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 of cells 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.

Impact of pro‐oxidant agents on the morula‐blastocyst transition in bovine embryos

“…Both pro-oxidants, AAPH and BSO, induced the degeneration of some blastocysts, as previously described (Feugang et al, 2003). The timing of appearance of the first signs of degeneration was not different between the two prooxidants (Table 2).…”

“…Total cell number and the proportion of apoptotic nuclei detected by the TUNEL technique were not different between blastocysts resisting to oxidative stress at day 8 and control blastocysts of the same age. Moreover, we previously showed that hatching rates were not affected (Feugang et al, 2003). The quality of resisting embryos using those limited criteria seems unaffected by the treatment.…”

“…The antiapoptotic factor Bcl-2 has been shown to be present in bovine blastocysts (Kölle et al, 2002;Yang and Rajamahendran, 2002). However, the increase in apoptosis observed at day 7 is unlikely to be at the origin of blastocyst degeneration as it was mainly observed in the ICM cells while the process of degeneration started from the trophectoderm (Feugang et al, 2003).…”

Trichostatin A Modulates Apoptotic-Related Gene Expression and Improves Embryo Viability in Cloned Bovine Embryos

“…In NT embryos, the percentage of embryos with DNAfragmented nuclei is apparently higher than in in vitro fertilization (IVF) embryos for cows (Fahrudin et al, 2002;Feugang et al, 2003) and pigs (Hao et al, 2003). During mouse embryonic development, multiple caspases and antiand pro-apoptotic genes are expressed (Cohen, 1997;Cryns and Yuan, 1999;Salveson and Dixit, 1997), and in cows, antiand pro-apoptotic genes such as Bcl2, Bcl-xL, and Bax are involved in regulation of apoptosis in preimplantation embryos (Augustin et al, 2003;Kolle et al, 2002;Lonergan et al, 2003;Yang et al, 2002).…”

Culture systems for bovine embryos

“…The expression patterns of genes such as superoxide dismutase (SOD) enzymes, heat shock protein (hsp) and Bcl2/Bax are the results of high level of ROS in culture systems (Lequarre et al, 2001;Lonergan et al, 2003c). It has been reported that culture conditions associated with an increased level of prooxidants induce blastocyst degeneration only observed on Day 8 post-insemination (pi), whereas these blastocysts were morphologically acceptable for a transfer on Day 7 pi (Feugang et al, 2003;Feugang et al, 2004). This suggested that the low fetal development and high incidence of abnormalities (i.e., chromosomes aberrations) observed after embryo transfer are more likely due to a progressive accumulation of toxic effects during culture (Lonergan et al, 2004).…”