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 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.

scholarly journals Cytoplasmic polyadenylation controls cdc25B mRNA translation in rat oocytes resuming meiosis

Reproduction ◽  
2006 ◽  
Vol 132 (1) ◽  
pp. 21-31 ◽  
Author(s):  
Eran Gershon ◽  
Dalia Galiani ◽  
Nava Dekel
Keyword(s):  
Neutralizing Antibodies ◽  
Germinal Vesicle ◽  
Mrna Translation ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Germinal Vesicle Breakdown ◽  
Cytoplasmic Polyadenylation ◽  
Dual Specificity ◽  
M Phase ◽  
Mrna Polyadenylation

Resumption of meiosis in oocytes represents the entry into M-phase of the cell cycle and is regulated by the maturation-promoting factor (MPF). Activation of MPF is catalyzed by the dual specificity phosphatase, cdc25. In mammals, cdc25 is represented by a multigene family consisting of three isoforms: A, B and C. A recent report that female mice lacking cdc25B exhibit impaired fertility suggests a role for this isoform in regulating the G2- to M-transition in mammalian oocytes. Supporting the above-mentioned observation, we demonstrate herein that microinjection of neutralizing antibodies against cdc25B interfered with the ability of rat oocytes to undergo germinal vesicle breakdown (GVB). We also show accumulation of cdc25B in GVB oocytes and a transient reduction in its amount at metaphase I of meiosis. The accumulation of cdc25B was associated with its mRNA cytoplasmatic polyadenylation and was prevented by the protein synthesis inhibitor cyclohexamide as well as by the polyadenylation inhibitor cordycepin. Immunofluorescence staining revealed translocation of cdc25B to the metaphase II spindle apparatus. Taken together, our findings provide evidence that cdc25B is involved in resumption of meiosis in rat oocytes. We further demonstrate for the first time, a periodic accumulation of cdc25B throughout meiosis that is translationally regulated and involves cdc25B mRNA polyadenylation.

scholarly journals A nuclear factor required for specific translation of cyclin B may control the timing of first meiotic cleavage in starfish oocytes.

1993 ◽  
Vol 4 (12) ◽  
pp. 1295-1306 ◽  
Author(s):  
S Galas ◽  
H Barakat ◽  
M Dorée ◽  
A Picard
Keyword(s):  
Protein Synthesis ◽  
Metaphase Plate ◽  
Germinal Vesicle ◽  
Cyclin B ◽  
Protein Synthesis Inhibitor ◽  
Nuclear Factor ◽  
Synthesis Inhibitor ◽  
Cdc2 Kinase ◽  
Hormonal Stimulation ◽  
Germinal Vesicle Breakdown

In most animals, the rate of cyclin B synthesis increases after nuclear envelope breakdown during the first meiotic cell cycle. We have found that cyclin B-cdc2 kinase activity drops earlier in emetine-treated than in control starfish oocytes, although the protein synthesis inhibitor does not activate the cyclin degradation pathway prematurely. Moreover, protein synthesis is required to prevent meiotic cleavage to occur prematurely, sometimes before chromosomes have segregated on the metaphase plate. In normal conditions, increased synthesis of cyclin B after germinal vesicle breakdown (GVBD) balances cyclin degradation and increases the time required for cyclin B-cdc2 kinase to drop below the level that inhibits cleavage. Taken together, these results point to cyclin B as a possible candidate that could explain the need for increased protein synthesis during meiosis I. Although direct experimental evidence was not provided in the present work, cyclin B synthesis after GVBD may be important for correct segregation of homologous chromosomes at the end of first meiotic metaphase, as shown by a variety of cytological disorders that accompany premature cleavage. Although the overall stimulation of protein synthesis because of cdc2 kinase activation is still observed in oocytes from which the germinal vesicle has been removed before hormonal stimulation, the main increase of cyclin B synthesis normally observed after germinal vesicle breakdown is suppressed. The nuclear factor required for specific translation of cyclin B after GVBD is not cyclin B mRNA, as shown by using a highly sensitive reverse transcription followed by polymerase chain reaction procedure that failed to detect any cyclin B mRNA in isolated germinal vesicles.

Manganese inhibits spontaneous nuclear maturation in bovine cumulus-enclosed oocytes

2001 ◽  
Vol 81 (2) ◽  
pp. 223-228 ◽  
Author(s):  
Sylvie Bilodeau-Goeseels
Keyword(s):  
Follicular Fluid ◽  
Germinal Vesicle ◽  
Atomic Absorption Spectrophotometry ◽  
Inhibitory Effect ◽  
Manganese Concentration ◽  
Maturation Stage ◽  
Bovine Oocyte ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Oocyte Meiosis

The objective of this work was to examine the effects of manganese concentration on nuclear maturation of bovine cumulus-enclosed (CEO) and denuded oocytes cultured for 7 h or 22 h. Following culture, oocytes were then fixed and stained for assessment of nuclear maturation stage. The addition of MnCl2 significantly suppressed nuclear maturation after 7h of culture (15, 69, 84 and 70% of oocytes were still at the germinal vesicle stage after culture with 0, 50 μM, 0.5 mM and 5mM MnCl2, respectively; P < 0.001). However, MnCl2 was without significant effect on denuded oocytes cultured for 7 h. When CEO and denuded oocytes were cultured with manganese for 22 h, the percentages of mature oocytes were reduced (96 , 20, 15, 3% and 80, 39, 53 and 16% for CEO and denuded oocytes cultured with 0, 50 μM, 0.5 mM and 5 mM MnCl2, respectively; P < 0.0005). The inhibitory effect of 50 μM MnCl2 was transient and reversible because it did not maintain oocytes in meiotic arrest after 22 h of culture. In addition, 72% of the CEO cultured with 50 μM MnCl2 for 7 h and subsequently cultured without manganese for 18 h were mature. The concentration of manganese (6 ± 1 μM) in follicular fluid (as determined by atomic absorption spectrophotometry) was below inhibitory concentrations. In conclusion, manganese inhibited germinal vesicle breakdown in bovine CEO; however, only the effect of the lowest concentration tested (50 μM) was reversible. Key words: Bovine, oocyte, meiosis, manganese, follicular fluid

Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii)

1999 ◽  
Vol 11 (5) ◽  
pp. 247 ◽  
Author(s):  
Karen E. Mate ◽  
Janine M. Buist
Keyword(s):  
Kinase Inhibitor ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Tammar Wallaby ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Macropus Eugenii ◽  
Nuclear Maturation

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42–48 h in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 g mL –1 porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4–8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18–30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro. The addition of cycloheximide, a protein synthesis inhibitor, at concentrations of 1–100 g mL –1 , prevented maturation of tammar wallaby oocytes in vitro. This effect was reversible, as oocytes washed free of cycloheximide after 4 h of incubation were able to progress to MII. The addition of cycloheximide to wallaby oocytes at MI of meiosis prevented normal progression to MII suggesting that proteins critical for nuclear maturation are synthesized throughout the maturation process. Genistein, a protein kinase inhibitor decreased maturation of wallaby oocytes in a dose dependent manner. However, the concentration required to significantly inhibit maturation of wallaby oocytes (60 g mL –1 ) was greater than that required for eutherian species. Most wallaby oocytes were able to undergo germinal vesicle breakdown (GVBD) in the presence of high concentrations of genistein but produced abnormal chromatin configurations and were unable to progress to MII. Future studies will examine whether cytoplasmic changes occur in marsupial oocytes in vitro and their temporal relationship to nuclear maturation.

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.

MAPK/ERK kinase (MEK) signalling is required for resumption of meiosis in cultured cumulus-enclosed pig oocytes

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 7-16 ◽  
Author(s):  
B. Meinecke ◽  
C. Krischek
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Mek Inhibitor ◽  
Free Medium ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Mitogen Activated Protein ◽  
Drug Free ◽  
Medium Culture

Resumption of meiosis of mammalian oocytes is facilitated by the maturation promoting factor (MPF) and accompanied by activation of mitogen activated protein kinases (MAPK) which are phosphorylated by the MAPK kinase (MEK). In this study we examined the effects of PD 98059, which inhibits the activity of MEK, on in vitro maturation of pig oocytes. Cumulus-oocyte complexes (COCs) were cultured in the presence or absence of the drug (50 μM) for various time periods. To elucidate the influence of cumulus cells, COCs were first cultured in inhibitor-free medium, subsequently denuded, and incubated further in PD 98059 supplemented medium. Reversibility of drug action as tested following PD 98059 treatment of COCs by transferring them to drug-free medium. Culture of COCs in medium supplemented with PD 98059 prevents resumption of nuclear maturation in the majority of COCs. This inhibition was reversible and accompanied by a non-activation of both MAP and MPF. Addition of the MEK inhibitor to extracts of in vitro matured oocytes revealed that the kinase activities were not directly influenced by the inhibitor, suggesting a link between MAP and MPF kinases. Preincubation of COCs in inhibitor-free medium for 6 h followed by further culture of COCs or denuded oocytes in the presence of PD 98059 for various periods resulted in elevated MAP and MPF kinase activities, indicating an early and transient MEK signalling in the oocyte itself. These results support the idea that MAP and MPF are involved in the induction of germinal vesicle breakdown in porcine oocytes.

Nutrient pathways regulating the nuclear maturation of mammalian oocytes

2015 ◽  
Vol 27 (4) ◽  
pp. 572 ◽  
Author(s):  
Stephen M. Downs
Keyword(s):  
Cell Metabolism ◽  
Polar Body ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cell Types ◽  
Mature Oocyte ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation

Oocyte maturation is defined as that phase of development whereby a fully grown oocyte reinitiates meiotic maturation, completes one meiotic division with extrusion of a polar body, then arrests at MII until fertilisation. Completion of maturation depends on many different factors, not the least of which is the proper provision of energy substrates to fuel the process. Interaction of the oocyte and somatic compartment of the follicle is critical and involves numerous signals exchanged between the two cell types in both directions. One of the prominent functions of the cumulus cells is the channelling of metabolites and nutrients to the oocyte to help stimulate germinal vesicle breakdown and direct development to MII. This entails the careful integration and coordination of numerous metabolic pathways, as well as oocyte paracrine signals that direct certain aspects of cumulus cell metabolism. These forces collaborate to produce a mature oocyte that, along with accompanying physiological changes called cytoplasmic maturation, which impart subsequent developmental competence to the oocyte, can be fertilised and develop to term. This review focuses on nuclear maturation and the metabolic interplay that regulates it, with special emphasis on data generated in the mouse.

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.

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