66 Orai1 IS REQUIRED TO MAINTAIN CALCIUM OSCILLATION AT FERTILIZATION IN PORCINE OOCYTES

Abstract

During mammalian fertilization, the sperm induces an oscillation in the oocyte's intracellular free calcium concentration that stimulates oocyte activation. The train of calcium spikes is maintained by an influx of calcium across the plasma membrane, probably through a mechanism known as store-operated calcium entry. Despite their importance, little is known about the identity and regulation of the calcium entry channels that mediate calcium influx at fertilization. Previously we have shown that the Orai1 protein is localised in the plasma membrane of pig oocytes and plays an important role in calcium entry after depletion of the intracellular calcium stores. In this study, we investigated the function of Orai1 in signal transduction during fertilization. In Experiment 1, Orai1 levels were down-regulated by injecting immature pig oocytes with small interfering RNA (siRNA) against Orai1; control cells were injected with scrambled siRNA. The injected oocytes were then matured in vitro for 44 h. In Experiment 2, Orai1 was overexpressed by microinjection of mRNA encoding Orai1 conjugated to the enhanced green fluorescent protein (EGFP-Orai1) 36 h after the beginning of maturation; these oocytes were incubated for an additional 8 h. At the end of the incubation period the oocytes were loaded with the calcium indicator dye fura-2 and inseminated. Changes in the intracellular free calcium concentration were monitored by means of a fluorescence imaging system. Some of the fertilized oocytes were cultured for 7 days, at which time embryo development together with the total nuclear number of the embryos were recorded. Data were subjected to 1-way ANOVA; differences between treatments were analysed using the Tukey test. The level of Orai1 protein was significantly lower in Orai1 siRNA-injected oocytes compared to the control group, as determined by Western blot, indicating successful down-regulation of Orai1. During fertilization, control oocytes displayed a series of calcium transients that lasted up to 8 h. However, in Orai1 siRNA-injected oocytes (9 out of 10 measured) only 1 calcium rise could be observed; these oocytes were unable to generate repetitive calcium signals after gamete fusion. Overexpression of Orai1 also disrupted the pattern of the normal fertilization calcium signal; the oscillation ceased after just a few Ca²⁺ rises in 8 out of 10 oocytes. The percentage of cleaved oocytes was lower in the siRNA-injected group compared to both non-injected or control siRNA-injected oocytes (38.4 ± 4.6% vs 70.5 ± 2.8% and 70.4 ± 2.4%; P < 0.05). The frequency of blastocyst (1.9 ± 0.8% vs11.0 ± 3.1% and 9.9 ± 1.4%) and the total cell number per blastocyst (19.8 ± 0.8 vs 36.2 ± 0.7 and 33.5 ± 5.5) was also significantly lower in oocytes with down-regulated Orai1 levels. These results indicate that an Orai1-mediated calcium influx is required to maintain proper calcium oscillation at fertilization and is also essential to support subsequent embryo development.