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In the amphibian Xenopus laevis, the elongation factor 1 alpha proteins (EF-1 alpha) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF-1 alpha gene (EF-1 alpha S) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF-1 alpha (conversion of EF-1 alpha-GDP to EF-1 alpha-GTP) is assured by the EF-1 beta gamma complex. We show here that in Xenopus laevis embryos, contrary to the situation for EF-1 alpha, EF-1 beta, and EF-1 gamma mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF-1 beta and EF-1 gamma genes from the zygotic genome occurs several hours after that of the somatic EF-1 alpha S gene. Therefore, during early Xenopus development the expression of these three elongation factors is not co-ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the early Xenopus embryo are discussed.

Exogenous β casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in vivo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/min/oocyte, respectively, for β casein phosphorylated by casein kinase II and β casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of β casein.

Protein phosphatase activities were analyzed in vivo in Xenopus oocytes. The dephosphorylation of microinjected beta casein was inhibited when the tumor promoter okadaic acid was microinjected into oocytes. Inhibition was dose dependent and reversible; 50% of activity was recovered 15-30 minutes after microinjection.

The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.

Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements.

Elongation factor-1 (EF-1) is a major substrate for cdc2 kinase in Xenopus oocytes. The guanine-nucleotide exchange factor EF-1βγδ, appears to have a highly complex macromolecular structure containing several GTP/GDP exchange proteins, valyl-tRNA synthetase, and a putative anchoring protein EF-1γ. During meiotic cell division, the factor becomes phosphorylated by cdc2 kinase, not only on EF-1γ, but also on two different phospho-acceptors on EF-1δ. Phosphorylation is concomitant with changes in protein synthesis in vivo. Xenopus oocytes, and potentially all cells, contain a multitude of heteromeric forms of the complex which postulates that EF-1βγδ is not a “house keeping” factor but a sophisticated regulatory element.

International audience; Elongation factor 1 mediates the elongation step of mRNA translation. The transfer of aminoacyl-tRNA to ribosomes under hydrolysis of GTP is catalyzed by a GTP binding protein, EF1Ia. A guanine-nucleotide exchange complex now referred as EF1beta gamma delta replaces GDP by GTP on EF1alpha (1). A complex, purified from Xenopus oocytes as a substrate for the meiotic and mitotic p34Cdc2 kinase, was shown to contain the guanine-nucleotide exchange activity (2). The Xenopus complex is composed of three main proteins, p30, p36 and p47. Surprisingly, microsequencing of two of its components, p36 and p30 suggested the presence of two related proteins (2). We have previously cloned and sequenced the cDNA encoding for p47 or EF1gamma (3) and p36 or EF1delta (4), we present here the molecular cloning of p30 or EF1beta. This result allows for the first time, sequence analysis of EF1beta and delta proteins, both present in the same complex.

International audience; A polyclonal antibody was raised against bacterially produced catalytic alpha subunit of protein phosphatase 2A (PP2AC) cloned from Xenopus ovarian library. The amount of PP2AC in Xenopus oocytes determined by Western blot analysis was 1 ng/microgram of cytosolic protein. The antibody depleted PP2AC from oocyte extracts in association with 6 components (40, 62, 65, 80, 85 and 90 kDa). Prophase- and metaphase-arrested oocytes contained identical amounts of PP2AC. Metaphase oocytes showed one specific change in the 62 kDa protein associated with PP2AC.

Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.

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Maturation promoting factor (MPF) activity was recovered from progesterone-matured Xenopus oocytes cytosol, fractionated by polyethylene glycol-20,000 or ethanolic precipitation. An improved stabilization of the biological activity was obtained by adding adenosine 5′-O-(3-thiotriphosphate) (ATP-γ-S) (50 μ M) to the preparation buffers. Neither Ca 2+ ions nor calmodulin inhibit the partially purified MPF.

Microinjection of cAMP-dependent protein kinase inhibitor (1.8 μM) increases the cAMP level of Xenopus oocyte. Its effect was observed in full-grown (stage VI) as well as in vitellogenic (stage IV) oocytes. In contrast the inhibitor I1 of protein phosphatase-1 blocks cAMP accumulation. Progesterone (1 μM) decreases the cAMP level in control and in PKI-treated oocytes of both stages. These results show that cAMP concentration is regulated by a cAMP-dependent phosphorylation indicating the presence of a feedback mechanism. The feedback control is disrupted when oocyte is induced to mature by progesterone.

Free intracellular Ca 2+ was monitored in isolated Xenopus laevis oocyte during induced maturation using the Ca 2+ -sensitive luminescent protein, aequorin. Internal free Ca 2+ was not precisely measured but data suggest it was quite low (in the micromolar range). No change in internal free Ca 2+ was detected during maturation induced either by progesterone or by p -chloromercuribenzoate. By contrast, the ionophore A 23187 gave an increase in the free Ca 2+ level when there was a raised external Ca 2+ (10 mM), conditions which also induce oocyte maturation. About 3 h after progesterone or p -chloromercuribenzoate stimulation, the oocyte membrane potential decreased by about 50 mV while the membrane resistance increased transitorily.

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.

Following progesterone treatment, a significant lag (4–8 hr) in the induction of germinal vesicle breakdown (GVBD) is observed in amphibian oocytes. Preincubation of Xenopus oocytes in the presence of subthreshold doses of progesterone decreases the lag (1–3 hr) and, therefore, facilitates oocyte maturation. Progesterone facilitation of GVBD is a dose-dependent reversible phenomenon. On the other hand, it is also reported that cyclic-AMP phosphodiesterase inhibitors increase the lag (8–15 hr) between progesterone stimulation and germinal vesicle breakdown.

L-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na+ dependent and presumably coupled to Na+ gradient. Our results indicate that progesterone (10(-6) M) Blocks abruptly, around the germinal vesicle breakdown, the saturable transport of L-leucine. p-Chloromercuribenzoate (10(-4) M) induces maturation and after a short lag of time strongly inhibits L-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.

Progesterone induces in vitro meiotic maturation in Xenopus oocytes. 1. 1. Early steps of maturation are sensitive to inhibition by phosphodiesterase inhibitors (methyl xanthines) or by cholera toxin, suggesting that elevated levels of intracellular cyclic-AMP block progesterone action. 2. 2. Progesterone causes a significant decrease in the amount of newly synthesized cyclic AMP from microinjected α- 32 P-ATP. 3. 3. Ca 2+ ions are involved both in the initiation of progesterone action and in the whole process of the first meiotic division.

Xenopus laevis oocytes were microinjected with low molecular weight phosphoesters such as 2-glycerophosphate, phosphotyrosine, phosphoserine, phosphothreonine, 4-nitrophenyl phosphate, and orthophosphate. These compounds were able to induce a considerable reduction in the time course of progesterone-induced maturation, with 2-glycerophosphate being the most effective. The basal level of cAMP and its drop during maturation were not affected by the microinjection of 2-glycerophosphate. The injection of alkaline phosphatase (EC 3.1.3.1.) from calf intestine at a low concentration (10 ng per oocyte) was able to decrease or abolish the effect of 2-glycerophosphate. At higher concentration (25 ng per oocyte) this enzyme totally blocked progesterone- or maturation-promoting factor-induced maturation. Alkaline phosphatase might behave in vivo as a phosphoprotein phosphatase active towards phosphotyrosine-containing proteins. In addition, our results indicate that phosphate or phosphoester-containing buffers should be avoided in the course of maturation-promoting factor purification.

1. 1. The uptake and metabolism of progesterone by defolliculated oocytes of Xenopus laevis and Pleurodeles waltlii were studied; in both amphibians progesterone was actively incorporated by oocytes. 2. 2. In Pleurodeles incorporation rate was proportional to progesterone molarity in incubation medium. 3. 3. Oocytes from Pleurodeles and Xenopus extensively metabolized progesterone, but metabolites were qualitatively and quantitatively different; in Pleurodeles 20β-hydroxy-pregn-4-ene-3-one, 5α-pregnanedione, 3α-hydroxy-5α-pregnane-20-one and 3β-hydroxy-5α-pregnane-20-one were identified; the 20β-hydroxy-pregn-4-ene-3-one was the main metabolite formed. 4. 4. Experiments with cell fractions showed that the 5α-reductase activity was associated with the microsomal fraction and the 20β-hydroxysteroid oxydoreductase with the 100,000 g supernatant. 5. 5. In Xenopus Δ 4 -androstenedione, 17α-hydroxy-progesterone and testosterone were isolated; Δ 4 -androstenedione was the major metabolite formed.

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ∼40,000) is observed. Enucleation of [32P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [32P] phosphoproteins.

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF-1 beta and EF-1 gamma. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.

This paper describes the purification of a 47 kDa protein from Xenopus laevis oocytes that becomes phosphorylated when the oocytes undergo meiotic maturation. This protein (p47) is part of a high molecular mass complex containing at least two other proteins of molecular mass 30 and 36 kDa. This complex can be isolated from stage VI oocytes before maturation. We obtained a pattern for phosphopeptides in p47 phosphorylated in vivo very similar to that of the purified protein phosphorylated in vitro by p34cdc2 (a H1 kinase which is a component of the M-phase promoting factor) and [γ-32P]ATP. Therefore, the purified p47, already described as a marker of MPF activity, is the first reported in vivo substrate for the cell division control kinase.

ATP-gamma-S microinjection into Xenopus oocyte prevents progesterone induced maturation. Inhibition is time and dose dependent; 50% inhibition occurs when 50 nl of 0.5 mM ATP-gamma-S solution are microinjected/oocyte 1 hr prior to the hormonal trigger. ATP-gamma-S inhibited oocytes can be induced to mature (100%) following microinjection of extracts containing maturation promoting factor (MPF). Our results suggest that the maturation protein(s) has been stabilized in ovo by ATP-gamma-S microinjection, in its phosphorylated inhibitory form.

International audience; The results presented here show that a decrease in the concentration of total polyamines, due to a decrease in putrescine and spermine, occurs during oogenesis in Xenopus laevis. The microinjection of spermine or spermidine decreases the hormonal responsiveness (maturation) of the fully-grown oocytes. This effect is synergistic with that already described for the microinjection of casein kinase II (Mulner-Lorillon, O. et al. (1987) Eur. J. Biochem. 171, 107-117), a polyamine dependent enzyme. Therefore a decrease in polyamine concentration, via its effect on endogeneous casein kinase II, could constitute one of the molecular changes required for the acquisition of competence to mature.

Resume Les proteines phosphorylees in vivo ont ete analysees au cours de la maturation meiotique de l'ovocyte de Xenopus laevis . L'induction de la meiose par la progesterone provoque l'augmentation precoce (en une heure) d'une seule phosphoproteine [ 32 P] de poids moleculaire 105.000. L'inhibiteur thermostable purifie des proteines kinases dependantes de l'AMPc (PKI), qui induit la maturation, provoque egalement l'augmentation precoce de la proteine phosphorylee 105.000 lorsqu'il est introduit dans l'ovocyte par microinjection. L'augmentation de la phosphorylation des proteines, qui a lieu au moment de la rupture de la vesicule germinative, est comparable quantitativement et qualitativement lorsque les ovocytes sont stimules par la progesterone ou par la microinjection de PKI. Afin de confirmer la relation inverse entre l'augmentation de phosphorylation de la proteine 105.000 et l'activite proteine kinase dependante de l'AMPc, la sous-unite catalytique C de la kinase a ete injectee dans les ovocytes. La sous-unite C, qui inhibe la maturation, n'augmente pas de facon significative la phosphorylation de la proteine 105.000, alors qu'elle augmente le niveau total de phosphorylations in ovo . Des experiences d'enucleation permettent de localiser une proteine phosphorylee de PM 105.000 a la fois dans le cytoplasme et dans le noyau. De plus, l'augmentation de phosphorylation de la proteine 105.000 a ete trouvee dans le compartiment cytoplasmique lorsque les ovocytes ont ete enuclees une heure apres la stimulation hormonale.

Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min.

Melanosomes were purified by sucrose gradient centrifugation from different Amphibian tissues: oocytes, skin and retina. Melanosomes contain different proportions of SDS-soluble proteins (25% in ovary, 60% in skin and retina). Partial characterization of Amphibian melanoproteins by polyacrylamide gel electrophoresis was achieved; in each case the banding patterns were qualitatively different.

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.

Full-grown Xenopus laevis oocytes were incubated in NaHCO3 buffer equilibrated with carbon dioxide (5 to 100%). Germinal vesicle breakdown never occurred in spite of the appearance of the characteristic white spot at the animal pole. The effect of carbon dioxide was analyzed during progesterone-induced maturation. Carbon dioxide did not inhibit the early steps of maturation whereas it inhibited germinal vesicle breakdown even when applied 4 hr after the initial hormonal trigger. When oocytes were treated transiently in NaHCO3 buffer equilibrated with carbon dioxide and further incubated in Tris buffer, drastic delay in the kinetic of germinal vesicle breakdown was observed. Inhibition of progesterone-induced maturation by carbon dioxide treatment is coincident with the time of maturation promoting factor appearance (MPF). On the basis of microinjection experiments of MPF into recipient oocytes, it was also shown that MPF expression is not inhibited by carbon dioxide and thus indicates that the late phase of MPF formation and/or MPF amplification is a carbon dioxide-sensitive period.