Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development.
Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24h culture period. Cumulus-oocyte complexes were first prematured for 24h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 microM). 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 microM 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 microM (2.9%) and 25 microM (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 24h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 microM 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 microM 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 microM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 microM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.
Albarracín
José Luis
JL
Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Morató
Roser
R
Izquierdo
Dolors
D
Mogas
Teresa
T
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects*; D002478:Cells, Cultured; D002970:Cleavage Stage, Ovum; D003470:Culture Media; D003599:Cytoskeleton / Q000187:drug effects*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D008540:Meiosis; D008677:Metaphase; D018613:Microscopy, Confocal; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development* / Q000648:ultrastructure*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D013997:Time Factors
D003470:Culture Media; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine