Abstract
The Ca2+ entry mechanism that sustains the Ca2+ oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca2+ stores were depleted using cyclopiazonic acid (CPA); others were inseminated. Changes in the oocytes' cytosolic free Ca2+ concentration were monitored, while interaction between the expressed fusion proteins was investigated using fluorescence resonance energy transfer (FRET). Store depletion led to an increase of the FRET signal in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that Ca2+ release was followed by an interaction between these proteins. A similar FRET increase in response to CPA was also detected in oocytes co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming punctae after store depletion. ML-9, an inhibitor that can interfere with STIM1 puncta formation, blocked store-operated Ca2+ entry (SOCE) induced by Ca2+ add-back after a CPA treatment; it also disrupted the Ca2+ oscillations in fertilized oocytes. In addition, oocytes overexpressing mVenus-STIM1 showed high-frequency Ca2+ oscillations when fertilized, arguing for an active role of the protein. High-frequency Ca2+ oscillations were also detected in fertilized oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, and both of these high-frequency Ca2+ oscillations could be stopped by inhibitors of SOCE. Importantly, in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, we were also able to detect cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in pig oocytes, SOCE is involved in the maintenance of the repetitive Ca2+ transients at fertilization.