284 KINETICS OF OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT OF PARTHENOGENETIC PORCINE EMBRYOS AFTER MEIOTIC INHIBITION WITH ROSCOVITINE

Abstract

Maturation of mammalian oocytes is a very important process for subsequent embryo development after fertilization. Prolonged maturation time by meiotic inhibitors could be an effective method for improvement in the meiotic and developmental competence of mammalian oocytes. Roscovitine, a cyclin dependent kinase inhibitor, is known to specifically inhibit M-phase promoting factor (MPF) kinase activity and prevent the resumption of meiosis. The aim of this study was to examine the effect of roscovitine on the maturation and subsequent development of porcine oocytes. Ovaries were collected from slaughtered prepubertal gilts and COCs were aspirated from 2- to 5-mm antral follicles. In control, porcine cumulus oocyte complexes (COCs) were cultured in the maturation medium (TCM-199 supplemented with 0.3% BSA, 1 μg/mL FSH, 1 μg/mL LH, and 10 ng/mL EGF) for 44 h. In the experimental group, COCs were cultured in the inhibition medium (TCM-199 supplemented with 0.3% BSA and roscovitine) for 24 h, and then further cultured in the maturation medium for 44 h. Matured oocytes from both groups were activated by electrical pulse (1.2 kV/cm for 30 μs), and then cultured in PZM-3 medium for 6 days. Apoptotic cells in blastocysts were detected by TUNEL assay and total cell number was examined by propidium iodide (PI) counterstaining. Data were analyzed by chi-square and Student's t-test. The first experiment was conducted to determine the effect of roscovitine (0, 12.5, 25, 50, and 100 μM) on meiotic inhibition of GV oocytes. This effect was dose-dependent, and a concentration of 50 μM was sufficient to prevent meiotic resumption in 79.2% (76/96, 5 replicates) of the porcine oocytes after 24 h of culture when compared to 0 (15.4%, 15/97), 12.5 (32.1%, 36/112), 25 (57.4%, 54/94), and 100 μM (77.8%, 77/99). The second experiment was carried out to examine the kinetics of maturation of roscovitine-treated porcine oocytes. The concentration of roscovitine used was 50 μM. A total of 75.8% (50/66, 3 replicates) of roscovitine-treated oocytes reached metaphase II stage compared with 70.8% (46/65) of control. The third experiment was performed to compare embryo development between control and treated group after parthenogenetic activation. No differences (P > 0.05) were found between the control and the treated group in cleavage rate (77.2%, 132/171 vs. 68.0%, 115/169), blastocyst rate (26.9%, 46/171 vs. 17.8%, 30/169), and total (33.7 ± 12.4 vs. 35.1 ± 12.6) and apoptotic (2.2 ± 2.4 vs. 2.2 ± 1.2) cell number per blastocyst (4 replicates). The results suggest that roscovitine can be used to prolong maturation time of porcine oocytes without reducing meiotic maturation but also without significantly decreasing their subsequent developmental competence. Further studies are necessary to improve the developmental competence of porcine oocytes treated with roscovitine.