Diacylglycerol-enhanced electrical activation of porcine oocytes matured in vitro

Abstract

This study was undertaken to examine the effect of second messengers added to the electroporation medium on electric pulse-induced artificial activation of meiotic Metaphase II porcine oocytes. Six separate experiments evaluated second messengers added to electroporation medium. When added to electroporation medium, neither phospholipase C (PLC: 0 to 2.5 Units/ml), D-myo-inositol triphosphate (IP 3: 0 to 10,000 μM), nor guanosine 5′-O-(3-thiotriphosphate; GTP-γ-S: 0 to 100 μM) had any effect (P> 0.05) on activation rates. However, addition of 1,2-dioctanoyl-sn-glycerol (DiC 8) increased activation rates in a dose-dependent response. At a level of 1,000 μM, DiC 8 resulted in a higher activation rate (P< 0,05) than 0.0, 0.1, 1 or 10 μM of DiC 8 with a pulse, and the 1,000 and 10,000 μM of DiC 8 no-pulse control groups. Effects of DiC 8 (1,000 μM) and IP 3 (100 μM) in combination or individually were investigated. At 1,000 μM, DiC 8 caused a higher rate of activation (P< 0.05) than 100 μM IP 3, but the result was not different from DiC 8 + IP 3. In another experiment, no difference (P> 0.05) was observed between DiC 8, GTP-γ-S and IP 3, but DiC 8 + GTP-γ-S + IP 3 + PLC yielded a higher (P< 0.05) activation rate than PLC or the rate of the controls. No significant development (blastocyst) was observed after 5 days of culture in any of the experiments. Protein profiles of activated oocytes, determined by 1D SDS-PAGE, were characteristic of pronuclear-stage embryos. These data indicate that the addition of DiC 8 to the electroporation medium synergistically enhances the rate of activation of electrically stimulated in vitro-matured porcine oocytes.