PARENT SESSION
SLIDE SESSION 5: REGULATION OF MEIOSIS
Chairs: Stephen Downs, Sue Varmuza, Michele Calder (Trainee)
Univ Ottawa-Monpetit 202
2:30 PM-4:30 PM

ROSCOVITINE-INDUCED INHIBITION OF BOVINE OOCYTE MATURATION IS ENHANCED IN BSA SUPPLEMENTED MEDIA AND PROGRESSION TO MII IS ACCELERATED FOLLOWING RELEASE FROM INHIBITION.

Calder, Michele¹, Antonello, Laura¹, Watson, Andrew¹, ¹

ABSTRACT- Roscovitine is a specific inhibitor of MPF (cdc2-kinase) and inhibits progression of oocytes to MII for at least 24h. Blockage is reversible by washing oocytes and placing them into maturation media (TCM-199 plus serum and gonadotropins). In experiment one, cumulus-oocyte complexes were placed into TCM-199 containing pyruvate and bicarbonate in either 1) 3 mg/ml BSA (Fraction V) or 2) 10% newborn calf serum with 0, 12.5, 25, 50 and 100M roscovitine. After 23-24h, COCs were vortexed to remove cumulus cells and fixed in 1% paraformaldehyde. Oocytes were stained with 50ng/ml DAPI for 30 minutes, mounted on slides under coverslips supported by vaseline and examined under UV excitation. Oocytes were rated as germinal vesicle (GV), condensed chromatin (CC), premetaphase II (individual chromosomes visible) or MII (two groupings of chromosomes). The number of oocytes remaining immature (GV or CC) after 24h culture was compared. Doses of roscovitine above 25M significantly inhibited maturation compared to DMSO carrier (P < 0.001), but maturation blockage was higher in BSA vs. FCS (P < 0.001). At 12.5M roscovitine, maturation was inhibited more in media supplemented with BSA compared to serum (P < 0.001). In experiment two, roscovitine pre-treated oocytes (24 h) were fixed at 12, 14, 16 and 18h after placement into maturation media and progression to MII was compared to control oocytes placed directly in maturation media and fixed at 16 and 18h. Greater proportions (62.3 and 74.6%) of roscovitine pre-treated oocytes (24h) were at MII by 16h and 18h maturation compared to control oocytes at 16h and 18h (35.1 and 50.3%, P < 0.05). In conclusion, a lower dose of roscovitine can be used in BSA-supplemented compared to serum-supplemented media and progression to MII after roscovitine release is hastened, which may necessitate an earlier fertilization time. This may also have implications for synchronizing ooplasm for embryo cloning approaches.

KEY WORDS: oocyte, maturation, roscovitine, bovine