Electrically induced calcium elevation,activation, and parthenogenetic development of bovine oocytes

The influence of electrical stimulation on the level of intracellular Ca²⁺ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca²⁺ concentration was determined with the Ca²⁺ indicator fura-2 dextran (fura-2 D). The Ca²⁺ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca²⁺ rise from baseline (≈? 12 nM), a short-duration Ca²⁺ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca²⁺ rise (from 12 nM to 1,000–2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca²⁺ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca²⁺ rise, and at 1.0 kVcm^?1 for 40 μsec, all oocytes displayed a long-duration Ca²⁺ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca²⁺ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca²⁺ rises. This mannitol-induced Ca²⁺ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca²⁺ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca²⁺ stimulation impaired development. Optimum development was obtained with (1) three pulses of 0.2 kVcm^?1 for 20 μsec, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or (2) three pulses of 1.0 kVcm^?1 for 20 μsec after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca²⁺ rise prior to the pulse. The results indicate that the level of Ca²⁺ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca²⁺ response, activation, and parthenogenetic development. © 1993 Wiley-Liss, Inc.