Abstract
During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt) were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.
Highlights
In the eggs of all animal species studied so far, fertilization induces an increase in cytosolic calcium ([Ca2+]cyt), which appears to be the primary intracellular signal responsible for the initiation of the development of the egg following fertilization [1,2,3,4,5,6]
Imaging [Ca2+]cyt during in Vitro Fertilization (IVF) of Isolated Egg Cells Developed in Situ
In the case of immature egg cells, this rise in [Ca2+]cyt was confined to a certain region of the cell away from the sperm entry site, whereas fully mature egg cells exhibited [Ca2+]cyt waves sweeping through the entire cell at the focal plane of sperm entry (Figures 2a,b)
Summary
In the eggs of all animal species studied so far, fertilization induces an increase in cytosolic calcium ([Ca2+]cyt), which appears to be the primary intracellular signal responsible for the initiation of the development of the egg following fertilization [1,2,3,4,5,6]. Employing a microinjection technique elaborated by Pónya et al [32] allowed for the injection of isolated wheat (Triticum aestivum, L.) egg cells with the calcium-sensitive ratio dye (fura-2 dextran) in liquid medium, making IVF (in vitro fertilization) possible following injection This method was combined with the electrofusion procedure elaborated by Kranz et al [33] for maize gamete fusion [33,34]. To be the main intracellular Ca2+ store in the female gamete of wheat and on the preliminary result that [Ca2+]cyt elevation was seen in egg cells incubated and fused in Ca2+ free medium (the calcium rise that was observed needed to have originated from an internal calcium store), the ER was assumed to be the origin of the repetitive [Ca2+]cyt transients observed in mature, fertilized wheat (T. aestivum, L.) egg cells To test this hypothesis, a pharmacological approach was employed to examine the origin of the fertilization-associated [Ca2+]cyt change in the egg cytoplasm. Wheat female gametes were treated with thapsigargin, an inhibitor of Ca2+-pumps [37,38], which proved to be able to block Ca2+-ATPases in the ER, while leaving the plasma membrane calcium-pumps unaffected, at least at a certain concentration (10 μM) of the drug added to the fusion medium
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