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
38
REVERSIBILITY IN THE CYTOSKELETON AND DEVELOPMENT OF PIG OOCYTES TREATED WITH ROSCOVITINE.
Ju, Jyh-Cherng 1, Tsay, Chih1, Ruan, Chiwai2, Yang, Xiangzhong3, 1 2 3
ABSTRACT- Germinal vesicle (GV) breakdown of mammalian oocytes is regulated by activation of maturation promoting factor (MPF). A specific cdc2 kinase inhibitor, roscovitine, was used to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and were cultured in NCSU#23 medium containing different levels of roscovitine (0,10, 20, 30, 40, 50 uM in Exp 1 and 0, 40, 60, 80, 100, 120 uM in Exp 2) for 44 h. The COCs were cultured for another 44 h after removal of the chemical. Twenty oocytes in each group were fixed at 44 h in fixative for immunocytochemical labeling of the cytoskeleton and the rest (~20/group) were fixed at the end of 88 h after culture. Results showed that inhibition of the oocyte in the GV stage was not effective when 10-50 uM (Exp 1) of roscovitine were used (10-13%). Large proportions of oocytes (70-87%) escaped from inhibition and reached metaphase II (MII) or advanced stages at 44 h of culture, which were similar to that in the control group (87%) except for that in the 50 uM group (61%, P<0.05). When oocytes were released from the inhibitor, similar proportions (72-83%) of oocytes reached MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Exp 2), higher inhibitory effect was observed at the levels of 80-120 uM with 70-74% oocytes being blocked in the GV stage when compared to the control (19%) and the 40-60 uM (24-33%) groups. Although 15-21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and achieved MII stage at 44 h after removal of the chemical. This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor, which might provide flexibility for studying porcine oocyte development.
KEY WORDS: Cdc2 kinase inhibitor, IVM, microtubule, microfilament