Nitric oxide (NO) is a free radical that functions as a cell signaling molecule but at high concentrations can be toxic. It is formed from arginine, which is consumed by the mouse blastocyst, but its effect on early embryo development has been little studied. In this study, the role of NO in mouse preimplantation development has been examined in terms of developmental rate and oxidative metabolism. Zygotes were cultured in one of four media; potassium simplex optimization medium (KSOM), KSOM with amino acids (KSOMaa), KSOM without glutamine (KSOM-glut), or KSOM with 0.5 mM arginine (KSOMarg) +/- l-NAME (a specific inhibitor of NO production). End points were Day 4 blastocyst rates, cell counts determined using bisbenzimide and oxygen consumption. In KSOM and KSOM-glut, the blastocyst rate was decreased by 1 mM l-NAME from 50.2% +/- 3.1% and 37.4% +/- 4.5% to 6% +/- 3% and 0%, respectively. In KSOMaa, cavitation rates were unaltered but the blastocysts contained fewer cells (P < 0.001). Blastocysts cultured in KSOM and KSOM-glut consumed significantly more oxygen than those cultured in KSOMaa (P < 0.001 and P < 0.05, respectively). However, the addition of 0.1 mM or 1 mM l-NAME to KSOMaa significantly increased the amount of oxygen consumed (P < 0.05 and P < 0.001, respectively). The data suggest a physiological role for NO in mouse preimplantation metabolism and development. One possibility is that NO may limit oxygen consumption at the blastocyst stage at the level of mitochondrial cytochrome c oxidase.

Previous studies have demonstrated a role for the signalling agent nitric oxide in regulating preimplantation embryo development. We have now investigated the biochemical mode of action of nitric oxide in mouse embryos in terms of mitochondrial function and Ca2+ signalling. DETA-NONOate, a nitric oxide donor, decreased day 4 blastocyst cell number and oxygen consumption, consistent with a role for nitric oxide in the inhibition mitochondrial cytochrome c oxidase. Using live cell imaging and the nitric-oxide-sensitive probe DAF-FM diacetate, nitric oxide was detected at all stages of preimplantation development and FRET analysis revealed a proportion of the nitric oxide to be colocalised with mitochondria. This suggests that mitochondria of preimplantation embryos produce nitric oxide to regulate their own oxygen consumption. Inhibiting or uncoupling the electron transport chain induced an increase in nitric oxide and [Ca2+]i as well as disruption of Ca2+ deposits at the plasma membrane, suggesting that mitochondrial disruption can quickly compromise cellular function through Ca2+-stimulated nitric oxide production. A link between antimycin-A-induced apoptosis and nitric oxide signalling is proposed.