The role of store-operated Ca2 entry (SOCE) in the maintenance of sperm-induced Ca2 oscillations was investigated in porcine eggs. We found that 10 μM gadolinium (Gd3 ), which is known to inhibit SOCE, blocked Ca2 entry that was triggered by thapsigargin-induced store depletion and also caused an abrupt cessation of the fertilization Ca2 signal. In a similar manner 3,5-bis(trifluoromethyl)pyrazole 2 (20 μM), and tetrapandin-2 (10 μM), potent SOCE inhibitors, also blocked thapsigargin-stimulated Ca2 entry and disrupted the Ca2 oscillations after sperm-egg fusion. The downregulation of Stim1 or Orai1 in the eggs did not alter the Ca2 content of the intracellular stores, whereas co-overexpression of these proteins led to the generation of irregular Ca2 transients after fertilization that stopped prematurely. We also found that thapsigargin completely emptied the endoplasmic reticulum, and that the series of Ca2 transients stopped abruptly after the addition of thapsigargin to the fertilized eggs, indicating that the proper reloading of the intracellular stores is a prerequisite for the maintenance of the Ca2 oscillations. These data strengthen our previous findings that in porcine eggs SOCE is a major signaling cascade that is responsible for sustaining the repetitive Ca2 signal at fertilization.
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Vol. 93 • No. 1