole of spindle attaching to the cortex, then formed contractile ring, which ensued the severed the spindle into two haves, finally polar body formed with the successful cytokinesis. 3. Fluorescent microscopy monitoring the DNA changesControl oocytes showed the polar body formation (polar body contain very dense DNA with minimal cytoplasm), but Cdc42T17N mRNA injected oocytes showed no polar body formation.4. Cdc42T17N mRNA injected and control oocytes morphological changes observationContractile ring was formed with one pole of spindle attaching to the cortex and polar body formed successfully at the end of cytokinesis. Cdc42T17NmRNA showed no contractile ring formation, although clearly one pole of spindle attached the cortex and spindle separated into haves, with time gone, the two haves of spindle combined together again, which indicated that chromosome replication and separation were not affected by Cdc42T17N mRNA injection.5. Cdc42 activity monitoring of both Cdc42T17N mRNA injected and control oocytesGFP-wGBD(the specific probe of active Cdc42) was used to monitor the activity of Cdc42. 2-4 minutes before cytokinesis onset Cdc42 activity began to show up and gradually increase especially around and over the spindle pole area then wrapped the spindle downward along the polar body emission. Cdc42T17N mRNA showed no obvious Cdc42 activity although spindle morphological changes mimic the control oocytes except the polar body formation abortion.6. Colocalization of Cdc42 activity and g-actin during the cytokinesis process14nL Alexa-phalloidin (4U/ml) and 10nL Rhodamine-tubulin (3mg/ml) co-injected oocyte, since the formation of contractile ring Cdc42 activity signals (green) colocalized with the g-actin signals, both showed ring like shape with similar temporal-spatial manner.7. Statistic analysisSPSS 11.5 software was emplyed, and P＜0.05 was considered different significantly.Compared with control oocytes,cytokinesis and polar body formation percentages of Cdc42T 17N mRNA injected oocytes decreased significantly.CONCLUSIONS1,In the GV stage, MPF has no activity and increase at GVBD, again decrease before contractile ring formation.2,Compared with control oocytes, Cdc42T17N mRNA injection and its expression does not affect the GV and progesterone induced GVBD.3,Compared with control oocytes, Cdc42T17N mRNA injection inhibit oocyte cytokinesis and polar body formation4,Compared with control oocytes, Cdc42T17N mRNA injection does not disturb chromosome replication and separation.5,Cdc42 activity colocalizing with g-actin signal during the cytokinesis with a ring like shape.