4-Aminopyridine acts as a weak base and a calcium mobilizing agent in triggering meiosis reinitiation and activation in oocytes of the Japanese clam Ruditapes philippinarum

Collogue

SBCF-ATIPEXNRS

Biologie

du Dkveloppement,

10.5

mars 1995

TRIGGERS MEIOSIS REINITIATION IN OF THE JAPANESE CLAM RUDITAPES PHIWPPINARUiU BY ELICITING AN INTRACELLULAR CALCIUM SURGE. LIPPAI Monica,GOBET Isabelle, TOMKOWIAK Martine, DUROCHER Yves, LECLERC Catherine, MOREAU Marc and GUERRIER Pierre. Laboratoire de Biologie moltkulaire et cellulaire, VMR 49, Ecole Normale Supkrieure, 46 Alltfe d’lralie, 69364 Lyon Ckdex 07 and Centre de Biologic du Dkveloppement. UMR 9926, Universitk Paul Sabatier, 118 route de Narbonne. 31062 Toulouse Ceder

4-AMINOPYRIDINE MOBILIZING AGENT AND ACTIVATION

Ovarian oocytes of the bivalve mollusc RuditapesphiIippinarum are arrested during first meiotic prophase. Release from this blockade is triggered by the neurohormone serotonin (5-hydroxytvptamine, 5HT) 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 Il9 and in the intracellular free Ca*+ concentration (GOBET L.DUROCHER Y..LECLERC C..

Ovarian oocytes of the prosobranch mollusc Parella vulgata and the pelecypod Rua’itapes philippinarum are arrested during prophase of the first maturation division. Release from this blockade, which is revealed by germinal vesicle breakdown (GVBD) drives these oocytes to a second arrest in metaphase I, at which time the oocytes became fertilizable. The respective roles of Ca*+ and H+ ion movements during this early step in meiosis reinitiation has not been yet fully established. Thus, while GVBD only depends on an intracellular alkalinisation in Patella, an intracellular calcium surge seems to be more directly involved in the case of bivalve oocyte maturation (GOBET I., DUROCHER Y.. LECLERC C., MOREAU M. and

THIMEROSAL OOCYTES

MOREAUM

andCUERRIER

P.(1994)Dev.Biol.,164.S40-549)

Here, we analyse 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 able to preclude or to reverse tubulin polymerization when applied respecdvely to prophase- or to metaphase-arrested oocytes. Our results strongly suggest that the common final target for 5HT and thimerosal actions consists in a transient rise in internal free Ca*’ level that we could follow using Fluo3/AM as a probe. The effect of thimerosal in promoting oocyte maturation and increasing intracellular free Ca*+ concentration was improved by excess KCl. In addition, thimerosal, but nof KCl, was found to facilitate 5HTinduced maturation at subthreshold hormone concentrations which, by themselves, did not produce an intracellular Ca*+ surge. These data suggest that thimerosal may increase sensitivity of the IP3 receptors and unmask plasma membrane voltage-sensitive Ca*+ channels.

CHROMOSOMAL MEIOTIC SOUSTELLE Equipe biologie

LOCALIZATION AND INITIATION RECOMBINATION IN THE YEAST Saccharomyces cerevisiae Christine and NICOLAS Alain G.M.R. Institut Curie section 26, rue d’lJlm - 75231 PARIS Cedex 0.5

Meiotic recombination t.he proper disjunction during the reductional

OF

RVDITAPES

plays an essential role in of homologous chromosomes division of meiosis (~oeder G.S., (1990). Trends genet., 6, 385-389). Several "hot spots I' have been caracterized in the genome of the yeast. The intergenic region upstream of the ARG4 gene contains an initiator of recombination (Nicolas N.P. and Szostak J.W. (1989). Nature, A., Treco D., Schultes 338, 35-39) that undergoes a transient DNA double strand break (DSB) during meiosis. The repair of this DSB leads to meiotic recombination (Sun H., Treco D. and Scostak J.W. (1989). Nature, 338, 87-90). The existence of a "cold" region (lcM=lOkb) and a "hot" region (lcM=lkb) on both sides of the ARG4 region suggests the existence of a non random distribution of the initiation points along the chromosomes. The molecular origin of this phenomenon is unknown (chromatin state, intergenic and coding regions, nuclear localization?...). We know that DSB regions correspond to hypersensitive sites to micrococcal nuclease (ohta K., Shibata T . and Nicolas A. (1994). EMBO J., 13, 5754-5763). To determine if "position effects" exist to influence the frequencies of DSB and recombination, we have constructed inversions of the ARG4 region (hot region in place of the cold one and vice-versa). The result shows that the position of DSB sites is conserved in the inverted fragment (10kb) and that there isn't or little variation in the DSB frequency. This suggests that the inverted fragment is autonomous with regard to the DSB formation and is therefore not influenced by the chromosomal environment.

PHILIPPlNARUM

GOBET DUROCHER

Isabelle, LIPPAI Monica, TOMKOWIAK Martine, Yves, LECLERC Catherine, MOREAU Marc and GUERRIER Pierre Laboratoire de Biologie moltkulaire et cellulaire. UMR 49, Ecole Normale S@rieure. 46 All&e d’ltalie, 69364 Lyon Ckdex 07 and Centre de Biologie du Dkeloppement, UMR 9926, Universitk Paul Sabatier, 118 route de Narbonne. 31062 Toulouse Ckdex

GUERRlER

P. (1994) Dev. Biol.. 164.540-549).

In this work, we reveal the presence of acidic vesicles in the cortex of these oocytes and report that bafilomycin Ai and N,N’ dicyclohexylcarbodiimide, two inhibitors of the vacuolar-type H+-ATPase, applied to Ruditapes oocytes, produce a significant inhibition of their response to serotonin, the neurohormone responsible for meiosis reinitiation in this species. 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 study the effect and mode of action of 4-aminopyridine, a drug reputed to be a K+ channel antagonist and report that it 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 Ca*+ surge. This dual effect may explain why Ruditapes oocytes no longer stop in metaphase under these conditions and complete their maturation alike other bivalve species which are directly fertilizable at the germinal vesicle stage.

GeneratIon related to imaging. de

ACTS AS A WEAK BASE AND A CALCIUM IN TRIGGERING MEIOSIS REINITIATION IN OOCYTES OF THE JAPANESE CLAM

of spontaneous oocyte growth

SrigiMe LeRvre: ‘INSERM Unifh “AVCR, tikhov

calcium oscillations by immature mouse and meiotic competence: evidence bv

oocytes confocal

Arletfe Pestv: Eva Nagyova” and Jacques Tesfarf 355, 32 rue des Carnets, 9214&Clamati, FRANCE. 277 21, Tchequie.

In viva, the mammal oocyte acquires its meiotic competence during its growth in the follicle. Fully-grown oocytes undergo spontaneous meiotic maturation as soon as released from the follicle into the cullure medium. Using confocal laser scanning microscopy, we monitored [CaL+]i in living mouse wcytes loaded with the calciumsensitive fluorescent dyes flue-3IAM or Nuca Green TM (specific for nuclear calcium) at the time of follicle release. + We observed that the rate of immature growing oocytes exhibiting regular spontaneous Ca2+ oscillations is related to the age of the mouse and the oocyte meiotic competence (from day 11: (10% to day 26: 57,7%). + Our data confirm that spontaneous regular Ca2+ oscillations (interspike interval 64.2 f 6.2 set) occured in 75.6% (n=197) of the ftuo-3 loaded control immature fully-grown mouse occytes (day 30) during the first half hour following release from the follicle and 88,7% of these oscillating oocytes underwent germinal vesicle breakdown (GVB) after one hour of culture. Moreover, there is a clear correlation between the germinal vesicle chromatin appearance and the oocyte abilily to exhibit calcium oscillations. The observation of a clear polarization of the Ca*+ waves in 30% of the oocytes permit us to hypothesize upon the existence of a predetermined localization for the initial point of the calcium wave. We noliced that, inside the GV of these polarized oocytes (n=53), the nucleolus was always nearer by the nuclear membrane adjacent to the site of wave occurence (25.5*1.9 m vs 37.3kO.9 pm, P