Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes.

Abstract The presence of the store-operated Ca 2+ entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921 bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca 2+ influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca 2+ influx after store depletion and subsequent Ca 2+ add-back; the Ca 2+ oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca 2+ entry and had an inhibitory effect on the fertilization Ca 2+ signal. The abnormal Ca 2+ signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca 2+ entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca 2+ channel that is responsible for mediating Ca 2+ entry after the mobilization of intracellular Ca 2+ in oocytes. Orai1 and a functional store-operated Ca 2+ entry pathway are required to maintain the Ca 2+ oscillations at fertilization and to support proper embryo development.