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Procaine effects on Xenopus oocytes
S-PHASE BY 6-DMAP TREATMENT OF MATURING OOCYTES AND MITOTIC EMBRYOS FROM MiRINii IMVERTEBRATES. NEANT Isabel~ and DUBE Fraqois VniversW du Qltebec d Rimouski. Rimouski, PQ, Canada GSL 3AI and VMR49, ENS-Lyon, 69 364 Lyon cedex 07, France.
Fertilization stirs up dormant oocytes, arrested at one of four specific stage of meiotic maturation. Experiments were undertaken with oocytes from three marine invertebrate species, i. e. Spisula solidissima, Myti&s edulis and Strongytocentrotus droebachiensis, respectively fertilized at prophase I, metaphase I and pronucleus stages. While early embryonic mitoses exhibit rapid switching from M-phase (mitosis) to S-phase (DNA synthesis), meiotic cycles proceed without DNA replication. We previously showed that 6dimethylaminopurine (6DMAP). a potent protein kinase inhibitor, induces a release from M-phase arrest in Myri1u.s oocytes, followed by transition into an interphasic stage that is accompanied by a continuous DNA synthesis. MPF activity drops down and the p34edc2 h omolog becomes phosphorylated on tyrosine, in association with posttranslationally modified cyclins (N&x eful., 1994. Ia J Dev. Bid., 38, 513.523). Moreover, 6-DMAP drives backwards dividing cells into interphase. It thus induces continuous SH-thymidinc incorporation during Spisula oocyte meiosis and allows only one round of DNA replication during mitosis of clam and sea urchin embryos, whereas p34cdc2 homologs become phosphorylated on tyrosine in all cases. 6. DMAP overcomes the inhibition controls for DNA replication in meiosis, suggesting that this controlling event relies on protein phosphorylation. A model is proposed in light of these results
CYTOSTATIC FACTOR INACTIVATION IS INDUCED BY A CALCIUM-DEPENDENT MECHANISM PRESENT UNTIL THE SECOND CELL CYCLE IN FERTILIZED BUT NOT IN PARTHENOGENETICALLY ACTIVATED MOUSE EGGS
ZERNICKA-GOETZ#* Magdalena, KUBIAK# J&, ClEMERYCH* Maria, TARKOWSKI* Andrzej and MARO# Bernard If Departement de Biologic du Developpement, Institut Jacques Monod, C.N.R.S. - Universite Paris VII, Tour 43 - 2, place Jussieu, 75005 Paris. France * Department of Embryology, Institute of Zoology, University of Warsaw, Krakow&e Przedmiescie 26/28, (Xl-927 Warsaw 64, Poland Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained. arrested in M-phase, the oocytes fused with- fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte aetivation was still present af the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involvedin calcium release triggered by a sperm component at fertifization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage.
CORTO,
A NOVEL
DROSOPHiLA
N G%ftique ef Physiologia tuminy, Case 9W,‘I3288
NUCLEAR ANTIGEN. RQS$BT Roland, Laboratoire du D&eloppement, Fucuttt des Sciences Marseille, Cedex 9, France.
Law
dr de=
We report here on the isolation of a novel gene cloned froar an enhancer trap line and located in region 82E, on the third chromosome. This gene, named corfo, is expressed throughout the develoomenc of the fly iticluding oogenesis. The aminoa&l sequence deduced from conceptual translation of a full-length NJ340 cDNA shows no obvious similiraties to any known protein. The major characteristics are the basic nature (PI=IO) and tlmpresence of long homopolymeric stretches of glutamine, histidiue and alanine. Three rat polyclonal sera have been raised against two it&pendant Corm synthetic peptides and a BG& Corto fusion expressed in E. coli. These sera have allowed us to show that the pattern of expression of cortu mRNA and protein are identical all along the development In addition we have shown that the gene corm codes for a protein of 60 kDa distributed on the chromosome aims and the centrosomes. We are presently investigating the cellular function of this gene and its role during the fly development. Over-expression of a cOrt(l cDNA lead to au embryonic or late larval lethality depending on the time and the amount of product delivered. The adult escapers show phenotypes suggesting a reduction in the number of imagiaal cells. In addition we try to induce loss-of-function alleles bv+ a .Iiumu-start I mutagenesis. Our working hypothesis is presently hased on a role for the Corm protein during mitosis.
PROCAINE EFFECTS
ON XJ3NOPUS
BROWAEYS Edith l, FLAMEEFf VILAIN
Jean-Pierre
Itioratoire Technologies 2 Laboratoire 5 Rue Blake
OOCYTES
‘, RODEAU Jean-Luc 2 et
1
de Bioio ie du L%veloppement Vniversitt! des Scierxes t’~ de Lille S&S5 - Wlenewe d%sci Cedex de Neurobiolagie Cell&t-e, Centre de Neurochimie, CNM Paxcal - 67&W strusbourg Cedex
Full grown Xenopus oocytes undergo an alkalinization during progesterone stimulation. Proeaiue, a local auesfhetic, hasbeen used by sawem! a&rem to artificially alkali&e. the oocvte cvtosol and to in&me meiosis reinitiation. We have-reinvestigated the &eei of procaine on XemF oocytes using microelectrodes to measure intmceltular uH (OHiT, cd membmtte uoterttiat . conduct&e and voltage clamp eurreut. ~His&&eal studies have-&o be& carried out on procaine stimulated uoeytes. Bath perfusion with IO~mM procaine (pH 8.5) caused cell membrane depoktrization or, in voltage clamp conditions at a holding membrane potentiel of - 60 mV, elicited an inward current of -121 f 37 nA (SE&I ~ n = 27) associated with an increase in membrane conductance while pHi slowly decreased. It is the first report for an intracellular acidifmation~by piwaiae in Xenopus oacyte. When control pfxfwion~was reszlmed; dt the eleetrophysioto@al parameters returned rapi&y to their initial values, while PHi ceutmued to f&l Slightly before slowiy reeoveriug. Germinal vesicle -breakdown (GVBD), meiotic spindle and chromosomes coa*nsation were present ouly in progesterone treated ooeytes w&n exposed for at least 8 hours. Procaine treated oocytes did not show t&e usual white spot of matumclooeyte even when expoaiti@r was increasedug to-24hours. The germmal vesicle did not move toward the animal p&e but GVBD was observedin 49% of ooeytes expose&during 16 hours and in 715% of oocytes exposed during 24 hours. GVBD was accompanied by a meiotic spindle and condensed chromosomesin 37 % of the c&es. The “pseudo maturing” effect decreased when procaine was used at 10 mM in ND96 pH 7.5 whjle 20 mM pH 8.5 was rather toxic. Increasing the pH oft the medium to 8.5 without procaine addition never induced GV~BD in Xerwpus
oocytes.
Fertilization stirs up dormant oocytes, arrested at one of four specific stage of meiotic maturation. Experiments were undertaken with oocytes from three marine invertebrate species, i. e. Spisula solidissima, Myti&s edulis and Strongytocentrotus droebachiensis, respectively fertilized at prophase I, metaphase I and pronucleus stages. While early embryonic mitoses exhibit rapid switching from M-phase (mitosis) to S-phase (DNA synthesis), meiotic cycles proceed without DNA replication. We previously showed that 6dimethylaminopurine (6DMAP). a potent protein kinase inhibitor, induces a release from M-phase arrest in Myri1u.s oocytes, followed by transition into an interphasic stage that is accompanied by a continuous DNA synthesis. MPF activity drops down and the p34edc2 h omolog becomes phosphorylated on tyrosine, in association with posttranslationally modified cyclins (N&x eful., 1994. Ia J Dev. Bid., 38, 513.523). Moreover, 6-DMAP drives backwards dividing cells into interphase. It thus induces continuous SH-thymidinc incorporation during Spisula oocyte meiosis and allows only one round of DNA replication during mitosis of clam and sea urchin embryos, whereas p34cdc2 homologs become phosphorylated on tyrosine in all cases. 6. DMAP overcomes the inhibition controls for DNA replication in meiosis, suggesting that this controlling event relies on protein phosphorylation. A model is proposed in light of these results
CYTOSTATIC FACTOR INACTIVATION IS INDUCED BY A CALCIUM-DEPENDENT MECHANISM PRESENT UNTIL THE SECOND CELL CYCLE IN FERTILIZED BUT NOT IN PARTHENOGENETICALLY ACTIVATED MOUSE EGGS
ZERNICKA-GOETZ#* Magdalena, KUBIAK# J&, ClEMERYCH* Maria, TARKOWSKI* Andrzej and MARO# Bernard If Departement de Biologic du Developpement, Institut Jacques Monod, C.N.R.S. - Universite Paris VII, Tour 43 - 2, place Jussieu, 75005 Paris. France * Department of Embryology, Institute of Zoology, University of Warsaw, Krakow&e Przedmiescie 26/28, (Xl-927 Warsaw 64, Poland Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained. arrested in M-phase, the oocytes fused with- fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte aetivation was still present af the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involvedin calcium release triggered by a sperm component at fertifization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage.
CORTO,
A NOVEL
DROSOPHiLA
N G%ftique ef Physiologia tuminy, Case 9W,‘I3288
NUCLEAR ANTIGEN. RQS$BT Roland, Laboratoire du D&eloppement, Fucuttt des Sciences Marseille, Cedex 9, France.
Law
dr de=
We report here on the isolation of a novel gene cloned froar an enhancer trap line and located in region 82E, on the third chromosome. This gene, named corfo, is expressed throughout the develoomenc of the fly iticluding oogenesis. The aminoa&l sequence deduced from conceptual translation of a full-length NJ340 cDNA shows no obvious similiraties to any known protein. The major characteristics are the basic nature (PI=IO) and tlmpresence of long homopolymeric stretches of glutamine, histidiue and alanine. Three rat polyclonal sera have been raised against two it&pendant Corm synthetic peptides and a BG& Corto fusion expressed in E. coli. These sera have allowed us to show that the pattern of expression of cortu mRNA and protein are identical all along the development In addition we have shown that the gene corm codes for a protein of 60 kDa distributed on the chromosome aims and the centrosomes. We are presently investigating the cellular function of this gene and its role during the fly development. Over-expression of a cOrt(l cDNA lead to au embryonic or late larval lethality depending on the time and the amount of product delivered. The adult escapers show phenotypes suggesting a reduction in the number of imagiaal cells. In addition we try to induce loss-of-function alleles bv+ a .Iiumu-start I mutagenesis. Our working hypothesis is presently hased on a role for the Corm protein during mitosis.
PROCAINE EFFECTS
ON XJ3NOPUS
BROWAEYS Edith l, FLAMEEFf VILAIN
Jean-Pierre
Itioratoire Technologies 2 Laboratoire 5 Rue Blake
OOCYTES
‘, RODEAU Jean-Luc 2 et
1
de Bioio ie du L%veloppement Vniversitt! des Scierxes t’~ de Lille S&S5 - Wlenewe d%sci Cedex de Neurobiolagie Cell&t-e, Centre de Neurochimie, CNM Paxcal - 67&W strusbourg Cedex
Full grown Xenopus oocytes undergo an alkalinization during progesterone stimulation. Proeaiue, a local auesfhetic, hasbeen used by sawem! a&rem to artificially alkali&e. the oocvte cvtosol and to in&me meiosis reinitiation. We have-reinvestigated the &eei of procaine on XemF oocytes using microelectrodes to measure intmceltular uH (OHiT, cd membmtte uoterttiat . conduct&e and voltage clamp eurreut. ~His&&eal studies have-&o be& carried out on procaine stimulated uoeytes. Bath perfusion with IO~mM procaine (pH 8.5) caused cell membrane depoktrization or, in voltage clamp conditions at a holding membrane potentiel of - 60 mV, elicited an inward current of -121 f 37 nA (SE&I ~ n = 27) associated with an increase in membrane conductance while pHi slowly decreased. It is the first report for an intracellular acidifmation~by piwaiae in Xenopus oacyte. When control pfxfwion~was reszlmed; dt the eleetrophysioto@al parameters returned rapi&y to their initial values, while PHi ceutmued to f&l Slightly before slowiy reeoveriug. Germinal vesicle -breakdown (GVBD), meiotic spindle and chromosomes coa*nsation were present ouly in progesterone treated ooeytes w&n exposed for at least 8 hours. Procaine treated oocytes did not show t&e usual white spot of matumclooeyte even when expoaiti@r was increasedug to-24hours. The germmal vesicle did not move toward the animal p&e but GVBD was observedin 49% of ooeytes expose&during 16 hours and in 715% of oocytes exposed during 24 hours. GVBD was accompanied by a meiotic spindle and condensed chromosomesin 37 % of the c&es. The “pseudo maturing” effect decreased when procaine was used at 10 mM in ND96 pH 7.5 whjle 20 mM pH 8.5 was rather toxic. Increasing the pH oft the medium to 8.5 without procaine addition never induced GV~BD in Xerwpus
oocytes.