Abstract
The purpose of these experiments was to determine the effect of electroporation of IP3 into the cytosol of murine secondary oocytes and evaluate any alterations in [Ca^2+^]~i~ resulting from Ca^2+^ release from intracellular stores. In addition, we evaluated the effect of ethanol (ETOH) on the release of Ca^2+^ from intracellular stores. Oocytes were loaded with the Ca^2+^ indicator fluo‐3 by incubation in 100 μl drops of medium containing 2 μM fluo‐3/AM for 60 min at 37°C. Changes in fluorescence were monitored by use of an inverted microscope which had been connected to a spectrofluorometer. Fluorescent intensity measurements were acquired for a minimum of 416 sec time span or up to 1,248 sec, with integration readings of 1 sec duration obtained every 2 sec throughout the measurement period. The experimental design consisted of comparing the rise in [Ca^2+^]~i~ of fluo‐3 loaded secondary oocytes subjected to electroporation in PBS and Ca^2+^‐free PBS, each containing 25 μM IP3, to that elicited by PBS and Ca^2+^‐free PBS containing a final concentration of 7% ETOH. Non‐pulsed control secondary oocytes were placed in PBS + 25 μM IP3 during monitoring of [Ca^2+^]~i~ fluorescence. Pulsed control secondary oocytes were placed in Ca^2+^‐free PBS, subjected to electroporation pulse, and monitored for [Ca^2+^]~i~ fluorescence.
Electroporation of IP3 was accomplished by placing fluo‐3 loaded secondary oocytes between the electrodes of a glass slide fusion chamber which had been overlaid with 300 μl of PBS + 25 μM IP3 or Ca^2+^‐free PBS + 25 μM IP3. A 5 sec, 3 volt, alternating current (AC) alignment pulse followed by a single, square wave, direct current (DC) fusion pulse of 1.56 kV·cm^–1^ for 99 μsec was applied to the electrodes. For ETOH treatment, fluo‐3 loaded secondary oocytes were placed in PBS or Ca^2+^‐free PBS and allowed to equilibrate for 7 min in the dark. No pulse was applied to ETOH treatment secondary oocytes. Micropipets were used to keep the secondary oocyte in a fixed position throughout the measurement period. After a 20 sec baseline fluorescent reading was obtained, fluorescent measurement was interrupted and 150 μl of PBS (or Ca^2+^‐free PBS) was removed and replaced with 150 μl of 14% ETOH in PBS (or Ca^2+^‐free), bringing the final concentration after equilibration to 7% ETOH. Fluorescent intensity measurement resumed immediately following the addition of 14% ETOH. A dramatic and immediate rise in [Ca^2+^]~i~ was observed upon application of electropolation pulse and [Ca^2+^]~i~ was maintained at an elevated level for a minimum of 14 min. Repetitive [Ca^2+^]~i~ oscillations were obtained in mouse secondary oocytes after electroporation of 25 μM IP3 in Ca^2+^‐free PBS that occurred for 20.5 min with a gradual increase in the interval between [Ca^2+^]~i~ oscillation peaks over time. After ETOH treatment, a dramatic rise in mouse secondary oocyte [Ca^2+^]~i~ in PBS and Ca^2+^‐free PBS was observed. There was no significant different (P > 0.05) in [Ca^2+^]~i~ between PBS + ETOH and Ca^2+^‐free PBS + ETOH, indicating the rise in [Ca^2+^]~i~ resulted from a release of Ca^2+^ from intracellular stores. The ability to consistently produce repetitive [Ca^2+^]~i~ oscillations may aid in the study of post‐fertilization development and cell cycle events. Current studies are being conducted to determine if IP3 can be used to enhance the rate of electric pulse induced parthogenesis and subsequent development. © 1993 Wiley‐Liss, Inc.