Activation of porcine oocytes with calcium ionophore: effects of extracellular calcium.

The present study examined the mechanism of A23187-induced activation in pig oocytes, with special reference to the effects of extracellular calcium on oocyte activation. The following endpoints were evaluated: intracellular free calcium concentration ([Ca2+]i), intracellular pH ([pH]i), cortical granule (CG) exocytosis, pronuclear formation, and blastocyst development. In experiment one, when oocytes were exposed to 50 microM A23187 for 5 min in a medium with, or without, calcium, a significant (P < 0.004) increase in the [Ca2+]i was observed in medium with calcium but not in medium without calcium. An increased [pH]i (0.08 unit in medium with calcium and 0.13 unit in medium without calcium), cortical granule exocytosis and pronuclear formation were observed in oocytes treated with A23187 irrespective of the presence or absence of calcium in the medium. In experiment two, the effects of treatment time (0, 0.5, 1, 2, and 5 min) on nuclear activation of oocytes with A23187 were further examined in medium with, or without, calcium. It was found that a 2 min treatment activated more (71-74%) oocytes than the other treatments. Treatment for 5 min in medium without calcium resulted in chromatin condensation in some oocytes. Microtubules were not found in these oocytes. In experiment three, developmental ability was examined of the oocytes treated with A23187 in medium with, or without, calcium. In vitro fertilized oocytes were used as a positive control. It was found that 16%, 6% and 38% of the oocytes treated with A23187 in medium with calcium, in medium without calcium, and in vitro fertilized oocytes developed to blastocysts after culture for 7 days, respectively. These results indicate that A23187 can induce pig oocyte activation in calcium-free medium without a typical increase in the [Ca2+]i and that A23187-induced pig oocyte activation is accompanied by an increase in [pH]i. Oocytes activated with A23187 can develop to blastocysts regardless of activation in medium with, or without, calcium.