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Heat shock and tumor necrosis factor- induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism

¹ Department of Animal Sciences, University of Florida, PO Box 110910, Gainesville, Florida 32611-0910, USA and ² Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife-PE, Brazil

Correspondence should be addressed to P J Hansen; Email: hansen{at}animal.ufl.edu

Heat shock and tumor necrosis factor- (TNF-) induce apoptosis through different mechanisms, with heat shock acting to cause mitochondrial depolarization and caspase-9 activation, while TNF- acts through a receptor-mediated process to activate caspase-8. In some cells, however, TNF- can also cause mitochondrial depolarization and caspase-9 activation. In the present study, we tested the hypothesis that heat shock at 41 °C and TNF- induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism. Treatment of embryos with either heat shock (41 °C) or TNF- 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-. 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 °C, and 15.1% for embryos cultured with 10 ng/ml TNF-. 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 °C, and 8% for embryos cultured with 10 ng/ml TNF-. Although TNF- 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-. The percent of cells that were TUNEL positive was increased by TNF- 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- involve activation of caspase-9-dependent pathways. It is likely that TNF- 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.

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				A. M Brad, K. E M Hendricks, and P. J Hansen The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage Reproduction, December 1, 2007; 134(6): 789 - 797. [Abstract] [Full Text] [PDF]