High developmental competence of cattle oocytes maintained at the germinalvesicle stage for 24 hours in culture by specific inhibition of MPF kinaseactivity

Roscovitine, a potent inhibitor of M-phase Promoting Factor (MPF) kinase ac tivity, was used to maintain cattle oocytes at the germinal vesicle stage f or a 24-hr culture period. A concentration of 25 mu M of roscovitine was su fficient to reach the maximum level of meiotic resumption inhibition with 8 3 +/- 6% of the oocytes remaining at the germinal vesicle stage after the 2 4 hr of culture. The histone H1 kinase activity was maintained at a basal l evel after culture under roscovitine inhibition at any of the concentration s tested (12.5, 25, 50, and 100 100 mu M). This inhibitory effect of roscov itine was fully reversible since 89 +/- 4% of the oocytes cultured for 24 h r in the presence of 25 mu M of roscovitine reached the metaphase II stage after a further culture of 24 hr in permissive medium (TCM199 supplemented with 10 ng/ml EGF). The cleavage rate as well as the development to the bla stocyst stage was not different for oocytes cultured for 24 hr under roscov itine (25 mu M) inhibition and then matured for 24 hr in the presence of EG F as compared to oocytes not submitted to prematuration culture (82 +/- 8% cleavage and 41 +/- 4% blastocysts at 8 days post insemination for control oocytes compared to 90 +/- 7% and 36 +/- 7% respectively for roscovitine-tr eated oocytes). Roscovitine meiotic inhibition was also effective in the pr esence of EGF, and the final developmental potential as well as the kinetic s of blastocyst formation were not affected after such prematuration treatm ent. The EGF induced cumulus expansion was also inhibited by roscovitine. T hese results indicate for the first time the feasibility of culturing cattl e oocytes under meiotic inhibition without decreasing their resulting devel opmental potential. (C) 2000 Wiley-Liss, Inc.