Deciphering the H-Ras pathway in Xenopus oocyte.

Xenopus oocytes are arrested in prophase of the first meiotic division. In response to progesterone, they re-enter meiosis and arrest again in metaphase of the second meiotic division. This process, called meiotic maturation, is under the control of the Cyclin B-Cdc2 complex, M phase promoting factor (MPF). Injection of a constitutively active Xenopus H-Ras protein activates MPF, suggesting that Ras proteins could be implicated in the progesterone transduction pathway. The aim of this study was (1) to elucidate the pathway triggered by H-Ras leading to MPF activation in Xenopus oocytes and (2) to investigate whether endogenous H-Ras is involved in the physiological process of meiotic maturation. We generated three constitutively active double mutants, each of them recruiting a single effector in mammalian cells, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) or RalGDS. Our results show that the activation of a PI3K-related enzyme is crucial for H-Ras-induced MPF activation, whereas the recruitment of either MAPK or RalGDS is not. However, although the H-Ras/PI3K pathway is functional in Xenopus oocytes, it is not the physiological transducer of progesterone responsible for meiotic resumption.

Two distinct mechanisms control the accumulation of cyclin B1 and Mos in Xenopus oocytes in response to progesterone.

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.

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.

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.

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.

Direct activation of cdc2 with phosphatase: identification of p13suc1-sensitive and insensitive steps.

In Xenopus oocytes, activation of MPF during prophase-metaphase transition is associated with the tyrosine dephosphorylation of the cdc2 protein. In vivo and in cell-free extracts kinase activation can be inhibited by excess p13suc1, a subunit of the protein kinase. Here we have demonstrated that affinity-purified cdc2 from Xenopus prophase oocytes may be activated in vitro by exposure to potato acid phosphatase. In vitro, excess p13 does not inhibit tyrosine dephosphorylation of prophase cdc2, but nonetheless binds and prevents the activation of the enzyme. By contrast, fully activated enzyme from metaphase Xenopus eggs is insensitive to excess p13. These observations define a p13-sensitive state in the activation of fully active cdc2 that follows tyrosine dephosphorylation.

Human retinoblastoma protein (Rb) is phosphorylated by cdc2 kinase and MAP kinase in Xenopus maturing oocytes.

Xenopus oocyte meiotic maturation combines features of G0/G1 and G2/M transitions of the cell cycle. To study the in ovo Rb kinase activity, we have microinjected human Rb into oocytes. Microinjected human Rb localizes into the nucleus, is hypophosphorylated in prophase oocytes, becomes hyperphosphorylated during meiotic maturation and is dephosphorylated as the cell reenters interphase. Inactivation or overexpression of the cyclin D-cdk4/6 complex in an oocyte extract does not affect the Rb kinase activity. This kinase activity could be attributed to both cdc2-cyclin B and MAP kinase, opening new perspectives of investigation in somatic cells.

Inhibition of glycosphingolipid synthesis induces p34cdc2 activation in Xenopus oocyte.

In Xenopus prophase-blocked oocytes, it is assumed that progesterone interacts with the plasma membrane to initiate a signalling cascade that ultimately leads to MPF activation. Progesterone regulates negatively the cAMP pathway through an inhibition of adenylate cyclase. However, the mechanisms linking the initial action of the hormone with adenylate cyclase activity remain to be elucidated. Here, we demonstrate that PDMP, an inhibitor of glucosphingolipid synthesis, triggers oocyte meiotic maturation in a cAMP- and cycloheximide-dependent manner, whereas exogenous ceramide is unefficient. We propose that sphingolipid metabolism and targeting represent an important regulatory process of oocyte meiosis.

Membrane-anchored cyclin A2 triggers Cdc2 activation in Xenopus oocyte.

In Xenopus oocyte, the formation of complexes between neosynthesized cyclins and Cdc2 contributes to Cdc2 kinase activation that triggers meiotic divisions. It has been proposed that cytoplasmic membranes could be involved in this process. To investigate this possibility, we have injected in the oocyte two undegradable human cyclin A2 mutants anchored to the endoplasmic reticulum (ER) membrane. They encode fusion proteins between the truncated cyclin A2-Delta152 and a viral or cellular ER-targeting domain. We show that both mutants are fully functional as mitotic cyclins when expressed in Xenopus oocytes, bind Cdc2 and activate M-phase promoting factor.

In vitro binding of free cdc2 and raf kinase to membrane vesicles: a possible new regulatory mechanism for cdc2 kinase activation in Xenopus oocyte.

The G2-M transition of the cell cycle is under the control of the M-phase promoting factor (MPF) formed of cdc2 kinase and cyclin B. The Xenopus prophase-blocked oocyte contains a stockpile of cyclin B2-cdc2 complexes that are maintained inactive by a double inhibitory phosphorylation on Thr-14 and Tyr-15 of cdc2. Free cdc2 molecules that are not associated with cyclin, are present in excess as compared to cyclin B2-associated cdc2. This pool of free cdc2 is permanently recruited to associate with neosynthetized cyclin B2 in the resting prophase oocyte, to feed up the pre-MPF stockpile. During re-entry into meiosis, free cdc2 could generate with newly synthesized cyclin B a small level of active MPF, that could serve as starter to initiate the conversion of pre-MPF into MPF. It was, therefore, of high interest to investigate whether free cdc2 interacts with other proteins and what could be its intracellular localization. To address these questions, we developed an in vitro system of membrane vesicles. We demonstrate here that free cdc2 is recovered in association with the external layer of membrane vesicles, whereas cyclin B2-associated cdc2 is not. Cyclin is able to associate in vitro with cdc2-containing membrane vesicles. This association does not induce the inhibitory cdc2 phosphorylations. However, it does not lead to active complexes, suggesting that membrane vesicles prevent cdc2 activation. C-Raf1, another kinase activated during reentry into meiosis, is also totally recovered in association with the membrane vesicles.

Function and regulation of cdc25 protein phosphate through mitosis and meiosis.

Activation of the cyclin B-cdc2 kinase mitotic inducer involves dephosphorylation of two inhibitory residues, tyrosine 15 and threonine 14, cdc25 is the specific phosphatase that directly dephosphorylates and activates the cdc2 kinase, cdc25 activity is regulated by phosphorylation. Both phosphatases 1 and 2A could act as cdc25-specific inhibitory phosphatases. Although the cyclin B-cdc2 complex plays a role in activating cdc25, it is highly probable that a distinct protein kinase is involved as a trigger in cdc25 activation. The implication of raf kinase as a cdc25-specific activating kinase in human cells and Xenopus oocytes is discussed.

Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B.

Activation of maturation-promoting factor at the onset of mitosis requires the tyrosine dephosphorylation of one of its components, the cdc2 protein kinase. cdc25 is the specific tyrosine phosphatase that activates cdc2. We find that Xenopus oocytes contain a relative of cdc25, p72. In Xenopus embryos the abundance of p72 does not oscillate during the cell cycle. However, p72 directly associates with cdc2-cyclin B in a cell cycle-dependent manner, reaching a peak at M phase. The M phase kinase that associates with p72 is catalytically active. These results suggest that the mechanism by which cdc25 triggers cdc2 activation involves a periodic physical association between cdc25 and the cyclin B-cdc2 complex and also that mitotic control can be affected by mechanisms other than transcriptional regulation of the cdc25 gene.

Xenopus cyclin D2: cloning and expression in oocytes and during early development.

We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truncated version of cyclin D2 was also isolated. Microinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.

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.

Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study.

Xenopus oocytes were stained by anti-tubulin and anti-MAP1 antibodies during the first meiotic cell division. In the prophase-blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti-MAP1 antibody and polyclonal anti-centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone-induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti-MAP1 and anti-centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium- and antimitotic drug sensitive. Anti-MAP1 and anti-tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti-centrosome antibody.

Distribution of microtubules during the first meiotic cell division in the mouse oocyte: effect of taxol.

Mouse oocytes were stained by antitubulin antibody and by an anti-MAP1 antibody (JA2) during the first meiotic cell division. At germinal vesicle stage, JA2 antibody exclusively stains the nucleoplasm. When the nuclear envelope breaks down (GVBD) (2-3 h of culture), numerous foci of microtubules appear around the disrupting nuclear envelope; they are decorated by anti-MAP1 antibody. After 10-12 h of culture, the antigen component detected with anti-MAP1 antibody is present in the meiotic spindle. The antimitotic agent taxol (10 microns) induces microtubule formation, mainly at the periphery of the germinal vesicle. At GVBD, taxol provokes the formation of a large microtubular array stained with both antibodies, which is associated with the condensed chromosomes. Furthermore, numerous cytoplasmic asters become visible in the cytoplasm. At metaphases I and II, taxol induces an important enlargement of the metaphase spindles and increases the number of cytasters.

Estramustine phosphate inhibits germinal vesicle breakdown and induces depolymerization of microtubules in mouse oocyte.

The first meiotic cell division (meiotic maturation) of the dictyate stage of mouse oocytes, removed from the follicle, resumes spontaneously in vitro. This study indicates that estramustine phosphate reversibly blocks meiotic maturation by inhibiting germinal vesicle breakdown. Oocyte cryostat sections were stained with anti-tubulin and two antibodies (anti-MAP1: JA2 and 5051) which decorated the metaphase spindle during meiotic maturation. It was found that (1) estramustine phosphate depolymerized microtubules in ovo and dispersed non-tubulin antigens associated with microtubules of the metaphase spindle at various stages of maturation, and (2) estramustine phosphate decreased the ability of taxol to induce cytoplasmic asters. These results suggest that germinal vesicle breakdown and microtubule polymerization may be linked.

Tyrosine phosphorylation of p34cdc2 is regulated by protein phosphatase 2A in growing immature Xenopus oocytes.

Growing stage IV Xenopus oocytes are unresponsive to progesterone treatment. They contain a store of preMPF composed of tyrosine phosphorylated p34cdc2 and cyclin B2. The endogenous store of preMPF cannot be recruited by cdc25 protein phosphatase or cyclin protein microinjections. This is in contrast with full-grown stage VI oocytes where microinjections of these proteins are known to activate the autoamplification of MPF. When cyclins are microinjected into stage IV oocytes, they associate with endogenous free p34cdc2 and the illegitimate complexes undergo phosphorylation on tyrosine 15. High doses of human cyclin A allow, however, part of the neoformed complexes to be activated as an histone-H1 kinase; this partial activation of p34cdc2 is sufficient to induce germinal vesicle breakdown in these small oocytes. Co-injections of cyclin A or cyclin B together with okadaic acid (10 microM in the microinjection solution), an inhibitor of protein phosphatase 2A (PP2A), lead to the full activation of neoformed p34cdc2/cyclin complexes. These results indicate that small oocytes possess an active tyrosine kinase that inactivates new p34cdc2/cyclin complexes. Inhibition of PP2A by okadaic acid prevents this inactivation reaction and conversely allows the illegitimate complex to be activated. Neither the activating phosphorylation on threonine 161 nor the inactivating phosphorylation on tyrosine 15 take place in stage IV enucleated oocytes. Altogether, our results show that the accumulation of inactive p34cdc2/cyclin B2 during the long-lasting prophase of the oocyte is positively controlled by PP2A through the tyrosine phosphorylation of p34cdc2.