Specific inhibition of beta-tubulin synthesis in Xenopus oocytes using anti-sense oligodeoxyribonucleotides.
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C Jessus
Publications and source records attributed to C Jessus.
Specific inhibition of endogenous beta-tubulin synthesis in Xenopus oocytes by anti-messenger oligodeoxynucleotides.
An oligodeoxynucleotide containing 27 nucleotides, complementary to a highly conserved sequence of beta-tubulin mRNAs, led to a nearly complete inhibition of beta-tubulin synthesis in Xenopus oocytes after microinjection. Inhibition persisted 24 hours post-injection and was specific for beta-tubulin as the synthesis of alpha-tubulin as well as that of other proteins from the oocyte was not affected. Complete inhibition of beta-tubulin synthesis did not prevent progesterone-induced meiotic maturation and formation of the chromosome spindle. This result indicates that the pool of endogenous tubulin already present in fully-grown oocytes is sufficient to allow normal meiotic maturation. This finding correlates with previous experiments showing that the turn-over of tubulin is very slow in the oocyte.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Interaction between rat brain microtubule associated proteins (MAPs) and free ribosomes from Xenopus oocyte: a possible mechanism for the in ovo distribution of MAPs.
The binding of microtubule associated proteins (MAPs) to free 80 S ribosomes isolated from Xenopus laevis oocytes inhibits in vitro tubulin assembly (Jessus et al., 1984). The inhibition of tubulin polymerisation was shown to be dependent upon GTP. The dose of GTP needed to induce 50% of the maximal effect was 0.5 mM. Furthermore, the inhibition is enhanced by pretreatment of the ribosomes with ATP-gamma-S, and partially abolished after phosphatase treatment, which strongly suggests that protein phosphorylation regulated the inhibitory effect. When fluorescent purified MAPs are microinjected into Xenopus laevis oocyte, they cap 1 h later the basal nuclear envelope; in contrast, when the fluorescent MAPs-ribosome complex is injected, the fluorescent MAPs remain in the cytoplasm and never reach the region underlying the nuclear envelope.
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In vitro inhibition of tubulin assembly by a ribonucleoprotein complex associated with the free ribosome fraction isolated from Xenopus laevis oocytes: effect at the level of microtubule-associated proteins.
The 100 000 X g supernatant prepared from defolliculated Xenopus laevis oocytes inhibits, in a dose-dependent manner, the in vitro polymerization of rat brain neurotubulin. The oocyte inhibitory factor is thermolabile, totally inactivated by RNAse and partially by trypsin. A preliminary purification of the inhibitor showed that it is associated with the free oocyte ribosomes (80 S). The saturable binding of microtubule-associated proteins, essentially MAP2, to this 80 S ribonucleoprotein fraction is responsible for the inhibition of the in vitro tubulin assembly.
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