P-144: Glucose-6-Phosphate Dehydrogenase Activity in Ovine Oocytes in Association with Developmental Characteristics and Expression of Bax Gene

Background: Recent studies have revealed that oocyte competence can be assessed through the presence of the glucose-6-phosphate dehydrogenase (G6PDH) enzyme, as indicated by brilliant cresyl blue (BCB), a dye that can be degraded by G6PDH. In the present study, we aimed to examine the validity of BCB test to select developmentally competent oocyte in ovine and its association with stage-specific expression profile of an apoptosis regulatory gene (Bax). Materials and Methods: Ovine cumulus-oocyte complexes (COCs) were exposed to 26 μM BCB diluted in mDPBS for 90 min. After BCB exposure, COCs were divided into 2 groups: BCB+ (blue cytoplasm, low G6PD-activity) and BCB- (colourless cytoplasm, high G6PD-activity). Oocytes in control group were incubated without exposure to BCB dye. After IVM, oocytes were subjected to IVF followed by embryo culture for 9 days. Concerning the transcript level, real-time PCR was used to quantify the expression of Bax mRNAs between Control, BCB+ and BCB- oocytes at different maturational stages: Germinal vesicle (GV) and Metaphase II (MII). Results: There were significantly more MII oocytes in BCB+ (78.9%) and control (73.8%) groups than in BCBgroup (52.4%, p<0.05). Both, BCB+ (71.7%) and Control (67.3%) groups showed significantly higher cleavage rates compared to BCB- group (50.5%, p<0.05 ), whereas no difference was found between Control and BCB+ group. Blastocyst rate was also significantly higher for BCB+ (34.5%) group compared to control (20.8%) and BCB- (4.4%, p<0.05) groups, with control group being significantly higher than BCB- group. Moreover, the expression of Bax gene at mRNA level showed no significant differences among the groups, neither at GV nor at MII stage. Conclusion: Using BCB test, we successfully incorporated the G6PDH-activity into selecting a subset of oocytes with superior developmental capacity with no correlation with transcription level of the Bax gene.