Behavior of γ-tubulin during spindle formation in Xenopus oocytes: requirement of cytoplasmic dynein-dependent translocation

Zygote ◽  
2005 ◽  
Vol 13 (3) ◽  
pp. 219-226 ◽  
Author(s):  
Tomoya Kotani ◽  
Masakane Yamashita
Keyword(s):  
Oocyte Maturation ◽  
Late Stage ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Germinal Vesicle Breakdown ◽  
Spindle Formation ◽  
Bipolar Spindle

Vertebrate oocytes do not contain centrosomes and therefore form an acentrosomal spindle during oocyte maturation. γ-Tubulin is known to be essential for nucleation of microtubules at centrosomes, but little is known about the behaviour and role of γ-tubulin during spindle formation in oocytes. We first observed sequential localization of γ-tubulin during spindle formation in Xenopus oocytes. γ-Tubulin assembled in the basal regions of the germinal vesicle (GV) at the onset of germinal vesicle breakdown (GVBD) and remained on the microtubule-organizing centre (MTOC) until a complex of the MTOC and transient-microtubule array (TMA) reached the oocyte surface. Prior to bipolar spindle formation, oocytes formed an aggregation of microtubules and γ-tubulin was concentrated at the centre of the aggregation. At the late stage of bipolar spindle formation, γ-tubulin accumulated at each pole. Anti-dynein antibody disrupted the localization of γ-tubulin, indicating that the translocation described above is dependent on dynein activity. We finally revealed that XMAP215, a microtubule-associated protein cooperating with γ-tubulin for the assembly of microtubules, but not γ-tubulin, was phosphorylated during oocyte maturation. These results suggest that γ-tubulin is translocated by dynein to regulate microtubule organization leading to spindle formation and that modification of the molecules that cooperate with γ-tubulin, but not γ-tubulin itself, is important for microtubule reorganization.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

254 DYNAMICS OF GOLGI APPARATUS DURING BOVINE OOCYTE IN VITRO MATURATION: EFFECTS OF INHIBITORS ON CDC2A AND CYTOPLASMIC-DYNEIN ATPase ACTIVITY

2009 ◽  
Vol 21 (1) ◽  
pp. 225
Author(s):  
S. E. Racedo ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Golgi Apparatus ◽  
Atpase Activity ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Bovine Oocyte ◽  
Germinal Vesicle Breakdown

The process of maturation encompasses a complex series of molecular and structural events. Completion of the nuclear changes to produce a metaphase II (MII) oocyte does not reflect the molecular and structural maturity of an oocyte, which is sometimes termed cytoplasmic maturation. The Golgi apparatus phosphorylates, fragments, and changes the localization during oocyte maturation. GM130 and phospho-GM130 are used as markers for the Golgi apparatus and phosphorylated Golgi apparatus, respectively. The goal of this study was to analyze the dynamics of the Golgi apparatus and its association with microtubules in bovine oocytes at different stages of in vitro maturation [IVM; i.e. germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and MII]. The roles of CDC2A kinase (also known as p34cdc2) and cytoplasmic-dynein ATPase on Golgi dynamics were studied by using specific inhibitors. The distribution of the markers was assessed by immunocytochemistry and laser confocal microscopy. To unravel the role of CDC2A and cytoplasmic dynein ATPase on the dynamics of the Golgi apparatus, the inhibitors roscovitine (ROS) and sodium-orthovanadate (SOV), respectively, were used. In the first experiment, the nuclear maturation rate was checked in the presence of the inhibitors at different times and for different incubation times to explore whether oocytes were able to reach the MII stage. At the GV and GVBD stages, the Golgi apparatus is observed as fragments named mini-Golgies and at the MI and MII stages as punctate foci throughout the cytoplasm. Our results showed 2 well-defined movements of the Golgi apparatus toward opposite directions, depending on the maturation stage. The first movement was observed between 5 and 9 h of IVM (i.e. the GVBD stage), when the Golgi apparatus relocalized from the ooplasm to the periphery. The second movement was observed between 9 and 15 h of IVM (i.e. the MI stage), when the Golgi apparatus moved from the cortex to throughout the cytoplasm and remained there up to the MII stage. The use of inhibitors on CDC2A and cytoplasmic-dynein ATPase at selected time points revealed that CDC2A played a crucial role on the distribution of this organelle during the first movement, whereas the final localization at the GVBD stage was dependent on cytoplasmic-dynein transport. The second movement of the Golgi apparatus was disturbed by the SOV treatment, but not by the use of ROS, suggesting a role of cytoplasmic-dynein-dependent transport during the distribution and organization of the punctate foci at the MI stage. The phosphorylation status of Golgi was not affected at the different incubation times with inhibitors, except in those oocytes incubated with ROS for 24 h, suggesting a role of CDC2A. In conclusion, we describe the involvement of CDC2A during the first movement of the Golgi apparatus and the importance of cytoplasmic-dynein ATPase activity in the first and second relocalization of Golgi during bovine oocyte maturation. DAAD.

Ha-rasVal-12,Thr-59 activates S6 kinase and p34cdc2 kinase in Xenopus oocytes: evidence for c-mosxe-dependent and -independent pathways

1990 ◽  
Vol 10 (1) ◽  
pp. 310-315
Author(s):  
C B Barrett ◽  
R M Schroetke ◽  
F A Van der Hoorn ◽  
S K Nordeen ◽  
J L Maller
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Antisense Oligonucleotides ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Protein Product ◽  
Germinal Vesicle Breakdown ◽  
S6 Kinase ◽  
Kinase Activation ◽  
Histone Kinase

Treatment with insulin or progesterone or microinjection of the transforming protein product of Ha-rasVal-12,Thr-59 (p21) is known to induce germinal vesicle breakdown in Xenopus oocytes. We have investigated the effect of p21 on S6 kinase and the H1 histone kinase of maturation-promoting factor in the presence and absence of antisense oligonucleotides against the c-mosxe proto-oncogene. Injection of p21 led to a rapid increase in S6 phosphorylation, with kinetics similar to those previously observed with insulin. Microinjection of c-mosxe antisense oligonucleotides inhibited germinal vesicle breakdown induced by p21 and totally abolished S6 kinase activation by insulin or progesterone but only partially inhibited activation by p21. However, the activation of p34cdc2 protein kinase by all three stimuli was blocked by antisense oligonucleotides. The results suggest that in oocyte maturation c-mosxe functions downstream of p21 but upstream of p34cdc2 and S6 kinase activation, although not all p21-induced events require c-mosxe.

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.

Involvement of catecholamines in the regulation of oocyte maturation in frogs

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 271-281 ◽  
Author(s):  
Inés Ramos ◽  
Susana Cisint ◽  
Claudia A. Crespo ◽  
Marcela F. Medina ◽  
Silvia N. Fernández
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Significant Inhibition ◽  
Follicle Cells ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Dose Dependent ◽  
Metabolic Behaviour

The present study investigates the role of catecholamines in the regulation of Bufo arenarum oocyte maturation. The metabolic changes in the oxidation of carbohydrates and the meiotic resumption evinced by the germinal vesicle breakdown were used as indicators of cytoplasmic and nuclear maturation, respectively. The results obtained suggest that noradrenaline (norepinephrine) could be one of the factors responsible for the metabolic behaviour that characterises cytoplasmically immature oocytes. The use of adrenaline (epinephrine), on the other hand, induced a metabolic change which made oocytes cytoplasmically mature. The effect of both catecholamines, which was dose-dependent, was observed in ovarian oocytes (surrounded by follicle cells) as well as in coelomic oocytes (free from follicle cells), suggesting the presence of adrenergic receptors in the gamete. The results obtained using adrenergic agonists and antagonists suggest that the effect of adrenaline would be due to an interaction with β2-receptors. Although catecholamines have an influence on the determination of the stage of cytoplasmic maturation of the oocytes, they do not affect nuclear maturation by themselves. Nevertheless, pretreatment of follicles with adrenaline caused a significant inhibition in progesterone-induced nuclear maturation even though this effect was markedly weaker when using noradrenaline.

scholarly journals Antioxidants reversibly inhibit the spontaneous resumption of meiosis

1999 ◽  
Vol 276 (4) ◽  
pp. E684-E688 ◽  
Author(s):  
M. Takami ◽  
S. L. Preston ◽  
V. A. Toyloy ◽  
Harold R. Behrman
Keyword(s):  
Oocyte Maturation ◽  
Oxygen Radicals ◽  
Germinal Vesicle ◽  
Nordihydroguaiaretic Acid ◽  
Butylated Hydroxytoluene ◽  
Germinal Vesicle Breakdown ◽  
Tert Butyl ◽  
Tert Butyl Hydroperoxide ◽  
Lauryl Gallate

We previously showed that the cell-permeant antioxidant 2(3)- tert-butyl-4-hydroxyanisole (BHA) inhibited germinal vesicle breakdown (GVBD) in oocyte-cumulus complexes (OCC) of the rat. The objective of the present studies was to assess other antioxidants and whether such inhibition was reversible. Spontaneous GVBD in OCC incubated for 2 h was significantly inhibited ( P < 0.005) by nordihydroguaiaretic acid (NDGA; GVBD = 19.4%), BHA (GVBD = 25.7%), octyl gallate (OG; GVBD = 52.2%), ethoxyquin (EQ; GVBD = 58.8%), 2,6-di- tert-butyl-hydroxymethyl phenol (TBHMP; GVBD = 59%), butylated hydroxytoluene (BHT; GVBD = 59.5%), and tert-butyl hydroperoxide (TBHP; GVBD = 60.0%). Other antioxidants that produced lower but significant ( P < 0.05) inhibition of oocyte maturation included propyl gallate (PG; GVBD = 70.3%), 2,4,5-trihydroxybutrophenone (THBP; GVBD = 71.4%), and lauryl gallate (LG; GVBD = 71.4%). Antioxidants that had no effect on oocyte maturation at the same concentration (100 μM) included ascorbic acid, vitamin E, and Trolox. Inhibition of GVBD was evident for up to 8 h of incubation of OCC and denuded oocytes (DO) with BHA or NDGA and was reversed by washing. NDGA was less potent than BHA for inhibition of GVBD in DO, unlike that seen with OCC. Oocyte maturation was induced by incubation of follicles for 3 h with human chorionic gonadotropin (hCG), and this response was inhibited by BHA or NDGA. These findings support the conclusion that cell-permeant antioxidants inhibit spontaneous resumption of meiosis, which may implicate a role of oxygen radicals in oocyte maturation.

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2129-2139 ◽  
Author(s):  
Marion Peter ◽  
Jean-Claude Labbé ◽  
Marcel Dorée ◽  
Elisabeth Mandart
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Mapk Pathway ◽  
Germinal Vesicle ◽  
Mapk Cascade ◽  
Germinal Vesicle Breakdown ◽  
Mapk Activation

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.

scholarly journals Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts

2005 ◽  
Vol 389 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Tomoya Kotani ◽  
Masakane Yamashita
Keyword(s):  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Middle Part ◽  
Microtubule Organization ◽  
Activity Inhibition ◽  
Green Fluorescent

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-γ-tubulin antibody. The results indicate possible roles of γ-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.

Sign in / Sign up

Export Citation Format

Share Document