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

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.

Download Full-text

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

PeerJ ◽

10.7717/peerj.8180 ◽

2020 ◽

Vol 8 ◽

pp. e8180 ◽

Cited By ~ 1

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.

Download Full-text

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

10.7287/peerj.preprints.27837v1 ◽

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

Download Full-text

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

Microscopy and Microanalysis ◽

10.1017/s1431927611000274 ◽

2011 ◽

Vol 17 (3) ◽

pp. 431-439 ◽

Cited By ~ 25

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.

Download Full-text

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

Microscopy and Microanalysis ◽

10.1017/s1431927600037314 ◽

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.

Download Full-text

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

Reproduction ◽

10.1530/rep.1.00336 ◽

2005 ◽

Vol 129 (2) ◽

pp. 229-234 ◽

Cited By ~ 13

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.

Download Full-text

The GTPase SPAG-1 orchestrates meiotic program by dictating meiotic resumption and cytoskeleton architecture in mouse oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e16-02-0132 ◽

2016 ◽

Vol 27 (11) ◽

pp. 1776-1785 ◽

Cited By ~ 7

Author(s):

Chunjie Huang ◽

Di Wu ◽

Faheem Ahmed Khan ◽

Xiaofei Jiao ◽

Kaifeng Guan ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Polar Body ◽

Germinal Vesicle ◽

Mapk Signaling ◽

Cortical Granule ◽

Germinal Vesicle Breakdown ◽

Atp Production ◽

M Phase ◽

Polar Body Extrusion ◽

Actin Expression

In mammals, a finite population of oocytes is generated during embryogenesis, and proper oocyte meiotic divisions are crucial for fertility. Sperm-associated antigen 1 (SPAG-1) has been implicated in infertility and tumorigenesis; however, its relevance in cell cycle programs remains rudimentary. Here we explore a novel role of SPAG-1 during oocyte meiotic progression. SPAG-1 associated with meiotic spindles and its depletion severely compromised M-phase entry (germinal vesicle breakdown [GVBD]) and polar body extrusion. The GVBD defect observed was due to an increase in intraoocyte cAMP abundance and decrease in ATP production, as confirmed by the activation of AMP-dependent kinase (AMPK). SPAG-1 RNA interference (RNAi)–elicited defective spindle morphogenesis was evidenced by the dysfunction of γ-tubulin, which resulted from substantially reduced phosphorylation of MAPK and irregularly dispersed distribution of phospho-MAPK around spindles instead of concentration at spindle poles. Significantly, actin expression abruptly decreased and formation of cortical granule–free domains, actin caps, and contractile ring disrupted by SPAG-1 RNAi. In addition, the spindle assembly checkpoint remained functional upon SPAG-1 depletion. The findings broaden our knowledge of SPAG-1, showing that it exerts a role in oocyte meiotic execution via its involvement in AMPK and MAPK signaling pathways.

Download Full-text

The Distribution and Possible Role of ERK8 in Mouse Oocyte Meiotic Maturation and Early Embryo Cleavage

Microscopy and Microanalysis ◽

10.1017/s1431927612013918 ◽

2013 ◽

Vol 19 (1) ◽

pp. 190-200 ◽

Cited By ~ 3

Author(s):

Shang-Wu Yang ◽

Hao Huang ◽

Chen Gao ◽

Lei Chen ◽

Shu-Tao Qi ◽

...

Keyword(s):

Polar Body ◽

Germinal Vesicle ◽

Early Embryo ◽

Mouse Oocyte ◽

Meiotic Maturation ◽

Germinal Vesicle Breakdown ◽

First Polar Body ◽

Embryo Cleavage ◽

Polar Body Extrusion ◽

Oocyte Meiotic Maturation

AbstractIt is well known that extracellular signal-regulated kinase 8 (ERK8) plays pivotal roles in various mitotic events. But its physiological roles in oocyte meiotic maturation remain unclear. In this study, we found that although no specific ERK8 signal was detected in oocyte at the germinal vesicle stage, ERK8 began to migrate to the periphery of chromosomes shortly after germinal vesicle breakdown. At prometaphase I, metaphase I (MI), anaphase I, telophase I, and metaphase II (MII) stages, ERK8 was stably detected at the spindles. By taxol treatment, we clarified that the ERK8 signal was stained on the spindle fibers as well as microtubule asters in MI and MII oocytes. In fertilized eggs, the ERK8 signal was not observed in the two pronuclei stages. At prometaphase, metaphase, and anaphase of the first mitosis, ERK8 was detected on the mitotic spindle. ERK8 knock down by antibody microinjection and specific siRNA caused abnormal spindles, failed chromosome congression, and decreased first polar body extrusion. Taken together, our results suggest that ERK8 plays an important role in spindle organization during mouse oocyte meiotic maturation and early embryo cleavage.

Download Full-text

WAPL orchestrates porcine oocyte meiotic progression via control of spindle assembly checkpoint activity

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00740-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Changyin Zhou ◽

Yilong Miao ◽

Xue Zhang ◽

Bo Xiong

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Polar Body ◽

Spindle Assembly ◽

Protein Levels ◽

Meiotic Progression ◽

Oocyte Meiosis ◽

Chromosome Alignment ◽

Porcine Oocyte ◽

Polar Body Extrusion

Abstract Background In mitotic cells, WAPL acts as a cohesin release factor to remove cohesin complexes from chromosome arms during prophase to allow the accurate chromosome segregation in anaphase. However, we have recently documented that Wapl exerts a unique meiotic function in the spindle assembly checkpoint (SAC) control through maintaining Bub3 stability during mouse oocyte meiosis I. Whether this noncanonical function is conserved among species is still unknown. Methods We applied RNAi-based gene silencing approach to deplete WAPL in porcine oocytes, validating the conserved roles of WAPL in the regulation of SAC activity during mammalian oocyte maturation. We also employed immunostaining, immunoblotting and image quantification analyses to test the WAPL depletion on the meiotic progression, spindle assembly, chromosome alignment and dynamics of SAC protein in porcine oocytes. Results We showed that depletion of WAPL resulted in the accelerated meiotic progression by displaying the precocious polar body extrusion and compromised spindle assembly and chromosome alignment. Notably, we observed that the protein level of BUB3 was substantially reduced in WAPL-depleted oocytes, especially at kinetochores. Conclusions Collectively, our data demonstrate that WAPL participates in the porcine oocyte meiotic progression through maintenance of BUB3 protein levels and SAC activity. This meiotic function of WAPL in oocytes is highly conserved between pigs and mice.

Download Full-text

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

Nature Communications ◽

10.1038/s41467-021-26826-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dalileh Nabi ◽

Hauke Drechsler ◽

Johannes Pschirer ◽

Franz Korn ◽

Nadine Schuler ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Polar Body ◽

Spindle Assembly ◽

V Protein ◽

Oocyte Meiosis ◽

Spindle Microtubules ◽

Polar Body Extrusion ◽

Metaphase I

AbstractProper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Download Full-text

Txndc9 Is Required for Meiotic Maturation of Mouse Oocytes

BioMed Research International ◽

10.1155/2017/6265890 ◽

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.

Download Full-text