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C Thibier

Publications and source records attributed to C Thibier.

17 recordsLinked to original sources
PubMed2000

Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopus oocytes.

Xenopus prophase oocytes reenter meiotic division in response to progesterone. The signaling pathway leading to Cdc2 activation depends on neosynthesized proteins and a decrease in PKA activity. We demonstrate that Eg2 protein, a Xenopus member of the Aurora/Ipl1 family of protein kinases, accumulates in response to progesterone and is degraded after parthenogenetic activation. The polyadenylation and cap ribose methylation of Eg2 mRNA are not needed for the protein accumulation. Eg2 protein accumulation is induced by progesterone through a decrease in PKA activity, upstream of Cdc2 activation. Eg2 kinase activity is undetectable in prophase and is raised in parallel with Cdc2 activation. In contrast to Eg2 protein accumulation, Eg2 kinase activation is under Cdc2 control. Furthermore, by using an anti-sense strategy, we show that Eg2 accumulation is not required in the transduction pathway leading to Cdc2 activation. Altogether, our results strongly suggest that Eg2 is not necessary for Cdc2 activation, though it could participate in the organization of the meiotic spindles, in agreement with the well-conserved roles of the members of the Aurora family, from yeast to man.

Animals↗
PubMed1997

In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes.

Metaphase II arrest of Xenopus oocyte is characterized by the presence of M-phase-promoting factor (MPF) and of a microtubular spindle, both of which are stable in the presence of protein synthesis inhibitors. We studied in vivo this equilibrium state that is settled during meiotic maturation. At time of germinal vesicle breakdown (GVBD), cdc2 kinase and MAP kinase activities are stimulated. A component of the cyclin ubiquitin ligase, CDC27, is phosphorylated at the same time and remains phosphorylated until fertilization, indicating that an important component of the ligase complex is modified as early as GVBD. During a first period extending from GVBD until the cortical anchorage of the metaphase II spindle, homogeneous pools of cdc2 kinase and mitogen-activated protein (MAP) kinase activities are present in oocyte and are strictly dependent on protein turnover, since protein synthesis inhibition induces their total inactivation and drives oocytes into interphase. The metaphase II spindle, once anchored into the cortex, is no more sensitive to protein synthesis inhibition, likewise MAP kinase activity. During this cellular arrest, cdc2 kinase is divided into two distinctly regulated pools. The first one contains cyclin B that actively turns over and is subjected to a microtubular checkpoint. The second one is stable. Alteration of intracellular compartmentation of metaphase II oocytes either by gentle centrifugation or by cold shock inactivates MAP kinase and targets all cyclin B molecules for full destruction. We therefore suggest that MAP kinase participates to the cytostatic activity by preventing part of cyclin B molecules from entering the ubiquitination/degradation machinery which is still turned on in metaphase II oocytes.

Animals↗
PubMed1997

On cyclins, oocytes, and eggs.

Oocyte and egg are suitable model systems for studying cell division since meiotic maturation resembles a G2/M transition and early embryonic divisions are precisely timed and occur without zygotic transcription. The analysis of oocytes and eggs from different species provides the opportunity to understand the roles of proteins that the critical to the progression and maintenance of the cell cycle. Among them, cyclins are certainly worthy of investigation. Mitotic cyclins (cyclins A and B) are clearly implicated in meiosis and early embryonic cell cycles. More recent studies have revealed that G1-type cyclins (cyclins E and D) could also play a role in both processes and cyclin H has been suggesed to participate to CAK activity (cdc2-activating kinase) in oocytes. The study of cyclins in oocytes and eggs clearly offer insights into their roles during the cell cycle.

Animals↗
PubMed1994

Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I.

Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.

Animals↗
PubMed1994

Association of p34cdc2 kinase and MAP kinase with microtubules during the meiotic maturation of Xenopus oocytes.

p34cdc2 protein is found in prophase, metaphase and activated Xenopus oocytes at a similar level whereas its kinase activity oscillates within meiosis. Using an anti-PSTAIRE antibody that recognizes Xenopus p34cdc2, it was demonstrated that the major part of p34cdc2 was associated with microtubules isolated in vitro from Xenopus oocytes. Conversely, tubulin was recovered in association with p34cdc2 in p13-Sepharose pellets. The abundance of the fraction of p34cdc2 which was associated with microtubules did not oscillate during the meiotic maturation and the activation process. By contrast, the histone H1 kinase activity of p34cdc2 estimated in microtubular oocyte pellets was much higher in metaphase than in prophase oocytes. Cyclin B, which is associated in vivo with p34cdc2 in prophase and metaphase oocytes, was also present in the microtubular fractions. However, cyclin was not necessary for the binding of p34cdc2 to microtubules since p34cdc2 from activated eggs, where cyclin was missing, still copurified with microtubules. Purified MAP2, but not tubulin, was able to bind to p34cdc2, demonstrating that the association between p34cdc2 and microtubules was mediated by microtubule-associated proteins. During the meiotic maturation of Xenopus oocytes, several protein kinases were activated, among them MAP kinase. MAP kinase also associated with microtubules. It was demonstrated that both p34cdc2 kinase and MAP kinase purified from Xenopus oocytes were able to phosphorylate in vitro rat brain MAP2. However both protein kinases phosphorylated different domains of MAP2, suggesting that they might regulate microtubules in different ways.

Animals↗
PubMed1992

A predominant basic alpha-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation.

Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four alpha-isoforms and four beta-isoforms. We have identified a basic alpha (alpha b)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 35S methionine labelled oocytes we demonstrate that the disappearance of the alpha b isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this alpha-isoform to a more acidic pI. Moreover, in vitro experiments using 35S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the alpha b-isoform, suggesting that the observed posttranslational modification might be regulated by p34cdc2. However, the nature of this modification remains to be elucidated.

Animals↗
PubMed1991

Intracellular localization of MAP2-related protein (O-map) in prophase I and metaphase II oocytes of Xenopus.

An antibody directed against rat brain microtubule-associated protein 2 (MAP2) immunoprecipitated a protein of 240 kDa from a Xenopus oocyte extract. In contrast, in Xenopus brain extract, this antibody recognizes a protein of 280 kDa. The oocyte MAP2-related protein (called O-MAP) is present in both prophase I and metaphase II-blocked oocytes as demonstrated by immunoblotting experiments; it is in vivo phosphorylated. Immunocytochemical studies using the anti-rat brain MAP2 antibody demonstrated that the O-MAP colocalizes within the cortical microtubular array present in both prophase I and metaphase II oocytes. However, O-MAP is not associated with the microtubular structures which are organized during the oocyte prophase-metaphase transition, i.e., a giant cytoplasmic network and both the meiotic spindles. O-MAP therefore appears as a microtubule-associated protein oocyte specific and may play a role in the regulation of microtubule stability and the organization of the oocyte cytoskeleton.

Animals↗
PubMed1988

Presence of microtubules in isolated cortices of prophase I and metaphase II oocytes in Xenopus laevis.

An extensive array of microtubules has been shown to exist in the cortex of Xenopus laevis oocytes both at the prophase I and metaphase II stages. The cortical microtubules were visualized after the oocyte cortex was squashed and immunostained using anti-tubulin antibody. They are cold- and nocodazole-sensitive; their stability to both treatments decreases after meiotic maturation. Biochemical extraction of manually isolated oocyte cortices, in a microtubule-stabilizing buffer, confirms these cytological observations.

Animals↗
PubMed1988

Taxol reveals cortical sites of microtubule assembly in Xenopus oocytes. Role of the nucleus.

A binding colchicine assay together with an immunostaining study with an anti-tubulin antibody showed that taxol, when added to the incubation medium, induces the formation of new microtubules in the Xenopus oocyte cortex. The capacity of the tubulin assembly in the submembranous cytoplasm decreases in progesterone-matured oocytes. In contrast, in enucleated matured oocytes this change does not occur. Altogether, these results show that taxol provokes tubulin assembly exclusively in the cortex of prophase oocyte, whereas in normal matured oocytes both cortical and cytoplasmic cytaster microtubules can be induced by taxol. The swelling of the oocyte nucleus therefore controls the spatial distribution of nucleation centers for tubulin assembly during meiotic maturation.

Alkaloids↗
PubMed1987

Levels of microtubules during the meiotic maturation of the Xenopus oocyte.

The total level of tubulin and the ratio of polymeric tubulin to tubulin dimer were measured by a colchicine filter-binding assay during meiotic maturation of the Xenopus oocyte. Although the total level of tubulin remains unchanged (0.12 +/- 0.03 micrograms/oocyte), the level of polymeric tubulin decreases during maturation (25% in prophase oocytes versus 20% in metaphase oocytes). The percentage of polymerized tubulin was estimated after drug (nocodazole and taxol) treatments and cold treatment in prophase and progesterone-matured oocytes; in all cases the microtubules present in mature oocyte are less stable than prophase microtubules. The presence of the nucleus modifies neither the level nor the stability of prophase microtubules. Our quantitative results as well as cytological arguments suggest that full-grown Xenopus oocytes may contain a cortical microtubular array.

Animals↗
PubMed1985

Identification of microtubule-associated proteins (MAPs) in Xenopus oocyte.

Microtubules were isolated from prophase-blocked oocytes of Xenopus laevis with the use of the anti-tumor drug taxol. In addition to tubulin, 5 microtubule-associated proteins (MAPs) were characterized. Among them, 2 high molecular mass proteins (200-300 kDa) are phosphorylated in ovo. The oocyte MAP extract promotes the assembly of rat brain 6 S purified tubulin.

Alkaloids↗
PubMed1983

Regulation of 17 beta-hydroxysteroid oxydoreductase of the Xenopus laevis follicle by gonadotropin and prolactin.

Ovarian follicles of Xenopus laevis, incubated in vitro actively metabolized radioactive 4-androstene-3, 17-dione to testosterone, indicating the presence of a 17 beta-hydroxysteroid-oxydoreductase (17 beta-HSOR). This enzymatic activity was found to be solely localized in the follicular envelopes. In vitro treatment of follicles with hCG for 2 hr induced a 40% decrease of the 17 beta-HSOR activity. The gonadotropin-induced inhibition was dose-dependent and only observed with gonadotropic LH-like hormones. Concomitant treatment with prolactin completely prevented the hCG effect.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

In vitro effects of progesterone and estradiol-17 beta on choleragen activated Xenopus oocyte adenylate cyclase.

The basal cAMP levels of full-grown defoliculated Xenopus oocytes were shown to average 1.2 pmol per oocyte. Follicle cAMP concentration was not significantly different from that of denuded oocyte and was found, when expressed as unit of volume to be independent of follicle size. In vitro gonadotropin treatment of follicle does not affect cAMP accumulation. During the course of progesterone induced maturation we failed to detect any modification of the oocyte cAMP content. Cholera toxin treatment increased cAMP levels; estradiol-17 beta potentialized cholera toxin action and progesterone antagonized it. Estradiol-17 beta was also found to reduce the amount of cholera toxin necessary to inhibit 50% of the progesterone induced meiosis maturation.

Adenylyl Cyclases↗
PubMed1982

Microinjected progesterone reinitiates meiotic maturation of Xenopus laevis oocytes.

Microinjection of progesterone dissolved in paraffin oil induces the reinitiation of meiotic maturation in the Xenopus oocyte; 50% maturation is obtained when 50 nl of a 50 microM solution is microinjected into the oocyte. The kinetics of the response to microinjected progesterone are similar to the kinetics of response to externally applied hormone. When an aqueous solution of progesterone is microinjected instead of an oil solution, maturation is never observed, a result which confirms previous work. Leakage of the steroid into the external medium was estimated to range from 1.6 pmol/hr when microinjection was performed in oil to 3.6 pmol/hr when it was performed in aqueous solution. Metabolism of the hormone microinjected in oil is weak (less than 20%) as compared to that after aqueous microinjection (greater than 80%). Progesterone microinjected in oil decreases the cAMP content as does externally applied hormone. We therefore conclude that progesterone acts initially on an intracellular site in order to trigger meiotic maturation of the Xenopus oocyte.

Animals↗
PubMed1980

Stimulation of prostaglandin accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells in vitro.

LHRH and LHRH agonists have been reported to exhibit somewhat paradoxical antireproductive effects. The current studies were undertaken to examine the actions of these substances on one parameter of ovarian function which is required for ovulation, i.e. prostaglandin production. Granulosa cells were obtained from immature rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h in vitro. LHRH, [D-Ala6, des-Gly-NH2(10)]LHRH-ethylamide (Analog I) and [D-Leu6, des-Gly-NH2(10)]LHRH-ethylamide (Analog II) were observed to stimulate prostaglandin accumulation by granulosa cells. Analog I, for example, at 100 ng/ml increased PGE from 0.19 +/- 0.06 in the control to 7.7 +/- 2.01 ng/2 x 10(6) cells (n = 7; P less than 0.01) after 5 h. TRH, on the other hand, had no effect on prostaglandin accumulation. When added with LH or FSH, the stimulation by maximal concentrations of Analog I and the gonadotropins appeared additive. Although the stimulation of prostaglandin accumulation by LH appears to be mediated by cAMP, no effect of Analog I on the amount of cAMP could be detected. cAMP was determined in cells plus medium at times of 1.5 min to 5 h and at concentrations of 10-2000 ng/ml Analog I, but did not change from the control. LHRH and LHRH agonists, therefore, stimulate the accumulation of ovarian granulosa cell prostaglandins and do so in a manner apparently distinct from that of LH or FSH.

Animals↗
PubMed1979

Cyclic AMP synthesis in Xenopus laevis oocytes: inhibition by progesterone.

[alpha-32P]ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.

1-Methyl-3-isobutylxanthine↗