Abstract:
Cyclin-dependent kinase inhibitors (CDKIs) have been used for prematuration culture the aim at improving oocyte competence. However, CDKIs seem to accelerate nuclear maturation (Hashimoto et al., 2002 Biol. Reprod. 66, 1696?1701). The aim of the present work was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at low dose (Ponderato et al, 2001 Mol. Reprod. Dev. 60, 579?585) on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 ?M BLI (B) or 6.25 ?M BLI + 12.5 ?M ROS (BR) in TCM-199 for 24 h. After prematuration, oocytes were submitted to in vitro maturation (IVM in TCM-199 + 0.5 ?g mL?1 FSH, 50 ?g mL?1 LH, 10% FCS) for another 24 h. Oocytes were fixed every 3 h (40?50 oocytes/time point/group in 4 replicates) to assess nuclear status. In Experiment 2, oocytes were submitted to prematuration, but the inhibitors were diluted in TCM-199 or DMEM. IVM lasted 21 h in DMEM (same hormone supplementation as in TCM-199 + 5% FCS and 50 ng mL?1 EGF). After IVM, all groups (140?150 oocytes/group in 7 replicates) were in vitro fertilized. Oocytes and sperm (2 _ 106 sperm cells mL?1) were co-cultured for 18 h. Embryos were cultured in CR2aa in co-culture with granulosa cells for 8 days. All cultures were in microdrops under oil, at 38.5�C under 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. Data were analyzed by GLM and GENMOD procedures (SAS program; SAS Institute, Inc., Cary, NC, USA), for Experiments 1 (4 replicates) and 2 (7 replicates), respectively. Cell numbers were analyzed by ANOVA and Tukey test. In Experiment 1, at 0 h, C and B oocytes were all (100%) at germinal vesicle stage (GV). BR had less GV oocytes (89 � 1%, P < 0.05), indicating that BR was less effective in maintaining meiotic block for 24 h. After 3 h IVM, B and BR had less oocytes in GV (85 � 2 and 80 � 1%, respectively; P > 0.05) than C (100%, P < 0.05), suggesting an acceleration of oocyte maturation. At 12 h, however, most oocytes were at intermediate stages (metaphase I to telophase I) in all groups (78 � 1?83 � 2%, P > 0.05). After 21 and 24 h, all groups had similar metaphase II (MII) rates (77 � 1?89 � 1 for 21 h and 85 � 2?96 � 8 for 24 h P > 0.05). These results suggest that after 12 h, meiosis acceleration was less evident and oocytes proceeded nuclear maturation at similar rates. In Experiment 2, cleavage (79 � 3?84 � 3%, P > 0.05) and Day 7 blastocyst rates (26 � 4?37 � 4%, P > 0.05) were similar for all groups. After 8 days in culture, all groups presented similar blastocyst rates (35 � 4?40 � 4%, P > 0.05), except for the group prematured with BR in DMEM, which presented lower blastocyst rates (32.3 � 4%) only when compared with C (40 � 4%, P < 0.05). Hatching rates were similar (10 � 3?16 � %3, P > 0.05) as were total cell numbers (141 � 5?170 � 10). In conclusion: (a) BR is less effective in maintaining meiosis block; (b) B and BR accelerate the first half of meiosis progression in about 3 h; and (c) BR used in DMEM during prematuration may negatively affect developmental rates. Financial support was provided by Fapesp, Brazil.