title: Effect of prooxidant agents added at the morula/blastocyst stage on bovine embryo development, cell death and glutathione content.
abstract: Two prooxidant agents, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), a generator of free radicals in the culture medium, and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, were used to reinforce from the morula stage (day 5 post-insemination, p.i.) the oxidative stress encountered by bovine embryos in culture. Exposure to increasing concentrations of both prooxidants from the morula stage did not affect blastocyst formation but some blastocysts were found degenerated on day 8 in a dose-dependent manner (0, 0.001, 0.01, 0.1 mM AAPH gave respectively 0, 10%, 32%, 48% degeneration, while 0, 0.1, 0.2, 0.4 mM BSO led respectively to 0, 14%, 30%, 41% degeneration). Hatching rates and cell numbers of surviving blastocysts were not affected. Morulae and early blastocysts exposed from day 5 to day 6 p.i. appeared more resistant than expanded blastocysts (75-80% survival vs 20-65%; p < 0.05). Treatment with BSO significantly decreased the level of reduced glutathione in day 7 blastocysts (0.02 vs 0.42 pmol per embryo in the control) while AAPH had no effect (0.38 pmol per embryo). The proportion ofcells showing membrane lesions was increased in degenerated blastocysts from day 7.5 p.i. In AAPH-treated, but not in BSO-treated embryos, cell membrane permeabilisation seems to occur before blastocyst degeneration. DNA fragmentation evaluated by the TUNEL technique was increased in day 7 blastocysts by both prooxidants (2.8 +/- 0.4 in the control group vs 4.5 +/- 0.4 and 6.0 +/- 0.4 respectively in the AAPH- and BSO-treated groups). Addition of an inhibitor of caspase-3, DEVD-CHO, partially prevented DNA fragmentation, indicating that prooxidant treatment induced a caspase-dependent pathway of apoptosis.
authors.affiliation: Université catholique de Louvain, Institut des Sciences de la Vie, Louvain-la-Neuve, Belgium.
authors.forename: Jean-Magloire
authors.initials: JM
authors.lastname: Feugang
authors.forename: Anne
authors.initials: A
authors.lastname: Van Langendonckt
authors.forename: Hichem
authors.initials: H
authors.lastname: Sayoud
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Rees
authors.forename: Serge
authors.initials: S
authors.lastname: Pampfer
authors.forename: André
authors.initials: A
authors.lastname: Moens
authors.forename: Franz
authors.initials: F
authors.lastname: Dessy
authors.forename: Isabelle
authors.initials: I
authors.lastname: Donnay
mesh_terms: D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000187:drug effects*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000187:drug effects*; D020287:In Situ Nick-End Labeling; D009028:Morula / Q000187:drug effects*; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects
chemical_list: D000578:Amidines; D004791:Enzyme Inhibitors; D016877:Oxidants; D019328:Buthionine Sulfoximine; C046728:2,2'-azobis(2-amidinopropane); D005978:Glutathione