Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Animal Science

Committee Chair

Zoltan Machaty

Committee Member 1

Ryan Cabot

Committee Member 2

Shihuan Kuang

Committee Member 3

Laurie A. Jaeger


As the most abundant mineral in organisms, ionized calcium (Ca2+) plays a crucial role in cellular signaling, regulating a great number of physiological activities. In this study, the Ca2+ entry mechanism that sustains the long-lasting Ca2+ oscillations in fertilized pig eggs was investigated. Porcine eggs matured in vitro were used for the experiments. In some eggs, the intracellular Ca2+ stores were depleted using cyclopiazonic acid (CPA, a SERCA pump blocker), others were inseminated with boar spermatozoa. To identify the mechanism that mediates the Ca2+ influx, various inhibitors of store-operated Ca2+ entry (SOCE) were used. We found that the inhibitors blocked the Ca2+ influx stimulated by the depletion of the intracellular Ca2+ pool; they were also able to disrupt the sperm-induced Ca2+ oscillations. Then the involvement of the key SOCE components, STIM1 and ORAI1, and their potential interaction in regulating Ca2+ influx was evaluated by expressing these proteins tagged with various fluorophores in the eggs. Changes in the eggs’ cytosolic free Ca2+ concentration were detected by a Ca2+ imaging system, while interactions between the expressed fusion proteins was investigated using Fluorescence Resonance Energy Transfer (FRET). Eggs overexpressing mVenus-STIM1 showed high-frequency Ca2+ oscillations when fertilized and high-frequency oscillations were also detected in fertilized eggs co-expressing mVenus-STIM1 and mTurquoise2-ORAI1. In addition, these high-frequency Ca2+ oscillations could be stopped by ML-9, an inhibitor of STIM1, arguing for an active role of STIM1 and ORAI1 in the process. Store depletion led to an increase of the FRET signal in eggs co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming puncta after store depletion. A similar FRET increase in response to a CPA treatment was also detected in eggs co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that the Ca2+ release was followed by an interaction between the expressed proteins. Most importantly, in such eggs we were also able to observe cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in porcine eggs, the interaction of SOCE components STIM1 and ORAI1 is responsible for the maintenance of the repetitive Ca2+ increases at fertilization. In another line of experiments, the role of Mg2+ in the regulation of Ca2+ signaling was investigated. We found that the Ca2+ influx stimulated by the mobilization of intracellularly stored Ca2+ was significantly smaller in pig eggs when Mg2+ was present in the extracellular medium. Furthermore, the addition of Mg2+ to the extracellular medium disrupted the ongoing Ca2+ oscillations in a concentration-dependent manner. TRPM7, a non-selective ion transporter with a kinase domain, may be involved in mediating the negative effects of Mg2+ on Ca2+ signaling. When the eggs were treated with the TRPM7 inhibitor NS8593, the Ca2+ oscillations stopped. Disruption of the signal was associated with an elevated cytosolic Ca2+ level in the eggs. This Ca2+ rise was later proved to be the result of Ca2+ influx, as it could be inhibited by the SOCE blocker ML-9. Based on these results, we concluded that Mg2+ negatively regulate Ca2+ signaling in eggs by indirectly inhibiting SOCE. Overall, the current study provided additional evidences that SOCE is essential during signaling in fertilized pig eggs. Clarifying the exact mechanism by which the sperm-induced Ca2+ signal is regulated has far-reaching implications in assisted reproductive technologies.