Electrofusion‐induced intracellular Ca2+ flux and its effect on murine oocyte activation

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Abstract
These experiments were designed to monitor influx of extracellular Ca2+ into the murine ooplasm following a 1.56 kV · cm−1 direct current (DC) electrofusion pulse and subsequently to determine its effect on rate of activation. Pulse media consisted of nonelectrolyte (0.3 M mannitol) and electrolyte (phosphatebuffered saline; PBS) media each containing 0.0, 0.05, or 0.9 mM Ca2+ (groups T1–T3 and T4–T6, respectively). Cumulus‐free oocytes were incubated in 100 μl drops of PBS containing 2 μM of the calcium indicator fluo‐3/AM for 60 min at 37°C. Fluo‐3/AM‐loaded oocytes were equilibrated for 7 min in assigned treatment media (T1–T6) prior to application of DC pulse. Change in fluorescent intensity was monitored for 6.5 min after DC pulse by photon counting spectrofluorometry. Fluorometric measurements demonstrate a dramatic rise in intracellular free Ca2+ (Ca2+i) following DC pulse is associated with Ca2+ ion concentration in the pulse medium. Significantly (P < 0.01) higher Ca2+i levels were observed when 0.9 mM Ca2+ was added to the pulse medium (T3 and T6) compared with pulse medium containing lower Ca2+ ion concentrations (T1, T2, T4, and T5; P > 0.05). Differences (P < 0.01) were observed in peak Ca2+i levels 18 sec after pulse with mean percent change in fluorescence of 5.1%a, 33.9%b, 112.7%c, 1.2%a, 9.3%a, and 99.9%c for T1–T6, respectively (values with different superscripts are significantly different at P < 0.01). Increased oocyte membrane permeability to Ca2+ ion after DC pulse was observed for a minimum of 5 min after delivery of the 1.56kV · cm−1 pulse. Nonloaded oocytes receiving the same pulse treatments were cultured in 100 μl drops of Whitten's medium for 8 h prior to evaluation of oocyte activation. Activation was defined as the formation of one pronucleus and one polar body, two or more pronuclei, or two cells each containing a nuclear structure. Activation rates of 28.2%b, 31.1%b, 70.4%d, 35.6%b, 57.3%c, 71.8%d 8.7%a (P < 0.01) were observed for T1–T6 and control nonpulsed oocytes, respectively. These data demonstrate that supplementation of 0.9 mM of Ca2+ ion to pulse medium results in a dramatic rise in Ca2+i after DC pulse. This influx of Ca2+ ion has a positive effect on oocyte activation rate.