Activation and penetration in vitro of pig oocytes treated with calcium ionophore

The present study examined the effects of pre-treatment of pig oocytes with different concentrations (0-50 microM) of calcium ionophore A23187 (CaA) on their activation, development and penetration in vitro. Although untreated oocytes were not activated and did not cleave in culture, high proportions of treated oocytes did so and 20% of oocytes developed to the blastocyst stage when treated with 6.25 microM CaA for 2 min. However, these proportions were reduced in a concentration-dependent manner. When inseminated in vitro with 1 x 10(6) spermatozoa/ml, the penetration rate of oocytes treated with 6.25 microM CaA was similar to that of untreated oocytes. However, fewer oocytes treated with 12.5 and 50 microM CaA were penetrated than untreated oocytes. On the other hand, the proportion of monospermy of oocytes treated with 6.25 microM CaA was higher than the values in oocytes not treated or treated with 50 microM CaA. The time required for zona dissolution of oocytes treated with 6.25 and 12.5 microM CaA was not different from that in untreated oocytes, but oocytes treated with 50 microM CaA required a longer time than untreated oocytes, indicating that zona solubility by protease does not reflect penetrability of oocytes in vitro. When oocytes were inseminated with different concentrations (1-10 x 10(6) cells/ml) of spermatozoa, the highest penetration rate was observed at 1 x 10(6) cells/ml in untreated oocytes and a similar result was obtained in oocytes treated with 6.25 microM CaA. There was no difference in the rate of monospermy in untreated oocytes among different concentrations of spermatozoa, but in treated oocytes, higher proportions of monospermy were observed at 0.5-5 x 10(6) than 10 x 10(6) cells/ml. At 1 x 10(6) cells/ml, the proportion of monospermy was higher in treated than untreated oocytes. These results suggest that pre-treatment of pig oocytes with 6.25 microM CaA, an appropriate concentration, inhibits polyspermic penetration in vitro when insemination occurs with spermatozoa at a concentration of 1 x 10(6) cells/ml.