Rab6a is a novel regulator of meiotic apparatus and maturational progression in mouse oocytes

Rab6a is a novel regulator of meiotic apparatus and maturational progression in mouse oocytes


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ABSTRACT Rab family GTPases have been well known to regulate intracellular vesicle transport, however their function in mammalian oocytes has not been addressed. In this study, we report


that when Rab6a is specifically knockdown, mouse oocytes are unable to progress normally through meiosis, arresting at metaphase I. Moreover, in these oocytes, the defects of chromosome


alignment and spindle organization are readily observed during maturation and resultantly increasing the aneuploidy incidence. We further reveal that kinetochore-microtubule attachments are


severely compromised in Rab6a-depleted oocytes, which may in part mediate the meiotic phenotypes described above. In addition, when Rab6a function is altered, BubR1 levels on the


kinetochores are markedly increased in metaphase oocytes, indicating the activation of spindle assembly checkpoint. In sum, we identify Rab6a as an important player in modulating oocyte


meiosis, specifically the chromosome/spindle organization and metaphase-anaphase transition. SIMILAR CONTENT BEING VIEWED BY OTHERS RAN-GTP ASSEMBLES A SPECIALIZED SPINDLE STRUCTURE FOR


ACCURATE CHROMOSOME SEGREGATION IN MEDAKA EARLY EMBRYOS Article Open access 01 February 2024 PAK2 IS ESSENTIAL FOR CHROMOSOME ALIGNMENT IN METAPHASE I OOCYTES Article Open access 22 February


2023 CENP-V IS REQUIRED FOR PROPER CHROMOSOME SEGREGATION THROUGH INTERACTION WITH SPINDLE MICROTUBULES IN MOUSE OOCYTES Article Open access 11 November 2021 INTRODUCTION To ensuring


successful fertilization and early embryonic development, the oocyte undergoes specialized cell divisions named meiosis I and II. Meiosis I begins with germinal vesicle breakdown (GVBD)


after stimulation by pituitary luteinizing hormone (LH) and ends with first polar body (PB1) extrusion1,2. During meiosis, microtubules first organize into a barrel-shaped bipolar spindle,


with all chromosomes aligned at the metaphase plate, following which recombined homologous chromosomes are segregated at anaphase/telophase3. Oocytes are finally progress uninterruptedly to


meiosis II and become arrested for a second time waiting for fertilization4. Notably, it has been suggested that chromosome segregation is error prone during mammalian oocyte meiosis5,6. Any


mistakes in this process can result in the generation of aneuploid embryos, contributing to pregnancy loss or severe birth defects7. Similar to mitosis, spindle assembly checkpoint (SAC)


mechanism in oocytes prevents the premature metaphase-anaphase transition until all chromosomes successfully attach to the bipolar spindle with proper tension8,9. The core components of SAC


are the Mad and Bub protein families, which inhibit the activation of the anaphase promoting complex (APC) and therefore the degradation of cyclin B and anaphase onset10. Rab (Ras-related


proteins in brain) GTPases are evolutionarily conserved, essential components of vesicle trafficking pathways. Over 70 human Rab and Rab-like members of the Ras superfamily have been


identified11. For example, Rab1, which is located at endoplasmic reticulum (ER) exit sites and the pre-Golgi intermediate compartment, mediates ER–Golgi trafficking12. Rab5, localized to


early endosomes and phagosomes, has been well recognized to involve in membrane tethering and docking13,14. Most recent findings have also suggested that Rab5 GTPase participates in


chromosome congression and spindle assembly in both mitotic cells and meiotic oocytes15,16. Two isoforms of Rab6 GTPase, Rab6a′ and Rab6a, differ in only three amino acids and are expressed


in mammalian cells17,18. Rab6 has been shown to regulate a retrograde transport route connecting early endosomes to ER18,19. Rab6a′ functions in a pathway involved in mitotic arrest through


the interaction with dynein/dynactin complex at the kinetochores20. During _Drosophila_ oogenesis, Rab6 is required for the polarization of the oocyte microtubule cytoskeleton and for the


posterior localization of oskar mRNA21. However, so far, the function of Rab6 during mammalian oocyte meiosis remains unknown. Here, by employing siRNA knockdown analysis, we discovered the


involvement of Rab6a in meiosis of mouse oocyte, particularly in controlling meiotic progression and meiotic structures and report our findings below. RESULTS RAB6A KNOCKDOWN ADVERSELY


AFFECTS MATURATIONAL PROGRESSION OF MOUSE OOCYTES To explore the function of Rab6a, fully-grown oocytes were injected with specifically-designed siRNA (Rab6a-siRNA); a negative control siRNA


was included as control. After microinjection, the oocytes were arrested at GV stage for 20 hours with milrinone to promote mRNA degradation. Immunoblotting confirmed that the significant


reduction of Rab6a proteins in oocytes (Fig. 1A,B). After 3 hours culture, both control and Rab6a-knockdown groups resumed meiosis normally, indicated by the similar GVBD rate (Fig. 1C).


However, Pb1 extrusion was decreased in Rab6a-siRNA oocytes compared to controls (35.9 ± 7.6% vs. 87.4 ± 4.3% control, _p_ < 0.05; Fig. 1D), indicating that Rab6a-depleted oocytes failed


to complete meiosis I and form the first polar body (Fig. 1E, blue arrowheads). In some oocytes where a meiotic division appears to have been completed, the symmetrical division was


frequently observed (Fig. 1E, red asters). To further define the developmental stage of those Rab6a-siRNA oocytes without polar body, we performed nuclear staining and quantitative analysis.


As shown in Fig. 1F, we found that more than 35% of oocytes injected with Rab6a-siRNA were blocked in meiosis I, which was dramatically increased compared to controls. Together, these


results suggest that alteration of Rab6a function adversely impacts oocyte maturation and meiotic divisions. RAB6A FUNCTIONS IN CHROMOSOME ALIGNMENT AND SPINDLE ORGANIZATION The above data


prompted us to ask whether depletion of Rab6a affects the meiotic apparatus in oocytes. For this purpose, Rab6a-siRNA and control oocytes were immunolabeled with anti-tubulin antibody to


visualize the spindle and co-stained with propidium iodide (PI) for chromosomes. Confocal microscopy revealed that most control oocytes at metaphase presented with a typical barrel-shape


spindle and well-aligned chromosomes on the metaphase plate (Fig. 2Aa). In striking contrast, we found a high percentage of chromosome congression failure (20.7 ± 3.6% vs. 8.9 ± 2.4%


control, _p_ < 0.05; Fig. 2C) and spindle defects (13.2 ± 2.1% vs. 6.1 ± 2.0% control, _p_ < 0.05; Fig. 2D) in Rab6a-depleted oocytes, showing diverse disorganized spindles (Fig.


2Ba,b, arrows) with several scattered chromosomes (Fig. 2Ba,b, arrowheads). As shown in Fig. 2Ab,c, during normal oocyte meiosis I, accompanying with chromosomes moving evenly away from the


equator toward opposite poles, homologous chromosomes are accurately segregated at anaphase and telophase stages. By contrast, in a small number of anaphase/telophase oocytes depleted of


Rab6a, the aberrant chromosome separation was readily detected (Fig. 2Bc, arrowheads), a lagging chromosome phenotype that was about 4 times more prevalent than in control oocytes. Taken


together, these results indicate that Rab6a is required for chromosome movement and spindle assembly in meiotic oocytes. INCREASED INCIDENCE OF ANEUPLOIDY IN RAB6A-DEPLETED OOCYTES Since


Rab6a knockdown led to the chromosome misalignment and missegregation during meiosis, we postulated that the numerical abnormalities of chromosomes may be induced in matured Rab6a-siRNA


oocytes. To test this hypothesis, we analyzed the karyotype of MII oocytes by chromosome spreading combined with kinetochore labeling. As shown in Fig. 3, aneuploidy was observed in 34.2% of


oocytes injected with Rab6a-siRNA compared with 10.5% of controls (Fig. 3A shows representative images of euploidy and aneuploidy, respectively; Fig. 3B). These observations suggest that


loss of Rab6a disrupts the assembly of meiotic spindle and movement of meiotic chromosomes, consequently contributing to the generation of aneuploid eggs. RAB6A KNOCKDOWN IMPAIRS THE


KINETOCHORE-MICROTUBULE ATTACHMENTS IN OOCYTE Next, we decided to search for a potential mechanism that would explain the requirement of Rab6a for meiotic regulation in oocytes. On meiotic


entry, dynamic microtubules form a bipolar spindle, which is responsible for capturing and congressing chromosomes. These events require proper attachment between spindle microtubules and


kinetochores, large protein structures built on centromeric chromatin22. We therefore asked whether kinetochore-microtubule (K-MT) attachments are defective in Rab6a-depleted oocytes. To do


this, metaphase I oocytes were immunolabeled with CREST to detect kinetochores, with anti-tubulin antibody to track microtubules as described previously15. By confocal microscopy we found


that the predominant pattern of K-MT in normal oocytes is amphitelic attachment, in which the kinetochore of one chromosome is connected to the spindle pole and the kinetochore of the other


chromosome is connected to the opposite spindle pole (Fig. 4A, chromosomes 1 and 2). Of note, quantitative analysis revealed that the proportion of amphitelic attachment was significantly


reduced after Rab6a knockdown (38.2 ± 4.1 vs. 75.1 ± 3.0% control, _p_ < 0.05), whereas the percentages of undefined attachment (Fig. 4B, chromosomes 5), loss attachment (kinetochores


unattached to either pole; Fig. 4B, chromosomes 6 and 7) and merotelic attachment (one kinetochore attached to both poles; Fig. 4B, chromosomes 8) in Rab6a-siRNA oocytes were all accordingly


increased in comparison to control cells (Fig. 4C). These attachments are regarded as errors that must be corrected because chromosome missegregation would be produced if they persisted


until anaphase. Collectively, the results are indicative of, in Rab6a-depleted oocytes, the erroneous K-MT attachments could result in the meiotic defects observed in our experiments. RAB6A


KNOCKDOWN PROVOKES THE SPINDLE ASSEMBLY CHECKPOINT Cells have a sophisticated safety mechanism known as the spindle assembly checkpoint (SAC) to ensure that chromosomes have time to


correctly line up on the spindle before the cell can divide23. To successfully complete meiosis, chromosomes in oocyte must congress to the spindle equator and generate amphitelic K-MT


attachments. In the absence of such attachments oocyte will delay meiotic exit24. The mechanism that monitors and responds to K-MT attachment is the SAC; and two proteins, called Bub1 and


BubR1, play an essential role in this process23,25. Considering the K-MT misattachments and meiosis I block in Rab6a-depleted oocytes, we reasoned that these defects may arise from the SAC


activation. To test this possibility, we analyzed the SAC activity during oocyte meiosis by immunolabeling of BubR1, which is an integral part of checkpoint complex and in its absence, SAC


control is lost26. As shown in Fig. 5A, in normal oocytes, BubR1 was localized to unattached kinetochores at pre-metaphase I and almost lost at metaphase I when kinetochores are properly


attached. However, when Rab6a was abated, BubR1 expression on the kinetochores was markedly increased to approximately normal levels in meiosis I-arrested oocytes (Fig. 5A,B), indicative of


the activation of SAC. Together, these results imply that the effects of Rab6a on meiotic structures and progression are likely to be mediated through SAC signaling in oocytes. DISCUSSION


Rab family GTPases have been implicated in vesicle formation, vesicle delivery along cytoskeleton elements and docking at target membranes through the recruitment of effectors27. Here we


discover a novel role for Rab6a during meiosis: the involvement in chromosome/spindle organization and metaphase/anaphase transition in mammalian oocytes. Our cytological analysis revealed


the failure of chromosome congress and spindle assembly in oocytes when Rab6a function was altered. In particular, lagging chromosomes were frequently observed in Rab6a-siRNA anaphase


oocytes. Karyotypic analysis also confirmed the increased aneuploidy rate of these oocytes (Figs 2 and 3). Accurate alignment and segregation of homologous chromosomes or sister chromatids


is a crucial event in meiosis. Chromosome movement depends on the establishment of physical and biochemical interactions between spindle microtubules and specialized chromosomal regions, the


kinetochores28. Any errors in this process may result in aneuploid egg formation, which causes early embryo death, spontaneous abortion and genetic diseases29,30. In line with this notion,


we found that Rab6a knockdown apparently impaired the K-MT attachments in meiotic oocytes (Fig. 4). These data collectively indicate that loss of Rab6a function could compromise K-MT


interactions, whereupon lead to the chromosome misalignment and aneuploidy production in oocytes. In addition, we found that, when Rab6a was abated, the microtubule network was impaired in


fully-grown immature oocytes and the actin cap failed to form in metaphase oocytes (unpublished data), indicating that Rab6a plays an important role in the maintenance of cytoskeletal


structure during oocyte maturation. On the other hand, these findings imply that disorganization of microtubule and actin filament may contribute, at least in part, to the symmetric division


(Fig. 1E) and spindle defects (Fig. 2B) we observed in Rab6a-siRNA oocytes. Moreover, another important phenotype is that Rab6a-depleted oocytes were blocked in metaphase (Fig. 1). It has


been extensively reported that the kinetochore functions as a structural platform and as a signaling hub that coordinates chromosome attachment, SAC activity and cell cycle progression from


metaphase to anaphase31. The SAC is an evolutionarily conserved regulatory mechanism that responds to the presence of improper K-MT attachments and inhibits cell progression to prevent


errors in chromosome separation32. A large body of evidence has shown that the SAC exists in mouse oocytes and is able to recognize unattached kinetochores as in mitosis24. Importantly, our


confocal scanning showed that BubR1 is constantly present in Rab6a-siRNA oocytes even at metaphase stage (Fig. 5). By using a conditional knockout approach, Touati _et al.,_ recently


demonstrated that meiotic SAC was defective in _BubR1_ null oocytes and accordingly, meiosis I was accelerated and chromosomes were not aligned at the metaphase plate26. Interestingly, they


also found that BubR1 is required for the establishment of stable spindles in oocytes. The above observations strongly suggest that the activation of SAC signal is probably a major factor


contributing to the meiosis I arrest in Rab6a-depleted oocytes. On the basis of our findings, two important questions are raised: how Rab6a affects the K-MT attachments and SAC signaling in


oocyte meiosis and what proteins are the potential effectors mediating this pathway? Although the definite molecular mechanism remains unknown, genetic and biochemical assays have revealed


some critical clues during these processes. For example, a direct role of the dynein/dynactin complex in the transport of several kinetochore proteins and the spindle checkpoint inactivation


has been found33,34. Rab6a′ is proposed to be able regulate the dynamics of the dynein/dynactin complex at the kinetochores and consequently trigger the spindle checkpoint through


interaction with p150Glued and GAPCenA35. Future experiments aimed to characterize the interaction between Rab6a and kinetochore proteins will help to clarify the above questions.


Regardless, due to the limitation of oocyte number and technical reason, we have not yet been able to directly screen the potential targets of Rab6a in mouse oocytes. In conclusion, our data


support a model: Rab6a knockdown in oocytes may compromise the interaction between kinetochore and microtubule, which in turn leads to the activation of SAC signal, and, as a result,


chromosome misalignment and spindle defects are established during meiosis, causing the production of aneuploid eggs and persistent defects in embryos. MATERIALS AND METHODS All chemicals


and reagents were obtained from Sigma unless otherwise stated. ICR mice were used in this study. All experiments were approved by the Animal Care and Use Committee of Nanjing Medical


University and were performed in accordance with institutional guidelines. ANTIBODIES Rabbit polyclonal anti-Rab6a and Sheep polyclonal anti-BubR1 antibodies were purchased from Abcam


(Cambridge, MA, USA; Cat#: ab95954 and ab28193); Human anti-centromere CREST antibody was purchased from Antibodies Incorporated (Davis, CA, USA; Cat#:15–234). Cy5-conjugated donkey


anti-human IgG and FITC-conjugated donkey anti-goat IgG were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA, USA; Cat#: 709-605-149 and 705-095-147). FITC-conjugated goat


anti-rabbit IgG was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Mouse monoclonal anti-β-actin antibodies and mouse monoclonal FITC-conjugated anti-α-tubulin antibodies were


purchased from Sigma (St. Louis, MO, USA; Cat#: A5441 and F2168). OOCYTE COLLECTION AND CULTURE Female ICR mice (4–6 weeks) were sacrificed by cervical dislocation after intraperitoneal


injections of 5 IU pregnant mare serum gonadotropin (PMSG) for 46 hours. Cumulus-enclosed oocytes were retrieved by manual rupturing of antral ovarian follicles. Fully-grown denuded oocytes


were collected by removing cumulus cells with repeatedly mouth-pipetting. Oocytes were cultured in M16 medium under mineral oil at 37 °C in a 5% CO2 incubator for _in vitro_ maturation.


SIRNA KNOCKDOWN Fully-grown immature oocytes were microinjected with Rab6a-targeting siRNA to knock down Rab6a proteins. siRNA was diluted with water to give a stock concentration of 1 mM


and 2.5 picoliter solution was injected. A siRNA negative control was injected as control. To facilitate the siRNA-mediated mRNA degradation, oocytes were arrested at GV stage in M2 medium


containing 2.5 μM milrinone for 20 hours and then cultured in milrinone-free medium for further experiments. Rab6a-siRNA sequence: 5′- GGAGCAACCAGUCAAUGAATT-3′; 5′-UUCAUUGACUGGUUGCUCCTT-3′


Control siRNA sequence: 5′-UUCUCCGAACGUGUCACGUTT-3′; 5′-ACGUGACACGUUCGGAGAATT-3′. IMMUNOFLUORESCENCE AND CONFOCAL MICROSCOPY Oocytes were fixed with 4% paraformaldehyde in PBS (PH 7.4) for


30 minutes, permeabilized with 0.5% Triton X-100 for 20 minutes and then blocked in 1% BSA-supplemented PBS for 1 hour at room temperature. The processed samples were incubated overnight at


4 °C with primary antibodies as follows: anti-BubR1 antibody (1:250) and FITC-conjugated α-tubulin antibody (1:200). FITC- or Cy5-conjugated secondary antibodies were then applied for 1 hour


at room temperature as appropriate. Chromosomes of oocytes were evaluated by costaining with propidium iodide (red) or Hoechst 33342 (blue) for 10min. Samples were examined under a laser


scanning confocal microscope (LSM 700; Zeiss, Oberkochen, Germany). For quantification of oocytes with meiotic defects, the gross morphology of spindle/chromosomes was assessed. To detect


kinetochores, oocytes were labeled with human CREST auto-immune antibody (1:500) according to the protocol previously described15. To assess the kinetochore-microtubule (K-MT) attachments,


oocytes were briefly chilled at 4 °C to induce depolymerization of non-kinetochore microtubules just prior to fixation. To measure the intensity of fluorescence, Image J software (NIH) was


used as previously described36. WESTERN BLOT ANALYSIS A pool of 100 oocytes was lysed in Laemmli sample buffer containing protease inhibitor and then subjected to 10% SDS-PAGE, resolved and


electroblotted onto a PVDF membrane. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% low fat dry milk for 1 hour and then incubated with anti-Rab6a antibody


(1:1,000) overnight at 4 °C. After multiple washes in Tris-buffered saline containing 0.1% Tween 20 and incubation with anti-rabbit horseradish peroxidase linked antibody, the protein bands


were visualized using an ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA). The membrane was then stripped and reblotted with anti-β-actin (1:5,000) antibody


for loading control. CHROMOSOME SPREAD Chromosome spreading was performed as previously described37. Oocytes were exposed to Tyrode’s buffer (pH 2.5) for to remove zona pellucidae and then


fixed in a drop of 1% paraformaldehyde with 0.15% Triton X-100 on a glass slide. Samples were labeled with CREST (1:500) for 1 hour to detect kinetochores and chromosomes were counterstained


with Hoechst 33342. Laser scanning confocal microscope was used to examine chromosome numbers in oocytes. STATISTICAL ANALYSIS Data are presented as mean ± SD, unless otherwise indicated.


Differences between 2 groups were analyzed by Student’s _t_ test. Multiple comparisons between more than 2 groups were analyzed by 1-way ANOVA test using Prism 5.0. _P_ < 0.05 was


considered to be significant. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Hou, X. _et al._ Rab6a is a novel regulator of meiotic apparatus and maturational progression in mouse oocytes.


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(2009). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by National Key Scientific Research Projects (2014CB943200),


National Natural Science Foundation of China (No. 31301181) and Natural Science Foundation of the Jiangsu Higher Education Institutions (No. 13KJA310001). AUTHOR INFORMATION Author notes *


Hou Xiaojing and Zhang Jiaqi contributed equally to this work. AUTHORS AND AFFILIATIONS * State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China Xiaojing


Hou, Jiaqi Zhang, Ling Li, Rujun Ma, Juan Ge, Longsen Han & Qiang Wang * Center of Reproductive Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China Rujun Ma


Authors * Xiaojing Hou View author publications You can also search for this author inPubMed Google Scholar * Jiaqi Zhang View author publications You can also search for this author


inPubMed Google Scholar * Ling Li View author publications You can also search for this author inPubMed Google Scholar * Rujun Ma View author publications You can also search for this author


inPubMed Google Scholar * Juan Ge View author publications You can also search for this author inPubMed Google Scholar * Longsen Han View author publications You can also search for this


author inPubMed Google Scholar * Qiang Wang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS X.H. and Q.W. designed research; X.H., J.Z.,


L.L., R.M., J.G. and L.H. performed research; X.H., J.Z. and Q.W. analyzed data; X.H. and Q.W. wrote paper. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial


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CITE THIS ARTICLE Hou, X., Zhang, J., Li, L. _et al._ Rab6a is a novel regulator of meiotic apparatus and maturational progression in mouse oocytes. _Sci Rep_ 6, 22209 (2016).


https://doi.org/10.1038/srep22209 Download citation * Received: 10 December 2015 * Accepted: 08 February 2016 * Published: 26 February 2016 * DOI: https://doi.org/10.1038/srep22209 SHARE


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