171 Protective Effect of Sodium Pyruvate Against H₂O₂-Induced Oxidative Stress in Bovine and Swine Oocytes

Abstract

A previous study from our laboratory (Ramos-Ibeas et al. 2017 Mol. Cell. Biochem. 429, 137-150, 10.1007/s11010-017-2942-z) described the protective effect of sodium pyruvate (SP) against H₂O₂-induced oxidative stress in somatic and embryonic stem cells. In this study, we tested the same experimental conditions on bovine and swine oocytes during in vitro maturation (IVM). To this aim, cumulus-enclosed oocytes (COC) recovered from bovine and swine ovaries were treated for 1 h with different concentrations of H₂O₂ and then matured in serum-free TCM-199 with and without SP supplementation. All experiments were done in 3 replicates using 12 to 20 oocytes per experimental group; data were analysed by two-way ANOVA. As a first step, we determined the dose-response curve of H₂O₂ treatment during IVM, using the reaching of metaphase 2 as endpoint. We found that swine COC were more sensitive to H₂O₂ damage than bovine COC: indeed, they tolerated a range of concentrations from 128 to 512 μM, whereas the range for bovine COC was between 1024 and 4096 μM. The COC from both species were treated for 1 h with 128 μM (swine) or 2048 μM (bovine) H₂O₂ and then matured with or without different concentrations of SP. Control COC, unexposed to H₂O₂, were also matured with and without SP supplementation. We found that the addition of 25 mM SP to swine COC reduced the maturation rate both in control and H₂O₂-treated groups. Instead, reducing the addition of SP to 10 mM we observed a beneficial effect because the metaphase II level in treated oocytes increased from 38% (15/40) to 67% (24/36) and the difference was statistically significant. In bovine COC, the addition of 25 mM SP did not affect control oocytes during IVM but decreased maturation rate in treated oocytes, whereas 10 mM SP had no effect on the control group and showed a tendency to increase maturation rate in treated oocytes. In a second set of experiments, to determine if cumulus cells acted as a barrier for SP to reach the oocyte, the COC were denuded after H₂O₂ treatment and matured with or without SP. In swine denuded oocytes (DO) matured with SP, the rate of metaphase II increased significantly both in control [from 71% (34/48) to 92% (44/48)] and in treated groups [from 48% (23/48) to 72% (36/50)]. In bovine DO, the supplementation of SP did not increase the maturation rate significantly (25/41 v. 29/41 in control groups, 12/36 v. 18/40 in treated groups). In conclusion, we found that toxic H₂O₂ concentrations were approximately 10-fold higher for bovine than for swine, indicating that bovine COC were more resistant to oxidative stress. This could explain why SP supplementation had a significant protective effect in swine but only a minor effect in bovine. Moreover, the difference in SP effect between bovine and swine COC could be due to their morphology because bovine cumulus is often larger and more compact than swine cumulus and this difference could influence how SP reaches the inner oocyte and how it is metabolized.

This work was funded by projects EpiHealth FP7 n. 278418, EpiHealthNet FP7-PEOPLE-2012-ITN n. 317146 and Fecund FP7 n. 312097.