Calcium influx-mediated signaling is required for complete mouse egg activation

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca²⁺) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca²⁺ release from inositol 1,4,5-trisphosphate (IP₃)-sensitive intracellular stores. We tested the hypothesis that Ca²⁺ influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca²⁺ source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca²⁺ influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca²⁺ occurred, in the absence of Ca²⁺ influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca²⁺ chelator, BAPTA/AM, demonstrated that Ca²⁺ influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca²⁺ transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca²⁺-free medium remained arrested in metaphase II. Inhibition of store-operated Ca²⁺ entry had no effect on ICSI-induced egg activation, so Ca²⁺ influx through alternative channels must participate in egg activation signaling. Ca²⁺ influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca²⁺. These results suggest that Ca²⁺ influx at fertilization not only maintains Ca²⁺ oscillations by replenishing Ca²⁺ stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.