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 921bp. 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.

Calcium influx in mammalian eggs

“…Sperm-derived PLCz cleaves PIP 2 and generates InsP 3 that triggers Ca 2C release from the endoplasmic reticulum. Results in pig oocytes seem to indicate that STIM1 senses store depletion and transmits the empty signal to the plasma membrane where it opens ORAI1 channels and induces storeoperated Ca 2C entry (Lee et al 2012, Wang et al 2012. Recent data from mouse oocytes did not support the involvement of a store-operated entry mechanism (Miao et al 2012, Takahashi et al 2013.…”

“…Both indirect immunocytochemistry and overexpression of fluorescently tagged ORAI1 revealed that ORAI1 was present mostly in the cell cortex consistent with plasma membrane localization. Expression of ORAI1 decreased in pig eggs during maturation with STIM1 levels remaining fairly constant (Wang et al 2012); this was different in mouse eggs where ORAI1 expression levels were unaltered during maturation while STIM1 was expressed only at very low levels in the GV-stage oocyte (Gó mez-Fernández et al 2012). Despite these species-specific differences in the expression levels of these signaling proteins, the effect of the changes seems to be the same in both species: to enhance the small store-operated Ca 2C influx measured in GV-stage oocytes to a much higher level by the time the oocytes complete maturation and are ready for fertilization.…”

Ion currents modulating oocyte maturation in animals

“…Recently, they have been identified in pig and mouse oocytes and their involvement in store-operated calcium entry has also been demonstrated [GomezFernandez et al 2009;Koh et al 2009;Yu et al 2009]. In porcine, Orai1 expression decreased during oocyte maturation along with the increasing ability to generate a large calcium influx after store depletion [Wang et al 2012]. In addition, down-regulation of Orai1 expression by siRNA microinjection blocked calcium influx after store depletion and subsequent calcium add-back.…”

“…Over-expression of Orai1 in the oocytes modified store-operated calcium entry and had an inhibitory effect on the calcium fertilization signal. Co-over-expression of Orai1 and STIM1 led to a dramatic increase in calcium entry after store depletion [Wang et al 2012].…”

Ion currents involved in gamete physiology

“…Their up regulation has been shown to induce a dramatic increase in Ca 2+ entry after intracellular store depletion. Interestingly, in pig Orai1 expression decreases during oocyte maturation (Wang et al, 2012) whereas its down regulation inhibit Ca 2+ influx after store depletion and the Ca 2+ oscillations induced by the fertilizing sperm. On the other hand up regulation of Orai1 in the oocytes also modifies store-operated Ca 2+ entry and plays an inhibitory effect on the fertilization Ca 2+ signal, that results moreover in a long term negative effect on following embryo development.…”