title: Heat shock and tumor necrosis factor-alpha induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism.
abstract: Heat shock and tumor necrosis factor-alpha (TNF-alpha) induce apoptosis through different mechanisms, with heat shock acting to cause mitochondrial depolarization and caspase-9 activation, while TNF-alpha acts through a receptor-mediated process to activate caspase-8. In some cells, however, TNF-alpha can also cause mitochondrial depolarization and caspase-9 activation. In the present study, we tested the hypothesis that heat shock at 41 degrees C and TNF-alpha induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism. Treatment of embryos with either heat shock (41 degrees C) or TNF-alpha increased the proportion of blastomeres that were TUNEL positive and the proportion of embryos exhibiting elevated caspase-9 activity. Furthermore, the caspase-9 inhibitor, z-LEHD-fmk, blocked the increase in TUNEL-positive nuclei caused by both heat shock and TNF-alpha. For embryos at day 6 after insemination, for example, the percent of blastomeres positive for TUNEL was 3.6% for control embryos, 11.1% for embryos cultured at 41 degrees C, and 15.1% for embryos cultured with 10 ng/ml TNF-alpha. In the presence of z-LEHD-fmk, the percent of cells positive for TUNEL was 3.7% for control embryos, 6.1% for embryos cultured at 41 degrees C, and 8% for embryos cultured with 10 ng/ml TNF-alpha. Although TNF-alpha did not cause a measurable increase in caspase-8 activity, there was a tendency (P = 0.07) for treatment of embryos with z-IETD-fmk, an inhibitor of caspase-8, to partly reduce the magnitude of the increase in TUNEL-positive cells caused by TNF-alpha. The percent of cells that were TUNEL positive was increased by TNF-alpha from 9.7 to 19.7% in the absence of inhibitor and from 13.0 to 15.6% in the presence of z-IETD-fmk. Results indicate that induction of apoptosis by both heat shock and TNF-alpha involve activation of caspase-9-dependent pathways. It is likely that TNF-alpha also activates apoptotic pathways involving caspase-8 but that the degree of activation is small and caspase-9-dependent pathways are required for full activation of apoptosis.
authors.affiliation: Department of Animal Sciences, University of Florida, PO Box 110910, Gainesville, Florida 32611-0910, USA.
authors.forename: Bárbara
authors.initials: B
authors.lastname: Loureiro
authors.forename: Amber Mary
authors.initials: AM
authors.lastname: Brad
authors.forename: Peter James
authors.initials: PJ
authors.lastname: Hansen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000032:analysis; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D053453:Caspase 9 / Q000032:analysis / Q000378:metabolism; D061945:Caspase Inhibitors; D002417:Cattle / Q000502:physiology*; D047108:Embryonic Development / Q000187:drug effects; D004789:Enzyme Activation; D005260:Female; D006358:Hot Temperature; D020287:In Situ Nick-End Labeling; D009842:Oligopeptides / Q000494:pharmacology; D011247:Pregnancy; D046509:Tissue Culture Techniques; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D061945:Caspase Inhibitors; D009842:Oligopeptides; D014409:Tumor Necrosis Factor-alpha; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D053453:Caspase 9