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ated Ca2+ entry is functional in eggs and it may serve to refill the endoplasmic
reticulum. The analysis of the precise link between the Ca2+ influx and the series of
Ca2+ transients in fertilized mouse eggs has revealed that the rising phase of each
transient is followed by an increased influx of extracellular Ca2+ (McGuinness et al.
1996 ). The influx is smaller but still detectable between the transients. Another
study also found that the repetitive Ca2+ oscillations in mouse eggs were associated
with a persistent Ca2+ entry triggered during the initial Ca2+ release (Mohri et al.
2001 ). The entry seemed to be store operated, and it was thought to be responsible
for refilling the stores, facilitating additional Ca2+ release cycles, and thus sustaining
the Ca2+ oscillations.
In somatic cells, store-operated Ca2+ entry is mediated by the collaboration of
stromal interaction molecule (STIM) and Orai proteins. STIM1 and STIM2 are sin-
gle-pass transmembrane proteins in the membrane of the endoplasmic reticulum
(Liou et al. 2005 ; Roos et al. 2005 ). With a canonical EF hand directed toward the
lumen, they are able to sense the Ca2+ content of the store. When Ca2+ is released from
the stores, STIM1 moves to the plasma membrane and stimulates Ca2+ entry. The
influx channels are formed by Orai proteins (Orai1, Orai2, and Orai3). Of the three
isoforms, Orai1 seems to be the most potent; it is located in the plasma membrane,
and upon interaction with STIM1, it allows extracellular Ca2+ to enter the cytosol
(Feske et al. 2006 ; Vig et al. 2006 ; Zhang et al. 2006 ). The presence of STIM1 has
been shown in frog, mouse, and pig eggs (Koh et al. 2007 ; Gómez- Fernández et al.
2009 ; Yu et al. 2009 ). Upon store depletion STIM1 proteins form small clusters
(puncta) that redistribute in regions of the endoplasmic reticulum close to the plasma
membrane. In frog eggs that display a single Ca2+ transient at fertilization, store-
operated Ca2+ entry is inactivated during oocyte maturation (Yu et al. 2009 ). In the
pig, however, it remains functional, and the downregulation of STIM1 by means of
siRNAs completely abolishes the sperm-induced Ca2+ spikes (Lee et al. 2012 ). The
channel component Orai1 has also been shown to be present in eggs. Both indirect
immunocytochemistry and overexpression of fluorescently tagged Orai1 have
revealed that the protein is present mostly in the cell cortex consistent with plasma
membrane localization (Yu et al. 2009 ; Wang et al. 2012 ; Gómez-Fernández et al.
2012 ). In pig eggs, Orai1 knockdown inhibits Ca2+ entry following store depletion
and, more importantly, abolishes the train of Ca2+ spikes following gamete fusion.
These data imply that, at least in porcine eggs, Ca2+ influx during fertilization is medi-
ated by STIM1 and Orai1 proteins and their interaction is essential to sustain the
repetitive Ca2+ signal.
Surprisingly, known inhibitors of store-operated Ca2+ entry, or the expression of
protein fragments that interfere with STIM1-Orai1 interaction, fail to prevent the
sperm-induced Ca2+ oscillations in mouse eggs (Miao et al. 2012 ; Takahashi et al.
2013 ). This seems to indicate that although extracellular Ca2+ is essential to main-
tain the sperm-induced Ca2+ spikes, the Ca2+ influx during fertilization in mice is not
regulated by the store. In mice, a Ca2+ influx mechanism has been described that is
under the control of protein kinase C (PKC; Colonna et al. 1989 ). Fluorescently
labeled PKC translocates to the plasma membrane repeatedly in fertilized mouse
eggs, and the pattern of translocation follows that of the Ca2+ transients and, also,
Z. Machaty et al.