The effect of cdk kinase inhibitors on resumption of meiosis and activation of porcine oocytes

Butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle breakdown (GVBD) in porcine oocytes in a concentration-dependent manner. GVBD was almost totally inhibited over the course of 44 h of culture when 100 pM BL I was included in TCM 199 containing either 3 mg/ml BSA or 5 % fetal calf serum (FCS). In contrast, 50 pM BL 1 was fully effective in combination with BSA only. The FCS supplemented medium diminished the inhibitory effect of BL 1 (50 pM) and 42 % of oocytes underwent GVBD. In porcine oocytes eultivated in BL I-supplemented medium immediately after isolation neither p34cdc2 kinase nor MAP kinase was activated. The füll reversibility of the inhibitory step secures that during the subsequent maturation culture oocytes reached metaphase II with correctly arranged microtubuli in the meiotic spindle and well-expanded cumuli. Oocytes at late diakinesis stage (after 24 hr culture in control medium) did not continue in meiotic maturation (50 p.M BL I) while the p34cdc2 kinase activity was inhibited and the first meiotic spindle was not normally formed. In contrast, oocytes at late metaphase I anaphase I stage (after 30 hr culture in control medium) either formed one nucleus (38 %) or continued to metaphase II (62 %) during the 12 hr culture in 50 pM BL I. In metaphase II oocytes BL I induced parthenogenetic activation in a dose dependent manner (25, 50 and 100 uM, respectively), either alone (7 %, 35 %, 48 %) or in combination with ionomycin (27 %, 68 % and 82 %). A haploid pronucleus was observed (90 %) when Ca* influx was stimulated in the presence of BL I, although pronuclear formation without meiotic progression was observed when BL I was used alone. Combination of BL I and ionomycin can be an approach to improve the efficiency of mammalian oocyte activation.