Abstract
ABSTRACTEgg activation at fertilization in mammals is initiated by prolonged Ca2+ oscillations that trigger the completion of meiosis and formation of pronuclei. A fall in mitogen-activated protein kinase (MAPK) activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK pathway inactivation during fertilization of mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca2+ oscillations, but MAPK does not completely inactivate until after pronuclear formation. The MAPKs present in eggs are Mos, MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) and MAPK3 and MAPK1 (ERK1 and ERK2, respectively). Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in the signal from a Mos–luciferase reporter is not associated with egg activation. Furthermore, Mos overexpression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels or MEK activity.
Highlights
IntroductionMammalian oocytes are arrested at the second metaphase of meiosis
Before fertilization, mammalian oocytes are arrested at the second metaphase of meiosis
Monitoring mitogen-activated protein kinase (MAPK) activity with the ERK1 split-luciferase reporter MAPK activity reporter (MAPKAR) In order to measure changes in MAPK activity within intact mouse eggs, we designed a genetically-encoded bioluminescent reporter that is capable of monitoring the activity of the terminal protein kinase in the MAPK pathway, namely ERK
Summary
Mammalian oocytes are arrested at the second metaphase of meiosis. Received 23 October 2013; Accepted 25 March 2014 stimulated by the sequential action of two upstream protein kinases, Mos and MAP2K1 and MAP2K2 (hereafter referred to as MEK1/2) (Verlhac et al, 1994; Verlhac et al, 1996; Dupreet al., 2011) Both MPF and the MAPK pathway become active during the final stages of oocyte maturation as the oocytes transition from arrest at the germinal vesicle stage through meiosis to arrest at meiotic metaphase II (Verlhac et al, 1994; Verlhac et al, 1996; Dupreet al., 2011). Unlike MPF, the exact timing of MAPK inactivation has not been measured and there are no evident causal links between the signaling pathways triggered by the sperm and the delayed decline in MAPK activity
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