GM130, A Cis-Golgi Protein, Regulates Meiotic Spindle Assembly And Asymmetric Division In Mouse Oocyte

GM130 is identified as a part of cis-Golgi matrix. Together with its interacting partner proteins, including P115, giantin, GRASP65, and Rab GTPases, GM130 is involved in the regulation of ER-to-Golgi transport, glycosylation and maintenance of Golgi structure. Recently, increasing evidence has indicated that GM130 has unexpected roles in the control of cell polarization, migration and division. Gm130 plays key roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that in GV oocytes, GM130 was distributed throughout the cytoplasm and seemingly concentrated around the germinal vesicle. After GVBD, an accumulation of dotted structures of GM130 was shown in the central part of the oocyte. A proportion of GM130 began to migrate to the spindle poles before the condensed chromosomes were aligned at the equator of the spindle. By MI, GM130 was mainly presented at the spindle poles. As oocytes progressed to anaphase/telophase (AT), GM130 was diffusely distributed and associated with the midbody between the PB and the oocyte. At MⅡstage, GM130 again became localized to the spindle poles. GM130 was localized to the spindle poles at both metaphaseⅠand metaphaseⅡstages and associated with the midbody at telophaseⅠstage. The association of GM130 with spindle poles was further confirmed by its colocalization with the centrosome-associated proteins, MEK1/2. The immunoblotting results showed that GM130 was expressed at all stages By nocodazole treatment, we clarified that GM130 localization was consistently dependent on spindle assembly. Then we investigated the possible function of GM130 by specific morpholino microinjection. This treatment caused abnormal spindle formation and the major defect was elongated spindle. In GM130 MO-injected group, the PB extrusion rate was significantly lower than that in control MO-injected group. And, most of extruded polar bodies were extremely big. Our results showed that knockdown of GM130 impaired the localization of MTOCs proteins y-tubulin and Plkl. But, the formation of F-actin caps was not sensitive to GM130. To determine more precisely the nature of the GM130 depletion-caused defect, we carried out live cell imaging experiments in which we coinjectedβ5-tubulin-GFP mRNA with GM130 MO into GV oocytes and filmed the course of meiotic maturation by time-lapse microscopy. Using live cell imaging we observed that depletion of GM130 affected spindle migration and resulted in elongated spindle. Spindles distal pole moved closer to the cortex, followed by a cleavage furrow developing and a large polar body extrusion. While, in the oocytes without PB, the spindle also elongated at a similar time, but any pole could not attach to the cortex and the cleavage furrow could not be observed. We further found that depletion of GM130 blocked p-MEK1/2 accumulation at the spindle poles. And, it was shown that GM130 detached from the spindle poles in oocytes treated with MEK specific inhibitor U0126. Taken together, our results suggested that GM130 might act as a centrosome-associated protein to regulate spindle assembly and might cooperate with the MAPK pathway to play roles in spindle organization, migration and asymmetric division during mouse oocyte maturation. This study divided into two parts: First GM130, a cis-Golgi protein, regulates meiotic spindle assembly.[Purpose]To identify the role of GM130 regulating meiotic spindle assembly.[Method]Oocyte collection and cultureThe oocytes were collected in M2 medium supplemented with or without 2.5 mM milrinone. Milrinone was used to keep oocytes at germinal vesicle (GV) stage. Then oocytes were washed thoroughly and cultured in M2 under liquid paraffin oil at 37℃in an atmosphere of 5% CO2 in air. At different times of culture, oocytes were collected for immunostaining, drug treatment, microinjection and Western blot.Immunofluorescence and Confocal microscopyOocytes were fixed with 4% paraformaldehyde/PBS (pH 7.4) for at least 30min. After being permeabilized with 0.5% Triton X-100 at room temperature for 20min, oocytes were blocked in 1% BSA supplemented PBS for 1h and then incubated with mouse anti-GM130 antibody (BD Transduction Labs; 1:100), Rabbit polyclonal anti-phospho-MEK1/2 (ser217/221) antibody (Cell Signaling Technology, Beverly, MA), anti-α-tubulin antibody (Sigma; 1:200), Mouse polyclonal anti-a-tubulin-FITC (Sigma; 1:100), mouse anti-y-tubulin antibody(Sigma;1:100) and mouse monoclonal anti-Plkl antibody(Sigma; 1:100) respectively, overnight at 4℃. After three washes with PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min 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), Cy5-anti-human IgG (Jackson; 1:100) or FITC-anti-mouse IgG (Zhong Shan Jin Qiao; 1:100) for 1 h at room temperature and then washed three times with PBS containing 0.1% Tween-20 and 0.01% Triton X-100. The oocytes were co-stained with Hoechst 33342 or PI. Finally, the oocytes were mounted on glass slides and examined with a confocal laser scanning microscope (Zeiss LSM 510 META, Germany).Immunoblotting analysisA total of 200 mouse oocytes were collected in SDS sample buffer and heated for 4min 30s at 100℃. The proteins were separated by SDS-PAGE and then electrically transferred to polyvinylidene fluoride membranes. Following transfer, the membranes were blocked in TBST (TBS containing 0.1% Tween 20) containing 5% skimmed milk for 2h, followed by incubation overnight at 4℃with 1:1000 Mouse anti-GM130 antibody and 1:1000 mouse monoclonal anti-β-actin antibody (for GM130, incubation buffer was 5% BSA in TBST). After washing 3 times in TBST, 10min each, the membranes were incubated for 1h at 37℃with 1:1000 horseradish 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).Nocodazole treatment of oocytesFor nocodazole treatment, 10mg/ml nocodazole in DMSO stock was diluted in M2 medium to give a final concentration of 20μg/ml for oocytes for 10min. After treatment, oocytes were washed thoroughly and used for immunofluorescence. Control oocytes were treated with the same concentration of DMSO in the medium before examination.Microinjection of GM130 or control morpholino antisense oligos MOMicroinjections were performed using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) and completed within 30 minutes. A volume of 2 mM GM130 MO (GENE TOOLS, LLC, 5'-GGGCCACATCACCACGATCCCGGCA-3') was microinjected into the cytoplasm to delete GM130. 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 for 21 h in M2 medium containing 2.5 mM milrinone before being transferred to the M2 medium for culture. Each experiment consisted of three separate replicates and approximately 300 oocytes were injected in each group.[Results]Expression and subcellular localization of GM130 during mouse oocyte meiotic maturation.We cultured oocytes for 0h,2h,8h,12h, the time points when most oocytes reached the GV, GVBD, MI, and MⅡstages respectively, to examine the expression of GM130 during mouse oocyte meiotic maturation. The immunoblotting results showed that GM130 was expressed at all stages. To investigate the subcellular localization of GM130 during meiotic maturation, mouse oocytes were processed for immunofluorescent staining at different stages of maturation. As shown in Figure 1B, in GV oocytes, GM130 was distributed throughout the cytoplasm and seeminglyconcentrated around the germinal vesicle. After GVBD, an accumulation of dotted structures of GM130 was shown in the central part of the oocyte. A proportion of GM130 began to migrate to the spindle poles before the condensed chromosomes were aligned at the equator of the spindle. By MI, GM130 was mainly presented at the spindle poles. As oocytes progressed to anaphase/telophase (AT), GM130 was diffusely distributed and associated with the midbody between the PB and the oocyte. At MⅡstage, GM130 again became localized to the spindle poles in a crescent shape. The apparent association of GM130 with spindle poles prompted us to ask whether GM130 was colocalized with other known centrosome associated proteins, such as p-MEK1/222. As shown in Figure 1C, GM130 followed the same localization pattern as that of p-MEK1/2 and their signals overlapped at MⅠand MⅡstages. Co-localization of GM130 with p-MEK1/2 confirmed that the localization of GM130 was at the two spindle poles.Localization of GM130 in mouse oocytes treated with nocodazole.To clarify the correlation between GM130 and microtubule dynamics, spindle-perturbing drug nocodazole was employed. At MⅠstage, after treated with 20μg/ml for 10min, a microtubule-depolymerizing drug, microtubules were completely disassembled and no intact spindles could be observed. At MⅠstage, GM130 was mainly presented at the spindle poles as described above. But, in this case, the localization of GM130 was altered and dispersed into the cytoplasm. After the treated oocytes were thoroughly washed and cultured to allow microtubule reassembly, the GM130 again presented at the poles of the reformed spindle apparatus.Depletion of GM130 caused abnormal spindle at MI stage.To further dissect the roles of GM130 in mouse oocyte meiosis, we knocked down GM130 by its specific MO injection. Western blot and volume analysis showed that the expression level of GM130 was significantly reduced. After microinjection, the oocytes were arrested at the GV stage for 21 h in M2 medium containing 2.5 mM milrinone to prevent meiosis resumption. Then, the oocytes were transferred to the M2 medium for culture. And, after 8h of culture the oocytes were collected. In the GM130 MO injection group, the oocytes exhibited various morphologically defective spindles, The major defect was elongated spindle (55%, n=120), others including spindles with no poles, one pole, multi poles and unformed spindles with astral microtubules as well as many cytoplasmic asters.(Fig.3C). As shown in Figure 3D, the rate of abnormal spindles (80.5%, n=154) in the GM130 MO injection group was significantly higher than that in the control MO injection (p<0.05).Dissociation ofγ-tubulin and Plk1 from spindle poles in GM130-knockdown oocytes. The results above showed that GM130 might be involved in the spindle organization and pole tethering. We have recently shown that centrosome or centrosome- associated proteins, such as y-tubulin and Plkl, were important regulators in microtubule organization and spindle formation during mouse oocyte meiosis. Employing MO injection, we found that GM130 depletion affected the localization of these proteins. Plkl and y-tubulin were localized to the spindle poles in control MO-injected oocytes at MI stage; while Plkl and y-tubulin detached from spindle poles and were detected on the spindle fibers or dispersed to the cytoplasm in GM130 MO-injected oocytes.[Conclusion]GM130 is 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.Second GM130 regulates MI spindle migration and the asymmetric division during mouse oocyte meiotic maturation.[Purpose]To identify the role of GM130 regulating MI spindle migration and the meiotic asymmetric division[Method]Coinjection ofβ5-tubulin-GFP mRNA with GM130 or control morpholino antisense oligos MOMicroinjections were performed using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) and completed within 30 minutes. To examine how GM130 depletion perturbed the meiotic division, we coinjectedβ5-tubulin-GFP mRNA, synthesized as previously reported with 4mM GM130 MO or control MO into GV oocytes as described above. Each oocyte received approximately 10 p1β5-tubulin-GFP mRNA with GM130 MO orβ5-tubulin-GFP mRNA with control MO. After microinjection, the oocytes were arrested at the GV stage for 21 h in M2 medium containing 2.5 mM milrinone before being transferred to the M2 medium for culture. Each experiment consisted of three separate replicates and approximately 300 oocytes were injected in each group.Imaging ExperimentsMicrotubule dynamics were filmed on Perkin Elmer precisely Ultra VIEW VOX confocal Imaging System. We used a narrow band pass EGFP filter set and a 30% cut neutral density filter from Chroma. Exposure time was set ranging between 300 and 800 ms depending on tubulin-GFP fluorescence level. The acquisition of digital time-lapse image was controlled by IP Lab (Scanalytics) or AQM6 (Andor/Kinetic-imaging) software packages. The measurement of S and D was done using Image J. Confocal images of spindles in live oocytes were acquired with a 20 x oil objective on a spinning disk confocal microscope (Perkin Elmer).U0126 treatment of oocytesFor U0126 treatment, 10mM U0126 in DMSO stock was diluted in M2 medium to give a final concentration of 50μM. The GV oocytes were incubated in M2 medium containing 50μM U0126 to MI stage. Control oocytes were treated with the same concentration of DMSO in the medium before examination.[Results]GM130 depletion decreased PB extrusion and caused large PB formationAfter GM130 or control MO injection, oocytes were cultured in fresh M2 medium for 16h. We noted that a large proportion of oocytes extruded an abnormally large PB in the GM130 MO injected group, and in some cases the enlarged PB contained a spindle with normal morphology. In many cases, the oocyes arrested at MI stage. In GM130 MO-injected group, the PB extrusion rate (49.4%, n=154) was significantly lower than that in control MO-injected group(79.5%, n=161, p<0.05). And, most of extruded polar bodies (60.5%, n=76) were extremely big. The rate of oocytes with large PB in GM130 MO injected group was, significantly higher than that in control MO-injected group (5.5%, n=128).The phenomenon that GM130 depletion affected the asymmetric division of mouse oocytes attracted our attention. The key to asymmetric division in mouse oocytes is the spindle migration which is dependent on the actin cytoskeleton.To test whether the formation of the F-actin cap was sensitive to GM130 deletion, we analyzed oocytes at 7h and 14h after GVBD. In control MO-injected oocytes, an F-actin cap was prominent accompanied by the juxtaposing MⅠchromosomes. In contrast, GM130 MO-injected oocytes exhibited centrally localized MⅠchromosomes and lacked any cortical F-actin cap. Following overnight incubation (14h after GVBD), the MⅡchromosomes always lied juxtaposing these F-actin caps in control MO-injected oocytes, while GM130 MO-injected oocytes with large PB exhibited these F-actin caps and all caps (14/14) were located at the junction of the ooctye and large PB. In GM130 depleted oocytes cultured for 14h after GVBD without PB extrusion, we found that 40% of the oocytes (7/16) exhibited no F-actin cap while 60% (9/16) did. These phenotypes correlated, respectively, with MⅡchromosomes that were localized centrally or near the cortex. Therefore, formation of these F-actin caps was not sensitive to GM130, but was controlled by spindle (or more accurately chromosomes) positioning.GM130 depletion perturbed the asymmetric division of mouse oocytesTo determine more precisely the nature of the GM130 depletion-caused defect, we carried out live cell imaging experiments in which we coinjectedβ5-tubulin-GFP mRNA with GM130 MO or control MO into GV oocytes and filmed the course of meiotic maturation by time-lapse microscopy. In control oocytes expressing β5-tubulin-GFP, the meiotic spindle was visible about 4 hr after GVBD and migrated toward the cortex followed by a rapid extrusion of the first PB at about 9hr culture after GVBD. In oocytes co-injected withβ5-tubulin-GFP mRNA and GM130 MO, we found various morphologically defective spindles. The spindle was also visible about 4 hr after GVBD, most of them remained in the central part of the oocytes at 7hr after GVBD. We exhibited the film of typical oocytes with large PB, and oocytes without PB extrusion. The spindle began to elongate at about 7hr after GVBD, and its distal pole moved closer to the cortex, followed by a cleavage furrow developing and a large polar body extrusion. While, in the oocytes without PB, the spindle also elongated at a similar time, but any pole could not attach to the cortex and the cleavage furrow could not be observed.To quantitatively compare the behavior of the spindle in GM130 MO and control MO-injected oocytes, we measured the spindle length, and the distance of the spindle pole to the further cortex (D) over time. In control oocytes, the two poles moved together as a result of spindle migration, and the length of the spindle remained constant, even at anaphase. Both the control MO and GM130 MO-injected oocytes had a similar spindle length at 4hr post-GVBD. At 7hr after GVBD, however, spindle length was significantly larger in GM130 MO-injected oocytes than that in control MO-injected oocytes. When the spindle elongated greatly (about 1.6 time longer) with the pole closer to the cortex, the oocyte extruded a large PB. If the spindle only elongated slightly and could not arrive at the cortex, oocytes arrested at MI and could not extrude PB. The intriguing phenomenon that the spindle in GM130 MO-injected oocytes could not migrate alternatively elongate at AT stage attracted our attention. We measured the distance of the further spindle pole to the cortex. In contrast to the control oocytes, in which pole-to-cortex distance increased steadily during AT stage, while in GM130 MO-injected oocytes this distance remained constant during meiotic progression.Detachment of GM130 and p-MEK1/2 from the spindle poles in U0126 treated oocytes and GM130 depleted oocytes, respectivelyIn our previous study, we have revealed that p-MEK1/2 plays critical roles in regulating the microtubule organization and polar body extrusion, and deletion of p-MEK1/2 results in lengthened MI spindles and large polar body formation23. In this work, the similar phenotypes in GM130 depletion oocytes prompted us to speculate the relationship between GM130 and p-MEK1/2. As shown in Figure 7, p-MEK1/2 and GM130 were localized to the two spindle poles in the control oocytes at MI stage. P-MEK1/2 was no longer accumulated at spindle poles, but detected on the spindle fibers in the GM130 MO injected oocytes. Treatment of U0126, the specific MEK inhibitor, caused disorganized spindles and GM130 dispersion into the cytoplasm in mouse oocytes.[Conclusion]GM130 is directly required for spindle relocation and may participate in the MAPK signaling pathway to regulate the spindle migration and meiotic asymmetric division.