scholarly journals Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation.

1995 ◽  
Vol 6 (9) ◽  
pp. 1111-1124 ◽  
Author(s):  
J Li ◽  
A N Meyer ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Oocyte Maturation ◽  
Xenopus Oocyte ◽  
Biological Significance ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Site Directed Mutagenesis ◽  
Germinal Vesicle Breakdown ◽  
Rapid Kinetics ◽  
Phosphopeptide Mapping

Maturation-promoting factor, consisting of cdc2 protein kinase and a regulatory B-type cyclin, is a universal regulator of meiosis and mitosis in eukaryotes. In Xenopus, there are two subtypes of B-type cyclins, designated B1 and B2, both of which are phosphorylated. In this study, we have investigated the biological significance of this phosphorylation for Xenopus cyclin B1 during meiotic maturation. We have used a combination of site-directed mutagenesis and phosphopeptide-mapping to identify serine residues 2, 94, 96, 101, and 113 as presumptive phosphorylation sites, and together these sites account for all cyclin B1 phosphorylation in oocytes before germinal vesicle breakdown (GVBD). Single Ser-->Ala mutants as well as multiple site mutants have been constructed and characterized. Phosphorylation of cyclin B1 appears to be required for Xenopus oocyte maturation, based on the significantly diminished ability of the quintuple Ala mutant to induce oocyte maturation. Furthermore, partial phosphorylation of these five sites is sufficient to meet this requirement. Phosphorylation of cyclin B1 is not required for cdc2 kinase activity, for binding to cdc2 protein, for stability of cyclin B1 before GVBD, or for destruction of cyclin B1 after GVBD or after egg activation. A quintuple Glu mutant was also constructed, with serine residues 2, 94, 96, 101, and 113 mutated to Glu. In contrast to the quintuple Ala mutant, the quintuple Glu mutant was able to induce oocyte maturation efficiently, and with more rapid kinetics than wild-type cyclin B1. These data confirm that phosphorylation, as mimicked by Ser-->Glu mutations, confers enhanced biological activity to cyclin B1. Possible roles of cyclin B1 phosphorylation are discussed that might account for the increased biological activity of the quintuple Glu mutant.

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.

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.

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.

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 On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis.

1991 ◽  
Vol 114 (4) ◽  
pp. 755-765 ◽  
Author(s):  
H Kobayashi ◽  
J Minshull ◽  
C Ford ◽  
R Golsteyn ◽  
R Poon ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Oocyte Maturation ◽  
Posttranslational Modifications ◽  
Germinal Vesicle ◽  
Stage Iv ◽  
Cyclin B1 ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Previous Level ◽  
Made In

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.

PLCz-induced Ca2+ oscillations are enhanced after germinal vesicle breakdown during mouse oocyte maturation

2016 ◽  
Author(s):  
Jessica Sanders ◽  
Ethan Bateson ◽  
Yuansong Yu ◽  
Michail Nomikos ◽  
Antony Lai ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Mouse Oocyte ◽  
Germinal Vesicle Breakdown

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase

1993 ◽  
Vol 13 (11) ◽  
pp. 6653-6660
Author(s):  
L M Chuang ◽  
M G Myers ◽  
J M Backer ◽  
S E Shoelson ◽  
M F White ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Sh2 Domain ◽  
Glutathione S Transferase ◽  
Hormonal Stimulation ◽  
Germinal Vesicle Breakdown ◽  
Sh2 Domains ◽  
Igf I ◽  
Phospholipase C Gamma

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.

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.

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