scholarly journals Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APCCdh1

2006 ◽  
Vol 176 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Petros Marangos ◽  
Emmy W. Verschuren ◽  
Ruby Chen ◽  
Peter K. Jackson ◽  
John Carroll
Keyword(s):  
Meiotic Division ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Germinal Vesicle Breakdown ◽  
Prophase I ◽  
Mouse Oocytes ◽  
Dependent Inhibition ◽  
Morpholino Oligonucleotides ◽  
Mitotic Inhibitor

Mammalian oocytes are arrested in prophase of the first meiotic division. Progression into the first meiotic division is driven by an increase in the activity of maturation-promoting factor (MPF). In mouse oocytes, we find that early mitotic inhibitor 1 (Emi1), an inhibitor of the anaphase-promoting complex (APC) that is responsible for cyclin B destruction and inactivation of MPF, is present at prophase I and undergoes Skp1–Cul1–F-box/βTrCP-mediated destruction immediately after germinal vesicle breakdown (GVBD). Exogenous Emi1 or the inhibition of Emi1 destruction in prophase-arrested oocytes leads to a stabilization of cyclin B1–GFP that is sufficient to trigger GVBD. In contrast, the depletion of Emi1 using morpholino oligonucleotides increases cyclin B1–GFP destruction, resulting in an attenuation of MPF activation and a delay of entry into the first meiotic division. Finally, we show that Emi1-dependent effects on meiosis I require the presence of Cdh1. These observations reveal a novel mechanism for the control of entry into the first meiotic division: an Emi1-dependent inhibition of APCCdh1.

scholarly journals The dynamics of cyclin B1 distribution during meiosis I in mouse oocytes

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 153-162 ◽  
Author(s):  
Petros Marangos ◽  
John Carroll
Keyword(s):  
Fluorescent Protein ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
Living Mouse ◽  
Green Fluorescent ◽  
Meiosis I

Cdk1-cyclin B1 kinase activity drives oocytes through meiotic maturation. It is regulated by the phosphorylation status of cdk1 and by its spatial organisation. Here we used a cyclin B1-green fluorescent protein (GFP) fusion protein to examine the dynamics of cdk1-cyclin B1 distribution during meiosis I (MI) in living mouse oocytes. Microinjection of cyclin B1-GFP accelerated germinal vesicle breakdown (GVBD) and, as previously described, overrides cAMP-mediated meiotic arrest. GVBD was pre-empted by a translocation of cyclin B1-GFP from the cytoplasm to the germinal vesicle (GV). After nuclear accumulation, cyclin B1-GFP localised to the chromatin. The localisation of cyclin B1-GFP is governed by nuclear import and export. In GV intact oocytes, cyclin export was demonstrated by showing that cyclin B1-GFP injected into the GV is exported to the cytoplasm while a similar size dextran is retained. Import was revealed by the finding that cyclin B1-GFP accumulated in the GV when export was inhibited using leptomycin B. These studies show that GVBD in mouse oocytes is sensitive to cyclin B1 abundance and that the changes in distribution of cyclin B1 contribute to progression through MI.

scholarly journals YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Alaa A. Eisa ◽  
Santanu De ◽  
Ariana Detwiler ◽  
Eva Gilker ◽  
Alexander C. Ignatious ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Oocyte Maturation ◽  
Messenger Rna ◽  
Germinal Vesicle ◽  
Protein Isoforms ◽  
Cell Cycle Protein ◽  
Meiotic Arrest ◽  
Germinal Vesicle Breakdown ◽  
Prophase I ◽  
Mouse Oocytes

Abstract Background Immature mammalian oocytes are held arrested at prophase I of meiosis by an inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1). Release from this meiotic arrest and germinal vesicle breakdown is dependent on dephosphorylation of CDK1 by the protein, cell cycle division 25B (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the cytoplasm of immature oocytes and is thus maintained in an inactive form. The importance of YWHA in meiosis demands additional studies. Results Messenger RNA for multiple isoforms of the YWHA protein family was detected in mouse oocytes and eggs. All seven mammalian YWHA isoforms previously reported to be expressed in mouse oocytes, were found to interact with CDC25B as evidenced by in situ proximity ligation assays. Interaction of YWHAH with CDC25B was indicated by Förster Resonance Energy Transfer (FRET) microscopy. Intracytoplasmic microinjection of oocytes with R18, a known, synthetic, non-isoform-specific, YWHA-blocking peptide promoted germinal vesicle breakdown. This suggests that inhibiting the interactions between YWHA proteins and their binding partners releases the oocyte from meiotic arrest. Microinjection of isoform-specific, translation-blocking morpholino oligonucleotides to knockdown or downregulate YWHA protein synthesis in oocytes suggested a role for a specific YWHA isoform in maintaining the meiotic arrest. More definitively however, and in contrast to the knockdown experiments, oocyte-specific and global deletion of two isoforms of YWHA, YWHAH (14-3-3 eta) or YWHAE (14-3-3 epsilon) indicated that the complete absence of either or both isoforms does not alter oocyte development and release from the meiotic prophase I arrest. Conclusions Multiple isoforms of the YWHA protein are expressed in mouse oocytes and eggs and interact with the cell cycle protein CDC25B, but YWHAH and YWHAE isoforms are not essential for normal mouse oocyte maturation, fertilization and early embryonic development.

scholarly journals Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes

2021 ◽  
Author(s):  
Natsumi Takei ◽  
Keisuke Sato ◽  
Yuki Takada ◽  
Rajan Iyyappan ◽  
Andrej Susor ◽  
...  
Keyword(s):  
Translational Control ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Spindle Formation ◽  
Rna Granules ◽  
Mouse Oocytes ◽  
Oocyte Meiosis ◽  
Tudor Domain ◽  
Mitotic Inhibitor ◽  
Meiosis I

ABSTRACTAfter completion of meiosis I, the oocyte immediately enters meiosis II and forms a metaphase II (MII) spindle without an interphase, which is fundamental for generating a haploid gamete. Here, we identify tudor domain-containing protein 3 (Tdrd3) as a novel regulator of oocyte meiosis. Although early mitotic inhibitor 2 (Emi2) protein has been shown to ensure the meiosis I to II transition and the subsequent MII spindle formation by inhibiting the anaphase-promoting complex/cyclosome (APC/C), how it accumulates after meiosis I has remained unresolved. We isolated Tdrd3 as a protein directly binding to Emi2 mRNA. In GV-stage mouse oocytes, Emi2 mRNA assembled into RNA granules containing Tdrd3, while cyclin B1 mRNA, which was translated in early meiosis I, formed different granules. Knockdown of Tdrd3 attenuated Emi2 synthesis in meiosis II without affecting cyclin B1 synthesis in meiosis I. Moreover, Tdrd3-deficient oocytes entered interphase and failed to form an MII spindle after completion of meiosis I. Taken together, our results indicate the importance of Tdrd3-mediated translational control of Emi2 mRNA, which promotes Emi2 synthesis in meiosis II, for the progression of meiosis.

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 Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

2006 ◽  
Vol 174 (6) ◽  
pp. 791-801 ◽  
Author(s):  
Suzanne Madgwick ◽  
David V. Hansen ◽  
Mark Levasseur ◽  
Peter K. Jackson ◽  
Keith T. Jones
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
Morpholino Knockdown ◽  
Short Time ◽  
Mitotic Inhibitor ◽  
Antisense Morpholino ◽  
Meiosis I

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 971-980 ◽  
Author(s):  
H. Alexandre ◽  
A. Van Cauwenberge ◽  
Y. Tsukitani ◽  
J. Mulnard
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Chromatin Condensation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

The Role of Tissue Transglutaminase in the Germinal Vesicle Breakdown of Mouse Oocytes

2001 ◽  
Vol 286 (2) ◽  
pp. 229-234 ◽  
Author(s):  
Sung Woo Kim ◽  
Zee-Won Lee ◽  
ChangKyu Lee ◽  
Kyung Soon Im ◽  
Kwon-Soo Ha
Keyword(s):  
Tissue Transglutaminase ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

032. Cdh1: A CELL CYCLE PROTEIN INVOLVED IN FEMALE MEIOSIS AND PREVENTION OF ANEUPLOIDY

2010 ◽  
Vol 22 (9) ◽  
pp. 10
Author(s):  
K. T. Jones
Keyword(s):  
Meiotic Division ◽  
Polar Body ◽  
Single Copy ◽  
Cyclin B1 ◽  
Cell Cycle Protein ◽  
Prophase I ◽  
First Polar Body ◽  
A Cell

Mammalian oocytes are arrested at the dictyate stage of prophase I in the ovary. In growing follicles, oocytes can become responsive to Luteinising Hormone and will undergo meiotic resumption just before ovulation. During the first meiotic division, homologous chromosomes are segregated, a process that is very error prone in human oocytes. By ovulation the oocyte has extruded its first polar body and has re-arrested at metaphase of the first meiotic division. Recent work from our lab has established that the protein Cdh1 is involved uniquely in both in the process of prophase I arrest and the correct segregation of homologs in meiosis I. Thus in cultured oocytes, in vitro antisense knockdown of Cdh1 induces both meiotic resumption and high rates of aneuploidy as a result of non-disjunction during first meiosis. Cdh1 causes prophase I arrest by inducing cyclin B1 degradation and maintaining low levels of the kinase CDK1, whose activity induces meiotic resumption. Cdh1 is an activator of the Anaphase-Promoting Complex (APC), a ubiquitin ligase that earmarks proteins such as cyclin B1 for proteolysis. Cdh1 prevents aneuploidy by causing the degradation of Cdc20, a protein that is responsible for activating the APC once all homologs are correctly aligned at metaphase. Thus loss of Cdh1 seems to prematurely activate APC(Cdc20) activity. It is interesting that a single protein can affect two important meiotic transitions in oocytes. However to explore its functions more fully, and confirm that an in vitro knockdown is faithfully replicated by in vivo loss, a targeted knockout of Cdh1 is needed. Therefore we have generated an oocyte specific Cdh1 knockout by ZP3 promoter driven Cre- recombinase activity in oocytes carrying loxP insertions in the single copy Cdh1 gene. This talk will therefore focus on the effects of an in vivo Cdh1 knockout.

The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Jaroslav Kalous ◽  
Michal Kubelka ◽  
Jan Motlík
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Experimental Conditions ◽  
Germinal Vesicle Breakdown ◽  
Mapk Activation ◽  
Mapk Phosphorylation ◽  
Kinase Activation ◽  
Mouse Oocytes ◽  
Significant Difference ◽  
Epidermal Growth

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 μM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocyte–cumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 μM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.

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