Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol

Abstract

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor "EX527", and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

Keywords: Cryopreservation; Embryos; Lipid; Mitochondria; Resveratrol.

Fig. 1.

Effects of resveratrol treatment on blastocyst characteristics. A, B: expression levels of SIRT1 (A) and pAMPK (B) proteins. C: ATP content. D: ROS content. E: mitochondrial DNA copy number. F: lipid content. At least 50 embryos were used for blastocyst productions in each group. In Figs. 1-A, B, D and F, the average fluorescence intensity of control blastocysts (treated with vehicle only) was defined as 1.0. Con.: vehicle group, Res.: resveratrol group.

Fig. 2.

Effects of resveratrol treatment of short duration (24 h) on expression levels of SIRT1 and pAMPK, and on mitochondrial function. Expression levels of SIRT1 (A) and pAMPK (B), ATP content (C), ROS content (D), and lipid content (E) in embryos cultured with or without resveratrol for 24 h; ^ab P < 0.05. In Figs. 2-A, B, D, and E, the average fluorescence intensity of control embryos was defined as 1.0. Experiments were repeated at least three times. Con.: vehicle group, Res.: resveratrol group.

Fig. 3.

ATP content (A) and lipid content (B) in embryos cultured under four conditions. Con: ethanol at the same concentration as used with resveratrol and EX527 treatment, Res: 0.5 µM resveratrol, Res + Ex: 0.5 µM resveratrol and 1 µM EX527 (specific inhibitor of SIRT1), Ex: 1 µM EX527 (^ab P < 0.05). In Fig. 3-B (lipid content), the average fluorescence intensity of control embryos was defined as 1.0. Ten 8-cell stage embryos were cultured for 1 day in each group. Experiments were repeated at least four times.

Fig. 4.

Lipid content in embryos cultured in four types of media for 1 day. Con: vehicle media only, Res: 0.5 µM resveratrol, Res + Etm: 0.5 µM resveratrol and 10 µM etomoxir, Etm: 10 µM etomoxir (^ab P < 0.05). The average fluorescence intensity in control embryos was defined as 1.0. Experiments were repeated four times.