Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes.
Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.
Sellier
C
C
Laboratoire de Biologie du Développement, EA 1033, IFR 118, Université des Sciences et Technologies de Lille, SN3, Villeneuve d'Ascq, France.
Bodart
Jean-Francois
JF
Flament
S
S
Baert
F
F
Gannon
J
J
Vilain
J-P
JP
D000136:Acid-Base Equilibrium / Q000502:physiology*; D000643:Ammonium Chloride / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D005260:Female; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology*; D016258:Genes, mos / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000502:physiology; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008540:Meiosis / Q000187:drug effects; D008677:Metaphase / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D010766:Phosphorylation; D011374:Progesterone / Q000378:metabolism* / Q000494:pharmacology; D017231:Proto-Oncogene Proteins c-mos / Q000378:metabolism; D015398:Signal Transduction; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D029867:Xenopus Proteins; D000643:Ammonium Chloride; D011374:Progesterone; D017231:Proto-Oncogene Proteins c-mos; D016200:Maturation-Promoting Factor; D019950:Mitogen-Activated Protein Kinase 1; C448499:p42 MAP kinase, Xenopus