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R Bellé

Publications and source records attributed to R Bellé.

At least 55 records · Page 3Linked to original sources

A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte.

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.

Animals↗

ATP-gamma-S (adenosine 5'-0(3-thiotriphosphate)) blocks progesterone-induced maturation of the Xenopus oocyte.

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.

Adenosine Triphosphate↗

In vivo effects of microinjected alkaline phosphatase and its low molecular weight substrates on the first meiotic cell division in Xenopus laevis oocytes.

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.

Alkaline Phosphatase↗

[A 45 kDa phosphorylated protein, resistant to alkaline treatment, appears at the time of rupture of the nuclear envelope during the 1st meiotic division of the Xenopus oocyte].

Xenopus oocytes were prelabeled with 32PO4 and induced to mature by progesterone treatment (1 microM). At the time of the breakdown of the germinal vesicle (nucleus), an alkali stable 45 kDa phosphoprotein appears in the 165 000 X g oocyte supernatant. Phospho-amino acid analysis shows that the 45 kDa protein is phosphorylated at threonine residues.

Animals↗

Stabilization of the maturation promoting factor (MPF) from Xenopus laevis oocytes. Protection against calcium ions.

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-gamma-S) (50 microM) to the preparation buffers. Neither Ca2+ ions nor calmodulin inhibit the partially purified MPF.

Adenosine Triphosphate↗

cAMP-dependent protein kinase regulates in ovo cAMP level of the Xenopus oocyte: evidence for an intracellular feedback mechanism.

Microinjection of cAMP-dependent protein kinase inhibitor (1.8 microM) 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 microM) 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.

Animals↗

Early increase of a 105,000-dalton phosphoprotein during meiotic maturation of Xenopus laevis oocyte.

In ovo [32P] phosphoproteins were analyzed during meiotic maturation of Xenopus laevis oocytes. A phosphoprotein of 105,000-dalton was found to increase early (one hour) after progesterone induction of meiosis. The pure heat-stable inhibitor (PKI) of cAMP-dependent protein kinase, which induces maturation, was microinjected into oocytes. Again the early increase in the 105,000-dalton [32P] phosphoprotein occurred. The burst in protein phosphorylation, which takes place at the period of germinal vesicle breakdown, was quantitatively and qualitatively comparable in progesterone and PKI-stimulated oocytes. In order to confirm the inverse relationship between the 105,000 dalton [32P] phosphoprotein increase and cAMP-dependent protein kinase activity, purified C-subunit of the kinase has been microinjected into oocytes. C-subunit which inhibits maturation did not increase significantly the 105,000-dalton [32P] phosphoprotein whereas it increased the total level of in ovo phosphorylation. Enucleation experiments favour the localization of the 105,000-dalton protein in both the oocyte cytoplasm and nucleus. Furthermore the progesterone-induced increase in the phosphorylation of the 105,000-dalton protein was found in the cytoplasmic compartment after oocyte enucleation.

Animals↗

Free calcium in full grown Xenopus laevis oocyte following treatment with ionophore A 23187 or progesterone.

Free intracellular Ca2+ was monitored in isolated Xenopus laevis oocyte during induced maturation using the Ca2+ -sensitive luminescent protein, aequorin. Internal free Ca2+ was not precisely measured but data suggest it was quite low (in the micromolar range). No change in internal free Ca2+ was detected during maturation induced either by progesterone or by p-chloromercuribenzoate. By contrast, the ionophore A 23187 gave an increase in the free Ca2+ level when there was a raised external Ca2+ (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.

Aequorin↗

[Oocyte maturation in Xenopus laevis. The entry of progesterone is necessary for maturation].

In vitro progesterone-induced maturation of Xenope ovocyte was studied as a function of progesterone uptake and extracellular progesterone concentration. Maturation depends on the initial total amount of progesterone in incubation medium and does not depend on the extracellular hormonal concentration. This result strongly argues for an intracellular receptor site for progesterone.

Animals↗

[Oocyte maturation in Xenopus laevis. Arguments in favor of the role of calcium ions].

Influx and efflux of 45Ca++ ions in Xenopus leavis isolated full-grown oocytes were measured. Calcium influx was from 0.5 to 1 pmole/oocyte/h. Progesterone, which induces maturation does not influence Ca++ fluxes while PCMB caused a significant increase of Ca++ influx during the course of PCMB (p-Cl-HgBzO-) induced maturation. Our results show that the presence of Ca++ is not necessary in extracellular medium for germinal vesicle breakdown (GVBD) induced by progesterone. In contrast, the kinetic and the percent of germinal vesicle breakdown observed when maturation was induced by PCMB depend on the extracellular Ca++ concentration in Ca-EGTA buffers.

Animals↗

Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis.

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.

Animals↗