Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development

Abstract

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24 h culture period. Cumulus–oocyte complexes were first prematured for 24 h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 μM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 μM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 μM (2.9%) and 25 μM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24 h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 μM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 μM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 μM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 μM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.