The applicant proposes to examine the role of protein phosphorylation in regulating the first cycle of division of the fertilized mouse egg during which time there is a transition from meiotic to mitotic control. Specifically, the investigators will determine the role of MAP kinase in pronuclear envelope assembly/disassembly and in maintenance of meiotic arrest by sustaining high levels of cdc2/cyclin B kinase activity. In the first specific aim, a constitutively active mutant of MAP kinase (MEKE) will be injected into fertilized eggs and the effects of activated endogenous MAP kinase on pronucleus formation and pronuclear envelope breakdown will be observed. MEKE is a dual specificity kinase that phosphorylates both threonine and tyrosine residues and is the only known direct upstream activator of MAP kinase. In the second aim, the role of MAP kinase and maintenance of meiotic arrest will be examined. In one set of experiments, the phosphatase that is specific for MAP kinase (MKP) and causes MAP kinase inactivation will be injected into metophase II eggs. These eggs will then be examined for the phosphorylation of MAP kinase and maintaining the high levels of cdc2/cyclin B kinase activity, as well as resumption of the cell cycle. In the second set of experiments, MEKE will be injected into fertilized eggs that are reversibly arrested in M phase by an incubation medium containing a nocodazole. The cells will then be microinjected with MEKE and cells incubated in a nocodazole-free medium. The cells will then be assayed for endogenous MAP kinase activity and cell cleavage arrest.
| Kubelka, Michal; Anger, Martin; Pavlok, Antonin et al. (2002) Activation of pig and cattle oocytes by butyrolactone I: morphological and biochemical study. Zygote 10:47-57 |
