René Ozon

Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes

Micro‐injection of, or incubation with okadaic acid (OA), a specific phosphatase inhibitor, can induce formation of maturation‐promoting factor (MPF) and germinal vesicle breakdown (GVBD) in Xenopus laevis oocytes. Comparison of the dose‐response curves of OA on maturation, isolated enzymes and phosphatase activities in crude oocyte preparations suggests that inhibition of both polycation‐stimulated (PCS) and ATP,Mg‐dependent (AMD) phosphatases is sufficient but requires that a critical phosphorylation level is attained of one or several of their substrates, resulting in the formation of active MPF and meiotic maturation.

6-Dimethylaminopurine(6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte

The first meiotic cell division (meiotic maturation) of dictyate stage mouse oocytes removed from the follicle resumes spontaneously in vitro. We used the puromycin analog 6-dimethylaminopurine (6-DMAP) to test the respective roles of protein synthesis and protein phosphorylation in driving this process. While protein synthesis inhibitors do not block meiosis resumption, 6-DMAP was found to inhibit germinal vesicle breakdown (GVBD), by inhibiting the burst of protein phosphorylation without changing the rate of incorporation of [35S]methionine into proteins. This effect is reversible; it depends both upon drug concentration and the particular female. When added after GVBD and before the emission of the first polar body, 6-DMAP decreases the level of protein phosphorylation and induces decondensation of the chromosomes and reformation of the nuclear envelope. In contrast, 6-DMAP did not trigger these processes in metaphase II oocytes which only produce resting nuclei when treated by protein synthesis inhibitors. From these data, we conclude that (1) the early appearance and stability of mouse MPF in Metaphase I oocytes depend on protein phosphorylation rather than on protein synthesis, and (2) protein synthesis is necessary to maintain the condensation of the chromosomes in metaphase II oocytes.

Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte

Histone H1 kinase and protein phosphorylation have been studied in mouse oocyte. Histone H1 kinase activity increases when the oocyte enters M-phase at the time of GVBD and is paralleled with a burst of protein phosphorylation. This activity dramatically drops after parthenogenetic activation induced by puromycin. Okadic acid (OA), a potent inhibitor of protein phosphatases, induces GVBD when oocytes are arrested in the first meiotic prophase by dbc-AMP; the continuous presence of the phosphatase inhibitor, however, inhibits the polymerization of metaphase microtubules. Following activation of metaphase II-arrested mouse eggs by puromycin, OA can induce the breakdown of the nuclear envelope and the activation of histone H1 kinase. This indicates that in the absence of protein synthesis, and therefore of cyclin synthesis, inhibition of protein phosphatases may be sufficient to induce the entry into M-phase during the first cell cycle of the mouse parthenogenetic activated oocyte.

Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

In Xenopus oocytes, the c‐mos proto‐oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone‐induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos‐depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic‐like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes.

The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation: A 4-step scheme for meiotic maturation

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 microM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 microM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 microM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 microM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme--fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD--is presented and discussed.

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.

Electrical membrane properties of the Xenopus laevis oocyte during progesterone-induced meiotic maturation.

The membrane input resistance (R(m)), potential (E(m)), and capacitance (C(m)) of fully grown Xenopus oocytes removed from their follicles have been measured. Before hormonal maturation, R(m), E(m), and C(m) were found to be: 1.86 +/- 0.63 Momega, -49 +/- 17 mV, and 11.9 +/- 4.8 microF/cm2 of apparent surface area. Long-term recording from oocytes in the presence of 1 microM progesterone reveal that during early GVBD (germinal vesicle breakdown) the membrane potential depolarized to about -10 mV, the apparent specific capacitance decreased to 1.5 to 3 microF/cm2, and the membrane resistance transiently increased by a factor of about 3. These results indicate that a decrease in surface area, a transient increase in membrane resistance, and a change in the ionic mechanisms for the membrane potential occur during progesterone-induced meiotic maturation. The continuous monitoring of these parameters has further shown that the major changes occur at about 50 to 60% of the time from progesterone application to GVBD, and that at the sensitivities used, generally 1.25 mV/mm, no changes are seen immediately upon application of the hormone.

Characterization of MPF activation by okadaic acid in Xenopus oocyte

Okadaic acid (OA), a specific inhibitor of protein phosphatases, induces a rapid activation (30 min) of MPF when microinjected into the Xenopus oocyte. Neither protein synthesis inhibitors nor cAMP counteract the action of OA. These results indicate that the inhibition of protein phosphatase(s) is sufficient for the in vivo activation of MPF even after the full activation of cAMP-dependent protein kinase. In all experimental conditions (plus or minus inhibitors of protein synthesis; normal or elevated cAMP levels) OA induces a burst of protein phosphorylation together with the activation of MPF. Cytological analysis shows that OA provokes the breakdown of the nuclear envelope, the depolymerization of lamin and the condensation of the chromosomes. However, no metaphase spindles are organized, indicating that inhibition of protein phosphatases strongly affects the function of the microtubule organizing center.

Direct activation of protein phosphatase-2A0 by HIV-1 encoded protein complex NCp7:vpr

The effects of HIV‐1 encoded proteins NCp7, vpr and NCp7:vpr complex on the activity of protein phosphatase‐2A0 have been tested. We report that NCp7 is an activator of protein phosphatase‐2A0 and that vpr activated protein phosphatase‐2A0 only slightly. We also report that NCp7 and vpr form a tight complex which becomes a more potent activator of protein phosphatase‐2A0 than NCp7 alone. The ability of NCp7 to activate protein phosphatase‐2A0 is regulated by vpr. The C‐terminal portion of vpr prevents NCp7 from activating protein phosphatase‐2A0 while the N‐terminal portion of vpr potentiates the effect of NCp7 on the activity of protein phosphatase‐2A0. Our findings indicate that vpr may be acting as a targeting subunit which directs NCp7 to activate protein phosphatase‐2A0. In view of the fact that protein phosphatase‐2A functions as an inhibitor of G2 to M transition of the cell cycle and is involved in other key cellular processes such as the control of RNA transcription, the results presented in this report may explain how HIV‐1 causes cell cycle arrest which may lead to CD4+ T cell depletion and also how it disturbs normal cellular processes of its host cell.

Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes.

Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B·Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification.