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.

scholarly journals Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Dong Zhang ◽  
Shen Yin ◽  
Man-Xi Jiang ◽  
Wei Ma ◽  
Yi Hou ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Mitotic Arrest ◽  
Checkpoint Proteins ◽  
Meiotic Progression ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Oocyte Meiosis ◽  
And Function

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

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.

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 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 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.

Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium

1992 ◽  
Vol 103 (2) ◽  
pp. 389-396 ◽  
Author(s):  
C. Vincent ◽  
T.R. Cheek ◽  
M.H. Johnson
Keyword(s):  
Cell Cycle Progression ◽  
Polar Body ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Mouse Oocyte ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.

Antioxidants Stimulate Germinal Vesicle Breakdown, But Inhibit Chromosome Formation And Spindle Assembly In Porcine Oocytes

2000 ◽  
Vol 6 (S2) ◽  
pp. 964-965
Author(s):  
Qing-Yuan Sun ◽  
Randall S. Prather ◽  
Heide Schatten
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Meiosis ◽  
First Polar Body ◽  
Porcine Oocyte ◽  
Chromosome Formation

Mammalian oocytes are arrested at the diplotene stage of the first meiotic division. Release of oocytes from their follicles induces meiotic resumption characterized by germinal vesicle breakdown (GVBD), followed by the chromosome formation and metaphase I spindle organization and finally the extrusion the first polar body. Recently it was shown that cellpermeant antioxidants significantly inhibit spontaneous resumption of meiosis in mouse oocytes, which may indicate a role of oxygen radicals in oocyte maturation. The regulation of mouse oocyte meiosis resumption is different from that of large domestic animals in that GVBD is independent of Ca2+ and protein synthesis. The present study investigated the influence of two cell-permeant antioxidants, 2(3)-ter-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behavior and spindle assembly. Our findings revealed a different role of antioxidants in porcine oocyte meiosis resumption than in mouse oocyte maturation.

scholarly journals The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Vol 134 (1) ◽  
pp. jcs251025
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

JNK2 Participates in Spindle Assembly during Mouse Oocyte Meiotic Maturation

2011 ◽  
Vol 17 (2) ◽  
pp. 197-205 ◽  
Author(s):  
Xin Huang ◽  
Jing-Shan Tong ◽  
Zhen-Bo Wang ◽  
Cai-Rong Yang ◽  
Shu-Tao Qi ◽  
...  
Keyword(s):  
Polar Body ◽  
Specific Inhibitor ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Meiotic Maturation ◽  
Cytoplasmic Microtubule ◽  
Spindle Poles ◽  
First Polar Body ◽  
Oocyte Meiotic Maturation

AbstractIt is well known that c-Jun N-terminal kinase (JNK) plays pivotal roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that no specific JNK2 signal was detected in germinal vesicle stage. JNK2 was associated with the spindles especially the spindle poles and cytoplasmic microtubule organizing centers at prometaphase I, metaphase I, and metaphase II stages. JNK2 became diffusely distributed and associated with the midbody at telophase I stage. Injection of myc-tagged JNK2α1 mRNA into oocytes also revealed its localization on spindle poles. The association of JNK2 with spindle poles was further confirmed by colocalization with the centrosomal proteins, γ-tubulin and Plk1. Nocodazole treatment showed that JNK2 may interact with Plk1 to regulate the spindle assembly. Then we investigated the possible function of JNK2 by JNK2 antibody microinjection and JNK specific inhibitor SP600125 treatment. These two manipulations caused abnormal spindle formation and decreased the rate of first polar body (PB1) extrusion. In addition, inhibition of JNK2 resulted in impaired localization of Plk1. Taken together, our results suggest that JNK2 plays an important role in spindle assembly and PB1 extrusion during mouse oocyte meiotic maturation.

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