Age dependence of bovine oocyte activation

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca⁺⁺-Mg⁺⁺-H⁺ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1–5-mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12–16 hours, fixed, and stained with aceto-orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 μM showed that 1–100 μM levels resulted in 94–100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P < 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.