ABSTRACT
ERK-type MAP kinase activity is required for normal first meiotic (MI) metaphase spindle dynamics and first polar body formation at the MI/MII transition, and for MII arrest until egg activation. MEK and MAPK, however, remain active until meiosis is completed and pronuclei form, but whether MEK/MAPK activity affects MII spindle function during egg activation has been unknown. Polarized light microscopy revealed that the MII spindle rapidly (within approximately 15 min) lost birefringence upon treatment of the egg with U0126, indicating decreased organization at the molecular level upon MEK inhibition. In contrast, birefringence rapidly increased when MPF was inhibited with roscovitine, and this was similar to the increased birefringence previously shown after fertilization or parthenogenetic activation with Sr(2+). Confocal microscopy indicated that many spindles in U0126-activated eggs had failed to rotate or were dissociated from the egg cortex. Subsequently, abnormally-located midbodies were evident in U0126-induced parthenogenotes. Thus, MEK/MAPK activity is required to maintain the ordered structure of the MII spindle and for normal spindle dynamics during second polar body formation.
Subject(s)
MAP Kinase Kinase Kinases/physiology , Meiosis/physiology , Mitogen-Activated Protein Kinase Kinases/physiology , Ovum/metabolism , Animals , Butadienes/pharmacology , Cells, Cultured , Embryo Culture Techniques , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Female , GPI-Linked Proteins , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Male , Meiosis/drug effects , Membrane Glycoproteins/metabolism , Mesothelin , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Nitriles/pharmacology , Ovum/drug effects , Ovum/enzymology , Ovum/ultrastructure , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Spindle Apparatus/physiologyABSTRACT
The HCO(3)(-)/Cl(-) exchanger is quiescent in the unfertilized mouse egg but is highly active in regulating intracellular pH in the early embryo and required for normal development. We show here that the HCO(3)(-)/Cl(-) exchanger is active in first meiotic prophase (GV) oocyte but inactivated during meiotic metaphase before the MI to MII transition. Reactivation does not occur until the activated egg enters interphase. A quiescent HCO(3)(-)/Cl(-) exchanger is not simply a general feature of metaphase, because activity did not decrease during first mitotic metaphase. Inactivation of the HCO(3)(-)/Cl(-) exchanger during MI coincided with the activation of MAP kinase (MAPK), whereas its reactivation coincided with the loss of MAPK activity after egg activation. Maintaining high MAPK activity after egg activation prevented the normal reactivation of the HCO(3)(-)/Cl(-) exchanger. Inactivating MAPK in unfertilized MII eggs resulted in HCO(3)(-)/Cl(-) exchanger activation. Preventing MAPK activation during first meiotic metaphase prevented the inactivation of HCO(3)(-)/Cl(-) exchange. Conversely, activating MAPK in the GV oocyte resulted in inactivation of HCO(3)(-)/Cl(-) exchange. These results imply that the HCO(3)(-)/Cl(-) exchanger in mouse oocytes is negatively regulated by MAPK. Thus, suppression of pH-regulatory mechanisms during meiosis is a novel function of MAPK and cytostatic factor activity in the oocyte.
Subject(s)
Chloride-Bicarbonate Antiporters/metabolism , MAP Kinase Signaling System/physiology , Meiosis/physiology , Oocytes/physiology , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Bucladesine/metabolism , Butadienes/pharmacology , Calcium/metabolism , Cycloheximide/pharmacology , Demecolcine/pharmacology , Enzyme Inhibitors/pharmacology , Female , Humans , Hydrogen-Ion Concentration , Maturation-Promoting Factor/metabolism , Mice , Nitriles/pharmacology , Okadaic Acid/pharmacology , Oocytes/drug effects , Pregnancy , Strontium/metabolismABSTRACT
Mammalian eggs are arrested in metaphase II of meiosis until fertilization. Arrest is maintained by cytostatic factor (CSF) activity, which is dependent on the MOS-MEK-MAPK pathway. Inhibition of MEK1/2 with a specific inhibitor, U0126, parthenogenetically activated mouse eggs, producing phenotypes similar to Mos(-/-) parthenogenotes (premature, unequal cleavages and large polar bodies). U0126 inactivated MAPK in eggs within 1 h, in contrast to the 5 h required after fertilization, while the time course of MPF inactivation was similar in U0126-activated and fertilized eggs. We also found that inactivation of MPF by the cdc2 kinase inhibitor roscovitine induced parthenogenetic activation. Inactivation of MPF by roscovitine resulted in the subsequent inactivation of MAPK with a time course similar to that following fertilization. Notably, roscovitine also produced some Mos(-/-)-like phenotypes, indistinguishable from U0126 parthenogenotes. Simultaneous inhibition of both MPF and MAPK in eggs treated with roscovitine and U0126 produced a very high proportion of eggs with the more severe phenotype. These findings confirm that MEK is a required component of CSF in mammalian eggs and imply that the sequential inactivation of MPF followed by MAPK inactivation is required for normal spindle function and polar body emission.
