Characterization and in vitro control of MPF activity in zebrafish eggs.

We describe the characterization of maturation-promoting factor (MPF) in zebrafish eggs and used different defined conditions to maintain its activity in vitro. MPF activity levels are high in freshly ovulated mature eggs and decline rapidly within 5 min after either fertilization or parthenogenetic activation. The MPF activity of eggs matured in vitro declines faster when the eggs are incubated in Hank's culture medium supplemented with 0.5% BSA (H-BSA) than when incubated in Chinook salmon ovarian fluid (CSOF). MPF activity in nonactivated, aged eggs remains high in H-BSA supplemented with 75 microM MG132 or 10 mM caffeine, but neither MG132 nor caffeine can sustain high MPF activity in activated eggs. MG132-treated eggs showed delayed completion of metaphase and extrusion of the second polar body. Nuclear staining of the activated eggs confirmed the correlation between their cell cycle stage and MPF activity at each time point. An embryotoxic effect was found when matured eggs were held in 100 microM of MG132 or 20 mM caffeine for 1 h. Calcium-depleted medium and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid also showed detrimental effects on the embryos. Conversely, nonactivated, aged matured eggs maintained high MPF activity and developmental potential when CSOF was used as a holding medium.