Bicarbonate/chloride anion exchanger activity is cell cycle dependent during mouse oocyte meiotic maturation and egg activation.

摘要

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pHi) resulting from activation of a pH i regulatory transporter. As pHi regulation had not been studied in a mammalian model, I investigated pHi regulation in the mouse egg during meiotic maturation and egg activation. Steady-state pH i was measured using the pHi sensitive fluorophore SNARF-1-AM in germinal vesicle (GV) oocytes, ovulated eggs, and zygotes. No sustained changes in pHi occurred after germinal vesicle breakdown (GVBD), fertilization or during parthenogenetic egg activation.;HCO3-/Cl- exchanger activity was measured in unfertilized eggs and zygotes. Zygotes exhibited a marked intracellular alkalinization and Cl- efflux upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. HCO3-/Cl - exchanger activity was upregulated following in vitro fertilization (IVF) becoming maximal after 79 h. Activation of HCO3- /Cl- exchanger activity appeared to occur by activation of existing, inactive exchangers upregulation of activity was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. HCO3-/Cl- exchanger upregulation was also independent of PKC and cAMP-dependent pathways. Using cycloheximide-activated eggs, HCO3- /Cl- exchanger activation was independent of the repetitive Ca2+i transients.;HCO3-/Cl- exchanger activity, measured during the cell cycle, was robust in GV eggs, becoming downregulated during meiotic maturation. Low HCO3- /Cl- exchanger activity was a feature of meiotic metaphase only, as activity was not downregulated during metaphase of the first cell cycle. HCO3-/Cl- exchanger upregulation was dependent on an intact metaphase II spindle and could be blocked by the phosphatase inhibitor okadaic acid following Sr 2+ activation. Finally, HCO3-/Cl - exchanger activity could be activated in unfertilized eggs by the MEK inhibitor U0126. This suggests that HCO3- /Cl- exchanger activity is upregulated at fertilization in the mouse by a cell cycle-dependent mechanism that may involve the MAPK pathway.