133 ROLE OF CASPASE-9 AND STAGE OF DEVELOPMENT IN INDUCTION OF APOPTOSIS BY HEAT SHOCK AND TUMOR NECROSIS FACTOR-α IN BOVINE PRE-IMPLANTATION EMBRYOS

Heat shock and tumor necrosis factor-α (TNF-α) can increase apoptosis in bovine embryos in a developmental-dependent manner. It was hypothesized that addition of the caspase-9 inhibitor, z-LEHD-fmk, would block induction of apoptosis caused by heat shock of 41°C and TNF-α. Furthermore, it was hypothesized that the magnitude of induced apoptosis would increase with stage of development. Embryos were collected on day 4, 5, and 6 after in vitro insemination and were cultured for 24 h in the presence of either 100 μm z-LEHD-fmk reconstituted in 0.5% (v/v) dimethyl sulfoxide or vehicle dimethyl sulfoxide at either (1) 38.5°C for 24 h (control), (2) 41°C for 15 h followed by 38.5°C for 9 h, or (3) 38.5°C for 24 h with 10 ng/mL murine TNF-α. Embryos were then fixed, and the proportion of blastomeres undergoing apoptosis was determined using TUNEL labeling. Heat shock did not increase the percentage of blastomeres that were TUNEL-positive (% apoptosis) at day 4 (n = 100 embryos total). In contrast, heat shock increased % apoptosis at day 5 and day 6 (P < 0.04) and this effect was blocked by z-LEHD-fmk (temperature × inhibitor, P < 0.04). At day 5, % apoptosis in the absence and presence of z-LEHD-fmk was 3.8 ± 1.9% and 3.7 ± 1.7% at 38.5°C vs. 8.9 ± 1.5% and 4.1 ± 1.7% at 41°C (n = 75 embryos total). At day 6, % apoptosis in the absence and presence of z-LEHD-fmk was 3.6 ± 1.1% and 3.7 ± 1.2% at 38.5°C vs. 11.1 ± 1.1% and 6.1 ± 1.2% at 41°C (n = 168 embryos total). Mean cell number at the end of culture ranged from 21 to 26 cells at day 4, 43 to 73 cells at day 5, and 101 to 114 cells at day 6. Treatment with TNF-α also increased apoptosis at all days (P < 0.01), and z-LEHD-fmk blocked this effect (TNF × inhibitor, P = 0.05; n = 361 embryos total). Across days, % apoptosis was 3.6 ± 1.4% (control), 3.3 ± 1.3% (inhibitor), 11.1 ± 1.3% (TNF-α), and 6.0 ± 1.4% (TNF-α + inhibitor). Mean cell number at the end of culture ranged from 21 to 27 cells at day 4, 59 to 74 cells at day 5, and 105 to 115 cells at day 6. In conclusion, activation of caspase-9 dependent pathways is involved in the induction of apoptosis by heat shock and TNF-α. Moreover, the magnitude of induced apoptosis increases as embryos advance in development. This work was supported by USDA Grant No. 2004–34135–14715 and BARD Grant No. US–3551–04.