Ndc80 Regulates Meiotic Spindle Organization, Chromosome Alignment, and Cell Cycle Progression in Mouse Oocytes

2011 ◽  
Vol 17 (3) ◽  
pp. 431-439 ◽  
Author(s):  
Shao-Chen Sun ◽  
Ding-Xiao Zhang ◽  
Seung-Eun Lee ◽  
Yong-Nan Xu ◽  
Nam-Hyung Kim
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Meiotic Spindle ◽  
Immunofluorescent Staining ◽  
Mrna Levels ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
Chromosome Alignment

AbstractNdc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

scholarly journals OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes

2019 ◽  
Author(s):  
Di Xie ◽  
Juan Zhang ◽  
JinLi Ding ◽  
Jing Yang ◽  
Yan Zhang
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Immunofluorescent Staining ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Meiosis ◽  
Polar Body Extrusion ◽  
Spread Analysis

Background. OLA1 is a member of the GTPase protein family, unlike other members, it can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown. Methods. In this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity. Results. Immunofluorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.

scholarly journals OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8180 ◽  
Author(s):  
Di Xie ◽  
Juan Zhang ◽  
JinLi Ding ◽  
Jing Yang ◽  
Yan Zhang
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Immunofluorescent Staining ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Meiosis ◽  
Polar Body Extrusion ◽  
Spread Analysis

Background OLA1 is a member of the GTPase protein family; unlike other members, it possess both GTPase and ATPase activities, and can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response, protein synthesis and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown. Methods In this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity. Results Immunofluorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.

scholarly journals OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes

2019 ◽  
Author(s):  
Di Xie ◽  
Juan Zhang ◽  
JinLi Ding ◽  
Jing Yang ◽  
Yan Zhang
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Immunofluorescent Staining ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Meiosis ◽  
Polar Body Extrusion ◽  
Spread Analysis

Background. OLA1 is a member of the GTPase protein family, unlike other members, it can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown. Methods. In this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity. Results. Immunofluorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.

scholarly journals Translocation of phospho-protein kinase Cs implies their roles in meiotic-spindle organization, polar-body emission and nuclear activity in mouse eggs

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 229-234 ◽  
Author(s):  
Zhen-Yu Zheng ◽  
Qing-Zhang Li ◽  
Da-Yuan Chen ◽  
Heide Schatten ◽  
Qing-Yuan Sun
Keyword(s):  
Protein Kinase ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Activity ◽  
Spindle Poles ◽  
Oocyte Meiosis ◽  
First Polar Body

The protein kinase Cs (PKCs) are a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The phosphorylation of PKC in germ cells is not well defined. In this study, we described the subcellular localization of phopho-PKC in the process of mouse oocyte maturation, fertilization, and early embryonic mitosis. Confocal microscopy revealed that phospho-PKC (pan) was distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, phospho-PKC was localized in the vicinity of the condensed chromosomes, distributed in the whole meiotic spindle, and concentrated at the spindle poles. After metaphase I, phospho-PKC was translocated gradually to the spindle mid-zone during emission of the first polar body. After sperm penetration and electrical activation, the distribution of phospho-PKC was moved from the spindle poles to the spindle mid-zone. After the extrusion of the second polar body (PB2) phospho-PKC was localized in the area between the oocyte and the PB2. In fertilized eggs, phospho-PKC was concentrated in the pronuclei except for the nucleolus. Phospho-PKC was dispersed after pronuclear envelope breakdown, but distributed on the entire spindle at mitotic metaphase. The results suggest that PKC activation may play important roles in regulating spindle organization and stabilization, polar-body extrusion, and nuclear activity during mouse oocyte meiosis, fertilization, and early embryonic mitosis.

scholarly journals Txndc9 Is Required for Meiotic Maturation of Mouse Oocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Fanhua Ma ◽  
Liming Hou ◽  
Liguo Yang
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Redox Balance ◽  
Redox Status ◽  
Mouse Oocyte ◽  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
First Polar Body ◽  
Polar Body Extrusion

Txndc9 (thioredoxin domain containing protein 9) has been shown to be involved in mammalian mitosis; however, its function in mammalian oocyte meiosis remains unclear. In this study, we initially found that Txndc9 is expressed during meiotic maturation of mouse oocytes and higher expression of Txndc9 mRNA and protein occurred in germinal vesicle (GV) stage. By using confocal scanning, we observed that Txndc9 localized at both nucleus and cytoplasm, especially at spindle microtubules. Specific depletion of Txndc9 by siRNA in mouse oocyte resulted in decreasing the rate of first polar body extrusion and increasing abnormal spindle assemble. Moreover, knockdown of Txndc9 in germinal vesicle (GV) stage oocytes led to higher level of reactive oxygen species (ROS) and lower level of antioxidant glutathione (GSH) as compared with control oocytes, which indicated that Txndc9 may be involved in mediating the redox balance. In summary, our results demonstrated that Txndc9 is crucial for mouse oocyte maturation by regulating spindle assembly, polar body extrusion, and redox status.

scholarly journals Ral GTPase is essential for actin dynamics and Golgi apparatus distribution in mouse oocyte maturation

Cell Division ◽  
2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ming-Hong Sun ◽  
Lin-Lin Hu ◽  
Chao-Ying Zhao ◽  
Xiang Lu ◽  
Yan-Ping Ren ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Mouse Oocyte ◽  
Actin Dynamics ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
Ral Gtpase ◽  
Polar Body Extrusion ◽  
Cytoskeleton Dynamics

Abstract Background Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation. Results Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion. Conclusions In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

scholarly journals Comparison of the toxic effects of different mycotoxins on porcine and mouse oocyte meiosis

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5111 ◽  
Author(s):  
Yujie Lu ◽  
Yue Zhang ◽  
Jia-Qian Liu ◽  
Peng Zou ◽  
Lu Jia ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Animal Feed ◽  
Polar Body ◽  
Oocyte Quality ◽  
Mouse Oocyte ◽  
Toxic Effects ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
Porcine Oocyte ◽  
Polar Body Extrusion

Background Aflatoxin B1 (AFB1), deoxynivalenol (DON), HT-2, ochratoxin A (OTA), zearalenone (ZEA) are the most common mycotoxins that are found in corn-based animal feed which have multiple toxic effects on animals and humans. Previous studies reported that these mycotoxins impaired mammalian oocyte quality. However, the effective concentrations of mycotoxins to animal oocytes were different. Methods In this study we aimed to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA for mycotoxin research. We adopted the polar body extrusion rate of mouse and porcine oocyte as the standard for the effects of mycotoxins on oocyte maturation. Results and Discussion Our results showed that 10 μM AFB1 and 1 μM DON significantly affected porcine oocyte maturation compared with 50 μM AFB1 and 2 μM DON on mouse oocytes. However, 10 nM HT-2 significantly affected mouse oocyte maturation compared with 50 nM HT-2 on porcine oocytes. Moreover, 5 μM OTA and 10 μM ZEA significantly affected porcine oocyte maturation compared with 300 μM OTA and 50 μM ZEA on mouse oocytes. In summary, our results showed that porcine oocytes were more sensitive to AFB1, DON, OTA, and ZEA than mouse oocytes except HT-2 toxin.

LIM kinase activity is required for microtubule organising centre positioning in mouse oocyte meiosis

2017 ◽  
Vol 29 (4) ◽  
pp. 791 ◽  
Author(s):  
Xin Li ◽  
Yubo Zhu ◽  
Yan Cao ◽  
Qian Wang ◽  
Juan Du ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Direct Interaction ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Lim Kinase ◽  
Spindle Formation ◽  
Mouse Oocytes ◽  
Spindle Poles ◽  
Oocyte Meiosis ◽  
And Function

LIM kinase 1 (LIMK1) activity is essential for cell migration and cell cycle progression. Little is known about LIMK1 expression and function in mammalian oocytes. In the present study we assessed LIMK1 protein expression, subcellular distribution and function during mouse oocyte meiosis. Western blot analysis revealed high and stable expression of LIMK1 from the germinal vesicle (GV) to MII stage. In contrast, activated LIMK1 (i.e. LIMK1 phosphorylated at threonine 508 (pLIMK1Thr508)) was only detected after GV breakdown, with levels increasing gradually to peak at MI and MII. Immunofluorescence showed pLIMK1Thr508 was colocalised with the microtubule organising centre (MTOC) components pericentrin and γ-tubulin at the spindle poles. A direct interaction between γ-tubulin and pLIMK1Thr508 was confirmed by co-immunoprecipitation. LIMK inhibition with 1 μM BMS3 damaged MTOC protein localisation to spindle poles, undermined the formation and positioning of functional MTOC and thus disrupted spindle formation and chromosome alignment. These effects were phenocopied by microinjection of LIMK1 antibody into mouse oocytes. In summary, the data demonstrate that LIMK activity is essential for MTOC organisation and distribution and so bipolar spindle formation and maintenance in mouse oocytes.

RAB35 depletion affects spindle formation and actin-based spindle migration in mouse oocyte meiosis

2019 ◽  
Vol 25 (7) ◽  
pp. 359-372 ◽  
Author(s):  
Yu Zhang ◽  
Xiang Wan ◽  
Hong-Hui Wang ◽  
Meng-Hao Pan ◽  
Zhen-Nan Pan ◽  
...  
Keyword(s):  
Asymmetric Cell Division ◽  
Polar Body ◽  
Asymmetric Division ◽  
Mouse Oocyte ◽  
Meiotic Spindle ◽  
Spindle Formation ◽  
Oocyte Meiosis ◽  
Spindle Stability ◽  
Polar Body Extrusion ◽  
Cytoskeleton Dynamics

Abstract Mammalian oocyte maturation involves a unique asymmetric cell division, in which meiotic spindle formation and actin filament-mediated spindle migration to the oocyte cortex are key processes. Here, we report that the vesicle trafficking regulator, RAB35 GTPase, is involved in regulating cytoskeleton dynamics in mouse oocytes. RAB35 GTPase mainly accumulated at the meiotic spindle periphery and cortex during oocyte meiosis. Depletion of RAB35 by morpholino microinjection led to aberrant polar body extrusion and asymmetric division defects in almost half the treated oocytes. We also found that RAB35 affected SIRT2 and αTAT for tubulin acetylation, which further modulated microtubule stability and meiotic spindle formation. Additionally, we found that RAB35 associated with RHOA in oocytes and modulated the ROCK–cofilin pathway for actin assembly, which further facilitated spindle migration for oocyte asymmetric division. Importantly, microinjection of Myc-Rab35 cRNA into RAB35-depleted oocytes could significantly rescue these defects. In summary, our results suggest that RAB35 GTPase has multiple roles in spindle stability and actin-mediated spindle migration in mouse oocyte meiosis.

scholarly journals LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2553 ◽  
Author(s):  
Ru-Xia Jia ◽  
Xing Duan ◽  
Si-Jing Song ◽  
Shao-Chen Sun
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Actin Dynamics ◽  
Immunofluorescent Staining ◽  
Meiotic Maturation ◽  
Spindle Positioning ◽  
Oocyte Meiosis ◽  
Porcine Oocyte ◽  
Oocyte Meiotic Maturation

LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the cortex of porcine oocyte, which co-localized with actin. After LIMKi 3 treatment, the diffusion of COCs became weak and the rate of polar body extrusion was decreased. This could be rescued by moving oocytes to fresh medium. After prolonging the culture time of oocytes, the maturation rate of porcine oocyte increased in LIMKi 3 groups, indicating that LIMKi 3 may suppress the cell cycle during porcine oocyte maturation. We also found that after LIMKi 3 treatment actin distribution was significantly disturbed at porcine oocyte membranes and cytoplasm, indicating the conserved roles of LIMK1/2 on actin dynamics. Next we examined the meiotic spindle positioning in porcine oocyte, and the results showed that a majority of spindles were not attached to the cortex of porcine oocyte, indicating that LIMKi 3 may affect actin-mediated spindle positioning. Taken together, these results showed that LIMK1/2 inhibitor LIMKi 3 had a repressive role on porcine oocyte meiotic maturation.

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