Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs
Graphical abstract
Introduction
A large increase in the level of cytoplasmic Ca2+ is the key trigger for activation of development at fertilization in all animal species. In preparation for fertilization, mammalian germinal vesicle (GV)-intact, prophase I-arrested oocytes take up Ca2+ from the extracellular space and suppress Ca2+ leak from the endoplasmic reticulum (ER), resulting in a significant increase in ER Ca2+ stores as they resume meiosis and become arrested at metaphase II (MII eggs) [1], [2], [3]. In addition, they increase expression of the inositol 1,4,5-trisphosphate (IP3) receptor and alter ER localization such that it becomes enriched in the cortical region below the plasma membrane [4], [5], [6]. Together, these and other maturation-associated changes result in MII eggs that efficiently release Ca2+ in response to the fertilizing sperm. The initial Ca2+ release event at fertilization is induced by the activity of the sperm-specific phospholipase C zeta (PLCζ), which enters the egg’s cytoplasm following sperm-egg plasma membrane fusion [7], [8]. PLCζ catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and IP3; IP3 then activates the IP3 receptor to cause Ca2+ release from ER stores.
In mammals, periodic Ca2+ release from ER stores, known as Ca2+ oscillations, continue for several hours after fertilization. The persistence of these oscillations appears to be a critical driver of the success of embryo development [9]. In the absence of extracellular Ca2+, reductions in intraluminal ER Ca2+ levels that occur with each release event lead to cessation of the oscillations over time [10], [11], [12]. Hence, it is apparent that Ca2+ influx from the extracellular space, followed by accumulation into the ER, must occur during both oocyte maturation and following fertilization to support effective initial Ca2+ release, continued Ca2+ oscillations and optimal embryo development.
A common mechanism to replenish depleted ER Ca2+ stores via influx from extracellular sources is known as store-operated Ca2+ entry (SOCE) [13], [14]. The main molecular mediators of SOCE are STIM proteins, which sense ER Ca2+ levels, and ORAI proteins, which serve as plasma membrane Ca2+ channels. In mammals there are two STIM proteins, STIM1 and STIM2, and three ORAI proteins, ORAI1, ORAI2, and ORAI3. In response to a reduction in ER Ca2+, STIM proteins oligomerize and undergo redistribution within the ER to regions closely apposed to the plasma membrane (PM), known as ER-PM junctions. There, the STIM proteins interact directly with ORAI channels, inducing Ca2+ influx. This Ca2+ is subsequently pumped back into the ER by the action of sarco-endoplasmic reticulum Ca2+ ATPases (SERCA).
SOCE would be a logical mechanism to mediate Ca2+ entry following fertilization. Indeed, SOCE has been reported in both oocytes and MII eggs, though it appears more robust in oocytes [1], [15]. However, there is conflicting data in the literature regarding whether or not SOCE is an important physiological mediator of Ca2+ influx following fertilization. We and others demonstrated previously that chemical inhibitors of SOCE did not prevent Ca2+ influx following fertilization in the mouse [16], [17]. In contrast, several studies in both mouse and porcine oocytes suggest, instead, that SOCE is essential for this process [18], [19], [20], [21].
To definitively determine whether SOCE is required to support Ca2+ influx at fertilization in the mouse, we generated and analyzed conditional knockout mouse lines in which the oocytes lacked the major SOCE components. Here we show that there is no requirement for either STIM1, STIM2, or ORAI1 to support Ca2+ influx during oocyte maturation or to support persistent Ca2+ oscillations following fertilization. Experiments to determine what alternative channels mediate Ca2+ influx in mouse oocytes uncovered compelling evidence that spontaneous Ca2+ influx in oocytes and post-fertilization Ca2+ influx, which were previously attributed to SOCE, are instead mediated by either TRPM7, the melastatin-related transient receptor potential channel, or a TRPM7-like channel.
Section snippets
Mice
Stim1flox/flox and Stim2flox/flox mice [22] were crossed with a Zp3-cre transgenic line (Jackson Laboratory, Bar Harbor, ME, USA, Stock No. 003651) [23] to generate oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2. These mice were further intercrossed to obtain Stim1-Stim2 double cKO mice. All mice for these crosses were maintained on a predominantly C57Bl/6J genetic background, and the Zp3-cre transgene was bred through the male to avoid germline transmission of excised
STIM1 and STIM2 are dispensable for Ca2+ influx in mouse oocytes and eggs
Oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2 were generated by crossing lines carrying loxP-flanked (flox) alleles with a Zp3-cre transgenic line [22], [23]. Eggs were collected from superovulated females for immunoblot analysis to confirm loss of STIM1 and STIM2 proteins. STIM1 was detected in a lysate of 200 eggs from Stim1flox/flox (control) mice but was absent from eggs of Stim1flox/flox;Zp3-cre+ (cKO) mice, confirming that Zp3-cre causes efficient loss of STIM1
Discussion
A rise in intracellular Ca2+ is a universal activator of embryo development, and Ca2+ influx is required to sustain Ca2+ oscillations following mammalian fertilization [10], [11], [41]; however, the question of how this influx occurs is not fully resolved. We have recently shown that CaV3.2 channels contribute to Ca2+ influx in mouse oocytes and eggs, but additional Ca2+ influx mechanisms must also be functional [27]. Because Ca2+ content of the ER decreases and Ca2+ influx increases with each
Author contributions
MLB and CJW designed the experiments. MLB, EPB, and PS performed the experiments. MLB, PS, and YZ analyzed the data. MLB and CJW wrote the paper, and all authors edited the paper.
Acknowledgments
We thank Drs. Jim Putney and Gary Bird for helpful discussions and critical reading of the manuscript. We thank Masatsugu Oh-Hora for providing the Stim1-floxed mice, Stefan Feske for providing the Stim2-floxed mice, and Lutz Birnbaumer for providing Trpc-hepta knockout mice. We also thank Jean-Pierre Kinet and James Putney for providing access to Orai1 knockout mice on an ICR background. This work was supported by the Intramural Research Program of the NIH, National Institutes of Environmental
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