Upon fertilization, the mammalian oocyte undergoes a series of Ca2+ oscillations, which results in its activation and initiation of embryo development. It is believed that these periodic and extended Ca2+ responses are a consequence of intracellular Ca2+ release coupled to Ca2+ influx across the plasma membrane due to the activation of a process known as Store Operated Calcium Entry (SOCE). The underlying mechanism of SOCE is known to be controlled by Stim-Orai channels, where IP3-mediated emptying of ER calcium stores results in the aggregation of Stim1 molecules followed by interaction with Orai1 channels on the plasma membrane and activation of Ca2+ entry. Stim1 was shown to redistribute in the form of patches after mouse oocyte fertilization in a manner similar to its redistribution after pharmacological Ca2+ store depletion, while down-regulation of Orai1 expression in pig oocytes inhibited oscillations induced by fertilization. We have further shown that overexpression of Stim1 and Orai1 in mouse oocytes disrupts Ca2+ dynamics and inhibits egg activation. These data support a role for SOCE in Ca2+ signaling during fertilization. To further investigate the importance of Stim1 and Orai1 proteins in regulating events of egg activation, we are generating oocyte-specific Stim1 and Orai1 knockout mice by crossing Stim1-flox and Orai1-flox mice with ZP3-Cre transgenic mice. ZP3 is expressed only in oocytes leading to Cre expression only in this cell type and as such an oocyte-specific knockout. Studying rates of fertilization and Ca2+ entry in these knockout oocytes will provide insight into the role of these two proteins in the initiation and persistence of Ca2+oscillations after fertilization, and will help us determine whether there is an absolute requirement for Stim1 and/or Orai1 in regulating Ca2+ responses in early stages of fertilization.


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