Apoptose em embriões bovinos em estágio de préimplantação submetidos ao estresse térmico e ao fator de necrose tumoral-a (TNF-a)

Abstract

We tested the hypothesis that heat shock and tumor necrosis factor-α (TNF-α) induce apoptosis in bovine embryos through a caspase-9 dependent mechanism.Experiment 1 determined whether the caspase-9 inhibitor, z-LEHD-fmk, blocks the apoptotic effects of heat shock and TNF-α. Embryos were collected on day 4, 5, and 6 after in vitro insemination and cultured for 24 h in the presence of either 100 μM z-LEHD-fmk reconstituted in 0.5% (v/v) DMSO or vehicle (DMSO) at either 1) 38.5°C for 24 h (control), 2) 41°C for 15 h followed by 38.5°C for 9 h (heat shock), or 3) 38.5°C for 24h with 10 ng/mL murine TNF-α. The proportion of blastomeres undergoing apoptosis was determined using TUNEL labeling. The experiment was replicated 4-5 times/day using 172-248 embryos/day. In control embryos, heat shock and TNF-α increased the proportion of cells that were TUNEL-positive as compared to embryos cultured at 38.5°C for 24 h (P<0.001). For embryos incubated with z-LEHD-fmk, in contrast, neither heat shock nor TNF-α caused an increase in TUNEL labeling (treatment x inhibitor; P<0.05). Experiment 2 tested effects of different concentrations of z-LEHDfmk on apoptotic responses of embryos to TNF-α. Embryos ³16 cells were collected on Day 6 and cultured in the presence of different concentrations of z-LEHD-fmk (0, 1 10, and 100 μM ) ± 10 ng/mL TNF-α.. After 24 h at 38.5°C, embryos were washed, fixed, and analyzed by TUNEL. The experiment was replicated 4 times (176 embryos). Addition of 10 ng/mL TNF-α increased the percentage of cells that were TUNELpositive and this increase was blocked by all concentrations of z-LEHD-fmk (TNF-α x inhibitor; P<0.05). For Exp. 3, we analyzed whether heat shock increases caspase-9 activity and whether z-LEHD-fmk blocks this increase. Embryos at the morula or early blastocyst stage were collected on day 6 and transferred to a new drop with 100 μM z- LEHD-fmk or DMSO vehicle. Embryos were then cultured at either 38.5°C for 24 h or 41°C for 15 h followed by 38.5°C for 9 h. At the end of heat shock, embryos were washed and then incubated in a caspase-9 substrate (5 μM CaspaLux 9-M1D2) at room temperature for 1 h. Caspase-9 activity was then determined by evaluation of fluorescent intensity. Embryos were classified as having low, medium or high caspase activity. The experiment was replicated 5 times using 22-26 embryos/treatment. Heat shock increased the percent of embryos classified as having medium and high caspase-9 activity and z-LEHD-fmk blocked this effect (treatment P<0.05; inhibitor P<0.001). For Exp. 4, effects of TNF-α on caspase-9 activity were evaluated. Embryos at day 6 were cultured at 38.5°C for 3, 6, 9, 12, and 15 h with 10 ng/mL TNF-α or vehicle and then caspase-9 activity was measured as described before. The experiment was replicated 8 times using 37-39 embryos/treatment. The proportion of embryos classified as having medium or high caspase activity was increased by TNF-α at all time points except 3 h (treatment P< 0.05; inhibitor P<0.01). Exp. 5 was performed to analyze the effects of heat shock and TNF-α on mitochondrial membrane integrity. Embryos at Day 3.5 were incubated at 38.5°C for 24 h + TNF-α or were incubated at 41°C for 15 h followed by 9 h at 38.5°C (heat shock). After treatment, embryos were washed and incubated in 20 nM of DiOC6 at 38.5°C for 20 min in the dark. Mitochondrial depolarization was measured by quantifying the fluorescence units/area of each embryo. Heat shock caused loss in mitochondria membrane potential (P< 0.01). However, TNF- α did not show a significant decrease on membrane polarization. The experiment was replicated 5 times (71-78 embryos/treatment). In conclusion, activation of caspase-9 dependent pathways is involved in the induction of apoptosis by heat shock and TNF-α.