High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity.
Roscovitine, a potent inhibitor of M-phase Promoting Factor (MPF) kinase activity, was used to maintain cattle oocytes at the germinal vesicle stage for a 24-hr culture period. A concentration of 25 microM of roscovitine was sufficient to reach the maximum level of meiotic resumption inhibition with 83 +/- 6% of the oocytes remaining at the germinal vesicle stage after the 24 hr of culture. The histone H1 kinase activity was maintained at a basal level after culture under roscovitine inhibition at any of the concentrations tested (12.5, 25, 50, and 100 microM). This inhibitory effect of roscovitine was fully reversible since 89 +/- 4% of the oocytes cultured for 24 hr in the presence of 25 microM 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 blastocyst stage was not different for oocytes cultured for 24 hr under roscovitine (25 microM) inhibition and then matured for 24 hr in the presence of EGF 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-treated oocytes). Roscovitine meiotic inhibition was also effective in the presence of EGF, and the final developmental potential as well as the kinetics of blastocyst formation were not affected after such prematuration treatment. The EGF induced cumulus expansion was also inhibited by roscovitine. These results indicate for the first time the feasibility of culturing cattle oocytes under meiotic inhibition without decreasing their resulting developmental potential.
Mermillod
P
P
INRA, Station de Physiologie de la Reproduction, Nouzilly, France. mermillo@tours.inra.fr
Tomanek
M
M
Marchal
R
R
Meijer
L
L
D000818:Animals; D001755:Blastocyst / Q000187:drug effects; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005307:Fertilization in Vitro; D008540:Meiosis / Q000187:drug effects*; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism*; D009924:Organ Culture Techniques / Q000379:methods; D011494:Protein Kinases / Q000378:metabolism; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D013997:Time Factors
D004791:Enzyme Inhibitors; D011687:Purines; D000077546:Roscovitine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; C102333:histone H1 kinase