Active maturation-promoting factor is present in mature mouse oocytes.

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.

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Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis.

The Journal of Cell Biology ◽

10.1083/jcb.106.5.1445 ◽

1988 ◽

Vol 106 (5) ◽

pp. 1445-1452 ◽

Cited By ~ 11

Author(s):

R C Adlakha ◽

G L Shipley ◽

J Y Zhao ◽

K B Jones ◽

D A Wright ◽

...

Keyword(s):

Cell Cycle ◽

Hela Cells ◽

Physarum Polycephalum ◽

Germinal Vesicle ◽

Slime Mold ◽

G1 Phase ◽

Affinity Column ◽

Factor Activity ◽

Germinal Vesicle Breakdown ◽

Maturation Promoting Factor

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.

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Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1713 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1713-1717 ◽

Cited By ~ 20

Author(s):

R S Freeman ◽

S M Ballantyne ◽

D J Donoghue

Keyword(s):

Xenopus Laevis ◽

Antisense Oligonucleotide ◽

Xenopus Oocytes ◽

Germinal Vesicle ◽

Cyclin B1 ◽

Cyclin B ◽

Germinal Vesicle Breakdown ◽

The Relationship ◽

Maturation Promoting Factor

We have investigated the relationship between Xenopus laevis c-mos (mosXe) and the cyclin B component of maturation-promoting factor. Microinjection of Xenopus oocytes with in vitro-synthesized RNAs encoding Xenopus cyclin B1 or cyclin B2 induces the progression of meiosis, characterized by germinal vesicle breakdown (GVBD). By preinjecting oocytes with a mosXe-specific antisense oligonucleotide, we show that GVBD induced by cyclin B does not require expression of the mosXe protein. GVBD induced by cyclin B proceeds significantly faster than GVBD induced by progesterone or MosXe. However, coinjection of RNAs encoding cyclin B1 or cyclin B2 with mosXe RNA results in a 2.5- to 3-fold acceleration in GVBD relative to that induced by cyclin B alone. This acceleration of GVBD does not correlate with changes in the level of cyclin B1 and cyclin B2 phosphorylation.

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Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1713-1717.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1713-1717

Author(s):

R S Freeman ◽

S M Ballantyne ◽

D J Donoghue

Keyword(s):

Xenopus Laevis ◽

Antisense Oligonucleotide ◽

Xenopus Oocytes ◽

Germinal Vesicle ◽

Cyclin B1 ◽

Cyclin B ◽

Germinal Vesicle Breakdown ◽

The Relationship ◽

Maturation Promoting Factor

We have investigated the relationship between Xenopus laevis c-mos (mosXe) and the cyclin B component of maturation-promoting factor. Microinjection of Xenopus oocytes with in vitro-synthesized RNAs encoding Xenopus cyclin B1 or cyclin B2 induces the progression of meiosis, characterized by germinal vesicle breakdown (GVBD). By preinjecting oocytes with a mosXe-specific antisense oligonucleotide, we show that GVBD induced by cyclin B does not require expression of the mosXe protein. GVBD induced by cyclin B proceeds significantly faster than GVBD induced by progesterone or MosXe. However, coinjection of RNAs encoding cyclin B1 or cyclin B2 with mosXe RNA results in a 2.5- to 3-fold acceleration in GVBD relative to that induced by cyclin B alone. This acceleration of GVBD does not correlate with changes in the level of cyclin B1 and cyclin B2 phosphorylation.

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Induction of DNA replication in germinal vesicles and in nuclei formed in maturing mouse oocytes by 6-DMAP treatment

Zygote ◽

10.1017/s0967199400003646 ◽

1997 ◽

Vol 5 (3) ◽

pp. 213-217 ◽

Cited By ~ 3

Author(s):

J. Fulka ◽

N.L. First ◽

C. Lee ◽

J. Fulka ◽

R.M. Moor

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Germinal Vesicle ◽

The Other ◽

Immature Oocytes ◽

Mouse Oocytes ◽

Germinal Vesicles ◽

Immature Mouse ◽

Condensed Dna ◽

Metaphase Ii Oocytes

SummaryImmature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

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Replicating DNA does not block germinal vesicle breakdown in mouse oocytes

Journal of Experimental Zoology ◽

10.1002/jez.1402720310 ◽

1995 ◽

Vol 272 (3) ◽

pp. 245-248 ◽

Cited By ~ 12

Author(s):

J. Fulka ◽

R. M. Moor ◽

J. Fulka

Keyword(s):

Germinal Vesicle ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3192 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3192-3203 ◽

Cited By ~ 22

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.

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Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽

10.1242/dev.112.4.971 ◽

1991 ◽

Vol 112 (4) ◽

pp. 971-980 ◽

Cited By ~ 1

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.310-315.1990 ◽

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.

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