1 EFFECT OF OXYGEN TENSION DURING IN VITRO MATURATION AND FERTILIZATION ON BOVINE EMBRYO PRODUCTION AND CUMULUS–OOCYTE-COMPLEX mRNA ABUNDANCE

Abstract

Despite physiological oxygen tension being lower than in atmospheric gas, both in vitro maturation and fertilization are generally conducted under atmospheric oxygen tension (20%). The objective of this study was to evaluate the effect of two different oxygen concentrations (20 and 5%) during in vitro maturation (M) and fertilization (F) on bovine embryo production and to analyze differences in gene expression between cumulus–oocyte-complexes (COC) matured at 5% (M5) or 20% (M20) oxygen tension. A total of 1179 COC were matured, fertilized and cultured in vitro in 5 replicates divided in 4 groups according to the oxygen tension used (M5F5, M5F20, M20F5 and M20F20). Cleavage was assessed every 4 h from 24 to 48 h post-insemination (pi) and blastocyst yield was recorded from Day 6 to Day 8. For gene expression analysis, 5 pools of 10 COC (not denuded) of each group were snap frozen after maturation and stored at –80°C. After RNA extraction and RTPCR, qPCR was used to quantify relative abundance of 7 genes: the M-phase promoting factor subunit Cyclin B1, insulin-growth factor 2 (IGFR2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A (LDHA), glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GPX1), and the apoptosis related gene BAX. Histone 2A was used as housekeeping gene. Statistical analysis was performed analyzing two factors (M5 v. M20 and F5 v. F20) by two-way ANOVA (P ≤ 0.05) for embryo production and by one-way ANOVA (P ≤ 0.05) for gene expression. At every time point analyzed, maturation under low oxygen tension significantly improved both cleavage rate and blastocyst yield. However, the use of low oxygen tension at fertilization has a significant negative effect on developmental rates (48 hpi: M5F5: 71.6 ± 5.5; M5F20: 76.2 ± 2.7; M20F5: 40.1 ± 5.8; M20F20: 69.9 ± 0.8). (Day 6: M5F5: 12.6 ± 3; M5F20: 17.2 ± 2.4; M20F5: 2.5 ± 1.2; M20F20: 13.3 ± 4.) (Day 7: M5F5: 32.6 ± 4.9; M5F20: 38.8 ± 3.2; M20F5: 12.2 ± 1.2; M20F20: 34.2 ± 3.9.) (Day 8: M5F5: 35.5 ± 5; M5F20: 43.1 ± 5.1; M20F5: 15.6 ± 1.4; M20F20: 37.1 ± 4.2.) (Group: %Mean ± SEM). Four genes (Cyclin B1, GAPDH, IGFR2 and LDHA) were significantly upregulated in M5 COC, and three genes (GPX1, G6PD and BAX) did not show significant differences. In conclusion, low oxygen tension during maturation exerts a beneficial effect upon embryo development, whereas the opposite situation is observed during fertilization. Furthermore, the expression of a growth factor, a meiotic promoting factor and two enzymes of anaerobic glycolysis were significantly upregulated in COCs matured under low oxygen tension, which may be linked to the improvement in developmental rates.