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(19) ADMINISTRATION OF THE CASPASE INHIBITOR, z-DEVD-fmk, TO BOVINE PREIMPLANTATION EMBRYOS BLOCKS HEAT-INDUCED APOPTOSIS AND INCREASES EMBRYONIC SENSITIVITY TO HEAT SHOCK. Paula-Lopes, Fabiola1, Hansen, Peter1, 1 Dept. of Animal Sciences, Gainesville, FL ABSTRACT- Heat shock, which can compromise development of preimplantation bovine embryos, also increases the percentage of blastomeres labeled as TUNEL-positive and increases activity of group II caspases that cleave DEVD substrate (i.e., caspase 2, 3, and 7). It is hypothesized that the TUNEL labeling represents apoptosis. Furthermore, since the increase in TUNEL-positive blastomeres is limited, it is hypothesized that this apoptosis is beneficial to the survival and continued development of the embryo after heat shock. To test these hypotheses, effects of z-DEVD-fmk, an inhibitor of group II caspases were evaluated. On day 4 after insemination, embryos ≥ 16 cell stage were preincubated in KSOM containing vehicle (0.5-1% DMSO) or 200 KEY WORDS: bovine, embryo, apoptosis, heat shock |
M z-DEVD-fmk at 38.5°C for 15 h. Embryos were then maintained at 38.5°C or exposed to 41°C. In experiment 1, caspase activity was determined using PhiPhiLux-G1D2, a fluoroprobe that incorporates the group II caspase-recognition sequence DEVD. The pixel fluorescence intensity per unit area was determined in 39-59 embryos/treatment. In the absence of z-DEVD-fmk, heat shock increased caspase activity from 12.4 ± 2.4 at 38.5°C to 25.0 ± 2.3 arbitrary units at 41°C for 9 h. In the presence of z-DEVD-fmk, there was no increase in caspase activity (12.4 ± 2.5 at 38.5°C vs 11.5 ± 2.6 arbitrary units at 41°C) (treatment x temperature, p<0.01). In experiment 2, the percentage of TUNEL-positive cells was evaluated in 15-25 embryos/treatment. Heat shock for 9 h increased the proportion of TUNEL positive cells from 4.3 ± 3.2 % at 38.5°C to 26.0 ± 4.6%. However, z-DEVD-fmk blocked this effect of heat shock (2.2 ± 3.8 at 38.5°C vs 3.0 ± 3.6 at 41°C) (treatment x temperature, p< 0.01). In experiment 3, heat shock for 6-9 h reduced the percentage of embryos developing to the blastocyst stage from 19.6 ± 1.6 at 38.5°C to 10.0 ± 1.6 % at 41°C. The reduction in development caused by heat shock was magnified in the presence of z-DEVD-fmk (19.6 ± 1.6% at 38.5°C vs 3.0 ± 1.6% at 41°C) (315-324 embryos/treatment; treatment x temperature, p< 0.05). In conclusion, group II caspases mediate heat-induced apoptosis in bovine embryos. Moreover, inhibition of these caspases has a detrimental effect on embryonic resistance to heat shock. Support: USDA IFAFS 2001-52101-11318 and USDA TSTAR 2001-34135-11150.