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.

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.

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.

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.

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.

Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 440-445 ◽  
Author(s):  
Maria Eugenia Ortiz ◽  
Marta Inés Bühler ◽  
Liliana Isabel Zelarayán
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Nordihydroguaiaretic Acid ◽  
Ovarian Response ◽  
Similar Response ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Cox Inhibitors ◽  
Rhinella Arenarum ◽  
Dose Dependent

SummaryIn Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism – phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.

scholarly journals 290 MODULATION OF ARYLHYDROCARBON RECEPTOR ACTIVITY DURING IN VITRO MATURATION OF BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 295
Author(s):  
D. Nestler ◽  
M. Risch ◽  
B. Fischer ◽  
P. Pocar
Keyword(s):  
Oocyte Maturation ◽  
Target Genes ◽  
Critical Role ◽  
Germinal Vesicle ◽  
Protein Synthesis Inhibitor ◽  
Bovine Oocyte ◽  
Meiotic Arrest ◽  
Germinal Vesicle Breakdown ◽  
Arylhydrocarbon Receptor

The arylhydrocarbon receptor (AhR), a ligand activated transcription factor, has been extensively characterized from a toxicological point of view due to its ability to mediate the adverse effects of a variety of halogenated aromatic hydrocarbons. Recent reports on AhR knockout mice suggest that the AhR may play a role in ovarian physiology. We have previously demonstrated that AhR activity (as indicated by the up-regulation of the target gene cytochrome p450 1A1: CYP1A1) is stimulated during bovine oocyte IVM in the absence of exogenous ligands. Furthermore, exposure to specific AhR antagonists, besides down-regulating the expression of CYP1A1, significantly impairs the ability of the oocyte to complete maturation until the metaphase II stage (Pocar et al. 2004 Endocrinology 145, 1594–1601). The aim of the present study was to further investigate the mechanisms underlying the AhR activation during IVM. Several reports point to a critical role of phosphorylation in the regulation of the AhR-complex. Furthermore, the mitogen-activated protein kinase (MAPK, extracellular regulated kinase (ERK 1 and 2)) cascade has been shown to play a crucial role in regulating meiotic cell cycles during bovine oocyte maturation. A total of 572 bovine cumulus-oocyte complexes were used to investigate the potential role of the MAPK in modulating the activity of the AhR during IVM. The effect of the broad-spectrum serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the induction of CYP1A1 during oocyte maturation was investigated. As expected, exposure to 6-DMAP induced meiotic arrest (at the stage of germinal vesicle/germinal vesicle breakdown) and down-regulated the expression level of phosphorylated ERK 1 and 2. Interestingly, a significant down-regulation of the target genes CYP1A1 and CYP1B1 (9.5% and 26.8% of control, respectively) and an up-regulation of the AhR (199.4% of control) were observed at the mRNA level. This phenomenon was partially reversible after a period of further 24 h of culture in the absence of 6-DMAP. In this condition, besides a recovery of oocyte maturation and phosphorylation status of ERK 1 and 2 to levels comparable to control, a significant up-regulation of CYP1A1 mRNA was observed (68.3% of control). Finally, to confirm the role of serine/threonine kinases in modulating the activity of AhR during resumption of meiosis, we exposed the oocytes to cycloheximide, a protein synthesis inhibitor, also known to arrest oocyte maturation. Furthermore, although cycloheximide exposure induced meiotic arrest, no significant differences in the expression levels of AhR or its target genes compared to control were observed. Each experiment was replicated at least three times. Data were assessed using ANOVA followed by Duncans multiple rance test. The criterion for significance was set at P < 0.05. In conclusion, our results strongly suggest that 6-DMAP-sensitive kinase(s) is (are) involved in the regulation of AhR during bovine oocyte maturation. Further analyses are necessary to understand the biological significance of these observations.

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.

Role of prolactin in nuclear maturation and ovulation in amphibian oocytes

Zygote ◽  
2005 ◽  
Vol 13 (3) ◽  
pp. 265-268
Author(s):  
Inés Ramos ◽  
Susana Cisint ◽  
Marcela F. Medina ◽  
Claudia A. Crespo ◽  
Silvia N. Fernández
Keyword(s):  
Oocyte Maturation ◽  
Incubation Medium ◽  
Germinal Vesicle ◽  
Sexual Cycle ◽  
Breeding Period ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Bufo Arenarum

The present study was undertaken to determine the effect of prolactin (Prl) on Bufo arenarum oocyte maturation and ovulation, two characteristic events of the breeding period, the stage of the sexual cycle in which gamete growth is complete. We observed that Prl, at the doses assayed, did not affect nuclear maturation per se. In addition, when follicles were pretreated with Prl and progesterone was later added to the medium as a physiological nuclear maturation inducer, the percentage of germinal vesicle breakdown obtained with the steroid was unaffected by Prl. The analysis of the in vitro ovulation process demonstrated that pituitary homologous homogenate (PHH) produced a dose- and month-dependent stimulating effect. The maximum percentage of ovulated oocytes was obtained from the end of July to October, the period in which oviposition naturally occurs. Prl by itself did not affect the ovulation process, but when both the hormone and PHH were present in the incubation medium, a significant increase in ovulated oocytes was observed. The results suggest that Prl does not participate in oocyte maturation; however, its presence in the incubation medium would increase oocyte sensitivity to the action of the physiological ovulation inducers.

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