scholarly journals PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3295
Author(s):  
Xiaoyu Liu ◽  
Xiaoyun Liu ◽  
Dandan Chen ◽  
Xiuying Jiang ◽  
Wei Ma
Keyword(s):  
Transcriptional Control ◽  
Cell Cycle Progression ◽  
Catalytic Domain ◽  
Germinal Vesicle ◽  
Actin Dynamics ◽  
Germinal Vesicle Breakdown ◽  
Meiotic Progression ◽  
Oocyte Meiosis ◽  
Catalytic Inhibitor ◽  
Metaphase I

Phospholipase D2 (PLD2) is involved in cytoskeletal reorganization, cell migration, cell cycle progression, transcriptional control and vesicle trafficking. There is no evidence about PLD2 function in oocytes during meiosis. Herein, we analyzed PLD2 expression and its relationship with spindle formation and positioning in mouse oocyte meiosis. High protein level of PLD2 was revealed in oocytes by Western blot, which remained consistently stable from prophase I with intact germinal vesicle (GV) up to metaphase II (MII) stage. Immunofluorescence showed that PLD2 appeared and gathered around the condensed chromosomesafter germinal vesicle breakdown (GVBD), and co-localized with spindle from pro-metaphase I (pro-MI) to metaphase I (MI) and at MII stage. During anaphase I (Ana I) to telophase I (Tel I) transition, PLD2 was concentrated in the spindle polar area but absent from the midbody. In oocytes incubated with NFOT, an allosteric and catalytic inhibitor to PLD2, the spindle was enlarged and center-positioned, microtubules were resistant to cold-induced depolymerization and, additionally, the meiotic progression was arrested at MI stage. However, spindle migration could not be totally prevented by PLD2 catalytic specific inhibitors, FIPI and 1-butanol, implying at least partially, that PLD2 effect on spindle migration needs non-catalytic domain participation. NFOT-induced defects also resulted in actin-related molecules’ distribution alteration, such as RhoA, phosphatidylinosital 4, 5- biphosphate (PIP2), phosphorylated Colifin and, consequently, unordered F-actin dynamics. Taken together, these data indicate PLD2 is required for the regulation of microtubule dynamics and spindle migration toward the cortex in mammalian oocytes during meiotic progression.

scholarly journals Ca2+cyt negatively regulates the initiation of oocyte maturation

2004 ◽  
Vol 165 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Lu Sun ◽  
Khaled Machaca
Keyword(s):  
Cell Cycle ◽  
Oocyte Maturation ◽  
Cell Cycle Progression ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Cycle Progression ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Kinase Cascade ◽  
Cell Cycle Machinery

Ca2+ is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca2+ in mitosis. In contrast, there has been a long-standing debate as to whether Ca2+ signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca2+ (Ca2+cyt) plays a dual role during Xenopus oocyte maturation. Ca2+ signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca2+cyt signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca2+-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca2+cyt acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca2+cyt delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca2+ modulates both the cell cycle machinery and nuclear maturation during meiosis.

Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells

2002 ◽  
Vol 14 (7) ◽  
pp. 433 ◽  
Author(s):  
J. Ye ◽  
A. P. F. Flint ◽  
K. H. S. Campbell ◽  
M. R. Luck
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Conventional Culture ◽  
Oocyte Meiosis

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95–100%), but distributed from GVI to GVIV (GVI 56.1 ± 9.1%, GVII 15.3 ± 1.4%, GVIII 21.5 ± 7.1%, GVIV 7.1 ± 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 ± 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1–25 μg mL–1 resulted in 95–100% oocytes being arrested and synchronized at GVII. With >5 μg mL–1 CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 μg mL–1 CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6–12 h, metaphase I (MI) at 10–18�h and anaphase I/telophase I at 16–20 h. After 12 h preincubation with 5 μg mL–1 CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.

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 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.

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 Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca2+ store depletion from store-operated Ca2+ entry

2002 ◽  
Vol 156 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Khaled Machaca ◽  
Shirley Haun
Keyword(s):  
Oocyte Maturation ◽  
Xenopus Oocyte ◽  
Medical Science ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Egg Activation ◽  
Germinal Vesicle Breakdown ◽  
Store Depletion ◽  
Oocyte Meiosis ◽  
Maturation Promoting Factor

Department of Physiology and Biophysics, University of Arkansas Medical Science, Little Rock, AR 72205 During oocyte maturation, eggs acquire the ability to generate specialized Ca2+ signals in response to sperm entry. Such Ca2+ signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos–mitogen-activated protein kinase (MAPK)–maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca2+ influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.

Increase of cAMP upon release from prophase arrest in surf clam oocytes

2002 ◽  
Vol 115 (2) ◽  
pp. 311-320
Author(s):  
Jae-Hyuk Yi ◽  
Linda Lefièvre ◽  
Claude Gagnon ◽  
Michel Anctil ◽  
François Dubé
Keyword(s):  
Germinal Vesicle ◽  
Oocyte Activation ◽  
Spisula Solidissima ◽  
Germinal Vesicle Breakdown ◽  
Surf Clam ◽  
Oocyte Meiosis ◽  
High K ◽  
Direct Measurements ◽  
Camp Levels ◽  
Adenylyl Cyclase Inhibitor

Surf clam (Spisula solidissima) oocytes are spawned at the prophase I stage of meiosis, and they remain arrested at this stage until fertilization. Full oocyte meiosis reinitiation, first evidenced by germinal vesicle breakdown (GVBD), may be induced by artificial activators mimicking sperm, such as high K+ or serotonin. Previous reports indicated that treatments thought to increase the level of oocyte cAMP inhibited sperm- or serotonin-induced, but not KCl-induced, GVBD in clam oocytes. These observations extend the well known requirement for a drop in occyte cAMP levels in mammalian, amphibian or starfish oocytes and support the view that such a drop is universally important throughout the animal kingdom. We have re-examined the cAMP dependency of GVBD in clam oocytes and found that various treatments that raise oocyte cAMP levels did not, surprisingly, affect either KCl- or serotonin-induced GVBD. Such treatments, however, inhibited GVBD upon insemination of the oocytes, but this was due to the failure of sperm to fuse/penetrate the oocytes; thus, it was not an inhibition of oocyte activation as such. Direct measurements of oocyte cAMP levels after activation by serotonin, KCl or sperm showed that, contrary to expectations, there is a rise in cAMP levels before GVBD. Using SQ22536, an adenylyl cyclase inhibitor, the increase in oocyte cAMP level was partly prevented and GVBD proceeded, but with a significant retardation, indicating that the normal cAMP rise facilitates GVBD. Our work sheds light on the diversity of upstream pathways leading to activation of MPF and provides a unique model whereby the onset of meiosis reinitiation is associated with an increase, not a decrease, in oocyte cAMP levels.

scholarly journals Condensins I and II are essential for construction of bivalent chromosomes in mouse oocytes

2011 ◽  
Vol 22 (18) ◽  
pp. 3465-3477 ◽  
Author(s):  
Jibak Lee ◽  
Sugako Ogushi ◽  
Mitinori Saitou ◽  
Tatsuya Hirano
Keyword(s):  
Sister Chromatid ◽  
Meiotic Chromosome ◽  
Germinal Vesicle ◽  
Primary Role ◽  
Germinal Vesicle Breakdown ◽  
Chromosome Dynamics ◽  
Mouse Oocytes ◽  
First Time ◽  
Striking Contrast ◽  
Metaphase I

In many eukaryotes, condensins I and II associate with chromosomes in an ordered fashion during mitosis and play nonoverlapping functions in their assembly and segregation. Here we report for the first time the spatiotemporal dynamics and functions of the two condensin complexes during meiotic divisions in mouse oocytes. At the germinal vesicle stage (prophase I), condensin I is present in the cytoplasm, whereas condensin II is localized within the nucleus. After germinal vesicle breakdown, condensin II starts to associate with chromosomes and becomes concentrated onto chromatid axes of bivalent chromosomes by metaphase I. REC8 “glues” chromosome arms along their lengths. In striking contrast to condensin II, condensin I localizes primarily around centromeric regions at metaphase I and starts to associate stably with chromosome arms only after anaphase I. Antibody injection experiments show that condensin functions are required for many aspects of meiotic chromosome dynamics, including chromosome individualization, resolution, and segregation. We propose that the two condensin complexes play distinctive roles in constructing bivalent chromosomes: condensin II might play a primary role in resolving sister chromatid axes, whereas condensin I might contribute to monopolar attachment of sister kinetochores, possibly by assembling a unique centromeric structure underneath.

scholarly journals Cyclins regulating oocyte meiotic cell cycle progression†

2019 ◽  
Vol 101 (5) ◽  
pp. 878-881 ◽  
Author(s):  
Jian Li ◽  
Wei-Ping Qian ◽  
Qing-Yuan Sun
Keyword(s):  
Cell Cycle Progression ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Meiotic Maturation ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Meiotic Progression ◽  
Gonadotropin Surge ◽  
Oocyte Meiotic Maturation ◽  
Maturation Promoting Factor

Abstract Oocyte meiotic maturation is a vital and final process in oogenesis. Unlike somatic cells， the oocyte needs to undergo two continuous meiotic divisions (meiosis I and meiosis II) to become a haploid gamete. Notably, oocyte meiotic progression includes two rounds of unique meiotic arrest and resumption. The first arrest occurs at the G2 (germinal vesicle) stage and meiosis resumption is stimulated by a gonadotropin surge; the second arrest takes place at the metaphase II stage, the stage from which it is released when fertilization takes place. The maturation-promoting factor, which consists of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDK1), is responsible for regulating meiotic resumption and progression, while CDK1 is the unique CDK that acts as the catalytic subunit of maturation-promoting factor. Recent studies showed that except for cyclin B1, multiple cyclins interact with CDK1 to form complexes, which are involved in the regulation of meiotic progression at different stages. Here, we review and discuss the control of oocyte meiotic progression by cyclins A1, A2, B1, B2, B3, and O.

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.

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