Pierre Guerrier

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.

Activation of Barnea candida (mollusca, pelecypoda) oocytes by sperm or KCl, but not by NH4Cl, requires a calcium influx

Abstract Artificial activation of Barnea candida oocytes by NH 4 Cl and the addition of excess KCl are, respectively, independent and dependent upon external Ca 2+ concentration. The activating efficiency of NH 4 Cl increases when external pH is raised which suggests that the proportion of unionized penetrating NH 3 is the key factor for activation under these conditions. The external Ca 2+ -dependent period for KCl or sperm-induced activation does not exceed 3–4 min. The transmembrane Ca 2+ flux inhibitor D-600 reversibly inhibits KCl- or sperm-induced activation and the D-600-sensitive period closely corresponds to the Ca 2+ -dependent period. Ca 2+ ions alone can also trigger activation provided external Mg 2+ concentration is lowered. Finally, a direct demonstration is presented for the existence of a D-600-sensitive Ca 2+ uptake following KCl- or sperm-induced activation, by using the radioactive tracer 45 Ca. The significance of that Ca 2+ influx is discussed with respect to the possible involvement of an intracellular pH shift as a main factor in the activation of lamellibranch mollusc oocytes.

A novel method to produce triploids in bivalve molluscs by the use of 6-dimethylaminopurine.

Abstract To date, pressure shock, heat shock, and chemical treatment with cytochalasin B have been the major methods used to induce triploid bivalves. In this study, triploid bivalves were induced by a new chemical treatment using 6-dimethylaminopurine (6-DMAP). The capacity of 6-DMAP to produce triploid eggs and larvae was investigated in the Pacific oyster, Crassostrea gigas , the giant sea scallop, Placopecten magellanicus , and the blue mussel, Mytilus edulis . The triploid yields from the 6-DMAP treatments were compared with those of cytochalasin treatments. The highest percentage of triploid production was 90% in the Pacific oysters when the fertilized eggs were treated for 20 min with 300 μM 6-DMAP at 15 min after fertilization and 95% in the giant scallops when treated for 15 min with 400 μM 6-DMAP at 70 min after fertilization. Increasing durations of 6-DMAP treatments improved the efficiency of triploid induction. However, long incubations with 6-DMAP, which overlapped the period of first mitotic cleavage, led to the development of abnormal larvae. These included low percentages of normal veliger larvae in the Pacific oysters and developmental arrest at the trochophore stage in the blue mussels. The percentage of abnormalities increased with increased treatment duration. Triploid larvae of Pacific oysters produced by 6-DMAP or cytochalasin treatments had equivalent growth rates and were similar to those of control diploid larvae. However, triploid larvae showed high mortalities following these two chemical treatments. Overall, the results clearly demonstrate that 6-DMAP was an efficient and practical inducer of triploidy in bivalve molluscs. Moreover, the described procedure is the most simple and reproducible method ever reported. In addition, 6-DMAP is safer to handle than cytochalasin which is classified as a carcinogen.

Calmodulin in starfish oocytes: I. Calmodulin antagonists inhibit meiosis reinitiation

A heat-stable factor has been found in starfish (Patiria miniata and Marthasterias glacialis) oocytes that activates two calmodulin-dependent enzymes: bovine brain phosphodiesterase (10-fold increase) and sea urchin egg NAD-kinase (10- to 50-fold increase). The dose-response curves for activation of these enzymes were found to be parallel for the starfish egg extract and pure mammalian brain calmodulin. The active factor was purified by chromatography on DE 52 cellulose to which it remained bound and was eluted by 0.225 M ammonium sulfate. Active fractions were pooled, dialyzed, and run on a polyacrylamide gel. The starfish active factor comigrated with pure bovine brain calmodulin. A radioimmunoassay was performed on the purified factor; it cross-reacted with antibodies against pure calmodulin. That calmodulin may play a role in hormonally induced maturation of starfish oocytes is suggested by the fact that two calmodulin antagonists (trifluoperazine and vinblastine), which are also inhibitors of NAD-kinase, were found to block 1-methyladenine-induced oocyte maturation. The inhibition could be reversed by increasing the hormone concentration. Oocytes were sensitive to trifluoperazine only during the hormone-dependent period.

Changes in the pattern of protein phosphorylation during meiosis reinitiation in starfish oocytes

Abstract Endogenous protein phosphorylation in oocytes of Marthasterias glacialis was examined by incubating living oocytes with [32P]phosphate and cortical and endoplasmic fractions with [γ-32P]ATP. Individual phosphorylated proteins were detected by autoradiography after bidimensional and monodimensional electrophoresis, using SDS-polyacrylamide gradient gel slabs. An increased phosphorylation of several protein species was observed as early as 5 min following in vivo hormonal stimulation by 1-methyladenine. No dephosphorylation nor any change in protein staining of the gels was observed. In vitro phosphorylation patterns were consistent with those observed in vivo. They did not change upon in vitro 1-methyladenine addition and remained unaffected when the incubations were carried out in the presence of cAMP or beef heart protein kinase inhibitor. Cortical phosphorylation was inhibited by calcium ions. The results suggest that the hormone promotes alterations in the availability of phosphorylation sites in some proteins already present in the control oocytes which, as well as the corresponding activated cAMP-independent protein kinases, may play a significant role during formation of the maturation promoting factor.

Activation of calmodulin-dependent NAD+ kinase by trypsin

Sea urchin egg NAD+ kinase (ATP:NAD+ 2-phosphotransferase, EC 2.7.1.23), a calmodulin-dependent enzyme, can be activated by a moderate treatment with trypsin in a similar fashion to calmodulin. Stimulation by trypsin is dependent on its concentration (half-maximal dose: 1.5 microgram/ml) but independent of the presence of calcium. This suggests that limited proteolysis is able to activate NAD+ kinase as described for several other calmodulin-activated enzymes and that these enzymes may interact with calmodulin in a similar way.

Localization of electrical excitability in the early embryo of Dentalium

Abstract Electrophysiological properties of Dentalium trefoil embryos were examined. The blastomeres are electrically coupled, so to examine electrophysiological properties of individual blastomeres, we separated the vegetally located polar lobe and the two animal pole “QQ” cells by cutting with glass needles. The polar lobe membrane was found to be more excitable than that of the QQ cells, indicating a localization of ionic conductance mechanisms in the early embryo.