Synaptotagmin1Is Required For Spindle Stability And Metaphase-Anaphase Transition In Mouse Oocytes

Synaptotagmin1, a calcium sensor for exocytosis, forms the7S complex or so called SNARE protein complex, together with SNAP-25, syntaxin and synaptobrevin, to mediate docking and fusion of synaptic vesicles to the plasma membrane of the nerve terminal. Here we identified the unique localization, expression and function of Syt1during mouse oocyte meiotic maturation by using confocal microscopy. Western blotting, Morpholino-based knockdown and time-lapse live cell imaging. We showed that Syt1expression was gradually increased during oocyte maturation. Syt1was localized at the oocyte cortex from GV to MⅡ stages and at the spindle poles in MⅠ and MⅡ phases, with one third of a signal-free zone at the oocyte cortex where the chromosomes are located, which is similar to the distribution pattern of CGs from the Pro-MⅠ to MⅡ stages. Knockdown of Sytl resulted in Pro-MⅠ/MⅠ arrest and PB1extrusion decrease, with severely disrupted spindles and misaligned chromosomes. Knockdown of Syt1also caused abnormal localization of γ-tubulin, which became redistributed into the cytoplasm. Chromosome spreading showed failure of homologous chromosome segregation. The spindle assembly checkpoint protein Bub3was detected at the kinetochores even after10h of oocyte culture. Live cell imaging analysis revealed that knockdown of Syt1resulted in abnormal spindles with various morphologies and chromosomes arrested at the Pro-MⅠ/MⅠ stage. Defective spindles failed to support chromosome alignment along microtubules, which led to repetitive unsuccessful metaphase-anaphase transition and failure of PB1extrusion after extended culture. Taken together, we suggest that Sytl may act as a MTOC-associated protein to play important roles in mouse oocyte spindle organization/stability and that it is indispensable for the metaphase-anaphase transition to promote mouse oocyte meiotic maturation.[Purpose]To identify the role of Sytl regulating meiotic spindle assembly.[Method]Oocyte collection and cultureOocytes were collected in M2medium supplemented with or without2.5mM milrinone. Milrinone was used to maintain oocytes at the germinal vesicle (GV) stage. Then oocytes were washed thoroughly and cultured in M16under liquid paraffin oil at37℃in an atmosphere of5%CO2in air. At different times of culture, oocytes were collected for immunostaining, drug treatment, microinjection and Western blotting.Taxol treatment of oocytesFor taxol treatment,5mM taxol in DMSO stock was diluted in M16medium to achieve a final concentration of10μM, and oocytes were incubated for45min at37℃in an atmosphere of5%CO2in air. After treatment, oocytes were washed thoroughly and used for immunofluorescence experiments.Immunofluorescence and confocal microscopyOocytes were fixed with4%paraformaldehyde in PBS (pH7.4) for at least 30min. After being permeabilized with0.5%Triton X-100at room temperature for20min, oocytes were blocked in1%BSA supplemented PBS for1h and then incubated with rabbit anti-Syt1antibody (Abcam;1:200), rabbit anti-a-tubulin antibody (Sigma;1:200), mouse polyclonal anti-a-tubulin-FITC (Sigma:1:100), mouse anti-y-tubulin antibody (Sigma;1:100), and rabbit anti-Bub3antibody (Santa Cruz Biotechnology;1:50) respectively, overnight at4℃. After three washes with PBS containing0.1%Tween20and0.01%Triton X-100for5min each, the oocytes were labeled with FITC-conjugated goat-anti-rabbit IgG (Zhong Shan Jin Qiao;1:100), TRITC conjugated goat-anti-rabbit IgG (Zhong Shan Jin Qiao;1:100), TRITC conjugated goat-anti-mouse IgG (Zhong Shan Jin Qiao;1:100), Cy5-anti-rabbit IgG (Jackson;1:100) for1h at room temperature and then washed three times with PBS containing0.1%Tween-20and0.01%Triton X-100. Hoechst33342or PI (Propidium Iodide) was used to detect DNA. Finally, the oocytes were mounted on glass slides and examined with a confocal laser scanning microscope (Zeiss LSM510META or Zeiss LSM780META. Germany).Immunoblotting analysisA total of200or250mouse oocytes were collected in SDS sample buffer and heated for5min at100℃. The proteins were separated by SDS-PAGE and then electrically transferred to polyvinylidene fluoride membranes. Following transfer, the membranes were blocked in TBST (TBS containing0.1%Tween20) containing5%skimmed milk for2h, followed by incubation overnight at4℃with1:500rabbit anti-Sytl antibody and1:1000mouse monoclonal anti-β-actin antibody. After washing3times in TBST,10min each, the membranes were incubated for1h at37℃with:1000horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG, respectively. Finally, the membranes were processed using the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ).Microinjection of Syt1or control morpholino antisense oligos MOMicroinjections were performed using a Nikon Diaphot ECLIPSE TE300(Nikon UK Ltd., Kingston upon Thames, Surrey, UK) and completed within30minutes. A volume of2mM Sytl MO (GENE TOOLS, LLC,5’-GACTGGCACTCACCATTTTTGGTTC-3’) was microinjected into the cytoplasm to delete Sytl. The same amount of negative control MO(GENE TOOLS, LLC,5’-CCTCTTACCTCAgTTACAATTT ATA-3’) was also injected as control. After microinjection, the oocytes were arrested at the GV stage for24h in M2medium containing2.5mM milrinone before being transferred to the M16medium to be cultured for8hour and10hour. The collected oocytes were fixed to the next confocal images. Each experiment consisted of three separate replicates and approximately300oocytes were injected in each group.Data analysisFor each treatment, at least three replicates were performed. Statistical analyses were conducted by analysis of variance. Differences between treated groups were analyzed by Chi-Square using SPSS13.0software (SPSS Inc, Chicago, IL). Data are expressed as mean±SEM and P<0.05is considered significant.[Results]Expression and subcellular localization of Sytl during mouse oocyte meiotic maturationWe cultured oocytes for Oh,8h, and12h, when most oocytes had reached the GV, MI, and MⅡ stages, respectively, and collected oocytes to examine the expression of Syt1during meiotic maturation. The immunoblotting results showed that Sytl was expressed at all stages and gradually increased from GV to the MⅡ stages. To investigate the subcellular localization of Sytl during meiotic maturation, mouse oocytes were processed for immunofluorescent staining at different stages of maturation. Sytl was mainly accumulated at the oocyte cortex and partly distributed in the cytoplasm from GV to MⅡ stages with one third of the signal free zone at the oocyte cortex where the chromosomes are located, which is similar with the distribution pattern of CGs from the Pro-MI to MⅡ stages. Strikingly, there was accumulated Sytl immunostaining at the spindle poles in the MI and MⅡ phases. The apparent association of Sytl with spindle poles prompted us to ask whether Sytl was co-localized with other known MOTC proteins, such as y-tubulin. Sytl showed the same localization pattern as that of y-tubulin and both signals overlapped at the MI and MⅡ stages. Co-localization of Sytl with y-tubulin confirmed that Sytl is present at the two spindle poles.Localization of Sytl in mouse oocytes treated with taxolAfter determining that Sytl was localized at the spindle poles in the MI and MⅡ stages, we further investigated the correlation between Sytl and micro tubule dynamics. We used taxol. a microtubule-stabilizing agent, to treat oocytes. The microtubule fibers became excessively polymerized in taxol-treated oocytes, and numerous asters became assembled in the cytoplasm. In our experiments, Syt1was detected at the abnormal spindle poles as well as in the cytoplasmic asters.Knockdown of Sytl causes abnormal meiotic spindles and misaligned chromosomesTo further determine the roles of Sytl in mouse oocyte meiosis, we knocked down Sytl by its specific MO injection. Western blot analysis showed that the expression level of Sytl became significantly reduced, which confirmed the depletion efficiency of the designed Sytl specific MO sequence in mouse oocytes. After microinjection, the oocytes were arrested at the GV stage for24h in M2medium containing2.5mM milrinone to prevent meiosis resumption. The oocytes were then transferred to M16medium for culture. Oocytes were collected at8h or12h of culture. In the Syt1-MO injection group, oocytes exhibited various morphologically defective spindles and misaligned chromosomes; the major spindle defect was abnormal spindle poles (58%, n=130), including spindles with no poles, one pole and multiple poles. Others were elongated or displayed incomplete spindles with astral microtubules. Lagging chromosomes and absence of chromosome alignment were clearly observed. The rate of abnormal spindles in the Syt1-MO injection group (79.23%, n=130) was significantly higher than that in the control-MO injection group (28.47%, n=144)(χ2=70.594, p=0.000). Moreover, the rate of misaligned chromosomes in Syt1MO (63.90%, n=130) and control groups (28.47%, n=144) differed significantly (χ2=34.507,p=0.000).Dissociation of γ-tubulin from spindle poles in Syt1-knockdown oocytesThe above results indicated that Syt1might be involved in spindle organization and spindle pole congression. As is well known, the MTOC-specific protein, γ-tubulin, is important for microtubule nucleation and organization leading to spindle formation during mouse oocyte meiosis. After MO injection, we found that Syt1depletion affected the localization of γ-tubulin. γ-tubulin was localized to the spindle poles in the control-MO injected group (n=89) at the MⅠ stage; it failed to congress at the spindle poles, while it became distributed at the spindle fibers or dispersed into the cytoplasm in the Syt1-MO injected group(n=92).[Conclusion]Syt1is required for microtubule organization and it may act as a centrosome-associated protein or interact with other centrosome-associated proteins to regulate spindle assembly during mouse oocyte meiotic maturation.[Purpose] To identify Sytl functions on Pro-MI/MI arrest and the first polar body extrusion on mouse oocyte meiosis[Methods]Oocyte collection and culture, Immunofluorescence and confocal microscopy subscribed in the first sectionChromosome spreadingFor chromosome spreading, oocytes were left for10minutes in1%sodium citrate at room temperature and then fixed with fresh methanol:glacial acetic acid (3:1),10mg/ml PI was used for chromosome staining. Cells were examined with a Confocal Laser Scanning Microscope.Micro injection of Sytl or control morpholino antisense oligos MO and co-injection with β5-tubulin-GFP mRNA, H2B-RFP mRNA and Sytl-MO or control-MOMicroinjections were performed using a Nikon Diaphot ECLIPSE TE300(Nikon UK Ltd., Kingston upon Thames, Surrey, UK) and completed within30minutes. MO sequence (Gene Tools) was designed against the ATG and exonl splice sites of Syt1and mismatched control. Syt1-MO (GENE TOOLS, LLC,5’-GACTGGCACTCACCATTTTTGGTTC-3’) was microinjected into the cytoplasm to deplete Sytl. The same amount of negative control-MO (GENE TOOLS, LLC,5’-CCTCTTACCTCAgTTACAATTT ATA-3’) was also injected as control. To examine how Sytl knockdown disrupted the meiotic division process, we co-injected β5-tubulin-GFP mRNA and H2B-RFP mRNA, synthesized as previously reported1.2with2mM Sytl-MO or control-MO (the final concentration was up to1mM) into GV oocytes as described above. Each oocyte received approximately10pl Syt1-MO, control-MO,β5-tubulin-GFP mRNA and H2B-RFP mRNA with Sytl-MO or β5-tubulin-GFP mRNA and H2B-RFP mRNA with control-MO. After microinjection, the oocytes were maintained for24h in2.5mM milrinone M2medium, then thoroughly washed and transferred into fresh M16medium to culture. After4-5h culture, oocytes were observed. Each experiment consisted of three separate replicates and approximately200-300oocytes were injected in each group.Imaging experimentsMicrotubule and chromosome dynamics were recorded on a Perkin Elmer precisely Ultra VIEW VOX Confocal Imaging System. We used a narrow band pass EGFP and RFP filter set and a30%cut neutral density filter from Chroma. Exposure time was set ranging between300and800ms depending on tubulin-GFP and DNA-RFP fluorescence levels. The acquisition of digital time-lapse images was controlled by IP Lab (Scanalytics) or AQM6(Andor/Kinetic-imaging) software packages. Confocal images of spindles and chromosomes in live oocytes were acquired with a10x oil objective on a spinning disk confocal microscope (Perkin Elmer).Data analysisFor each treatment, at least three replicates were performed. Statistical analyses were conducted by analysis of variance. Differences between treated groups were analyzed by Chi-Square using SPSS13.0software (SPSS Inc, Chicago, IL). Data are expressed as mean±SEM and P<0.05is considered significant.[Results]Sytl knockdown results in Pro-MI/MI arrest and decreased PB1extrusionAfter Sytl or control MO injection oocytes were maintained in2.5mM milrinone M2medium for24h; the oocytes were then continuously cultured in fresh M16medium for10h or12h. We found that the majority of oocytes were arrested at the Pro-MI/MI stage in the Sytl-MO group while the oocytes in the control-MO group entered the anaphase I stage. The rate of Pro-MI/MI arrest in Sytl-MO group (91.80%, n=49) was remarkably higher than that in the control-MO group (31.48%, n=54)(χ2=39.055, p=0.000). The PB1extrusion rate (45.33%, n=154) was also significantly lower in the Sytl-MO group than in the control-MO group (60.76%, n=161, χ2=7.515.p=0.007).Sytl knockdown prevents chromosome segregation and activated the spindle assembly checkpointHomologous chromosomes failed to segregate and the majority of oocytes arrested at the Pro-MI/MI stage, with aberrant spindles in the Sytl-MO injected oocytes. To further confirm that Syt1knockdown oocytes were arrested in the Pro-MI/MI stage, we employed chromosome spreading experiments to confirm the failure in chromosome segregation. Oocytes in both the Sytl-MO and control-MO groups were cultured for10h. Our results showed that chromosomes were still in the bivalent state in the Sytl deleted oocytes (10/10); in contrast, univalent chromosomes were observed in the control oocytes (9/10), indicating the completion of anaphase. The Sytl knockdown oocytes failed to complete correct homologous chromosome segregation, which prompted us to explore the activity of SAC. We analyzed the localization of Bub3in oocytes, which is one principal component of SAC. Specific signals for Bub3were detected in Syt1knockdown oocytes which were arrested at the Pro-MI/MI stage. The control oocytes showed no signals for Bub3. These data indicate that the spindle assembly checkpoint was activated in Sytl knockdown oocytes.Sytl knockdown disturbs the metaphase-anaphase transition of mouse oocytesTo confirm more precisely the function of Sytl and meiotic cell cycle defects caused by Sytl-MO knockdown, we employed live cell imaging to observe the course of meiotic maturation by time-lapse microscopy. Oocytes were co-injected with β5-tubulin-GFP mRNA, H2B-RFP mRNA and Syt1-MO or control-MO. β5-tubulin-GFP mRNA was used to detect a-tubulin and H2B-RFP mRNA to detect DNA. In the control-MO group, a clear bipolar spindle was visible that slowly migrated to oocyte cortex; then, a clear anaphase/telophase stage was observed, followed by rapid polar body extrusion. In addition, we observed forty control oocytes with normal spindle morphology and well-aligned chromosomes in almost all of them, corresponding to the distinct meiotic stages, and most of them could complete first polar body extrusion. However, in the Sytl-MO injection group, we found various abnormal spindles. Chromosomes failed to separate and remained at the Pro-MI/MI stage until about13.5h. Then, impaired, unsuccessful and repetitive tubulin and DNA separation was observed. and part of the chromosome organizations detached from spindle microtubules during the impaired separation process. In addition, we could not observe first polar body extrusion. As shown in the supplementary, there were thirty Sytl-MO oocytes, and similar spindle and chromosome phenomena as described above were observed in all of them. Only two oocytes showed first polar body. Several Sytl-MO oocytes did not survive during the maturation process.[Conclusion]Sytl play an essential role in metaphase-anaphase transition during mouse oocyte meiotic maturation process.