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1.
Int J Dev Biol ; 39(3): 485-91, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7577439

ABSTRACT

Ovarian oocytes of the prosobranch mollusc Patella vulgata and the pelecypod Ruditapes philippinarum are arrested during prophase of the first maturation division. Release from this blockade, which is revealed by germinal vesicle breakdown, drives these oocytes to a second arrest in metaphase I, at which time the oocytes become fertilizable. The respective roles of Ca2+ and H+ ion movements during this early step in meiosis reinitiation has not been fully established yet. In this work we reveal the presence of acidic vesicles and report that bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, two inhibitors of the vacuolar-type H(+)-ATPase, applied to Ruditapes oocytes, produce a significant inhibition of their response to the natural neurohormone serotonin. Since sodium deprivation did not affect this response, this suggests that a v-type ATPase pump, possibly located in the membrane of these acidic vesicles, may play a subtle role in the cascade of events that releases oocytes from their prophase block. We then describe how 4-aminopyridine, a drug reputed to be a K+ channel antagonist, triggers both meiosis reinitiation and activation of Patella and Ruditapes oocytes. This agent acts as a weak base, its effect depending on external pH. Moreover, using the fluorescent probes BCECF and Fluo-3/AM, we observe that this drug both alkalinizes the endoplasm and promotes an intracellular Ca2+ surge. This dual effect may explain why Ruditapes oocytes no longer stop in metaphase under these conditions and behave like other bivalve species which are directly fertilizable at the germinal vesicle stage.


Subject(s)
4-Aminopyridine/pharmacology , Calcium/metabolism , Macrolides , Oocytes/cytology , Animals , Anti-Bacterial Agents/pharmacology , Bivalvia , Dicyclohexylcarbodiimide/pharmacology , Enzyme Inhibitors/pharmacology , Female , Hydrogen-Ion Concentration , Kinetics , Meiosis/drug effects , Metaphase , Oocytes/drug effects , Oocytes/physiology , Proton-Translocating ATPases/antagonists & inhibitors , Sodium/metabolism
2.
Int J Dev Biol ; 39(2): 401-7, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7669551

ABSTRACT

Ovarian oocytes of the bivalve mollusc Ruditapes philippinarum are arrested during first meiotic prophase. Release from this blockade is triggered by the neurohormone serotonin (5HT or 5-hydroxytryptamine), which promotes germinal vesicle breakdown and drives these oocytes to a second arrest in metaphase I. 5HT action involves binding to a specific G protein-coupled receptor which results in a transient rise in IP3 and in the intracellular free Ca2+ concentration. Here we analyze the cytological effects and mode of action of the sulphydryl reagent thimerosal which could also trigger meiosis reinitiation in Ruditapes. No metaphase I spindle formed under these conditions since thimerosal was found to be able to preclude or reverse tubulin polymerization when applied to prophase- or to metaphase-arrested oocytes, respectively. Our results strongly suggest that the common final target for 5HT and thimerosal actions consists in a transient rise in internal free Ca2+ level that we could follow using Fluo3/AM as a probe. The effect of thimerosal in promoting oocyte maturation and increasing intracellular free Ca2+ concentration was improved by excess KCI. In addition, thimerosal, but not KCI, was found to facilitate 5HT-induced maturation at subthreshold hormone concentrations which, by themselves, did not produce an intracellular Ca2+ surge. These data suggest that thimerosal may inhibit Ca2+ pumps of the endoplasmic reticulum and unmask the plasma membrane voltage-sensitive Ca2+ channels which also appear after 5HT-induced GVBD.


Subject(s)
Bivalvia , Calcium/metabolism , Meiosis/drug effects , Oocytes/cytology , Thimerosal/pharmacology , Aniline Compounds , Animals , Chromosomes/ultrastructure , Female , Fluorescent Dyes , Microtubules/drug effects , Oocytes/metabolism , Oocytes/ultrastructure , Potassium Chloride/pharmacology , Serotonin/pharmacology , Xanthenes
3.
Dev Biol ; 164(2): 540-9, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8045350

ABSTRACT

Prophase-arrested oocytes of Ruditapes philippinarum are triggered to undergo germinal vesicle breakdown under the influence of the neurohormone serotonin (5HT) and then arrest in metaphase 1. Our data show that these oocytes possess a single class of original 5HT receptors. Their binding parameters have been determined on semipurified membrane preparations incubated with [3H]5HT. No significant differences were observed when comparing 5HT-competent and -incompetent batches as well as prophase- or metaphase-arrested oocytes. Specific experiments including incubation with mastoparan or mas 7, GTP iontophoresis, and IP3 quantification strongly suggest that these receptors must be coupled with G-proteins to be functional. Peak change in IP3 mass occurs at 3 min and is likely to trigger the 5HT-dependent Ca2+ transient that begins at this time. In metaphase-arrested oocytes, binding of 5HT to its receptors no longer produces a Ca2+ surger. This is likely to result from a negative retrocontrol loop which would involve kinase C and exert its effect upstream of the Ca2+ surge. Indeed, the phorbol ester PMA proved able to reduce the Ca2+ response and to block 5HT action when applied during the first 3 min corresponding to the hormone-dependent period. Such an inhibition was reversed in the presence of 5 microM of the C kinase inhibitor GF109203X and could be bypassed by ionophore, ammonia, and thapsigargin, which trigger a receptor-independent Ca2+ surge.


Subject(s)
Meiosis , Oocytes/metabolism , Serotonin/metabolism , Signal Transduction , Animals , Binding Sites , Bivalvia , Calcium/metabolism , GTP-Binding Proteins/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Protein Kinase C/metabolism
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